Engineered PTCP-binding HER-family protein fusions and their
use for improving osteoprogenitor- mediated bone regeneration
By
MASSACH USETTS INSTITUTE
OF TECHNOLOLGY
Jaime J. Rivera Abreu
MAY 14 2015
B.S. Chemical Engineering
B.S. Biology
University of Puerto Rico at Mayagfiez, 2006
LIBRARIES
Submitted to the Department of Biological Engineering
in Partial Fulfillment of the Requirements for the Degree of
Doctor of Philosophy in Biological Engineering
at the
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
February 2015
C 2015 Massachusetts Institute of Technology. All rights reserved.
Signature redacted
Signature of author:
jiziiment
Certified by:
of Biological Engineering
October 24, 2014
Signature redacted
ii a G. Grffit
S.E.T.I. Pro
ssor o
iological
d Me
1i
Thesis Su
Signature
redacted
0
-
Approved by:
ing
rvisor
Forest M. White
Professor of Biological Engineering
Chair, Graduate Program Committee
2
This doctoral thesis has been examined by the following committee members:
Linda G. Griffith, Ph.D.
Department of Biological Engineering
Department of Mechanical Engineering
Massachusetts Institute of technology
Dane K. Wittrup (Chair), Ph.D.
Department of Biological Engineering
Department of Chemical Engineering
Massachusetts Institute of Technology
George M. Muschler, M.D.
Department of Biological Engineering
Cleveland Clinic
Alan Wells, M.D., DMSc
Department of Bioengineering
Department of Pathology
University of Pittsburgh
3
4
Engineered PTCP-binding HER-family protein fusions and their use for
improving osteoprogenitor-mediated bone regeneration
by
Jaime J. Rivera
Submitted to the Department of Biological Engineering
on October 24, 2014, in partial fulfillment of the
requirements for the degree of
Doctor of Philosophy in Biological Engineering
Abstract
Autologous bone marrow grafting has been shown to aid in the healing of
bone defects since the 1950s. Transplantation of freshly-aspirated autologous bone
marrow, together with a scaffold, is a promising clinical alternative to harvest and
transplantation of autologous bone for treatment of large defects. However, survival
and proliferation of the marrow-resident osteoprogenitors (CTPs) can be limited in
large defects by the inflammatory microenvironment. Ligands that can improve
CTP survival and other relevant upstream processes like colony formation and
proliferation should advance bone healing. One such ligand is the Epidermal
Growth Factor (EGF). EGF, when presented tethered (tEGF) on non-graftable
synthetic polymer substrates, induced growth and colony formation of CTPs with
additional cytoprotective effects not observed under soluble EGF stimulation.
The objective of this thesis work was to test whether tEGF can be a viable
alternative to enhance bone regeneration by tethering EGF onto a graftable,
osteoconductive matrix, beta-tricalcium phosphate (ITCP). Due to the lack
functional groups for bioconjugation on the ITCP surface, the tethering strategy
involved the use of a high-affinity PTCP binding peptide fused to the EGF domain.
This broadly-applicable tethering strategy led to retention of tethered EGF for more
than a week, while maintaining bioactivity in the bound state.
Novel methods were designed in order to study the effects of EGF-tethered
PTCP scaffolds on marrow stromal cell proliferation and on osteoprogenitor colony
formation from plated marrow. Results showed that tEGF can enhance both of
these processes. This motivated a experiment designed to test the performance of
EGF-tethered B-TCP scaffolds on a mid-sized, pre-clinical bone defect model (Canine
Femoral Multi-Defect Model; CFMD). The CFMD model revealed that both control
and EGF-tethered scaffolds promote bone formation to levels comparable to
mineralized cancellous allograft, with the tEGF condition showing signs of
advanced remodeling. However, due to a potential ceiling effect, a more
compromised bone defect model will be needed to accurately assess differential graft
performance. Altogether, this thesis demonstrates the capability of tEGF to
influence important biological processes related to bone healing, which shows
promise for its future use in bioactive graft formulations.
Thesis Supervisor: Linda G. Griffith
Title: S.E.T.I. Professor of Biological and Mechanical Engineering
5
6k
Acknowledgements
This thesis is dedicated to all those who come from underrepresented
backgrounds and put in the effort day in and day out to provide a better future for
themselves, their loved ones and, their offspring. To my father, who had multiple
jobs as I grew up in order to provide food and housing for us. He taught me
responsibility and hard work through example. To my mom, whose love and
compassion showed me how to be a better human being. Without the study habits
imparted by her I would have not accomplished this feat. To my brother, who
showed me discipline and the power of forgiveness. To the rest of my family for all
their love and support.
I want to thank Hector Castro for giving me a scholarship to be able to afford
to study at Santisima Trinidad high school. My college mentors Drs. Mildred
Chaparro and Doris Ramirez, whose MARC and SLOAN programs exposed me to
research and graduate school. Dr. David Suleiman for being the best teacher I ever
had. Mandana Sassanfar who gave me the opportunity to be part of the MITBiology summer research program. It was that summer, in Dr. Paul Matsudaira's
laboratory, where my passion for research grew as I saw myself forming part of
cutting-edge research. I thank those friends who have been of moral support and
whom which I have held many scientific conversations: Francisco Sanchez, Chris
Ng, David Weingeist, Arek Raczynski, Luis Rodrigues, Nancy Guillen, Angel
Santos, Luis Alvarez, Edgar Sanchez, Jorge Valdez, Amer Fejzic, and Maier
Avendaio. My college friends Joe, Matco, Juli, Cabo, Gus, Panchi, Gabo, Julio, Jolo.
I thank Doug Lauffenburger for the opportunity to be part of the BE
department. Aside from being a brilliant scientist and imparting an amazing vision
for the department during my interview weekend, he is a man of great patience and
wisdom. I want to thank my thesis committee for providing me with a wealth of
knowledge in various disciplines. Dane Wittrup for his knowledge of protein
engineering. Alan Wells for all his vast knowledge related to EGFR signaling, stem
cells and translational medicine. George Muschler for bringing vast orthopedic and
translational medicine experience that starts with research and extends into the
operating room. I want to thank the graduate students I collaborated with: Vivek
Raut, Melanie Rodrigues, and Austin Nuschke, whom which I spent countless hours
discussing science and planning experiments. I thank Linda Stockdale, my helping
hand for many years and Luis Alvarez, a great friend who showed me the ropes on
protein engineering. I'm grateful for MIT mentors like ex-Assistant Dean
Christopher Jones and Dean Blanche Staton provided moral support away from
home.
Last but not least, I want to thank my thesis advisor, Linda Griffith. Your
passion for science is what got me on board with this project and was a source of
constant energy throughout the years. Your grasp of engineering, biology and
translational medicine principles served as the best example of what it is to be a
scientist and a tissue engineer. I thank you for the privilege of being part of your
lab.
7
S8
Contents
1 I In tro d u ctio n ......................................................................................................................................
1.1
13
14
Bone graft alternatives for repair of critically-sized bone defects ............................
1.2 Bone marrow as a source of osteoprogenitors and its efficient clinical use for cell17
m ediated bone regeneration ............................................................................................................
1.3
Tethered EGF as a potential growth factor for bone augmentation .........................
23
1.4
Th esis objectiv es .......................................................................................................................
28
-
2 Enhancing primary human MSC proliferation on 3D beta-tricalcium phosphate (f-TCP)
scaffolds: facile surface tethering of epidermal growth factor using a newly discovered B
30
TC P b in d in g p ep tid e ..............................................................................................................................
2 .1
In tro d u ctio n ...............................................................................................................................
30
2 .2
R e su lts ........................................................................................................................................
33
33
2.2.1
I3TCP Binding Peptides Identified by Phage Display ..............................................
2.2.2
Binding affinities of EGF fusion proteins with single and concatameric BTCPbp .37
2.2.3
BPio-T-EGF in solution exhibits wild-type soluble EGF activity ..........................
2.2.4
Binding and Release of BPio-T-EGF tethered to BTCP scaffolds............................38
38
2.2.5 Tethered EGF stimulates an increase in hBMSC number on scaffolds following 740
d a y cu ltu re ...........................................................................................................................................
2.2.6 Tethered EGF does not alter plating efficiency of hBMSCs seeded onto f3TCP
sca ffo ld s ................................................................................................................................................
42
2 .3
D iscu ssio n ..................................................................................................................................
43
2.4
M aterials and M ethods ......................................................................................................
48
2.4.1
Fabrication of BTCP and BTCP-polymer composite scaffolds .................................
48
2.4.2
Phage display against TCP scaffolds .........................................................................
49
2 .4 .3
M u ta g en esis ...........................................................................................................................
49
2.4.4
P rotein Exp ression ................................................................................................................
50
2.4.5
Characterization of BPn-T-EGF binding to and elution from BTCP scaffolds..........51
2 .4 .6
C ell C u ltu re ............................................................................................................................
52
2.4.7
Validation of bioactivity of the EGF domain in fusion proteins ............................
53
2.4.8
hBMSC proliferation assays on BTCP scaffolds .........................................................
54
2.4.9
P lating efficiency assay..................................................................................................
56
9
3 Tethered EGF Enhances the Colony Forming Efficiency of Human Osteoprogenitors
Cultured on Beta-Tricalcium Phosphate Scaffolds.........................................................................58
3 .1
In trod u ctio n ...............................................................................................................................
58
3 .2
R e sults ........................................................................................................................................
62
3.2.1
Characterization of TCP substrates and protein binding .....................................
62
3.2.2 Tethered EGF enhances colony forming efficiency (CFE) without influencing
average cells per colony, cell density, or alkaline phosphatase activity ............................
63
3 .3
Discu ssion ..................................................................................................................................
66
3.4
M aterials and M ethods ......................................................................................................
70
3.4.1
BTC P Surface Fabrication ...............................................................................................
3.4.2
Synthesis and purification of EGF fusion protein with endotoxin removal.........71
3.4.3
Tethering EGF onto 6TCP coverslips ...........................................................................
71
3.4.4
Post-tethering Characterization ....................................................................................
72
3.4.5
Isolation and culture of human bone and marrow-derived cells.............................72
3.4.6
Staining, imaging and data analysis ...........................................................................
73
3.4.7
D ata validation and analysis.........................................................................................
73
3.4.8
S tatistical an alysis ...............................................................................................................
74
70
4 Evaluation of beta tricalcium phosphate scaffolds with tethered epidermal growth factor
in the canine femoral multi-defect model....................................................................................
75
4 .1
In trod uction ...............................................................................................................................
75
4 .2
R e su lts ........................................................................................................................................
79
4.2.1
BP-EGF binding, implanted dose, and stability..........................................................79
4.2.2
Cell and CTP Retention and Delivery on TCP Scaffolds..........................................79
4.2.3
Micro-CT Assessment of Bone Formation ..................................................................
81
4 .2 .4
H istology D ata .......................................................................................................................
83
4 .3
D iscu ssion ..................................................................................................................................
88
4.4
M aterials and M ethods ......................................................................................................
92
4.4.1
Animals and Experimental Design..............................................................................
4.4.2
Synthesis and purification of BP-EGF protein............................................................93
4.4.3
Tethering BP-EGF onto TCP.........................................................................................93
4.4.4
Quantification of tethered EGF and stability ...........................................................
4.4.5
Bone Marrow Aspiration and Preparation..................................................................95
10
...
.....
92
94
4.4.6
Assessment of Attachment and Retention of Marrow- Derived Cells and CTPs .... 95
4.4.7
Scaffold im plantation procedure...................................................................................
97
4.4.8
Sam ple Processing ................................................................................................................
98
4.4 9
M icro-CT Analysis ................................................................................................................
98
4.4.10
H istology A nalysis............................................................................................................100
4.4.11
Statistical A nalysis...........................................................................................................101
5
Conclusion ...................................................................................................................................
5.1
Sum m ary..................................................................................................................................102
5.2
Future Perspectives ...............................................................................................................
6
R eferences ...................................................................................................................................
11
102
104
107
12
Chapter 1
Introduction
Tissue engineers aim to achieve fundamental understanding of how tissues
work in order to methodically design treatments or engineer systems that foster
either the de novo formation of tissue, an improvement of tissue performance (i.e
treat a disease state), or allow tissues to fully regenerate after injury. The crucial
challenge is to successfully drive cell responses toward the particular phenotypes
characteristic of the specialized cells that worked together within the healthy
tissue. When injury occurs, the local stem cells within or surrounding the injured
tissue are activated and proliferate through the process of asymmetric self-renewal,
where they can create new stem cells as well as progeny that proliferate and
differentiate towards particular lineages, without depleting the local stem cell pool.
Soluble factors like cytokines, chemokines, and growth factors within the injured
tissue drive these multi-potent stem or progenitor cells to migrate into the injured
tissues to aid repair. Extracellular cues greatly influence how these cells behave in
the wound (i.e migration, proliferation), including lineage maturation.
There are many different soluble and matrix ligands associated with the
regeneration of damaged tissues, throughout early stages of inflammation (where
inflammatory cytokines are abundant), the proliferation/repair stage (where
deposition of matrix components like collagens occurs), and, the remodeling stage
(where fibrous tissue is broken down by proteases and other matrix components are
remodeled over time into a functional, native tissue structures). In cases where the
injuries are severe (on the mm or cm length scales), characterized by large tissue
loss and cell death, cells from surrounding healthy tissues need to migrate larger
distances, where oxygen and nutrient limitations become more pronounced, often
impairing cell survival. These mass transfer limitations not only affect cell viability
13
but also alter cells communication through paracrine signals, with negative impacts
cell differentiation and tissue malformation.
There are various potential interventions to guide cells within the wound to
re-form the native tissue. Some of these include: 1) transplanting a structural
lattice with particular topology and 3D structure (often mimicking the physical and
chemical nature of the native tissue); 2) augmenting the endogenous
stem/progenitor population in the cases where local stem and progenitor cell sources
are limiting (i.e large injuries) or local stem cell pool has been depleted (i.e disease
state); and/or 3) delivering soluble or tethered factors that will aid the endogenous
or transplanted stem/progenitor cells to survive, proliferate, and differentiate into
the desired cell type. Better understanding the way these cells (local or
transplanted stem/progenitor cells) respond to substrate three-dimensional
structure, porosity, surface chemistry and permeability (which can alter matrix
adsorption/deposition); mechanical properties (which can alter cell
mechanotransduction), and extracellular microenvironment (i.e growth factors and
matrix proteins, nutrients, hormones, oxygen tensions, metabolites etc), will
improve the success of synthetic, engineered microenvironments to effect bone
wound healing. The work in this thesis presents one way to modify clinical bone
grafting materials with bioactive cues to stimulate bone formation by transplanted
connective tissue progenitors.
1.1
Bone graft alternatives for repair of critically-sized bone defects
Bone grafting procedures can be traced all the way back to the 1660's, where
Job Van Meek'ren filled a bony defect in a soldiers cranium by successfully grafting
a piece of dog skull (1). Throughout the 1800's and early 1900's many animal and
clinical studies followed that showed bone grafting was a feasible way of repairing
bone defects (1). Nowadays, the consensus remains that autologous bone grafting is
14
the "gold standard" for treatment of critically-sized bone defects (2). The success of
these autogenous bone grafts is tied to the fact that such grafts provide an
osteoconductive structural support, osteoinductive factors and osteogenesis
potential (at least in the case of vascularized and cancellous autografts where a
fraction of osteoprogenitors survive transplantation) (3). Autologous bone grafts, -vascularized or non-vascularized, cancellous or cortical -- have strengths and
weaknesses in clinical use. On the positive side, autografts generally form better
bone unions, with enhanced vascularization, integration, and superior mechanical
stability in shorter time scales than allografts. The limitations include limited
availability of autograft bone (especially for large segmental defects), considerable
donor site morbidity, chronic pain, and debilitating symptoms (3-5).
Currently, allografts tend to be the second best option when autologous
sources of bone are insufficient to fill the need or the patient decides against
autologous bone harvest. Allografts provide an osteoconductive, structural support
matrix with some of the osteoinductive cues that can promote local or transplanted
osteoprogenitors to differentiate into osteoblasts and deposit bone matrix (6-8).
There are a variety of bone allografts available with varying anatomy (cortical,
cancellous or osteochondral), processing (fresh, frozen, freeze-dried or
demineralized), handling (powder, particulate, gel, etc.), and, sterilization (ethylene
oxide, gamma-irradiated, chemical soaking, etc.) procedures (3). The disadvantages
of using processed allograft typically include: higher rates of non-unions; loss of
angiogenic potency; compromised mechanical strength and osteoinduction; donor
variability; lot-to-lot variability; disease transmission (viral, HepB and HIV);
infection (bacterial and fungal); and, immune reaction with possible graft rejection
(3,9-11). Tissue bank registration of the disease state of the donors and application
of sterilization procedures have aided in the reduction of disease transmission and
infection. However, while these sterilization procedures prove to reduce disease
transmission and infections, they also reduce the osteoinductivity of allografts,
attributed to denaturation/degradation of osteoinductive factors during this process
15
(12-14). Other processing methods, like freeze-drying and demineralization, have
reduced immunological rejection, likely by denaturation/degradation of in-graft
HLA antigens (15). The lack of standardization of these sterilization procedures
across tissue banks has led to additional variability in the performance of allografts
(10).
Due to the aforementioned downsides and limitations of both autograft and
allograft use, there is much interest in developing synthetic (alloplastic) grafts -polymers, metal, bioactive glass, or calcium phosphates -- that mimic the
performance allo- and autografts. Most of the current resorbable, calcium
phosphate-based synthetic grafts used in the clinic (e.g. beta-tricalcium phosphate;
BTCP), are solely osteoconductive and do not provide the osteoinductive factors nor
the osteogenesis provided by osteoprogenitors. Since the first reports by Conolly and
Shindell in the 1980's that bone marrow transplantation alone or in combination
with bone grafts provided some osteogenesis potential, orthopedic surgeons have
been combining both allografts or alloplastic (synthetic) grafts with autologous bone
marrow aspirates for repair of critically-sized defects or non-unions. These marrowloaded synthetic grafts have potential for osteogenesis, but they still lacked the
osteoinductive cues present in auto- and allo-grafts to stimulate the population of
osteoprogenitors and supporting cells that reside within the bone marrow and other
tissues.
In 1965, Urist demonstrated osteogenesis in vivo from demineralized bone
matrix, a finding that led to work on extraction of soluble osteogenic factors from
natural bone matrix. Additional studies by Urist (8), Sampath ((16,17)), Wang
((7,18)), and Ozkaynak ((19)) led to the characterization of BMPs (e.g. BMP2A and
OP-1) and the demonstration of the capability of these molecules to induce bone
formation in vivo when implanted subcutaneously in rats (7). Since then bioactive
synthetic grafts that use osteoinductive factors in their formulations (like BMPbased products like OP-1 {BMP-7} and BMP2-Infuse {BMP-2}) prior to
16
osteoprogenitor transplantation have yielded great success in a variety of wounds.
However, their use in the clinic is limited by the high costs of current products on
the market and several recent complications due to off-label use (2,20,21). Other
downsides to current delivery of osteoinductive factors is that their release upon
hydration is mostly controlled by diffusion, which can lead to undesirable effects
like: bolus doses; rapid cell-mediated ligand depletion; and, leakage into blood
stream; which can potentially cause local cell receptor downregulation, subsequent
ligand desensitization, and, off-target ill-effects. Hence, proper delivery strategies
along with discovery of other bioactive ligands that can contribute to the relevant
processes leading to bone regeneration (22) need to be explored to establish more
biologically robust and cost-effective alternatives to bone grafting. Properly
engineered, synthetic bioactive grafts manufactured within GMP-certified facilities
will ultimately reduce lot-to-lot variability and improve clinical outcomes.
Additionally, better methods for osteoprogenitor isolation and transplantation
strategies will also go hand-in-hand with the clinical success of these synthetic,
bioactive bone grafts.
1.2
Bone marrow as a clinical source of osteoprogenitors for cellmediated bone regeneration
Percutaneous bone marrow injections have been used since the end of 1950's
in the repair of non-unions (23). Work from Friedenstein elucidated that this boneforming property of bone marrow was likely attributed to fibroblast-like cells within
the marrow (24). Through histological assessment, these cells were found to be
capable of forming osteogenic foci with surrounding mineralized, bone matrix when
transplanted into mice in diffusion chambers (24). Hence, the power of bone marrow
injections was attributed to these cells that had the capability to differentiate and
form bone matrix in vivo. Further work with autologous bone marrow, led mostly by
Connolly, demonstrated healing of critically sized bone defects in various animal
17
models and humans when used alone or in combination with demineralized bone
matrices (DBM) (25-27).
These early studies provided compelling evidence of the potential therapeutic
value of bone marrow or subpopulation of cells within it. Spurred by the
translational needs, the capability of plated bone marrow to form foci or colonies
was developed as a standard method of characterizing osteoprogenitor prevalence in
marrow. The heterogeneous population of stem and osteoprogenitor cells present
within bone marrow (and other tissues) and capable of forming foci/colonies in vitro
are referred to as Connective Tissue Progenitors (CTPs; (28)). Attempts of isolation
of subpopulations of CTPs that are essential to the process of bone formation in vivo
led to the discovery of a subpopulation of clonogenic, multipotent stromal cells now
referred to as mesenchymal stem and progenitor cells (MSCs) (29,30). MSCs were
isolated based on several surface markers present on fibroblast-like colony-forming
cells (30,31). The population was labeled as multipotent because in vitro assays
demonstrated multiple lineages (e.g. osteoblasts, chondrocytes, adipocytes, etc.)
could be derived from at least some of the cells after multiple passages, but it was
also noted that only a small percentage of cells in such culture-expanded
populations have true multi-lineage potential, hence the canonical "MSC"
nomenclature is now accepted to refer to a mixed population of skeletal stem cells
and committed progenitors (30). These cells are now known to be capable of
differentiating into additional lineages and can be isolated from various tissues
within the body including bone marrow, adipose tissue, placenta umbilical cord,
umbilical cord blood, peripheral blood, and dental pulp. These stem and progenitor
cells are of much interest today due to their therapeutic value to support tissue
regeneration in vivo and their immunomodulatory properties (32), however the use
of culture-expanded cells for bone regeneration has not become a clinical standard,
due to cost and other factors.
18
As MSCs gained popularity as potential therapeutics for a variety of diseases,
the need for standardization of isolation procedures and cell markers characterizing
MSCs became apparent, and a best practices consensus on how to isolate and
characterize MSCs has been described (31). In vitro, the clonogenic capability of
MSCs is obvious, as serial dilutions of these colony-forming cells yields a linear
correlation on colony forming efficiency (33) (bringing some doubt on the
hypothesized role that cell density may have on the formation of osteogenic foci
((24)). Interestingly, non-adherent populations within the marrow can be re-plated
and lead to additional colony forming units (34-36), highlighting the problem that
by plating marrow once, one cannot effectively close the mass balance on the total
number of CTPs in marrow. Substrate composition also influences CTP colony
forming efficiencies (35,36), likely due to a convolution of substrate adhesiveness,
cell attachment, migration, deposition of matrix and/or accumulation of cytokines
and growth factors that would promote the process of colony formation. All these
shine light on the complex, highly-interactive microenvironment that governs the
multiple steps in formation of osteogenic colonies in vitro.
Generating in vitro models that help predict in vivo performance has been
hard to establish. One can only extrapolate certain cell behaviors, which are
important biologically in the process of bone formation (e.g. activation, attachment,
survival, proliferation, migration, colony formation and/or osteogenic differentiation;
Figure 1-1 (22)). For example, although bone markers are important for
determining the osteogenic potential of cells within the marrow, no threshold or
expression level can be appropriately correlated with in vivo bone formation (37).
However, if bone marker expression is not present in vitro, most likely these cells
will not be able to follow the path towards osteogenic differentiation and bone
matrix deposition in vivo.
Optimization of autologous bone marrow or MSC/CTP transplantation along
graft vehicles requires consideration of some intrinsic limitations that the
19
microenvironment of the bone wound presents. The lack of oxygen, nutrients and
pro-apoptotic inflammatory microenvironment can induce the death of transplanted
bone marrow cells and osteoprogenitors within the first few days after
transplantation (-30-50% in first few days; (38)). One of the reasons for this quick
death is that diffusion distances required in healing of bone defects are much larger
than typical diffusion of nutrients and oxygen from bloodstream into surrounding
healthy tissue (-50x greater; (22)). Hence, due to the longer time scales involved in
vascularization of bone wound/grafts relative to the ability of osteoprogenitors to
survive early after transplantation, necrotic regions tend to form within the center
of the grafts with large amounts of apoptosis. This can cause a significant reduction
the number of osteoprogenitors that make it to the remodeling stage of wound
healing. In cases of high metabolic load (e.g. transplantation of large number of
osteoprogenitors), excessive necrosis can cause a chain of local inflammatory
response to cell debris that will further reduce cell survival. For this reason,
transplantation of large number of cells does not necessarily translate to more bone
formation. However, there is consensus that some minimum amount of cells (defectdependent) is needed to improve bone formation, as confirmed by better bone
healing of grafts loaded with BM, CTPs or MSCs relative to grafts without them
(37,39-43).
Culture methods or extracellular factors that can increase the tolerance level
of the transplanted cells and aid in survival of these should in theory help the
process of bone formation. Bone cells in general are quite resilient under low oxygen
tensions of the bone wound, but due to the large mass transfer limitations in the
wound, there is still expected to be significant amount of death near the center of
the defects. It is not clear whether rapid vascularization of the grafts can help
mitigate these effects, as the time scales for vascularization of large defects are on
the order of days, and cell survival is governed by events on shorter time scales.
Still, most osteogenic grafts have pore sizes that will allow for cell penetration and
blood vessel ingrowth as vascularization is absolutely required for healing. Other
20
alternatives to mitigate the mass transport problem in vivo focus on pre
conditioning cells to lower oxygen tensions (hypoxia) (44), (45) and pre-culturing
under osteogenic conditions has yielded some success in enhancing cell survival and
bone formation in vivo. This can only be done with culture-expanded cell
populations - arguably a less preferable alternative in terms of cost and risk (46).
Intraoperative alternatives to improve transplantation focus on better
concentration and selection of osteoprogenitors from marrow aspirates with removal
of unwanted soluble osteogenic inhibitors (serum or other soluble inhibitors that
may be present; (45)) or cells types (Immune cells, RBCs, etc.; (47,48)) that don't
contribute to the formation of the desired tissue. Additional cell types not directly
contributing to the process of bone formation would reduce the capability of CTP
colony formation and/or contribute to metabolic load without much benefit in bone
deposition. Efforts to evenly distribute these cells within the grafts is also of
interest, as this reduces local metabolic loads and also allows for more even
deposition of bone matrix which should allow for larger interconnectivity,
mechanical stability, and better integration to host tissue.
Overall, the force driving the intraoperative use of bone marrow aspirates is
the presence of osteoprogenitors in the marrow, which are linked to improved bone
regeneration outcomes (27,39,49-52). Muschler and co-workers defined a new
surgical technique for iliac crest marrow aspiration that generates maximal number
and concentration of osteoprogenitor without excessive dilution from peripheral
blood (53). Intraoperative use of BMAs typically involves mixing of the marrow with
the bone graft of choice to wick its surface and incorporate CTPs in the process. This
mixing, although simple to perform, has low CTP transplantation efficiencies. To
further enhance the use and efficacy of bone marrow aspirates, Muschler et al.
developed the Selective Cell Retention (SCR) technique (54). In SCR, heparinized
marrow is flowed over bone grafts to achieve a combination of flow-induced
adhesion and physical entrapment (in graft pores) of CTPs, enhancing CTP
prevalence by a factor -2x and increasing total CTP concentration by a factor of -4
21
when compared to simple mixing of the marrow with the grafts (54-56). This
consequently reduces the number and concentration of other cell populations that
are not known to contribute to osteogenesis, reducing the overall metabolic load in
the graft and thus potentially boosting the probability that CTPS will survive
(22,47,48). The in vivo efficacy of CTP concentration through SCR technology was
assessed in a canine femoral segmental defect model where SCR with DBM+CC
allograft treatment showed equivalency to an iliac crest autograft (55). The use of
SCR intraoperatively for CTP transplantation in bone grafts has yielded successful
results in other animals and clinical studies (54-59). The ability of intraoperativeSCR to improve bone augmentation with either allograft or synthetic scaffolds
makes it a versatile technique that is inexpensive and significantly improves
clinical outcomes, without significantly increasing time in the operating room.
Modifications to grafts that allow for better retention and selection of CTPs through
SCR should help improve outcomes, especially in large defects where the need for
osteoprogenitors is greater and the soft-tissue damage surrounding the bone defect
decreases local tissue sources of these. This can potentially be done by modification
of grafts with adhesive ligands like hyaluronic acid, which is know to select for
CTPs (60). Additional improvements to SCR related to the final distribution of these
cells within the grafts will aid in spatially distributing the metabolic load of the
transplanted cells and have a more uniform bone deposition along the bone graft
surface.
In summary, although these in vitro and in vivo studies using bone marrow,
MSCs or CTPs loaded onto bone grafts are highly context dependent (e. g. animal
&
model used, type of defect, cell culture conditions [substrate type: stiffness, ECM
growth factor deposition, passage number, cell doublings, seeding density, oxygen
tension, media composition], type of graft [resorption/dissolution rates, amount used,
handling properties, mechanical properties], seeding protocol [static or convective,
transplantation density, etc.]. External factors that improve cell survival (e.g
culture under lower oxygen tensions and nutrient/serum limitations), migration,
..--- - -...
- -
22
colony formation, proliferation without hampering osteogenic differentiation should
translate to better bone formation if other contributing factors are properly
controlled for (e. g. cell distribution in grafts, cell density, metabolic load,
contaminating cell populations, and soluble anti-osteogenic factors, etc). Bioactive
ligands within grafts that can allow for better retention of osteoprogenitors within
grafts through improvement of SCR should also be beneficial in one way or another
to the process of cell-mediated bone wound healing. Acquiring additional knowledge
on fundamental stem and progenitor cell biology and applying engineering
principles in the design of cell-loaded bone grafts will arguably lead to bioactive
synthetic grafts that approach performance levels comparable to autograft.
1.3 Tethered EGF as a potential growth factor for bone augmentation
Desired characteristics in choosing a growth factor for bone wound healing
include positive influences on CTP cell, adhesion, survival, migration, and
proliferation without impairment of differentiation. These processes are integral to
the process of bone regeneration in vivo (Figure 1-1; (22)). CTPs in bone marrow are
reported to be in a quiescent state in vivo (61-64), and are activated upon
extracellular stimulation (33,65-67). Friedenstein noticed that osteoprogenitor cells
from marrow formed foci in vitro that contained bone matrix, suggesting the need
for colony formation in order to induce the process of intramembranous ossification
in vivo. In vitro colony assays used to assess the bone-forming potential of marrowderived CTPS intrinsically require cell adhesion, and minimally adhesive substrates
are know to reduce colony forming efficiency (35,36); it is attractive to speculate
that some of the features of in vitro colony formation capture the processes in vivo
whereby the progenitors proliferate to create more bone-forming cells. Survival is
also an important part of the process of colony formation, as some sensitive cell
populations might not adapt to the new environmental conditions, particularly
those in vivo that the bone wound presents. Additionally, implanting cells into a
23
bone wound devoid of vasculature (hypoxic and lacking nutrients) and filled with
inflammatory cytokines, presents a survival challenge to any live cell, transplanted
or resident.
Since early characterization of bone-marrow derived CTP progeny (MSCs and
other subpopulations), it was noted that these cells possessed tyrosine kinase
receptors (RTK's), including EGFR, although EGFR expression was lower and more
variable than other receptors like PDGFR (68). It was noted that both soluble EGF
and PDGF were main factors in the formation of colonies from plated marrow (CFE)
and also capable of influencing their colony size (colony parameter that depends on
both cell migration and proliferation). Single-colonies possessing EGFR were also
capable of bone formation in vivo, which correlated well with the notion that bone
cells possess EGFR in vivo (69) and that EGFR plays an important role in bone
formation and osteoclast function (70-72). As MSCs gained traction for potential
therapeutic use, the number of investigations of growth factors like EGF capable of
promoting ex-vivo expansion of CTPs has grown. EGF has been demonstrated to
stimulate cell migration in MSCs (73) without hampering osteogenic differentiation
(73). However, soluble EGF interacts differently than tethered EGF. Soluble EGF
was not capable of improving colony formation from plated marrow under a variety
of adhesion contexts (36) and does not protect MSCs from pro-apoptotic TNF-family
ligands (74,75).
Due to the benefits of EGF on osteoprogenitors, delivery of EGF in grafts is an
attractive strategy for improving bone regeneration. However, most non-covalent
delivery methods rely on protein adsorption and lyophilization of ligands, which can
cause problems upon rehydration due to bolus release leading to rapid
internalization of EGF receptors on near-by cells and leakage into blood stream
which can cause some of off-target effects. With this in mind, Kuhl and Griffith ((76))
tethered EGF onto substrates and demonstrated stimulation of proliferation of cells
could be stimulated from the solid phase, eliminating many downsides to soluble
24
EGF delivery. Their work showed that EGF is competent to signal mitogenic and
morphological responses when tethered to substrates. Further work expanded on
this, revealing that not only EGF but insulin (77) and other growth factors like
FGF-2 can signal tethered (78). The mechanisms by which tethered EGF can still
induce proliferation were elucidated using in vitro models of EGFR dynamics. Using
an internalization deficient EGFR cell line model, it was observed that upon EGF
stimulation proliferation was still maintained (79). This was due to the fact that
EGF was still capable of activating EGFR, and that activated, surface-restricted
EGFR was capable of activation of the MAP kinase (MAPK) pathway, the main
pathway responsible for proliferative response of cells from EGF stimulation.
Additionally, it was observed that motogenesis was promoted when EGF was not
internalized, and this was attributed to more prolonged activation of intracellular
signaling ligands that occur only near the cell surface, like PLC-gamma (80). Aside
from this, EGF tethered to beads was able to form engage integrin receptors and
initiate actin-polimerization networks via the Arp2/3 complex (81). These
interactions seem to lead to increased cell attachment and cell spreading in MSCs
(74).
The biasing of intracellular signaling ligands and their differential temporal
activation leads to different phenotypes in cells. Aside from conservation of receptor
numbers due to lack of receptor internalization when EGF is tethered (tEGF), there
is prolonged phosphorylation of EGFR and ERK1/2 ligands (74,82-85). This
differential signaling seems to influence CTP and MSCs phenotypes related to the
process of bone ingrowth (Figure 1-1), like cell survival, migration, proliferation,
colony formation, and osteogenic differentiation. The pro-survival effects of tEGF
were evidenced by the protection of MSCs from TNF-family ligand-induced
apoptosis, whereas soluble EGF did not provide protection (74,75). tEGF was also
able to protect MSCs under the combined threats of hypoxia, serum-depravation,
and pro-apoptotic ligands, mimicking to a certain extent the threats to cell survival
that would be faced during early transplantation into bone wounds (75). Cell
25
adhesion and spreading (74) along with motogenesis (86) was enhanced under
similar substrates. When bone marrow aspirates from 39 donors were plated in
vitro under tEGF, CTP colony formation efficiency (CFE) increased regardless of the
adhesive background, without alteration of osteogenic marker expression (36).
However, soluble EGF was detrimental to colony formation under the same
conditions (36). Previous work has shown that expansion of MSCs can be readily
enhanced by addition of sEGF to media (68,73,87), but experiments performed with
other cell types (including fibroblasts), show that proliferation under tEGF is higher
that what can be achieved with soluble EGF concentrations above its EC50 (82,84),
an effect attributed to due to conservation of EGFR number under tEGF and no
subsequent loss of ligand sensitivity as seen with sEGF (88,89). Here in Chapter 2
we show that MSC proliferation can be enhanced under tEGF. The sustained
activation of EGFR under tEGF seems to contribute to enhanced MSC osteogenic
marker expression and matrix mineralization seen by Platt et al when osteogenic
cues are present in media (85), but other studies with MSCs or CTPs do not observe
alterations in osteogenic differentiation (36,75). These indicate that at minimum,
tEGF does not impede CTP nor MSC osteogenic differentiation.
All this scientific evidence regarding the effects of tEGF on osteoprogenitors
make it an attractive ligand for bone augmentation, due to its capability to
influence cell adhesion, survival, migration, colony formation, and, proliferation
without impeding differentiation of these CTPs.
26
TiiE JOURNAL
ENGINEERING PRINCIPLES OF CLINICAL
CELL-BASED TissuE ENGINEERING
o0
BONE & JOINT SURGERY - JBIS.ORG
VOLUME 86-A - NUMBlER 7 JULY 2004
Vascularization
'10,
Remodeling
Differentiation
Migration
Proliferation
Attachment
Figure 1 -1. (Figure and description taken from (22)). Stages of bone tissue ingrowth.
The sequential stages in the formation of new bone tissue are illustrated. Attachment
and/or activation of a stem cell (green) is followed by continued proliferation and migration
of the resulting progeny, forming a clone or colony of new cells. Less mature and more stemcell-like progenitors continue to proliferate and migrate at the periphery of the colony
(lighter green). Differentiation is characterized by the elaboration of an appropriate tissue
matrix, beginning in the center of the colony. In this case, the tissue formed first is woven
bone, although cells may also follow a pathway that results in cartilage formation or direct
apposition of new lamellar bone. Elaboration of a mature bone pheno- type does not occur in
the absence of a new or existing local blood supply (i.e., a sufficient local oxygen tension).
Remodeling involves the coupled process of osteoclastic bone resorption followed by
recruiting and activation of additional stem cells and progenitors from upstream
osteoblastic cells in bone marrow.
27
1.4 Thesis objectives
The efforts in this thesis are focused around the improvement of
osteoprogenitor mediated bone regeneration within bioactive synthetic bone grafts.
The overall aim is to impart osteogenesic and osteoinductive properties to synthetic
bone grafts by (a) inclusion of osteoprogenitor cells and (b) modification of scaffolds
with tethered EGF to enhance cell survival and function in vivo. The hypothesis is
that cell phenotypes seen in vitro under tethered EGF stimulation will translate
into better bone formation in vivo. Combination of bioactive ligands with better and
improved methods of osteoprogenitor isolation (53), concentration (47) and selection
(SCR; (54), along with the application of engineering principles governing wound
healing (22), should lead to synthetic grafts that better resemble the performance of
autograft bone.
One of the most common synthetic matrices for bone grafting is Betatricalcium phosphate. Composite grafts made up of BTCP/hydroxyapatite and
BTCP/collagen as well as pure BTCP have been widely used in animal models and
in the clinic for several bone healing applications (90-96). Its wide-spread use led us
to use it as the osteoconductive substrate for presentation of tethered EGF. Protein
adsorption is not valid means for delivery in vivo due to bolus release and lack of
prolonged retention in the tethered state. Hence, one main challenge to overcome is
the delivery of bioactive ligands, like tEGF, within these BTCP grafting materials.
The solution to this problem intrinsically necessitates either covalent modification
of bone grafts or means of high-affinity interactions on the surface that will prevent
rapid release of the growth factor upon implantation. Additionally, bioactivity needs
to be retained in the tethered state - a non-trivial challenge, as many proteins
denature when placed in contact with surfaces (97).
In this thesis, a high-affinity BTCP binding peptide discovered by Luis
Alvarez in the Griffith laboratory (PCT/US2011/063592) was used to reproducibly
28
tether EGF for periods of time relevant to the process bone wound healing. The
binding of BTCP binding peptide-EGF (BP-EGF) fusion proteins to BTCP substrates,
as well as release from the substrates, was characterized. Additionally bioactivity of
the EGF domain in BP-EGF was assessed both in solution and tethered form by
induction of MSC proliferation. The ability of EGF-tethered BTCP substrates to
induce colony formation of CTPs was evaluated using cells from bone marrow
aspirates and trabecular surface isolations from 8 patients. For these, customized
BTCP coverslips were used to accurately extract relevant colony parameters in
accordance with an established American standard for characterization of colony
forming unit assays (ASTM #F2944-12; (98)).
Finally, in order to assess the influence of tEGF on bone formation, EGFtethered BTCP scaffolds were tested in a canine femoral multi-defect model (CFMD).
Canines represent a good animal model for bone regeneration by various biological
metrics (99), making it suitable for preclinical evaluation of bioactive grafts. The
CMFD model provided multiple cylindrical defects (1.0cm x 1.5cm) into which the
TCP scaffolds were implanted, with assessment of both controls (naked BTCP
scaffolds) and treatment conditions (tEGF-BTCP scaffolds) within each femur. There
was no need for external fixation, hence the model is load-bearing. Control and
EGF-tethered scaffolds were implanted into defects and bone formation after 4
weeks was assessed by a combination of micro-CT and histology.
29
Chapter 2
Enhancing primary human MSC proliferation on 3D
beta-tricalcium phosphate (B-TCP) scaffolds: facile
surface tethering of epidermal growth factor using a
newly discovered B-TCP binding peptide
The main findings in this chapter include contributions from collaborators, and are
being submitted for publication with the following attributions:
Alvarez LM*, Rivera JJ*, Stockdale L, Saini S, Lee RT, Griffith LG. "Enhancing
primary human MSC proliferationon 3D beta-tricalciumphosphate (BTCP) scaffolds: facile surface tethering of epidermal growth factor
using a newly discovered B-TCP binding peptide"
*Denotes equal contribution
2.1 Introduction
Bone grafting procedures in the USA top the half-million mark annually and 2.2
million worldwide (2,31,100). They represent an approximate 1.5 billion dollar
industry in the USA alone (2,28,30,100). These procedures are a requirement for
healing of critically-sized bone defects, including non-unions, cavities and segmental
defects. Within the spectrum of bone grafting alternatives, autogenous cancellous
bone graft is the most common treatment of non-unions (40-50%) (2,53,101).
Autologous bone is the gold standard in treatment of non-mineralized matrix as it is
a vascularized graft that provides osteogenic cells with proper osteoinductive
stimulus that enhances cell-mediated repair. However, the available amount of
autologous bone is often insufficient to treat large defects and the primary
alternative graft approach, cadaver bone, has clinical shortcomings ranging from
risk of disease transmission to relatively poor long-term function.
30
Synthetic scaffolds that can recapitulate the ability of autologous bone to
promote bone regeneration would therefore be of great benefit in the clinic. Such
scaffolds would eliminate the need to harvest bone from patients and might allow
graft properties to be tailored for individual patient needs. Unfortunately, most
synthetic grafts, although osteoconductive, fall far short of the performance level of
autogenous bone or cancellous allografts, as they lack proper vascularization,
osteoprogenitor cells, and/or osteoinductive cues. Osteoprogenitor cells differentiate
into osteoblasts and produce the bone matrix (osteoid) that later mineralizes and is
remodeled into lamellar bone, hence these cells are essential for bone regeneration.
Osteoprogenitor cells arise from differentiation of connective tissue progenitors
(CTPs) (39,53,54,102), a heterogeneous population that includes multipotent
mesenchymal stem cells (MSCs) (31,54,55,57,60) (28,30,38,44,48,103).
Osteoinductive cues are important in synthetic grafts as they can help recruit and
stimulate near-by, tissue-resident stem and progenitor cells to participate in the
regeneration process. However, in many defect situations, the local environment is
relatively depleted of stem and progenitor cells and thus supplementation of the
graft with these essential cells is likely necessary to ensure healing.
CTPs are present in bone marrow aspirates, making marrow an attractive
therapeutic source of osteogenic precursors when stem and progenitor cells for graft
augmentation. Optimization of CTP isolation (22,53) and transplantation strategies
(22,39,54,102,104) has led to improved bone healing in animal models
(54,55,57,60,105). However, the hypoxic, nutrient-limited, and inflammatory
microenvironment of the bone wound can cause death of a substantial fraction of
transplanted cells within the first few days (38,44,48,69,71,72,103), reducing the
effective number of osteoprogenitors that contribute to the proliferative and
remodeling stage of wound healing (22,66,68,73,105). We thus hypothesize that
providing bioactive cues that stimulate survival and proliferation of connective
31
tissue progenitors within grafts, without interrupting terminal osteogenic
differentiation, will improve the outcome of bone healing (22,79,88,104).
Epidermal growth factor (EGF) stimulates survival, colony formation, and
proliferation of CTPs in vitro without interfering with subsequent osteogenic
differentiation (74,85,105). EGF binds to the EGF receptor (EGFR), an essential
regulator of both bone development and bone tissue homeostasis (69,71,72,74). In
vitro, EGF can induce proliferation, colony formation and colony growth of MSCs
(66,68,73,86,105). However, EGF drives EGFR internalization, resulting in
downregulation of EGFR and ligand desensitization when cells are exposed to
moderately high ligand concentrations (i.e., above the KD for ligand-receptor
interactions, ~1nM) (74,75,79,88).
Receptor downregulation can be avoided by presenting EGF in tethered rather
than soluble form, such that it binds to the EGFR but the EGF-EGFR complex is
physically restrained from internalization and downregulation by the tether that
links EGF to the culture substrate. Such tethered presentation restricts EGFR
signaling to the cell membrane, resulting in sustained EGFR activation and
different patterns of intracellular signaling and phenotypic responses compared to
soluble EGF (74,75,85,106). MSC spreading (74,90-93) and migration (86,94-96) are
enhanced with tethered compared to soluble EGF. A particularly attractive MSC
phenotype elicited by tethered EGF, but not soluble EGF, is protection of MSC from
pro-death hypoxia and inflammatory cytokine cues (74,75,107-110) while
preserving proliferative and osteogenic potential (74,75,85,106,111). This collection
of beneficial effects of tethered EGF on cultured MSCs and CTPs suggests that
modifying scaffolds with tethered EGF may enhance bone formation by
transplanted CTPs by protecting them from death and stimulating proliferation.
Beta-tri calcium phosphate (BTCP) scaffolds and composites thereof serve as a
substrate for tissue-resident or transplanted MSCs or CTPs, evidenced by their
32
performance in numerous animal studies (90-93,112) and its current use in a
variety of bone void fillers in the clinic (94-96,113). However, BTCP does not have
any intrinsic biological stimulatory activity and is not amenable to covalent
conjugation of biomolecules. Modification of BTCPscaffolds is thus limited to
adsorption/freeze drying strategies, which often result in bolus release due to the
relatively low affinity of biomolecules for the BTCPsurface.
High-affinity binding peptides, derived from combinatorial screens of peptide
libraries against the surface of interest, are an appealing means to tether proteins
to surfaces that lack functional groups for covalent modification (107-110,114). High
affinity peptides identified by screening are fused to the ligand of interest by a
peptide tether, thus creating a fusion protein that binds to the surface with high
affinity and presents the ligand in an accessible, bioactive form.
Here, we report the results of our screen of a commercial 12-mer phage display
library to identify high affinity BTCPbinding peptides. We used affinity maturation
strategies to obtain a peptide that binds tightly to BTCPafter fusing it to EGF and
other domains. Further, we describe an efficient scheme to concatamerize the
binding peptide and show that concatamerization up to a 10-mer increases the
affinity of the EGF fusion protein for BTCP. The bioactivity of EGF tethered to
clinically-approved BTCPscaffolds was demonstrated by assessing the plating
efficiency and in vitro proliferative response of human MSC over 7 days in culture.
2.2 Results
2.2.1 BTCP Binding Peptides Identified by Phage Display
-
Three rounds of panning yielded plaques for three of the six conditions
BTCP blocked with BSA, BTCP blocked with OBB buffer (non-mammalian blocking
33
buffer), and BTCP -PLGA composite blocked with OBB buffer. Mock conditions
(tubes only) and BTCP -PLGA blocked with albumin (BSA) did not yield plaques at
the 2nd and 3rd round respectively. The sequence Leu-Leu-Ala-Asp-Thr-Thr-His-HisArg-Pro-Trp-Thr was identified in a total of 28% (8/29) of the clones: 5 from BTCP
blocked with BSA; 2 from BTCP blocked with OBB protein buffer and 1 from
composite TCP -PLGA blocked with OBB buffer, (Figure 2-2A). The remaining 21
clones showed only modest sequence similarity based on the BLOSUM62 scores
(Figure 2-2A).
The 12-amino acid consensus sequence (Mw= 1448 Da) includes one
negatively charged residue (Asp), one positively-charged residue (Arg), and has a
predicted pI of 6.92. Interestingly, the sequence includes two histidines (nominal
pK of 6.1), which may become protonated in the low-pH environment of postsurgical inflammation or abstract protons from the calcium phosphate surface. The
peptide is predicted to be relatively soluble based on a grand average of
hydropathicity (GRAVY) score in the moderately negative range (-0.800). The
.
extinction coefficient (water) at 280 was determined to be 5500 M 1 cm-1
34
EGF
MBP
Tether
BP units
LLADTTHHRPWT
Figure 2-1. Structures of BTCP scaffolds and EGF - BTCP binding peptide
(BP-T-EGF) fusion protein. (Top) Macroscopic appearance of a 5mm BTCP Therilok
cross-shaped scaffold used for cell culture experiments. These scaffolds were crushed to a
coarse powder for phage panning to select binding peptides. (Middle) SEM micrographs of
the surface of BTCP scaffolds at low, intermediate and high resolution as indicated by scale
bars (Bottom) Structure of the fusion protein "BPio-T-EGF" comprising the 12-amino acid
BTCP-binding peptide fused to EGF by flexible protease-resistant tethers flanking a coil
domain (see supplementary materials for specific sequence).
35
A
B
BP n-T-EGF
-
- --- -PT
SSG
----- VPQ PY VPSHK
--PL
HNMAPA
2
10
-
-
-
-
-
5
-
Tethering Concentrations (iM):
-
PVL ----
1.62 4.88 14 64 ] 0.83 2.50 7.50
C
1X10 1
XK100,
Cn
0
0)
C,,
1.0x10-1
Ul
0
U
-
-
-
AA*P ----HTTPT
QYGVVSHLTT ----ESNP I L ISV ----- - -IGR I TIAL HP DPSPWLRSER ---- ESSMFQEGHR----KP F TRYGDVAI --- PFGARILSLN ---LSN$MSSLS - - -NMPAKI FAAM ----- EPTKEYETSYHR - - - -DINELYLRSLRA - --DYDSTHGAVFRL ---- -SKHERYPQSEEM ---HTHSSDGSLLGN - - -NYD SMS EPRSHG ---- -ANPI SVQTAMD - --
0.88 2.64 7.92
,
-ISFIS
-
TCPOBB1/1-12
TCPBSA19/1-12
TCPPLGA21/1-12
TCPOBB2/1-12
TCPBSA11/1-12
TCPBSA 10/1-12
TCPBSA18/1-12
TCPBSA14/1-12
TCPOBB/1-12
TCPPLGA29/1-12
TCPBSA13/1-12
TCPBSA17/1-12
TCPBSA15/1-12
TCPBSA1211-12
TCPPLGA26/1-12
TCPPLGA23/1-12
TCPPLGA22/1-12
TCPPLGA20/1-12
TCPPLGA28/1-12
TCPOBB/1-12
TCPOBB81-12
TCPOBB6/1-12
TCPPLGA25/1-12
TCPPLGA24/1-12
TCPBSA16/1-12
TCPOBB4/1-12
TCPOBB3/1-12
TCPPLGA27/1-12
TCPOBB7/1-12
10
1
A
N
-U
I
A
1.Qx10-4
Consensus
-
T+LLADTTHHRPWT - -
BP -T- EGF
IEBP3 -T- EGF
-1-
0
z
SBPIO -T- EGF
1
0
A
Bot Sensitivity limit
5
10
15
Tethering Concentration ([tM)
20
Figure 2-2. Identification of BTCP-binding peptide and concatamerization of
the sequence in EGF fusion proteins. Sequence alignment of multiple panning
experiments against BTCP material using orthogonal blocking and panning against
composite BTCP-PLGA substrates showing that the consensus sequence in 8 of 29 clones is
LLADTTHHRPWT (A). Anti-EGF immunoblot performed against scaffold-eluted BP,-TEGF fusion protein with different binding peptide repeats (n = 3, 5 and 10) tethered at
dilutions as indicated, illustrating greater tethering with increase n repeats of the binding
peptide in the concatamer (B). Quantification of anti-EGF immunoblot signals depicted in
2B shows that the 10-mer repeat of the 12-amino acid I3TCP-binding peptide imparts the
highest affinity binding to the BP,-T-EGF fusion protein (C).
36
2.2.2 Binding affinities of EGF fusion proteins with single and concatameric
13TCPbp
Based on the biophysics of interactions between the EGFR and tethered EGF,
it is desirable to present the EGF moiety using a spacer to enhance accessibility of
the ligand (36,74,76) . We therefore fused the binding peptide sequences to the Nterminus of human EGF (53 amino acids; MW=6.2KDa) with an intervening 106
amino acid sequence comprising a coiled-coil sequence (46 amino acids, MW=5.4
KDa) flanked on both ends by a flexible, protease-resistant spacer (25 amino acids;
MW=1.9KDa) along with several restriction enzyme sites for cloning (See
supplementary section for protein sequence). In previous work, we used paired highaffinity heterospecific coiled-coil sequences with the same protease-resistant spacer
in order to dimerize EGF and other EGFR family ligands, and had determined that
the fusion proteins and their dimers were active when constructed as either Nterminal or C-terminal fusions (106,113).
Further, we reasoned that the binding affinity of the peptide to BTCP might be
further enhanced by concatamerization of the binding sequence. We used
mutagenesis (see Methods) to concatamerize the 12-mer TCP binding peptide,
yielding protein fusions with 3, 5, and 10 repeats of the 12 amino acid BTCP binding
unit (LLADTTHHRPWT) flanked by other relevant protein domains as depicted in
Figure 2-1 (see Methods). We first examined the relative binding affinities of the
fusion proteins as a function of the number of repeats of the binding domain in the
fusion protein using a semi-quantitative approach based on eluting proteins
followed by western blot analysis (Figures 2-2B and 2-2C). We titrated the
adsorption concentrations across a range of 0 - 15 uM and found that binding
exhibited a profound dependence on the number of 12-mer repeats (3, 5 or 10) in the
binding domain (Figure 2-2B & 2-2C). Based on these results, we selected the fusion
37
protein with the 10x linear concatamer, referred to as BPio-T-EGF (See Figure 2-1),
to perform all subsequent cell interaction experiments.
2.2.3 BPio-T-EGF in solution exhibits wild-type soluble EGF activity
After selecting BPio-T-EGF as the best binder, we confirmed the purity and
activity of each recombinantly-produced 10-mer protein batch prior to use in cell
phenotypic assays. Western blots of samples subjected to SDS-PAGE showed that
the EGF is co-localized with the 73 kDa band (Figure 2-3A), as expected for intact
BPio-T-EGF. Biological activity of BPio-T-EGF compared to control wild type EGF
was assessed by analyzing activation of Erk-1 and Erk-2 (Erkl/2), a signaling
pathway that shows maximal phosphorylation 7-15 min after stimulation of EGFR
in MSC (36,74,76,85,115) (73,74,82-85,116,117). Compared to unstimulated controls,
a 2.5 to 3-fold increase in ERK1/2 phosphorylation was observed 10 min after
stimulation of MSC by either wild type EGF or BPio-T-EGF (Figure 2-3B). Results
were normalized to the loading control GAPDH (N=3 per condition; one-way
ANOVA p<0.0001; *pairwise comparisons with control were p<0.001 using Tukey's
multiple comparisons test; all data was log-transformed before analysis). There was
no statistical difference between the pairwise comparisons of wild type EGF and
soluble BPio-T-EGF (p>0.05; Tukey's test). Thus, the EGF domain in BPio-T-EGF
appears to be fully competent to activate the EGFR.
2.2.4 Binding and Release of BPio-T-EGF tethered to BTCP scaffolds
Comparable binding isotherms for BPio-T-EGF were observed for crosses of 3
mm and 5 mm using concentrations of 0.2-10 uM soluble protein (Figure 2-4A). The
resulting range of tEGF surface densities was estimated as 3,000 EGF/um 2 - 45,000
EGF/um 2 (see Methods), well within and above the value of 500-3,000 EGF/cm 2
found to provide maximal stimulation to epithelial and mesenchymal cells in
38
previous studies employing EGF tethered to polymer substrates (30,36,53,76,118).
However, because these previous studies employed tethering schemes that fostered
local clustering of tethered EGF, and the binding peptide approach would not
necessarily lead to such localized clustering, a tethering concentration of 2 uM (~
10, 000 EGF/ um2) was chosen for further studies. After a 7-day long incubation of
treated (2 uM) scaffolds in 1xPBS at 37'C, a -25% release of tethered BPio-T-EGF
protein was observed (N=4 per condition, Figure 2-4B). Another stability
experiment performed at lower temperatures (4C) using the same buffer revealed
there was no statistically significant release of BPio-T-EGF protein from 3mm BTCP
scaffolds (Normalized protein amounts were 1.02 +- 0.09 (day 0) and 1.02 +- 0.11
(day 5); both were normalized to t=0; N=3 per condition).
B
A
SDS-PAGE
5.
Western
Ladder Sample Ladder
CO)
Sample
C
A
4C)
C
I
(7
Ia
0
CD
rf+
3
0
C.)
0
-
2
N
(S5i(Oaj
U)
L
(2SK0.)f
M
BPio-T-EGF
(-73KDa)
0'
0
a-EGF
1*Ab
F]
Control
Figure 2-3: BPio-T-EGF fusion proteins activate EGFR
30nM
wtEGF
30nM
BP 10-T-EGF
Coomasie blue staining of
purified BPio-T-EGF by SDS-PAGE shows a single predominant band at the expected
molecular weight (73.5 KDa), and this band contains the EGF immunoreactive activity (A)
Soluble BPio-T-EGF elicits phosphorylation of ERK 1 and 2 in a comparable fashion to wild
type EGF (B).
39
A
B
LL
1,000-
-
n.s.
1.25-
5mm scaffold
3mm scaffold
U-
(N
(9
w
FL
0
C.,
0
CL
CO,
C
C0
I..
0
1=
1.00-
0
0
U-
0
0.50-
E
-L
E
100-
0.75-
06 N
1 OC 00b
E E
C,
C
C-
(D
0
0.25-
0
W
z
0.001
TA
sx
01
1
10
Tethering Concentration ([M)
Timed storage in IxPBS at 3700
Figure 2-4. Binding of BPio-T-EGF to BTCP scaffolds and elution over 7 days.
Binding isotherms of BPio-T-EGFfor 5mm and 3mm crosses (A). Analysis of released and
bound BPio-T-EGF protein for the 2uM tethering condition shows that assays are robust
(i.e., the combined bound and released at 7 days is statistically indistinguishable from the
initial amount) and that 75% of the BPio-T-EGF fusion protein remains bound to the
scaffold after a 7-day incubation in 1xPBS at 37C (B).
2.2.5 Tethered EGF stimulates an increase in hBMSC number on scaffolds
following 7-day culture
After establishing that the EGF domain of the BPio-T-EGF fusion protein
induced bioactivity when it was used in soluble form for MSC stimulation
(activation of signaling pathways downstream of activated EGFR), we investigated
phenotypic responses of low passage primary hBMSC cultured on BTCP scaffolds
modified with BPio-T-EGF fusion protein for three different densities of adsorbed
BPio-T-EGF fusion protein. We have previously shown that EGF tethered to
polymer substrates via polyethylene oxide (PEO) tethers can enhance proliferation
40
I
of hBMSC maintained in both ES and osteogenic media (22,102,106) We used day 7
as a metric for comparison in order to allow for several MSC population doublings
(36,74,75,86,106,115).
Scaffolds (BTCP crosses, see Methods) were pre-incubated with BPio-T-EGF
solutions at concentrations of 0.4 uM, 2 uM, and 9 uM in order to achieve a range of
surface densities (estimated as 4,000 - 45,000 BPio-T-EGF per lm2) and to
determine dose response. Human BMSCs were seeded onto the treated and control
scaffolds and cultured for 7 days in expansion medium. After 7 days, the relative
cell numbers were quantified using the Alamar Blue reagent, using cells seeded on
standard plates at different densities as a calibration to ensure the assay was in the
linear range. All scaffolds treated with BPio-T-EGF had a 2-2.3 fold greater number
of hBMSC number compared to surfaces without BPio-T-EGF (Figure 2-5A; N=3 per
condition; one-way ANOVA p-value<0.05 (p-value=0.02); all pairwise p-values of
BPio-T-EGF vs control were <0.05 using Tukey's multiple comparison test). No
statistical differences were observed between the different BPio-T-EGF surface
densities (All pairwise p-values>0.05 using Tukey's test). These results indicate
that EGF-tethered onto BTCP scaffolds does not impair expansion of hBMSCs, as
the final cell number was greater than the initial number (data not shown), but
these data are not sufficient to conclude that tethered EGF enhances proliferation,
as differences in initial plating efficiencies together with comparable expansion
rates may account for the observed differences at day 7. To parse these
mechanisms, we next examined plating efficiencies.
41
A
B
*
n.s.
3000
4
8 2000
1500
FF2-1
o2
T
n.s.
2500-
3
e 1000
E
CU
0
E
:
4x10 3
18x103
500
45x10 3
Control
12h
BPIO-T-EGF surface density (EGF per m2)
BP 1 -T-EGF
12h
Control
24hr
BP 10-T-EGF
24hr
Figure 2-5: Tethered BPio-T-EGF induces in vitro expansion of hBMSCs
cultured on 13TCP scaffolds. Cells were seeded on control and BPio-T-EGF- treated
scaffolds, cultured in expansion medium for 7 days and then subjected to Alamar blue assay
to assess cell numbers relative to controls. A 2-2.3 fold increase in total hBMSC number
was observed across three different tethered BPio-T-EGF surface densities (N=3 per
condition; one-way ANOVA p-value<0.05 (p-value=0.02); all pairwise p-values of tethered
EGF vs control were <0.05 using Tukey's multiple comparison test) (A). To parse effects
that tethered EGF might have on plating efficiency, MSCs were cultured on BTCP scaffolds
for 12-24 hr and the number of attached cells was then determined using a visual count of
nuclei following acid demineralization of agarose-embedded scaffolds. Results from 12-hr
and 24-hr cell culture revealed no statistical difference (n.s.; N;>3 per condition; One-way
ANOVA p-value>0.05) in the number of hBMSCs as a function of condition, indicating that
tethered EGF is not altering the plating efficiency (B).
2.2.6 Tethered EGF does not alter plating efficiency of hBMSCs seeded onto 13TCP
scaffolds
The Alamar Blue assay and other similar kinds of proliferation assays do not
have sufficient sensitivity to detect the relatively small number of cells present on
scaffolds immediately after seeding. Hence, we developed an approach to directly
count cells seeded on scaffolds by embedding scaffolds in agarose, demineralizing to
42
reveal an optically-clear mold, then staining cells and observing them with confocal
microscopy. Using this method, we found no statistical differences between the
direct count of P3 hBMSCs in the control or BPio-T-EGF conditions for both the
12hr and 24hr time point (Figure 2-5B). This suggests that the increase in hBMSC
number observed after a 7-day culture under BPio-T-EGF conditions was most
likely due to induction of proliferation and not due to differential plating efficiency.
2.3 Discussion
Here we demonstrated the use of a BTCP-binding peptide discovered through
phage display and concatamerized to improve its affinity, to reliably tether EGF
onto BTCP scaffolds for stimulation of hBMSCs. This high-affinity, concatamerized
binding peptide domain is of moderate size (120 amino acids, -14 kDa), and does
not contain unnatural amino acids or post-translational modifications like other
ceramic or apatite binders (84,116,117), making it compatible with standard protein
production and manufacturing strategies. This facile tethering approach only
requires incubation of the fusion protein (see Figure 2-1) with the BTCP substrate
and subsequent washings to remove excess unbound protein. This approach thus
allows for easy modification of BTCP bone void filler scaffolds with bioactive ligands
that can enhance the survival, proliferation or differentiation of tissue-resident or
transplanted osteoprogenitors (i.e. MSCs and CTPs; (30,53,118,119)
(22,102,119,120).
We focused on EGF as the fusion partner for the BTCP-binding peptide motif
because of the multifaceted roles EGFR plays in bone development and homeostasis
in vivo, together with previous studies demonstrating that EGF exerts beneficial
effects on migration, colony formation, and, proliferation of bone marrow-derived
CTPs when covalently tethered to model polymer surfaces (36,74,75,86,106). Simple
adsorption of EGF to the scaffold is not a reliable approach for bioactive delivery of
EGF for this application. We could not detect binding of EGF fusions lacking the
43
BTCP-binding domain, an observation consistent with reports that wild-type EGF
does not bind hydroxyapatite (76,84,121), a material with composition and surface
charge similar to BTCP (107-109,119,122-128), likely due to repulsion as both
possess negative surface charges around the same order of magnitude
(107,119,120,129,130).
We observed retention of BTCP-bound BPlo-T-EGF bioactivity across a range
of surface densities (estimated as 3,000 - 45,000 EGF/um2) as assessed by
enhancement of MSC proliferation (Figure 2-5A). We investigated this range based
on previous reports that tethered EGF enhances survival, proliferation, and colony
formation by MSCs and CTPs when tethered to polymer surfaces at densities of
500-5000 EGF per um 2 (36,74,75,86,106,131-134).
We did not observe a
statistically-significant effect of tethered EGF density on proliferation in the range
of 4000 - 45,000 EGF/um 2, but this is not surprising as tethered EGF densities as
low as -400 tethered EGF/um 2 induce maximal DNA synthesis in primary
hepatocytes and low as -700 tethered EGF/um 2 induces maximal response in
fibroblasts and keratinocytes (76,84,121,127,135)). We note that we attribute the
increase in cell number to proliferation rather than enhanced initial attachment, as
assessment of cell numbers present on the scaffolds by direct visual counting of
nuclei at 12 and 24 hr post-seeding showed no statistical difference among
conditions.
Although we did not directly measure the release and consumption of
tethered EGF in the presence of cells, the finding of enhanced proliferation over 7
days together with observations that 75% of the protein was still retained after 7
days of incubation in PBS at 37'C (Figure 2-4B) suggest that the tethered protein
remains active for at least several days in culture.
The success of using a filamentous phage display library to obtain high
affinity binders is not assured, as material properties, panning protocols, peptide
..... ......44
length and library size can be limiting factors. However, reasonably high affinity
binders to a variety of surfaces, including polymers, metals and, ceramics have been
identified through commercial and non-commercial libraries (107-109,122-128,136).
Variations in display strategies include the number of peptides presented per phage,
cyclized vs linear presentation, and peptide lengths (107-109,127,129,130).
Each
mode of presentation has advantages and disadvantages with respect to library size,
valency of display, and structural flexibility of peptides. Due to the polyvalent
nature of M13 p3 and p8 phage display, avidity can play a role in binding as p3
display offers 3-5 copies of the peptide and p8 display offers -2700 copies (131134,137,138). This polyvalency can lead to a higher apparent affinity of the phage
clones due to avidity, which leads to reduced binding once the monovalent peptides
are cloned and expressed.
Typical binder affinities of monovalent peptides from panning experiments
with commercial kits tend to be in the low uM range (113,127,135) (107,109,131133,136). Typically, 3 to 5 rounds of screening are sufficient to obtain binders with
desired affinities (107-109,127).
Additional rounds (>4) may introduce phage-
specific factors (e.g. phage replication) that reduce the sequence diversity with
minimal increase in affinity of the peptide pool (137-140) . After 3 rounds of panning
against BTCP using the 12-aa p3 M13 phage display kit (see Methods) we obtained
9-30 plaques per panning condition, except on tube controls which did not yield
plaques at round 3. After sequencing 9-10 phage plaques per condition, we found a
consensus BTCP binding sequence (LLADTTHHRPWT) that was present in 8 out of
29 clones. Importantly, this consensus sequence emerged across orthogonal BTCP
blocking conditions (BSA and OBB) as well as panning against a composite of BTCP
with the degradable polyester biomaterial PLGA, suggesting it is highly specific for
the target. We then created a fusion protein comprising this 12 amino acid peptide
fused to human EGF via protease-resistant spacer flanking a coil-coil motif
(113,141) and expressed the protein with a MBP motif to increase solubility of the
purified final protein product. This first generation fusion protein did not exhibit
45
the desired high affinity binding to BTCP, presumably because the 12 amino acid
peptide alone could not offset the thermodynamic driving force for the 76,000
molecular weight protein (including the MPB domain) to remain soluble.
We thus exploited a polyvalency approach in which created a linear
concatamerization of the binding peptide to increase the driving force for surface
adsorption. Sequential increases of the number of 12-mer binding peptide sequences
in the concatamer, from 3 to 5 to 10, were associated with increased affinity of the
fusion protein with the 6TCP surface (Figures 2-2B & 2-2C). The repeat number is
representative of the lower and higher bounds of p3 peptide copies per phage (i.e., 3and 5- mer (131-133,142)), while the 10-mer is a greater valency than the p3 display
could theoretically offer. Linear concatamerization has previously been used with
some success to increase binding peptide affinity (127), but is not a general
approach that works with all peptides tested. Intuitively, the domain spacing must
align with multiple repeats of the affinity target on the surface in order to maintain
multiple high affinity contacts. We did not systematically investigate the role of
domain spacing by varying the linker length between concatenated peptide
sequences, but speculate that further improvements in affinity could be achieved by
changing this variable.
Many features of the 12 amino acid binding sequence we identified are
consistent with affinity for calcium phosphate. LLADTTHHRPWT is a common
motif found in proteins with affinity for binding to phosphate-containing compounds
(ATP/ADP/AMP/UDP, Phosphoglyceric acid (PGA), DNA and tRNA, and others),
with 13 out of the top 15 hits on a BLAST search of this motif illustrating such
binding capabilities or ability to bind to divalent calcium. Amino acids present in
the mid-portion of our peptide (DTTHHR) -- including aspartic acid, threonine,
histidine, and arginine are all capable of forming hydrogen bonds and were
prominantly implicated in the interactions with phosphate groups within the active
site of several of the enzyme families from the BLAST results (PDB ID#s: 1JJV,
46
411V, 4DEC, and 3V4R; (139,140)). The TT motif has been implicated in the
interactions of dephospho-CoA kinases with the diphosphate group of adenosine diphosphate (ADP) through hydrogen bonding mechanisms (PDB ID#s: 1JJV, 411V;
(141)), suggesting a potential role for the TT motif in our binding peptide for
interaction with calcium phosphate. Further, amino acids with basic side or acidic
chains like arginine and aspartic acid can form salt bridges with the phosphate and
calcium ions, respectively. Aspartic acid residues, along with glutamate and
carboxy-glutamate residues in proteins are known to be able to form coordination
complexes with calcium ions in solution (142). Histidine residues can coordinate
with divalent cations like Ca+2 (139), and both histidine and aspartic acid residues
have been speculated to accelerate nucleation of calcium phosphates (CaP) by
removing protons from phosphate groups during the hydrogen bonding reaction
(143).
We speculate that the central 7aa polar domain in the binding peptide
dominates the energetics of interactions with the surface, with the flanking
hydrophobic amino acids (LLA on N-terminus; and PW on the C-terminus)
providing some conformational support or shielding water molecules away from the
surface to strengthen surrounding hydrogen bonds. However, mutational analysis
would be required to precisely detail the role of individual or group of amino acids.
Such analysis in other systems often reveals that only a few amino acids are crucial
for binding, and the surrounding amino acids often provide support (structural or
chemical) to optimize the interactions of those critical residues with the surface
(109,110,125,128,144,145).
Taken together, these results provide compelling motivation for ongoing
efforts to assess the colony-forming performance of BPio-T-EGF scaffolds with
human bone marrow samples, and to translate the in vitro findings to in vivo bone
healing models employing transplants of aspirated marrow, such as those in the
47
canine femoral defect model (112). Future studies might also address alterations in
the protein design to improve expression and purification properties.
2.4 Materials and Methods
2.4.1 Fabrication of BTCP and BTCP-polymer composite scaffolds
Scaffolds were fabricated at Integra Life Sciences from either 6TCP or a
composite of BTCP and polylactide-co-glycolide (PLGA) using the TheriForm 3D
rapid prototyping platform. Briefly, to create BTCP scaffolds, granulated 3TCP
powder was sintered and sieved to <106 um. Scaffolds were fabricated in the shape
of a cross by depositing binder in a programmed sequence onto a powder bed
containing a mixture of sintered BTCP powder and porogen (spray-dried lactose).
The scaffolds were then sintered to remove porogen and fuse the ceramic particles,
yielding crosses measuring 5.4x5.4x2.7mm (Figure 2-1, top). Scaffolds have an open
porous architecture (Figure 2-1, middle) with a pore volume of about 60% porous
and a mean pore diameter of 60um (range of 5-900pm). Chemically, the scaffolds
are approximately >95% BTCP with the remaining portion being other resorbable
forms of calcium phosphate. Composite BTCP- PLGA scaffolds were fabricated in a
similar fashion by mixing powders with a porogen. Composite scaffolds were fused
by exposure to chloroform vapor, leached, dried, and sterilized by ethylene oxide as
described elsewhere (112,113).
Scanning electron micrographs of the 3TCP scaffolds were obtained using a
Jeol 5600LV Scanning Electron Microscope. Samples were mounted on stubs,
sputter coated with Au/Pd (-5nm layer) and imaged at the desired magnifications
using an accelerating voltage of 5 kV.
48
2.4.2 Phage display against BTCP scaffolds
Pure 6TCP cross-shaped scaffolds (5.4x5.4x2.7mm) from Integra Life Sciences
were crushed into powder, autoclaved for 35 minutes at 1210C and stored under dry
sterile conditions prior to all experiments. The resulting sterile 6TCP powder was
blocked for 24 hours at 40C under moderate agitation with either sterile filtered
%
Odyssey Blocking Buffer (OBB; Licor; non-mammalian blocking buffer) or 5
bovine serum albumin in phosphate buffered saline (BSA, Sigma). Blocked BTCP
was pelleted at 2000 RPM for 2 minutes, washed 3X with PBS then subjected to
three rounds of phage display using the New England Biolabs linear 12-mer Ph.D.
kit (Andover, MA). Orthogonally blocked BTCP (i.e. blocked with BSA vs. OBB)
provided a control against panning against components of the blocking buffers.
Additional controls included a BTCP -PLGA composite cross-shaped scaffold
(5.4x5.4x2.7mm), crushed into powder, similarly blocked with BSA as well as a
mock tube to control against panning against tube components. After three rounds
of panning, ten plaques from each of the BTCP /BSA and BTCP -PLGA/OBB and
nine from the BTCP /OBB block condition were picked, amplified, then sequenced
(the mock condition and the BTCP -PLGA blocked with BSA did not produce plaques
after the second and third round, respectively). Sequences were analyzed for
consensus using JalView Multiple Sequence Alignment Editor.
2.4.3 Mutagenesis
We had previously created a pMAL-c5X vector (New England Biolabs)
expressing human epidermal growth factor in fusion with various epitopes (113).
Building on this, the highest-ranked sequence from all the sequenced third round
phage display panning (LLADTTHHRPWT) against BTCP was serially cloned into a
pMAL expression cassette using PCR mutagenesis and a short primer to generate a
library of multimer insertions fused to EGF via protease-resistant tethers flanking
a coil domain (Figure 2-1 bottom). PCR mutagenesis was performed with a
Quickchange Lightning II kit from Stratagene (Eugene, OR). PCR primers were
49
designed to prime wholly within the BTCP-binding peptide coding region thus
allowing multiple insertions during a single PCR mutagenesis round. Multimer
clones were sequenced to confirm DNA identity with target sequence, transformed
into BL21(DE3)pLysS E. coli and plated on ampicillin LB agar. We designate the
final gene product produced recombinantly as BPn-T-EGF where n denotes the
number of BTCP-binding peptide repeats in the binding domain and n ranged from
1-10.
2.4.4 Protein Expression
Protein was expressed in 1L BL21(DE3)pLysS E. coli cultures grown in
ampicillin-containing (50ug/mL) luria broth (LB) at 250 rpm, 370C until OD (600nm)
= 0.6, then induced with IPTG and incubated at 220C for 4 hours. Proteins were
harvested by pelleting cultures at 3700 RPM on an Allegra G3.8 rotor at 40C for 30
minutes then freezing the pellet at -800C overnight followed by cell lysis using
Bugbuster Reagent (EMD Chemicals) supplemented with PMSF and protease
inhibitor cocktail (Complete Protease Mini; Roche). Lysed cells were centrifuged at
3700 RPM on an Allegra G3.8 rotor at 40C for 1 hour. The supernatant was then
diluted 1:4 in tris-buffered saline and subjected to maltose binding protein affinity
chromatography in accordance with the manufacturer's instructions (New England
Biolabs). Eluted fractions were pooled and subjected to ultrafiltration through a
50,000 MWCO membrane U-tube concentrator (Novagen). The protein solution was
then sterile filtered through a 0.2 micron syringe filter. Purity was confirmed by
SDS-PAGE using coomassie staining. Typical purity of the full length fusion
protein (73 kDa) comprising MBP, ten concatamerized BTCP-binding peptide
motifs, the tether-coil domain, and EGF, i.e., BPiO-T-EGF, ranged from 75% to 90%,
with >99% of the protein being of recombinant nature. Contaminant protein was
BPio-T-EGF lacking the EGF motif (BPio-T). Similar results were observed for all
concatamer lengths of binding peptide. Stock protein quantification was performed
using a Nanodrop ND-2000 spectrophotometer at 280nm absorbance. The
50
absorbance values along with the predicted extinction coefficient obtained through
the ExPASy protparam tool (http://web.expasy.org/protparam;the full protein
sequence as input) were used to calculate the protein concentration. Concentrated
protein stocks were used immediately or stored for a week at 40C.
2.4.5 Characterization of BPn-T-EGF binding to and elution from BTCP scaffolds
BTCP scaffolds or sieved pure BTCP powder were incubated at room
temperature for two hours in purified BPo-T-EGF diluted in OBB buffer. After
incubation, scaffolds or powder were washed three times in three volumes of 20 mM
Tris-buffered saline at pH 7.4 followed by a final wash and storage in PBS. Relative
binding as a function of concatamer length and peptide concentration was
determined by eluting BTCP -bound BP,-T-EGF in 200 uL of pH 2.2 0.2M glycine
buffer + 1mg/mL BSA for two hours, then analyzing the eluate by Western blot
coupled with IR-dye immunofluorescence (10 Ab - Rabbit Anti-EGF {abcam;
cat#ab9695} ; 2' Ab Goat Anti-rabbit-AlexaFluoro680 {Invitrogen}). Control "-tEGF" protein incorporating all elements of the BPo-T-EGF except the BTCP-binding
peptide region was used as a negative control for non-specific binding in all
experiments. This negative "-t-EGF" control did not show detectable binding to
BTCP.
For the binding isotherms, 3mm and 5mm crosses were incubated with
protein solutions ranging from 0.2-9uM for -36 hours at 4C. After tethering, the
crosses were washed 2x with PBS then assayed for total protein by the BCA assay.
To ensure that samples were within the linear range of the BCA assay, crosses
tethered with BP,-T-EGF at the higher end of the concentration range were divided
into multiple segments and placed in individual wells of either 96-well plates or 48well plates as follows: 0.2uM (1:1), luM (1:2), and 2uM (1:3) conditions for the 3mm
scaffolds and the 0.4uM (1: 2) condition for the 5mm cross were performed in 96 well
plates; the 5uM condition for the 3mm cross (1:1) and the 2uM (1:1) and 9uM (1:2)
51
conditions for the 5mm crosses were performed in 48 well plates. Water (100uL, 96
well; 400uL, 48 well) water was added to the wells with scaffolds/fragments, which
were then crushed into semi-powder form (no large granules or scaffold blocks
visible). The BCA reagent was then added to all wells (100uL for 96 well and 400uL
for 48 well), the plates were sealed with plastic adhesive and incubated at 37C for
50 minutes. After incubation, the plates were centrifuged for 10 minutes at 2,000
RPM at room temperature. The well plates were then gently tilted and 10OuL out of
each well was moved to a new 96 well plate, being careful not to take any BTCP
powder in the process. The final plate was checked to ensure that all wells were free
from bubbles and the plate was read at 562nm. Background from wells without
crosses or protein were used to correct for background absorbance. We confirmed
that BTCP (in scaffold or powder form) did not change the background absorbance of
the BCA reagent solution. Absolute protein amounts in test wells were calculated
based standard curves using BP-T-EGF, where the ratio of the slope of the BP-TEGF over BSA standard curve was 1.5-1.6.
To close the mass balance, BP.-T-EGF protein concentrations remaining in
the supernate after the tethering process were measured via both Nanodrop
(absorption at 280 nm) for relatively concentrated samples (OD > 0.2) and the BCA
assay across a range of concentrations. Methods showed a strong correlation
(ANOVA p-value>0.05) with no statistical differences and coefficients of variation of
-15%.
2.4.6 Cell Culture
Human bone marrow mesenchymal stromal cells (hBMSCs) obtained at
Passage 1 (Texas A&M Health Science Center College of Medicine's Institute for
Regenerative Medicine) were culture expanded using expansion media (EX) (cMEM
with 2 mM L-glutamine, 16.5% fetal bovine serum, 100 units/ml of penicillin and
100 pg/ml streptomycin) at low seeding density (50cells/cm2) and frozen stocks were
52
made when cells reached 70-80% confluency to Passage 2 (freezing media: aMEM
with 2 mM L-glutamine, 16.5% Fetal Bovine Serum and DMSO mixed at a 65:30:5
v/v ratio). For the experiments, P2 stocks were expanded at low density (50cells/cm2)
and used for experiments when cells reached 70-80% confluency.
2.4.7 Validation of bioactivity of the EGF domain in fusion proteins
The intrinsic activity of the EGF domain in fusion proteins was assessed by
comparing the ability of soluble BP~o-T-EGF and wild-type EGF to stimulate
signaling activity downstream of EGFR in hBMSC. Passage 3 hBMSCs were seeded
onto Falcon 12-well tissue culture-treated polystyrene plates (Becton Dickinson and
Co., NJ USA) at a density of 18,000 per well and grown to a monolayer by culturing
in EM. The cells were then serum starved by incubation overnight in 2% dialyzed
FBS (Gibco - Life Technologies) and after overnight incubation the cells were
exposed to serum-free medium for 2hr prior to experiment. The medium was
aspirated and cells were exposed to serum-free media containing the corresponding
ligand or negative control (no ligand) for a period of 10 minutes. The media were
then aspirated, the plates were placed on ice and the wells were rinsed with ice cold
1xPBS and then incubated with ice-cold lysis buffer (Bio-Rad) that included both
protease inhibitors and phosphatase inhibitors used at 1x concentration (Complete
Protease Mini and PhosSTOP phosphatase inhibitor tablets; Roche). While on ice,
wells were scraped with rubber policemen and lysates collected and placed in the
wells of a sterile, low protein binding 1.2um filter 96-well plates (Multiscreen HTS,
low protein binding, Millipore) and placed on top of a regular 96-well plate and
centrifuged at 2,000 rpm for 5 minutes. The plate was then sealed with a microplate
sealing film (Thermo Scientific) and stored at -80'C or used immediately. The BCA
assay using BSA as a standard was used to measure lysate concentrations.
Western blots for phospho-ERK1/2 were performed by loading 10 micrograms
of lysate into each well of an SDS-PAGE gel (Nu-PAGE Novex 4-12% Tris-bis gel,
53
10-well; Invitrogen). Proteins in the gel were then transferred to a Nitrocellulose
membrane (Bio-Rad) using the X Cell II blot unit (Invitrogen). The membranes were
blocked using OBB for 1 hr at room temperature (RT). After the blocking step, the
blots were incubated overnight with the primary antibodies (10 Ab) targeting
phospho-ERK1/2 (Rabbit anti-phospho-ERK1/2 (Thr202/Tyr2O4) from cell signaling;
cat #4370) and the loading control GAPDH Ab (Mouse anti-GAPDH Ab from Abcam,
cat#AB9484) diluted in 1xPBS containing 0.1% Tween-20 (T-20). The p-ERK1/2
antibody was diluted 1:1000 and the GAPDH antibody was diluted 1:2000 (stock is
1mg/mL; use at 0.5-1 ug/mL). After 10 Ab incubation, the blots were washed 4x with
1xPBS containing 0.1% T-20 and incubated with the secondary antibody (20 Ab)
(1:10,000 dilution) tagged with IR-range dyes (700-800nm; Goat anti-mouse-IR800
dye from Odyssey and Goat anti-rabbit-AlexaFluoro680 from Invitrogen) in PBS
with 0.1% T-20 and 0.01% SDS. The blots were then incubated in the Ab solutions
for 45 minutes at RT with gentle shaking. Blots were washed 4X, placed in neat
1xPBS and scanned at in a LI-COR machine (700nm and 800nm channels). The
intensities from each band (p-ERK1/2 at 700nm or GAPDH at 800nm) were
measured using the LI-COR Image Studio LiteTM software. All the intensity values
for p-ERK1/2 were normalized to the GAPDH loading control and then normalized
to the no EGF controls (negative control). Results from all blots (N=3) were then
averaged and plotted. For the westerns against the EGF domain of BPn-T-EGF, a
similar procedure was performed but using a Rabbit Anti-EGF antibody (abcam;
cat#ab9695) as the 1* Ab and the goat anti-rabbit-AlexaFluoro680 (Invitrogen) as
the 2 0 Ab.
2.4.8 hBMSC proliferation assays on BTCP scaffolds
Cell proliferation was determined using the Alamar Blue (AB) assay
(Invitrogen) at 7 days post-seeding. Passage 3 hBMCSs were seeded at 2,00 cells per
well onto 5mm BTCP cross-shaped scaffolds (5.4x5.4x2.7mm; TherilokTM from
Integra Life Sciences). Cells in media were seeded into the center of the well
54
directly on top of the crosses (200uL total). This resulted in a seeding fraction of
-16-20% of total cells seeded for 3mm crosses and -31-42% for 5mm crosses,
approximately comparable to the cross sectional area of the crosses in the well. This
agrees well with previous observations that static seeding generally leads to low
seeding densities unless the scaffold occupies most of the well surface
(73,74,82,83,85,114).
Following seeding, cells were cultured overnight in EX media. Crosses were
then moved to new wells to eliminate contributions from cells that had attached to
the bottom of the well. The crosses were then cultured in new plates for a
cumulative time of 7 days in EX media. Each condition was performed in triplicate.
After the 7-day culture the cell-containing BTCP scaffolds (EGF-tethered and
controls) were moved into a sterile, low-protein binding 1.2um filter 96-well plate
(Multiscreen HTS, low protein binding; Millipore) and the Alamar Blue reagent
(Invitrogen) was then added to each well (mixed in a 1 part of the 10x AB reagent to
1 part EX media according to the manufacturer's instructions). The plate was then
incubated at 37*C, 5% CO2 for 4 hours with gentle mixing. After incubation, the
.
filter plate unit was attached on the bottom to a flat-bottom 96-well plate and
centrifuged at 1000 rpm for 3 minutes. This method allowed for complete recovery
of AB dye-media mixture. One-hundred microliters of the resulting AB dye-media
mixture collected was transferred to a new flat-bottom 96-well plate to be read by a
SpectraMax M2e multi-well fluorescent plate reader (Molecular Devices Corp.CA,
USA) at a 570 nm excitation wavelength and 585 nm emission wavelength as
recommended by manufacturer's instructions. The background fluorescence reading
(AB solution in media with a cell-free 6TCP scaffold) was subtracted from the AB
fluorescence reading for each well to obtain the net fluorescence. The average net
AB fluorescence units from each condition (N=3 per condition) were normalized to
no EGF ligand condition (control).
55
Following seeding, cells were cultured overnight in EX media. Crosses were
then moved to new wells to eliminate contributions from cells that had attached to
the bottom of the well. The crosses were then cultured in new plates for a
cumulative time of 7 days in EX media. Each condition was performed in triplicate.
After the 7-day culture the cell-containing BTCP scaffolds (EGF-tethered and
controls) were moved into a sterile, low-protein binding 1.2um filter 96-well plate
(Multiscreen HTS, low protein binding; Millipore) and the Alamar Blue reagent
(Invitrogen) was then added to each well (mixed in a 1 part of the 10x AB reagent to
1 part EX media according to the manufacturer's instructions). The plate was then
incubated at 37*C, 5% CO 2 for 4 hours with gentle mixing. After incubation, the
filter plate unit was attached on the bottom to a flat-bottom 96-well plate and
centrifuged at 1000 rpm for 3 minutes. This method allowed for complete recovery
of AB dye-media mixture. One-hundred microliters of the resulting AB dye-media
mixture collected was transferred to a new flat-bottom 96-well plate to be read by a
SpectraMax M2e multi-well fluorescent plate reader (Molecular Devices Corp.CA,
USA) at a 570 nm excitation wavelength and 585 nm emission wavelength as
recommended by manufacturer's instructions. The background fluorescence reading
(AB solution in media with a cell-free 13TCP scaffold) was subtracted from the AB
fluorescence reading for each well to obtain the net fluorescence. The average net
AB fluorescence units from each condition (N=3 per condition) were normalized to
no tEGF condition (control).
2.4.9 Plating efficiency assay
The Alamar Blue assay is relatively insensitive at low cell numbers. Hence to
determine the plating efficiency of cells on control and tethered EGF crosses at
early time points (12 and 24 hr), we developed a protocol to directly count cells that
were fixed and DAPI-stained by embedding crosses in agarose followed by a
demineralization step to dissolve the cross, allowing direct microscopic observation
of the cells in situ. In these experiments, we used smaller BTCP cross-shaped
56
scaffolds (dimensions: 3.6x3.6x1.8mm, Therilok II, Integra Life Sciences) in order to
have enough working distance to directly count all cells adhered onto scaffolds (with
10x objective) using a demineralization protocol. Crosses were fabricated by a
process identical to that used for larger crosses, sterilized by autoclaving, and then
were treated according to the EGF tethering protocol described above, using PBS as
a control.
Crosses were placed in individual wells of a 96-well plated and seeded with
11,000 cells in 200uL of EX media leading to -1,700-2,200 cells landing on the 3mm
scaffold (based on predicted -16-20% seeding efficiency for 3mm crosses placed in
96-well plates). Following culture for 12 or 24 hours in EX media, cells were rinsed
with 1x PBS, fixed for 2 hours with 2% glutaraldehyde/2% formaldehyde in 1x PBS,
then washed with multiple rinses in 1xPBS (3x; 30 seconds each). Crosses were
then embedded in agarose, demineralized in a mild HCl solution and left at 4C
until embedded scaffolds appeared translucent. The HCl solution was then
neutralized by multiple rinses with 1x PBS. The cells within the embedded scaffolds
were stained with DAPI and then imaged under a confocal microscope. Stacks were
taken using a 10x objective using the DAPI filter. Z-stacks covered a z-range of
1.8mm (TCP scaffold height=1.6mm) and an XY Z-stack sweep covered a total area
of -4x4mm range on the XY axis (total area scanned 4.4x4.4x1.8mm). Stacks along
the XY axes covering the complete cross were acquired and cells in each stack were
counted using the DAPI signal and the spots function in the IMARIS software.
57
Chapter 3
Tethered EGF Enhances the Colony Forming
Efficiency of Human Osteoprogenitors Cultured on
Beta-Tricalcium Phosphate Scaffolds
This chapter includes experiments performed in collaboration with the George
Muschler and Alan Wells labs, and the major findings are being submitted for
publication with the following attributions:
Rivera JJ*, Raut VP*, Stockdale L, Alvarez LM, Patterson TE, Boehm C, Wells A,
Muchler GF, Griffith LG. "Tethered EGF Enhances the Colony Forming
Efficiency of Human Osteoprogenitors Cultured on Beta-Tricalcium
Phosphate Scaffolds"
*Denotes equal contribution
3.1 Introduction
Stem and progenitor cell therapy has great potential for a variety of wound
treatments and diseases, with over 300 ongoing clinical trials listed in
clinicaltrials.gov (146). These cell sources may be derived from freshly isolated
tissue resident cells, or from culture expanded cell populations. Connective Tissue
Progenitors (CTPs) is a term that is used to define the heterogeneous population of
stem and progenitor cells that are resident in native tissues and that have the
ability to proliferate and give rise to progeny that can form one or more types of
connective tissues (bone, cartilage, fat, fibrous tissue, blood, ... cartilage)
(30,53,104,146-148). CTPs and their subsequent progeny are commonly isolated
and identified based on their property of adhering to tissue-culture polystyrene and
proliferation to form colonies when plated in vitro (28,30,53,146,149-151).
58
The prevalence of CTPs within a given population of cells can be estimated
using a colony forming unit assay, where the number of colonies observed per
million cells plated represents the observed prevalence (PcTPobs). The biological
properties of the observed colony founding CTPs can also be assessed based on the
proliferation, migration, differentiation and survival of their clonal progeny under
the conditions in which they are cultured. Methods for objective assessment of the
prevalence and biological performance of CTPs and other stem and progenitor
populations have recently been defined in an ASTM Standard Method using
automated image analysis (152). These methods greatly improve the accuracy of
these measurements.
ASTM methods also enable the assessment of changes in colony forming efficiency
(CFE) under different culture conditions. In any given sample there is a true
prevalence of CTPs (PCTPtrue) which represents the actual number of all CTPs that
could be induced to proliferate and form a colony of progeny under ideal conditions.
PCTPtrue is related to PcTPobs by the formula: PCTPtrue - PcTPobs x CFE, where CFE
represents the colony forming efficiency under the culture conditions used.
The prevalence of CTPs in freshly-aspirated bone marrow (BMA) is low
(~1:30,000 nucleated cells) and there is great patient-to-patient variation. As a
result, the concentration and prevalence of CTPs within most, if not all, bone defect
sites is likely to be suboptimal. A variety of methods have been investigated to
improve the concentration and prevalence of CTPs using methods such as density
separation (30,153), selective cell retention (54,55) and marker-based magnetic
separation (66,154).
Large, relatively avascular bone wounds present a particular clinical
challenge. Achieving optimal prevalence, activation and survival of CTPs in such
wounds - i.e., wounds characterized by hypoxia and pro-apoptotic inflammatory
signaling - is likely to be a key limiting factor in the success of cell therapy
59
approaches in these defects (22,48,74). Animal studies suggest that transplanted
stem cells suffer a high mortality post-transplant, even in modest defects
(38,44,103,155). The rate of oxygen decline following transplantation may also play
an important role in CTP survival and performance(156). Thus, strategies that
target CTPs to improve the activation and survival of CTPs may lead to improved
clinical performance.
An attractive target for improving the activation and survival of local and
transplanted CTPs is the epidermal growth factor receptor (EGFR), which is
present on CTPs(36,66,68,73,74,85,106) and known to be essential for multiple
stages of bone development and maturation (70,71,157,158). Soluble EGF (sEGF),
the canonical EGFR ligand in a group comprising transforming growth factor alpha
(TGFa), amphiregulin, betacellulin and others, enhances ex vivo expansion of
human bone marrow stromal cells (hMSCs) (66,73,74). EGF is both necessary and
sufficient to induce colony growth of freshly isolated Stro-1 positive marrow-derived
cells (36,66) and promotes cell migration of immortalized hMSCs (ihMSCs) (73,159).
Stimulation of EGFR by EGF or other EGFR ligands activates multiple downstream
intracellular signaling pathways including MAPK, AKT, and PLC-gamma and
influences cell activation, survival, migration, proliferation and differentiation
(85,106,159).
The balance of EGF-activated signaling pathways in CTPs and MSCs can be
shifted toward cell surface-associated pathways and sustained signaling if EGF is
presented in a substrate-tethered format instead of in the standard soluble form,
with dramatic consequences on cell phenotypic outcomes (74,85,106,160). EGF
tethered to the culture substrate via a polyethylene oxide tether (tEGF), mimicking
the matrix-embedded stimulation provided by tenascin-C (161), is more effective
than soluble EGF at promoting spreading of ihMSCs in serum free medium
(74, 159)and enhances formation of osteogenic colonies from plated human BMA
compared to sEGF (36). Importantly, tethered EGF protects MSCs against the pro-
60
death inflammatory cytokine Fas Ligand (FasL), but soluble EGF potentiates death
(74,75,160). Further, tEGF but not soluble EGF protects MSCs under the combined
insults of hypoxia, serum-deprivation and FasL treatment (75), and tEGF does not
inhibit differentiation (36,75,85,106).These phenotypic changes in the presence of
tethered compared to soluble EGF are associated with alterations in intracellular
signaling including prolonged phosphorylation of EGFR and downstream ERK
signaling and protection of EGFR from downregulation (74,75,85,159). An
additional advantage of the tethered mode of EGF delivery is control of the
distribution and local concentration of EGF in the pericellular environment and
reduction in the overall dose of growth factor when compared to a diffusion
controlled release of soluble EGF (76).
The tEGF construct described here represents an approach that is adapted
for clinically-relevant B-tricalcium phosphate (8TCP) scaffold materials. Scaffolds
containing BTCP are osteoconductive, and several BTCP- containing scaffolds are in
current clinical use for bone regeneration and transplantation of bone and marrowderived cells (90-94,96,162). Because BTCP does not contain functional groups for
covalent conjugation, we tethered EGF to TCP using a high-affinity
TCP binding
peptide fusion protein, BP-EGF (previously referred to as BP-1 0 -T-EGF;(163,164)).
We have shown that this protein-scaffold interaction is stable for at least a week
This work was designed to assess the in vitro effect of tEGF constructs on
colony forming efficiency (the relative number of colonies formed), proliferation (the
number of cells in the colonies), and differentiation (expression of an early
osteogenic marker, alkaline phosphatase) in human bone and marrow-derived CTPs
using the ASTM Standard Method F2944-12 (152).
61
3.2 Results
3.2.1 Characterization of BTCP substrates and protein binding
The PDMS support layer, surface topology and the coverage of BTCP granules
of final coverslips were investigated by SEM (Figure 3-2)). The thickness of the
PDMS layer (Figure 3-2A) was 40 um, a result consistent with the expected
thickness based on literature references to comparable coating protocols (165). The
mass of a PDMS-coated glass coverslip was 152
a BTCP-coated coverslip was 172
BTCP per coverslip is 20
2 mg (std dev; n = 5), while that of
4 mg (std dev, n=12); hence the average mass of
4 mg. At least 99% of the surface was covered with BTCP,
as assessed by brightfield transmission microscopy looking for uncoated PDMS
regions (images not shown).
As we have found in previous studies (163,164), after purification, the
recombinant protein was >99% pure (i.e., <1% bacterial origin) and contained on
average 75 3% of full-length (FL) fusion protein (BP-EGF) with the balance as
protein lacking the EGF motif (BP). We characterized the binding of two batches of
fusion protein to the BTCP substrates using a concentration of 2.7 PM (see Methods)
and found the mean amount of tethered EGF per 18mm diameter BTCP-coated
coverslip was 6.4
0.8 ug (N=12) (i.e., 0.32 ug BP-EGF/mg BTCP) with no statistical
difference between batches. The surface-bound protein remained stable after
incubation in PBS up to 5 days at 4*C and 37*C, with no loss of protein detected at
4*C and only -20-25% loss at 37*C.
The local density of tethered EGF at the cell-substrate interface was
estimated by dividing the amount of EGF bound per coverslip by the total surface
area of the BTCP particles (0.244 m 2 /gm, roughly half of which is accessible above
the PDMS film). Using these values, the 2.7 pM tethering concentration yields
~20,000
3000 (avg
std dev) EGF per pm 2 . This is about 2-3x the surface density
62
of previous work using tEGF on polymeric substrates (reported as 5,000-7,000 EGF
per
jim2)
using an approach that fosters ligand clustering (36,74).
3.2.2 Tethered EGF enhances colony forming efficiency (CFE) without influencing
average cells per colony, cell density, or alkaline phosphatase activity
The
PCTPObs
on control BTCP substrates for BMA samples ranged from 10 to
32 CTPs per million nucleated cells (3 patients; median =26; mean=22). The PcTPobs
on control BTCP substrates in the TS cell samples ranged from 20 to 175 CTPs per
million nucleated cells (5 patients; median=30; mean=60).
PcTPobs on tEGF surfaces was greater than on control surfaces in all 8
patient samples (Figure 3-3). The mean relative colony forming efficiency (rCFE)
(ratio of PCTPObs on tEGF surfaces to PcTPobs control surfaces) was 1.62
0.26 (p-
value < 0.0003, Figure 3-3), suggesting that tEGF surfaces resulted in a 62%
increase in CFU. The rCFE was consistent between BMA-derived CTPs (1.54
0.13,
n=3, p<0.02) and TS-derived CTPs (1.68 +/- 0.32, n=5, p<0.01) cell populations (see
Figure 3-3).
As illustrated in Figures 3-3B through 3D, there was no statistical
difference between the tethered EGF condition and control BTCP surfaces with
respect to mean cells per colony (p=0.15), mean colony density (p=0.31)(Figure 3-3C),
or area fraction of alkaline phosphatase (APAP) (p=0.84).
63
EGF
'
MBP
'BP
TCPBP units
Spacer
LLADTTHHRPWT
Figure 3-1: Schematic representation of the BTCP binding peptide-EGF (BPEGF) fusion protein. The EGF domain is linked to the 6-TCP binding peptide (BP). The
tether domain comprises two segments, a coil domain and a protease-resistant spacer
domain. The BP domain is a concatamer of 10 repeats of a 12-mer sequence identified by
phage display, and exhibits high affinity for OTCP surfaces. A maltose binding protein
domain (MBP) represents a non functional remnant of the initial fusion protein that was
used in purification and to enhance solubility (see (163,164) for more information).
(A)
(B)
(C)
(D)
64
Figure 3-2: Preparation and characterization of 2D BTCP surfaces. (A) A
Scanning Electron Microscope (SEM) image of a PDMS layer spin coated at 2,000 rpm onto
a glass coverslip and broken off to create delamination near the interface. From the scale
bar in this image we estimated the thickness of the PDMS layer to be 40 Pm (scale
bar=100pm; Magnification (MAG)=220x) (B) SEM image of a BTCP coverslip which
highlights the surface coverage by BTCP. The BTCP particle size used is ! 25 um (MAG:
100x; scale bar is 100um) (C) SEM image of a HCl-demineralized BTCP coverslip. This
image shows that the BTCP particles were exposed on the PDMS surface instead of being
fully submerged (MAG: 100x; scale bar is 100um). (D) Colony formation on a 18mm BTCP
coverslip. CTPs were plated on a BBTCP surface, fixed after 9 days of culture, and stained
using DAPI and VectaRed to label nuclei and stain regions of alkaline phosphatase activity
(dark spots).
(A)
2.5
0 Overall Mean
-e- Patient 1 (BM)
- - Patient 2 (BM)
-.- Patient 3 (BM)
Patient 4 (TS)
2.0
2
44-5 'C
0 1.5
1.0
1--5-
_o E--
8
0-+-
0.5
-+-
0.0
w/o
(C)
(B)
0
tEGF
Patient 5
Patient 6
Patient 7
Patient 8
w/ tEGF
(control)
1.6-
1.6-
1.6-
1.4-
1.4-
1.4-
1.2-
1.2-
1.0-
MOMEM
U:
(TS)
(TS)
(TS)
(TS)
8
0n
1.0.
8
0.6-
C
-
1.2-
1.0-
---
a
E~.
C
0.4-
0.0-
0.0-:1
w/o tEGF
(Control)
w/ tEGF
0.40.2-
0.2
0.2.
U0.6-
E
0
CU 0
(
%
CE 0.6.8
a) 0
1~
w/o tEGF
w/ tEGF
(Control)
0.1-.
w/o tEGF
w/ tEGF
(Control)
PTCP coverslip condition
Figure 3-3. Day 9 colony metrics from image analysis of control (no tEGF) and
tEGF ) BTCP coverslips (A) Tethered EGF improves CTP Colony Forming
65
Efficiency (CFE) on BTCP surfaces at Day 9. Tethered EGF increased CTP CFE by an
average of 62 26% (Std Dev; p-value<0.0003). Results were normalized to the no tEGF
condition for each of the 8 patients. The observed prevalence (Pobs) of colonies on control
(untreated) tTCP substrates for each patient is as follows (in order for patients 1-8): 13, 5,
16, 35, 8, 6, 6, and 4 colonies per million cells. B) Tethered EGF does not change
number of cells per colony at Day 9. No statistical difference was observed between
the tEGF and controls. Results were normalized to the no tEGF condition for each of the 8
patients. The mean number of cells per colony on control (untreated) BTCP substrates for
each patient is as follows (in order for patients 1-8): 93, 63, 83, 46, 11, 17, 18, and 18. (C)
Tethered EGF does not change relative colony density at Day 9. This figure
shows the mean number of cells per unit colony area (within colony boundary) on 3TCP
surfaces with and without tethered EGF. Results were normalized to the no tEGF condition
for each of the 8 patients. No statistical difference was observed between the tEGF and
controls. The mean cell density among colonies on control (untreated) BTCP substrates for
each patient is as follows (in order for patients 1-8): 263, 249, 182, 178, 57, 114, 166, and
172 cells per mm 2 . (D) Tethered EGF does not change AP expression in colony
cells at Day 9. The Area Positive of Alkaline Phosphatase (APAP) was quantified as the
mean AP-positive area per colony divided by the number of cells in the colony, mm 2/cell (see
methods). No statistical difference was observed between the tEGF and controls, although
large variation was seen patients. Results were normalized to the no tEGF condition for
each of the 8 patients. The absolute mean APAP among colonies formed in the control
(untreated) 6TCP substrates for each patient is as follows (in order for patients 1-8, ): 3800,
4100, 5000, 3500, 5700, 6900, 2700, and 2800. Patients 1-3 represent bone marrow
aspirates (BM) and patients 4-8 represent samples from trabecular surface (TS) for all the
data shown.
3.3 Discussion
Intraoperative transplantation of freshly-aspirated bone marrow combined
with an osteoinductive scaffold is an attractive clinical strategy for treatment of
bony defects. This approach has minimal morbidity, and can improve efficacy over
that of autograft. However, efficacy requires that osteogenic progenitors (CTP-Os)
66
survive transplantation and are effectively activated to proliferate and give rise to
progeny that can differentiate into a bone phenotype.
BTCP has demonstrated clinical efficacy as an osteoconductive scaffold
(22,57,93), and may therefore serve as a generalizable substrate on which to build
more effective bioactive surfaces. The data shown herein demonstrate that a
bioactive surface presenting tEGF by means of a BTCP binding peptide-EGF (BPEGF) fusion peptide construct increases colony forming efficiency (CFE) in freshly
isolated human CTP-Os derived from bone marrow aspiration and from bone
trabeculae by a mean of 62%. This finding has potentially important clinical
implications.
Colony formation requires the presence of a CTP-O, and is used as a
measure of the concentration and prevalence of CTP-Os in a given sample.
However, the detection of CTP-Os is inevitably limited by CFE. Colony formation
requires a series of events beyond just the presence of a CTP-O, including survival,
attachment, activation to enter the cell cycle, and proliferation at a rate sufficient to
form an observable colony. Therefore, the mechanism by which CFE was increased
could include an increase in attachment of potential CTP-Os that would not
otherwise attach in the first 48 hours after plating, or an activation of additional
CTP-Os that would not have begun proliferating in the absence of interaction with
the tEGF. Moreover, because there is as yet no definitive means by which one
might detect potential CTP-Os in the absence of attachment, activation and
proliferation, we are not aware of any means by which it would be possible to
distinguish between these two possible mechanisms based on the data that are
currently available.
A tEGF effect on CTP-O attachment would not be surprising. Several prior
studies have provided evidence for soluble or tethered EGF-mediated effects on
attachment and spreading of connective tissue progenitors and other cell types.
67
Most relevant is the report that immortalized human bone marrow-derived
mesenchymal stem cells (BMMSCs) showed enhanced attachment and spreading on
PEO brush substrates presenting tethered EGF compared to cells plated in the
absence of EGF or in the presence of soluble EGF (74), highlighting the differential
effects of tethered and soluble factor. Tethered EGF has also been reported to
enhance the attachment, spreading, and survival of corneal epithelial cells plated
on modified polyethylene terephthalate films (166). The EGFR-overexpressing
epidermoid carcinoma cell line A431 showed enhanced adhesion and spreading in
the presence of either soluble or tethered EGF, in a dose-dependent fashion, when
plated on certain densities of the synthetic adhesion ligand RGD, and the effects of
tethered EGF were more pronounced than those of soluble EGF (167). Although
tethered EGF is known to activate EGFR and exert effects through prolonged
activation of Erk and other intracellular pathways (74,85,166,167), Segall and
coworkers have also described more proximal effects of tethered EGF on actin
polymerization at the membrane, mediated by neural Wiskott-Aldrich Syndrome
protein (168). The population of CTP-Os in freshly aspirated human marrow is
heterogeneous with respect to expression of adhesion receptors and attachment
potential (169) and thus it is reasonable to speculate that tEGF may enhance
attachment of a subpopulation of CTP-Os expressing relatively fewer adhesion
receptors.
A tEGF effect on CTP-O activation would also not be surprising. Gronthos
and Simons (66) provided evidence that the presence of EGF in a serum free
environment was necessary and sufficient to enable colony formation in bone
marrow derived cells isolated using the STRO-1 antibody. Soluble EGF also
enhances the proliferation of human mesenchymal stem cells cultured in serumcontaining medium compared to serum alone when cells are cultured on tissue
culture plastic (73).
68
In theory, it is possible that exposure to tEGF could act exclusively as a
signal that would increase proliferation rate and not influence attachment and
activation. An increase in proliferation rate without effects on attachment or
activation might result in a shift in the size of a population of slow growing colonies
that might not reach an 8 cell stage by day 9. However, this mechanism is unlikely
in our view for two reasons. First, we observed no evidence for a shift in overall
colony size (cells per colony, NCeins) in the tEGF samples, which would be the
expected result of a generalize effect on proliferation. Second, in prior work using
these assay methods we have not found evidence that a prolonged period of
observation (culturing for long periods of time) results in observation of a greater
number of colonies. Therefore there is no evidence to suggest the presence of a
slowly growing subset of CTP-Os that might be unveiled by a slight shift in
proliferation rate.
The fact that we found no evidence of change in cells per colony (Nceiis)(a
metric of proliferation), colony density (a metric of morphology and migration) and
APAP (a metric of early differentiation) suggests that tEGF did not have profound
effect on proliferation, migration or differentiation among the CTP-Os. However,
further studies over longer period of time (enabling later stages of differentiation)
and with an expanded array of metrics (gene expression analysis, in vivo
implantation) would be required to confirm the absence of effects in these domains
of CTP-O performance.
Surface modifications using a binding peptide linked to a tethered bioactive
molecule represents a strategy that has the potential to be broadly applied, given
the diversity of BTCP implants that are currently used for bone regeneration. An
increase in CFE could have important clinical implications. Improved attachment
and/or activation of local or transplanted CTP-Os has the theoretical potential of
increasing the amount of new bone formation in a bone defect site as well as the
rate of bone formation and remodeling in a bone defect site. tEGF surface
69
modification may also influence the overall success or failure of bone regeneration
procedures by tipping the balance of competitive advantage to favor CTP-Os in a
wound site over other competing cell populations.
Finally, we note that it is highly encouraging that tEGF stimulated an
increase in CTP CFE for all 8 patients (8/8 positive outcomes) without affecting
osteogenic marker expression or colony size. While this is a relatively small sample
number, the variation in colony parameters among patients was large, suggesting
that this approach may be operating by a mechanism that is fundamentally similar
in all patients.
3.4 Materials and Methods
3.4.1 BTCP Surface Fabrication
Eighteen-millimeter diameter glass coverslips (thickness 0.15 mm) were used
as a foundation for creating a flat disc of densely-packed BTCP particles. A 10:1
mixture of SYLGARD 184 Silicone Elastomer base and curing agent (Dow Corning,
Midland, MI) was degassed in a vacuum oven at RT for 20 min, spin-coated (50 ul
drop; 2000 rpm: 30 seconds) onto coverslips pre-treated with Siliclad (Gelest Inc.,
Morrisville, PA), and then immediately coated with BTC particles by sieving the
source material (particle size < 106 um, a gift from Integra Orthobiologics,
Plainsboro, NJ) through a 25 um sieve (VWR Scientific) onto the sticky PDMS layer.
Loosely-bound particles were removed by gentle tapping, the PDMS was cured at
60'C for 2 hours and then the coverslips were sonicated upside down at 50 Hz for 2
minutes in deionized (DI) water (Aquasonic sonicator, VWR Scientific), rinsed with
70
-
DI water, dried overnight at RT, stored dry and used within 4 weeks of fabrication.
3.4.2 Synthesis and purification of EGF fusion protein with endotoxin removal
A BTCP-binding peptide (BP), discovered through phage display, was fused to
EGF (BP-EGF) and produced in E. coli (163,164) (see Supplemental Online Material
for sequence). An amylose resin was used to purify the final fusion protein.
Endotoxin was removed using a modified on-column method (170). Briefly, the
column-bound protein was washed with 25 column volumes of a 1% Triton X-114 in
20mM Tris-buffer pH 7.4 and then washed with 25 column volumes of the same
buffer without Triton X- 114, all at 4*C. This resulted in a final product with <80
EU/mg protein as assessed by the standardized Charles River Endosafe (CRE)
system. This is 100-fold lower (~6,000 EU/mg) than untreated samples and around
15-fold less than commercial wild type EGF.
3.4.3 Tethering EGF onto BTCP coverslips
BTCP coverslips were UV-sterilized (45 min, 60-75 J/m 2 , each side), soaked in
EtOH (2 min) and rinsed with sterile DI water and then sterile PBS. Each coverslip
was inverted and placed in contact with a 150 uL drop of sterile, ice-cold solution of
BP-EGF protein (2.7 pM in PBS which results in estimated tEGF surface density of
-20,000 EGF per pm 2 (see Results) that had been pre-pippetted onto a sterile, BSAblocked (1% BSA in PBS, overnight incubation) parafilm substrate, such that the
layer of liquid formed was confined between the coverslip and the substrate.
Tethering was carried out by incubation at 4'C for 36 hours in a sterile humidifying
chamber. Coverslips were then washed with PBS and used within 3 hr. All steps
were done under sterile conditions and all buffer and protein solutions were sterilefiltered before use.
71
3.4.4 Post-tethering Characterization
EGF-tethered coverslips (N=4) were rinsed twice with PBS, placed in fresh
PBS solution, and then stored at 4C for 0, 24 hours or 5 days, then removed and
rinsed twice with PBS. The amount of EGF associated with each coverslip was
assessed by the standard BCA assay (Piercenet, Rockford, IL) (171) as described
previously (163,164) using the fTCPBP-EGF protein for the standard curve.
3.4.5 Isolation and culture of human bone and marrow-derived cells
Cohort 1: 3 patients - Bone marrow aspirates from the iliac crest were
collected from patients undergoing elective hip or knee arthroplasty procedures and
prepared as previously described, isolating a buffy coat twice to minimize the
-
number of residual contaminating erythrocytes (28,48,154). Cohort 2: 5 patients
Discarded cancellous bone from the proximal femur of human subjects undergoing
elective hip arthroplasty procedures. To harvest cells from the trabecular surface
(TS), the cancellous bone was washed in a-MEM with antibiotic/antimycotic (Life
Technologies, Carlsbad, CA), cut into small pieces (1-2 mm in diameter) with an
osteotome, and washed thoroughly to remove cells from the marrow space. Cells
that remained adherent to the trabecular surface were then recovered by
collagenase type I (100U/ml, Sigma, Inc.) digestion (172). This cell suspension was
used as a source of TS-derived CTPs. All samples were obtained under an IRB
approved protocol with informed consent.
For plating and culture, coverslips were placed in 2.05 x 2.05 cm 2 Lab-TekTM
(Nunc, Logan, UT) glass chamber slides. Cells were plated in osteogenic medium (cMEM, 10% fetal bovine serum, 50 j.M ascorbate, 10 nM dexamethasone and
penicillin/streptomycin) at 500,000 nucleated cells per coverslip (NC/C) for BM or
200,000 NC/C for TS based on previously-observed CTP prevalence (53). Cultures
72
were maintained at 37'C in a humidified atmosphere of 5% CO 2 at 20% oxygen in
osteogenic media with medium changes every 3 days.
3.4.6 Staining, imaging and data analysis
After 9 days of culture, the medium was aspirated, and culture chambers
were vigorously rinsed with PBS to remove non-adherent cells. The remaining
adherent cells were fixed with 1:1 acetone/methanol for 10 minutes at RT and air
dried. Prior to staining, cells were hydrated with DI water for 10 minutes.
VECTASHIELDTM (Vector Labs, Burlingame, CA) mounting medium containing
DAPI (0.75 pg/L) was applied in the dark overnight, followed by a rinse using
deionized water. Alkaline phosphatase (AP) activity was assayed using SK5100
Vector Red kit (Vector Labs, Burlingame, CA) according to the manufacturer's
instructions. Dry coverlips were stored in the dark at RT until image acquisition.
Coverslips were imaged and analyzed as described previously (173). Briefly,
two-hundred forty individual images were collected at 461 x 344 pixels (1641.16 Jim
x 1224.64 pm), 8-bit gray level, using a 1Ox objective (pixel size = 3.56 pm) using
ImagePro software. Emission (EM) and excitation (EX) wavelengths for DAPI and
AP are as follows: 340-380nm EX and >425nm for DAPI, and 566nm EX and 575670nm EM for AP. Image processing and analysis were performed using software
developed in the Muschler laboratory using algorithms written in the C/C++
programming language and Motif X-Windows TM environment (173).
3.4.7 Data validation and analysis
The effect of tethered EGF on
PCTPObs,
cells per colony, cell density among
colonies and AFAP was evaluated in a manner defined by the ASTM Standard
Method F2944-12. A mean was calculated for PCTPObs for each patient with and
73
without the presence of tEGF. A mean was also calculated for all colonies formed in
each patient with and without tEGF to provide the number of cells (nuclei) within
the colony (NCeiis), colony area (A), cell density (D)(nuclei per mm2), and the area of
positive alkaline phosphatase stain per cell (APAP) (36). To adjust for the known
wide variation in PCTPObs between individual subjects, the raw extracted colony
parameters for each patient (w/ and w/o tEGF) were normalized to the raw control
condition (w/o tEGF). This normalization provides a measure of the relative effect of
adding tEGF surface modification to the culture conditions in each subject sample.
3.4.8 Statistical analysis
Statistical analyses were performed using standard ANOVA methods or
student's t-test using PRISM software. Power analyses indicated that a sample size
of at least 8 patient samples was sufficient to yield a significant difference with a =
0.05 and power > 0.80.
74
Chapter 4
Evaluation of beta tricalcium phosphate scaffolds
with tethered epidermal growth factor in the canine
femoral multi-defect model
This chapter comprises experiments conducted in collaboration with the labs of
George Muschler and Alan Wells, and the major findings herein are being
submitted for publication with the following attributions:
Raut VP*, Rivera JJ*, Luangphakdy V, Alvarez LM, Wells A, Griffith LG, Muchler
GF. "Tethered EGF Enhances the Colony Forming Efficiency of Human
Osteoprogenitors Cultured on Beta-Tricalcium Phosphate Scaffolds"
*Denotes equal contribution
Acknowledgments: We want to acknowledge Esteban Walker; James Herrick; Linda
Stockdale; Kristen Shogren; Cynthia Boehm; Thomas Patterson,, PhD; Sunil Saini, PhD;
Amit Vasanji, PhD; Kenneth Litwak, DVM; Christopher Heylman, PhD; Richard Rozik, and
Edward Kwee for consultation and technical assistance.
4.1 Introduction
A broad range of biomaterials aimed at regenerating or accelerating bone repair
have emerged from research efforts in the field of tissue engineering and
regenerative medicine. Formation of new bone by the progeny of native connective
tissue progenitor cells (CTPs) is a central feature of proposed treatment options.
Connective Tissue Progenitors (CTPs) are defined as the heterogeneous population
of stem and progenitor cells that are resident in native tissues and are capable of
proliferation to give rise to progeny that will differentiate to express one or more
75
connective tissue phenotypes (22,102,118). The strategy of using a cell-based
therapy to achieve bone regeneration inevitably includes the potential to use
biomaterials to "target" CTPs; i.e., to use their interactions with a biomaterial
surface to enhance their attachment, survival, migration, proliferation, or
differentiation. Many of these same cellular responses can be targeted using other
means such as mechanical or biophysical environment or soluble bioactive factors.
Biomaterial surfaces represent a unique opportunity due to the potential that
they offer to control the co-localization of signals and responding cells. Molecular
surface design can be used to tether bioactive molecules, such as EGF, to the
surface of a biomaterial. Tethering improves control over the distribution of the
bioactive material and associated cell response. Tethering reduces diffusion of the
signaling molecule away from the surface, and enables control over the
concentration and 3-dimensional conformation of the molecule (36,73,85,174). In
contrast, most current clinical therapies deliver growth factors by diffusion release
(e.g., BMPs). When growth factors are released by diffusion, their local
concentrations and rates of clearance are difficult to control, and doses are
relatively high due to loss by cell consumption and diffusion away from the site.
Finally, tethered growth factors, particularly EGF, can preferentially activate
different intracellular signaling pathways compared to soluble factors, thus
dramatically changing phenotypic responses (73-75,105,175). For example, tEGF
leads to survival of MSC in the face of pro-death inflammatory cues, whereas
soluble EGF potentiates death under the same conditions (74). This cellular
response argues for tEGF as an agent in stem cell-mediated regeneration.
Bone marrow is widely used as a clinical source of autogenous CTPs (176179). Bone marrow can be harvested from a bone marrow aspirate with very low
morbidity and minimal pre-processing compared to other sources of CTPs such as
fat and muscle (102,180). Bone regeneration is enhanced by transplanting
osteogenic cells along with the scaffold material, particularly when large defects are
76
involved (22,39,55,118,162,181-186). However, large bone defects are associated
with the loss of local tissue sources of CTPs (e.g. bone marrow, fat, periosteum, or
muscle), and a vascular system for nutrient transport. As a result, the survival and
performance of transplanted CTPs is challenged by a harsh environment of hypoxia
and pro-inflammatory signaling molecules (54,102,104,118,173).
EGF is one of many potential modulators of stem and progenitor cells in
musculoskeletal tissue engineering applications. EGF is a potent mitogen for
osteoblastic progenitors, and is both necessary and sufficient to induce colony
growth in bone marrow-derived cells (36,66). Receptors for the EGF ligand (EGFR)
are expressed by virtually every cell type, including stem and progenitor cells.
EGFR is a receptor tyrosine kinase that activates intracellular signaling cascades
that influence cell proliferation, migration, and differentiation, and is the
prototypical member of the EGFR family, which includes EGFR, Her-2, Her-3, and
Her-4. EGF-EGFR interaction induces EGFR homo- or hetero-dimerization and
auto-phosphorylation, which activates multiple downstream intracellular pathways.
Once bound to the receptor, EGF is rapidly internalized by receptor-mediated
endocytosis leading to receptor desensitization, thereby down-regulating the effects
of exogenous EGF (187). Compared to other growth factors, EGF is a relatively
small protein with a structure that is suitable for chemical modification (188,189).
Beta-tricalcium phosphate (TCP) is widely used clinically as bone void filler
(56,190-195). TriCalcium Phosphate (Ca3(PO4)2) is not a natural component of bone
but has chemical proportions of calcium and phosphate similar to bone mineral
(196), and is easily resorbed by osteoclasts in vivo, providing surrounding osteogenic
cells with a course of calcium and phosphate needed for new bone formation.
However, the lack of functional groups for bioconjugation (-OH, -SH, -NH2, -COOH,
etc.) limits the capability to covalently tether biomolecules to the TCP surface. To
circumvent this problem, we designed and produced a fusion protein containing a
novel high affinity TCP binding peptide (BP) domain fused to EGF via a flexible
77
protease-resistant tether domain (BP-EGF, see Figure 4-1; (197). This molecule
binds to TCP in a stable and bioactive manner (197). Further, this fusion protein
approach to tethering EGF enhances osteogenic colony formation from human
marrow cultured on TCP scaffolds (198).
This study was designed to test the hypothesis that modification of the surface of
an effective TCP scaffold with tethered EGF will have a positive effect on in vivo
bone regeneration in a biologically-relevant large animal model (58,60).
Integra TherilokTm 0-TCP Scaffold
(C)
Tethering Strategy:
Use of a high-affinity
A-TCP binding peptide fused to
the bioactive ligand (EGF)
jMBr
EGF
MBF
b
nt
P-TCP
binding
peptide
Tether
[
P-TCP
Figure 4-1: A schematic of presentation of tethered EGF on TCP surfaces through use of a TCP
binding peptide. (A) Photograph of a 3mm TCP scaffold lying on top of a ruler to show its size. (B)
SEM image of a 3mm TCP scaffold taken at a 35x magnification (scale bar is 500um). (C) Tethering of
EGF (tEGF) onto the TCP surface is achieved by fusing a TCP binding peptide domain (BP) to the Cterminus of EGF (BP-EGF; previously referred to as BP10-T-EGF (197)). A protease-resistant tether
provides mobility to the tethered EGF molecule.
78
4.2 Results
All subjects completed the procedure and outcome assessment without surgical
or postoperative complications.
4.2.1 BP-EGF binding, implanted dose, and stability
A mean of 6.3 micrograms of BP-EGF was tethered per TCP scaffold. As a result,
the implanted dose of TCP was 208 micrograms per defect site (6.3 micrograms X 33
scaffolds per defect).
BP-EGF surfaces were stable over 7 days in PBS. Retention of BP-EGF on
scaffolds over 7 days in PBS at 37 0 C was 100 +/- 6% at day 1 and 78 +/- 5% on day 7.
When tested on TCP powder at 4C in 1xPBS, retention was 98 +/- 1% on day 7
(p=0. 19).
4.2.2 Cell and CTP Retention and Delivery on TCP Scaffolds
Table 4-1 provides a summary of the cell and CTP retention and delivery on the
two scaffold constructs. The mean concentration of cells in the initial marrow
aspirate samples was 97 +/- 20 million cells/cc. The mean retention efficiency of cells
(REceis) in the TCP and tEGF-TCP groups was 38.6 +/- 4.7% and 44.8+/- 8.8%,
respectively. As a result, the mean concentration of cells in the TCP and tEGF-TCP
implants increased to 192 +/- 57 million cells/cc and 220 +/- 62 million cells/cc. This
represents an increase in cell concentration of 0.97 and 1.26 fold, respectively.
79
Nucleated cell concentration in bone marrow (million cells/CC): 97+/- 20
CTP concentration in bone marrow (thousand cells/CC): 23+/- 11
TCP group
tEGF group
192
220
Std. Deviation
57
62
Mean
56
54
Std. Deviation
31
36
Mean (%)
38.6
44.8
Std. Deviation
4.7
8.8
p-value (a<0.05)
0.12
Mean (%)
69.7
64.2
Std. Deviation
2.6
12.8
p-value (a0.05)
0.39
Mean (%)
1.9
1.5
Std. Deviation
0.28
0.64
p-value (a<0.05)
0.2
Mean
Cell Concentration
CTP
Concentration
(million cells/CC)
(thousand cells/CC)
Cell Retention
Efficiency
CTP Retention
Efficiency
Selection Ratio
Table 4-1: Selective Retention of cells and CTPs on TCP scaffolds with, or
without tethered EGF. These results demonstrate that tethered EGF does not
significantly influence the retention efficiency of cells or CTPs to the TCP scaffolds.
The mean concentration of CTPs in the initial aspirates was 23,000 +/- 11,000
CTPs/cc. The RE for CTPs (RECTPs) was 69.7 +/- 2.6% and 64.2 +/- 12.8%
respectively. As a result, the mean concentration of CTPs in the TCP and tEGFTCP groups increased to 56,000 +/- 31,000 CTPs/cc and 54,000 +/- 36,000 CTPs/cc of
scaffold, respectively. This represented a mean overall increase in CTP
concentration of 1.43 and 1.34 fold.
80
Both scaffolds demonstrated greater retention of CTPs than non-progenitor cells.
This is reflected in a selection ratio (REcTP/REcens) greater than 1.0 (1.9 +/- 0.28 for
TCP and 1.5 +/- 0.64 for tEGF-TCP respectively).
The tEGF modification resulted in no objective change in cell or CTP attachment
and retention behavior, as there were no significant differences were seen between
scaffold surfaces with respect to retention efficiency, selection ratio, changes in cell
or CTP concentration or changes in CTP prevalence.
4.2.3 Micro-CT Assessment of Bone Formation
Figure 4-2A illustrates the mean distribution of new bone formation in units of
percent bone volume (%BV) in the pericortical (PC) and intramedullary (IM) region
in each group using a 2D color map with a scale ranging from 0% (light blue) to 60%
(red). Depth in the defect is reflected along the x-axis, where the center of the
defect is at "0" and the periphery of the defect is at 5 cm (5 cm from the center of the
defect). The distance from the bottom of the defect is reflected in the y-axis. Figure
4-2B presents a 2D plot of mean %BV data relative to radial position within the
defect for both TCP and TCP-tEGF scaffolds across all heights in the PC and IM
regions.
The mean %BV for the TCP and tEGF-TCP groups over the entire defect was
46% vs. 38%, respectively.
In the PC region, the mean %BV was 48 % and 43% for
TCP and tEGF-TCP groups. In the IM region, the mean %BV was 45 % and 33%
respectively.
The mean % BV values had a very little variation between animals.
The mean %BV for all dogs was 42% (range 41.7 to 42.2). These data demonstrate
robust bone formation in all regions of the defect using both materials. As in prior
studies (54,58,60,112), %BV tended to be greatest in the PC region and to decrease
81
,
as one moved toward the center. In contrast to prior studies, %BV in the IM region
tend to increase as one moved toward the center of the defect, despite subtraction of
high density residual TCP.
TCP
PC
E
TCP + tEGF
11.5
11.5
11.0
11.0
10.5
10.5
10.0
10.0
9.5
9.5
9.0
9.0
8.5
8.5
E
Z
0
.0
E
0
8.0
0
1
2
3
4
5
8.0
0
1
2
3
~0
~10
%BV
~20
4
30
S
40
-
IM
6.5
6.5
6.0
6.0
5.5
5.5
5.0
5.0
4.5
4.5
4.0
4.0
3.5
3.5
3.0
3.0
4
0
5
1
2
3
4
Radial Distance from center [mm]
(A)
Mean %BV vs. Radial Position
65
TCP (PC)
60
55
50
TCP+tEGF (PC)
45
40
TCP (IM)
TCP+tEGF (IM)
35
30
25
20
0
1
2
3
Radial Position (mm)
(B)
A
82
4
5
5
Figure 4-2: Micro CT Analysis Results: (A) 2D Overlay plots illustrating mean %BV
as a function of radial and height. (B) Separating PC and IM regions.
4.2.4 Histology Data
Quantitative histomorphometry data is summarized in Table 4-2, describing the
mean tissue composition of TCP and TCP-tEGF defects with respect to void space,
hematopoietic marrow, vascular spaces, fibrous tissue, residual scaffold, woven bone
and new bone formation. Histological sections through the centerline of each defect
are presented in Figure 4-3 Both TCP and tEGF-TCP groups showed robust new
bone formation. An extended interconnected network of trabecular bone was
generally present with significant areas of unmineralized osteoid and frequent
vascular spaces. New bone was present on the surface of residual TCP scaffold.
Active remodeling by osteoclasts was seen throughout in the implant area.
Residual scaffold material occupied a small fraction of the total defect area (less
than 20%), indicating the advanced stage of bone remodeling. H&E slides showed
no evidence of inflammation in both TCP and tEGF-TCP groups.
The tissue composition was comparable in both groups with the exception that
TCP-EGF defects sites overall has 38% less residual TCP and 54% more fibrous
tissue. Data summarizing the contribution of each tissue type to the entire defect
and to the PC and IM regions of these defects is provided in Tables 4-2(A-G).
New bone area in the defect was comparable in both groups, 17.2% and 18.6%
(SE= 2.0%) for TCP and tEGF-TCP groups respectively. The difference between
TCP and tEGF-TCP groups was not statistically significant, though there was trend
in favor of tEGF-TCP. New bone formation was greater in the PC than in the IM
region overall in both groups, 20.8% and 22.3% in the PC region compared to 13.5%
and 14.9% in the IM region for the TCP and tEGF-TCP groups respectively (p <
0.0001), consistent with microCT data.
83
The area occupied by residual scaffold in the total defect was 14.7% and 9.1%
(SE = 2.8) for TCP and tEGF-TCP groups respectively, but not statistically different
(p=0. 19). Hematopoietic marrow was the most predominant tissue in both groups,
representing 37.2% of the tissue area in the TCP defects and 35.8% in the tEGFTCP defects. Fibrous bone marrow comprised a small portion of the tissue area in
both groups, 4.3% and 9.4% (SE = 2.4) in the TCP and tEGF-TCP groups
respectively. The area of vascular/ sinus tissue was 10+/- 1.4% and 8+/- 1.4% for
TCP and tEGF-TCP groups respectively. None of the histological differences
between TCP and tEGF-TCP groups were statistically significant.
(A): Percent New Bone Area
Region
Total Defect
IM
PC
Material
Mean
SE
TCP
17.2
2.0
TCP + tEGF
18.6
2.0
TCP
13.5
2.4
TCP + tEGF
14.9
2.4
TCP
20.8
2.4
TCP + tEGF
22.3
2.4
p-value
0.63
0.67
1 0.67
Comparison between IM and PC: p < 0.0001
(B) Percent Woven Bone Area
Region
Material
Mean
SE
TCP
0.53
0.07
TCP + tEGF
0.59
0.07
TCP
0.54
0.10
TCP + tEGF
0.38
0.10
Total Defect
IM
p-value
0.58
0.24
84
PC
TCP
0.52
0.10
TCP + tEGF
0.80
0.10
0.047
Comparison between IM and PC: p = 0.02
(C) Percent Residual Area
Region
Total Defect
IM
PC
Material
Mean
SE
TCP
14.7
2.8
TCP + tEGF
9.1
2.8
TCP
12.7
3.2
TCP + tEGF
10.5
3.2
TCP
16.6
3.2
TCP + tEGF
7.6
3.2
p-value
0.19
0.62
0.06
Comparison between IM and PC: p = 0.81
(D) Percent Hematopoietic Marrow Area
Region
Total Defect
IM
PC
Material
Mean
SE
TCP
37.2
3.7
TCP + tEGF
35.8
3.7
TCP
38.5
4.5
TCP + tEGF
32.2
4.5
TCP
35.9
4.5
TCP + tEGF
39.3
4.5
p-value
0.79
0.34
0.60
Comparison between IM and PC: p = 0.53
(E) Percent Fibrous Marrow Area
Region
Material
Mean
85
SE
p-value
TCP
4.3
2.4
TCP + tEGF
9.4
2.4
TCP
4.7
2.9
TCP + tEGF
13.2
2.9
TCP
4.0
2.9
TCP + tEGF
5.5
2.9
Total Defect
IM
PC
0.17
0.48
0.71
Comparison between IM and PC: p = 0.02
(F) Percent Vascular Sinus Area
Region
Total Defect
IM
PC
Material
Mean
SE
TCP
9.6
1.4
TCP + tEGF
8.3
1.4
TCP
12.3
1.8
TCP + tEGF
9.6
1.8
TCP
7.0
1.8
TCP + tEGF
6.9
1.8
p-value
0.51
0.29
0.98
Comparison between IM and PC: p = 0.01
(G) Percent Void Area
Region
Material
Mean
SE
TCP
16.4
2.1
TCP + tEGF
18.3
2.1
TCP
17.7
2.5
TCP + tEGF
19.0
2.5
15.2
2.5
17.6
2.5
Total Defect
IM
TCP
PC
TCP + tEGF
0.55
1
Comparison between IM and PC: p = 0.31
86
p-value
0.72
0.51
Table 4-2: Summary of Quantitative Histomorphometry Analysis. Tables 4-2AG summaries the quantitative comparison between TCP and EGF-TCP groups with respect
to new bone formation, woven bone, residual scaffold, hematopoietic marrow, fibrous
marrow, and vascular/sinus tissue.
0.31X
Animal 4
Animal 3
Animal 2
Animal 1
Site 1
tEGF
TCP
TCP
tEGF
Site 2
TCP
tEGF
tEGF
TCP
Site 3
TCP
tEGF
tEGF
TCP
Site4
tEGF
TCP
TCP
tEGF
Figure 4-3: Representative Goldner's stain histology images (at 0.31x). Each
histological section is 5 gm thick and cut along the bone axis through the central axis of the
defect site. Goldner's stains mineralized bone in green, unmineralized osteoid in red and
residual TCP scaffold in grey.
87
4.3 Discussion
This paper provides the first report of the in vivo performance of the BP-EGF
fusion protein as a method for surface modification of bone regeneration scaffolds
containing Beta-TCP (TCP). Commercially-available TCP scaffolds fabricated using
three dimensional printing (3DP) (TheriLokTM - Integra Life Sciences) were used as
a well-characterized platform for assessment of the growth factor effects. This
paper also provides the first report of the in vitro effect of BP-EGF fusion protein on
the attachment and retention of cells and CTPs in freshly isolated bone marrow
derived cells on TCPBP-EGF modified surfaces.
Much prior work has demonstrated that inclusion of adherent bone marrow
derived cells and CTPs improve the performance of bone regeneration scaffolds
compared to scaffolds without added cells (22,118). This assessment of TCP
scaffolds with and without BP-EGF modification was performed in a setting in
which cells and CTPs were added using the established technique of selective
retention (SR).
There are several theoretical advantages of using EGF as a bioactive agent in
the presence of transplanted cell populations, particularly CTPs. EGF is a known
mitogen for many progenitor cell populations (36,73,74,76) including bone marrow
derived CTPs (36). EGF has also been shown to activate intracellular pathways that
have potent anti-apoptotic effects, particularly when restricted to surface signaling
as with tEGF (74). This property is particularly valuable in the setting were cells
are transplanted in a stressed environment rich in pro-apoptotic signals (hypoxia
and pro-apoptotic cytokines) (74). EGF has also been shown to be a potential
activating agent in the activation of connective tissue progenitors (potential colony
founding cells), based on the fact that in serum free conditions exposure to EGF is
necessary and sufficient to induce colony formation (66).
88
There are several advantages of tethering EGF and other biomolecules to the
surface of an implanted biomaterial. Tethering provides the opportunity to control
the local concentration and distribution of a bioactive agent, and to control its
presentation in a bioactive confirmation (76,188,199). Soluble molecules like EGF
would normally diffuse rapidly away from the site of implantation; however,
tethering enables retention of the tethered growth factor in the desired location,
prolonging its potential biological effect (36,75,76,188). Tethering also offers the
opportunity to enhance the signaling of some ligands (including EGF) by pairing or
clustering ligands in such a way that dimerization of receptors is enhanced. Prior
work in an in vitro model in which the BP-EGF fusion protein was presented on a
2D surface of adherent beta TCP granules demonstrated an increase in colony
forming efficiency when seeded with human bone marrow derived cells, without a
change in expression of alkaline phosphatase (a marker of bone differentiation)
(198).
The assessment of both in vivo and in vitro performance presented in this paper
was accomplished using the established canine femoral multidefect (CFMD) model.
This model was selected as a screening platform based on several factors. The
CFMD provides a defect size that is large enough (10 mm diameter x 15 mm in
height) so that the survival of cells that are transplanted into the defect site are
limited by nutrient diffusion, and specifically the diffusion oxygen from the
vascularized periphery of the defect into the center (156). The CFMD model
provides 4 defect sites per animal, increasing the power of the data available,
allowing control for variation between animals, and reducing the burden of animal
life required for preliminary assessment in a large animal. The CFMD model has
also been characterized to have the potential to heal rapidly in the presence of a
highly effective bone graft construct. As a result, outcome can be assessed at 4
weeks, rather than much longer time periods that are needed for other common
large animal models, shortening the time needed to make effective comparisons and
potentially accelerating the rate at which systematic assessments can be made
89
(60,112). The CFMD model has been shown to be a sensitive model in which
biological differences between implanted synthetic scaffolds can be discerned in the
absence of added marrow (112). It has been used to characterize the performance of
autologous cancellous bone and allograft bone, the current clinical standards for
bone grafting. It has also been shown to be sensitive to the incremental effects of
bioactive agents, such as OP- 1 (58), as well as the effects of cell processing methods
that change in the cellular composition of implanted scaffold materials using both
microCT and histomorphometry as outcome measures (60). Moreover, the harvest
of bone marrow by aspiration from the proximal humerus of the coonhound can be
done using methods commonly used in human clinical practice, and with similar
yield and performance (54,58,60,112).
Despite these positive attributes, the limits of the CFMD model were reached in
this assessment.
Bone regeneration in both TCP-tEGF and TCP groups was so
robust compared to many previous scaffolds that it left relatively little room for
improvement; even though we saw a trend suggesting that tEGF improved
performance, this never reach statistical significance. We may therefore have
encountered a "ceiling effect" that may have limited our ability to detect a
biologically relevant effect of tEGF in increasing bone formation or accelerating
remodeling. Further assessment using a more stringent, larger bone defect in a
large animal model where healing is more significantly impaired by local tissue
compromise, inflammation and/or defect size is required to test the hypothesis that
tethered EGF enhances healing.
In vitro data collected during the preparation of the implant materials was
instructive regarding the performance of TCP scaffolds and BP-EGF modified TCP
surfaces. BP-EGF surfaces were found to be stable over 7 days on both TCP powder
and the formed TCP scaffolds.
90
Both TCP scaffolds and BP-EGF modified scaffolds performed well as surfaces
that allow for attachment and retention of cells and CTPs from freshly harvested
and heparinized bone marrow. When both TCP and BP-EGF scaffolds were loaded
with cells by passing a 6x excess volume of aspirated marrow derived cells in a
heparinized suspension through these matrices at a controlled rate, roughly 40-45%
of marrow derived cells were retained but 65-70% of colony founding CTPs were
retained. This resulted in an increase in the concentration of implanted cells and
CTPs (over 2 fold), as well as significant increase in the prevalence of CTPs (1.5 to
1.9 fold) among the cells implanted. This is consistent with the finding previously
reported using selective retention processing of both canine and human marrow (54)
Both microCT and histology image provide insight in to the composition of the
tissue within the defect. However, these two tools each have strengths and
weaknesses, and are not directly comparable due to the differences in sampling and
metrics. MicroCT has the advantage of being able to sample the entire volume of
every defect with a slice resolution of 45 pm, while histomorphometry was derived
from a 5 pm thick section taken through just the center of the defect. MicroCT data
in this study, was designed to be the "source of truth" for estimating the magnitude
and distribution of mineralized tissue, which should be most sensitive to differences
in new bone formation.
Histomorphometry, however, has the distinct advantage, of
directly evaluate the nature of the tissue. Histomorphometry can distinguish
between new bone formation and residual TCP as sources of mineral density, which
may appear the same on microCT. Histomorphometry can also distinguish between
cellular tissue, vascular structures, fat, and fibrosis and identify evidence of
inflammatory processes that are inaccessible using microCT alone. Histomorphometry, was designed as the "source of truth" for assessment of the relative
amount of residual scaffold in the defects and the relative composition of new
tissues formed with and without tEGF.
91
Correlation between histology data and microCT data provides some
confirmation that the thresholding decisions made in microCT analysis were
accurate.
The sum of the area of new bone formation and residual scaffold in both
scaffold groups seen in histomorphometry was approximately 30%. This is a good
match to for microCT data which was 42%, particularly when one remember that
histomorphometry data will be biased to underestimate the volume of bone
formation in this model because relative areas of the periphery of the defect sites
where bone formation is generally greatest is underrepresented in the center-cut
histomorphometry slices that were used.
Most important is that fact that histomorphometry revealed no evidence of
inflammatory reaction or compromise of tissue quality. Tissue formed in the
presence of local EGF signaling formed normal appearing bone, vascular
hematopoietic marrow, including intramedullary fat, with normal patterns of
osteoclast-mediated remodeling, without significant fibrosis. The trend for less
residual scaffold and lower mineral density in the IM region of the defect suggests
the possibility of a more advanced remodeling status in EFG treated sites, but these
differences were not significant.
4.4 Materials and Methods
4.4.1 Animals and Experimental Design
This study was conducted with an approval from the Cleveland Clinic
Institutional Animal Care and Use Committee, and in accordance with the
Principles of the Guide for the Care and Use of the Laboratory Animals. Four
skeletally mature research purpose-bred female coonhounds (31.6
2.2 kg), age
ranging from 1-3 years (mean = 1.9 years) were utilized. Materials were assessed in
the established CFMD model (58,60).
92
4.4.2 Synthesis and purification of BP-EGF protein
A TCP-binding peptide (BP), discovered through phage display, was fused to
EGF (BP-EGF) and produced in E. coli (197). The fusion protein contains 10
concatenated repeats of the binding protein motif fused to EGF via a flexible tether,
and herein we use the terminology "BP-EGF" as a short-hand acronym for the
-
longer more descriptive acronym "BPio-T-EGF" (Binding Protein, 10 repeats
Tether-EGF). An amylose resin was used to purify the final fusion protein and an
on-column method was used for endotoxin removal (170) . The column-bound
protein was washed with 25 column volumes of a 1% Triton X- 114 in 20mM Trisbuffer pH 7.4 and then with 25 column volumes of the same buffer without Triton
X- 114, at 4*C. Purified protein was tested for endotoxin using a standardized
Limulus Amebocyte Lysate (LAL) assay and the Charles River Endosafe (CRE)
system. The sensitivity of this assay kit was 0.05-5 Endotoxin units (EU)/ mL.
4.4.3 Tethering BP-EGF onto TCP
TheriLokTM TCP scaffolds and powder were provided by Integra Orthobiologics
(Plainsboro, NJ). TCP scaffolds were sterilized using steam autoclaving (121*C for
20 minutes at 15 psi). Polypropylene 96-well plates were used to incubate individual
scaffolds. Prior to incubation, the well plates were treated with 150 uL of sterile 1%
BSA (Calbiochem) overnight at 4'C, rinsed twice with 1X PBS, allowed to dry in a
hood, and stored at 4'C. BSA was used to minimize the non-specific adsorption.
BP-EGF protein solution was prepared at 2 pM on ice using 1X PBS as a
diluting agent. The scaffolds were first hydrated in 1X PBS under negative pressure
(2.4 mPa) for 5 minutes. Hydrated sterile TCP scaffolds were immersed in 125 pL of
BP-EGF solution in each well of the 96-well plate, and incubated at 4'C for 36 hours
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in a humidified chamber. Scaffolds were washed twice with 1X PBS immediately
before cell loading. TCP scaffolds incubated in 1X PBS were used as control.
The stability of BP-tEGF surface coating was assessed in vitro using the TCP
powder for the uniformity of quantification. Beta TCP powder (Integra
Orthobiologics, Plainsboro, NJ) was sieved to <106 um and sterilized in a steam
autoclave. Low-protein binding Durapore@ filter plates (Millipore, 1.2um pore size)
were used to facilitate washes without loss of TCP. Ten mg of TCP powder were
placed in the wells of the filter plate and 125pL of 2uM BP-EGF protein solution
was added. The 2uM tethering concentration and the corresponding surface dose of
tEGF was chosen based on previous in vitro experiments with low-passage human
MSCs, where this surface resulted in a -1.5-2 fold increase in cell number after a 7day culture in expansion media (197). The plates were then sealed with plastic
adhesive covers and placed at 4*C for 36 hours.
4.4.4 Quantification of tethered EGF and stability
The amount of BP-EGF bound to TCP scaffolds or TCP powder was assessed
by the bicinchoninic acid (BCA) assay (Piercenet, Rockford, IL) using BP-EGF
standards (171,200) with detailed methods as described (197). Binding and stability
were assessed at Days 0 and 7. To assess binding stability to TCP powder, the filter
plates containing the tethered TCP powder were rinsed twice with 200uL of 1xPBS.
For the Day 0 scaffolds, a measurement of bound protein was made immediately
after rinsing (N=3). For the Day 7 condition, 200uL of 1xPBS were added postrinsing. The wells were covered and stored at 4*C for 7 days. The wells were then
rinsed before protein assay. The residual BP-EGF absorbed on the walls of the filter
plate well during tethering (previously measured, approximately 6.5% of the total)
94
-
was subtracted from the raw protein measurements.
4.4.5 Bone Marrow Aspiration and Preparation
Bone marrow was aspirated percutaneously in 2 mL aliquots from the proximal
humerus using a Lee-Lok bone marrow aspiration needle (Lee Medical, Ltd,
Minneapolis, MN) and immediately mixed with 1 mL of normal saline solution
containing 1000 U Na- Heparin. Total volume of 30 mL heparinized bone marrow
was collected from each humerus. The concentration of nucleated cells was
determined by first lysing the red blood cells by mixing with 0.3% acetic acid and
then counting nucleated cells using a hemocytometer. Previous experience has
demonstrated that the canine proximal humerus is a reliable source of hematopoetic
bone marrow containing osteoblastic progenitors, with yields of nucleated cells and
CTPs that are comparable to human iliac crest aspirates (201).
4.4.6 Assessment of Attachment and Retention of Marrow- Derived Cells and CTPs
33 scaffolds were used to fill sterile polypropylene cylindrical chambers (1 cm
diameter, 1.5. cm height), (Figure 4-4A). Caps were applied at the top and bottom of
chamber. The chambers were then press fit into the inner diameter of a 10 mL
syringe. The syringes were mounted vertically on a syringe pump apparatus (Brain
Tree Scientific, MA, BS-9000). Six mL of heparanized bone marrow suspension was
pulled through the cage at a flow rate of 1.5 mL/minute (~2.4 mPa vacuum). The
concentration of cells and CTPs in the initial and effluent marrow suspension was
measured to determine the number of cells and CTPs retained in each matrix (54).
Cells from each sample were plated in 2 cm x 2 cm LabTek chamber (Nunc, Logan,
UT) (N=6) at 500,000 cells per chamber, and cultured for 6 days at 37'C in a
humidified atmosphere of 5% C02 and 20% oxygen. Osteogenic medium (a-MEM, 10%
fetal bovine serum, 50 pM ascorbate, 10 nM dexamethasone and
penicillin/streptomycin) was used. Media was changed at Day 3. The CTP
prevalence (number of colonies per 1 million nucleated cells) was measured using an
95
established CTP Colony Forming Unit assay (202). The scaffolds loaded with cells
were protected from desiccation in a closed sterile chamber until implantation.
Retained [cells]
1 Retained fCTPl
Initial fcells]
Initial JCTPJ
Initial- Effkuent= Retained
Effluent[cells]
Effluent [CTP
(B)
(D)
Figure 4-4: Preparation and Implantation of Scaffolds: (A) An open sterile
polypropylene cage (1cm dia, 1.5 cm height) containing 33 scaffolds, (B) Heparinized bone
marrow aspirate (BMA) was passed through the cage to allow cells and CTPs be exposed to
the implant surfaces. Non-adherent cells in the effluent were assayed for cells and CTPs
and discarded. (C) An open polypropylene cage after cell and CTP retention. The number of
cells and CTPs that were retained in the implant was determined by subtracting the
number of cells and CTPs from effluent sample from those in the initial sample. (D) Four
cylindrical defects (1cm dia, 1.5 cm height) were drilled. Scaffolds were then implanted in
the femoral defects of a canine. A zoomed in view is shown in the upper right corner.
96
Using the cell and CTP counts in the original sample (No, CTPo) and effluent
samples (NE, CTPE), the number of cells and CTPs retained in each graft, and the
retention efficiency (RE) for cells and CTPs were calculated (Figure 4-4B). The
number of cells and connective tissue progenitors retained in the matrix (NR and
CTPR) was determined by the following equations: NR = No
-
NE and CTPR =
CTPo - CTPE. The retention efficiency (RE) for cells and CTPs (REcens and
REcTP)was calculated as follows: REceiis = NR/No and RECTP = CTPR/CTPo. The
selection ratio (SR), relative efficiency of retention, for CTPs vs cells was calculated
as: SR = REcTP/ REcens). A selection ratio greater 1.0 implies positive selection or
enrichment of CTPs in the implant with respect to other marrow cells as a result of
preferential retention on the implant surface.
4.4.7 Scaffold implantation procedure
The surgical methods, animal care and sample processing have been previously
described (60,112). Briefly, a 10 cm skin incision was made along the longitudinal
axis of the femur. The femur was exposed in an extraperiosteal plane by blunt
separation of the biceps femoris and the vastus lateralis. A customized drill and
template system is used to create four identical 10 mm diameter x 15 mm long
unicortical cylindrical defects in the metaphysis and the proximal diaphysis of the
femur, as described in Figure 4-4C . A 1.0 cm diameter by 1.5 cm long stainless
steel spacer was placed in each defect to allow hemostasis to be achieved prior to
implantation.
Scaffolds from the TCP and tEGF-TCP groups were placed in each defect by
picking individual scaffold out of the chamber (Figure 4-4C) and placing them in an
orderly fashion to fill the defect (Figure 4-4D). Prior to closure, a customized
stainless steel plate was fixed to the femur using two 3.5 mm cortical screws passed
through the screw sites used to fix the drill template to the femur. This plate
97
reduces the risk of pathologic fracture by protecting the grafted region from
excessive bending and rotational stress.
4.4.8 Sample Processing
After 4 weeks implantation, the animals were sacrificed. The femur was
explanted and cleaned of soft tissue. The plate covering the defects was removed.
Individual graft sites were separated using a band saw. Each site was fixed in 10%
neutral buffered formalin. After 48 hours, the solution was replaced with 70%
ethanol to prevent demineralization. Samples were then sent for Micro-computed
Tomography (ImagelQ, Inc) and then to the Bone Histomorphometry Laboratory at
Mayo Clinic for histological assessment.
4.4.9 Micro-CT Analysis
Specimens was placed on the scanning platform of a GE eXplore Locus mCT (GE
Healthcare, Milwaukee, WI) in the cranial-caudal axis, parallel to the direction of
table travel, and 720 X-ray projections were acquired at 80 kV, 490 pA, with an
exposure time of 525 ms per individual projection. A dark-field (x-ray beam turned
off), a bright-field (x-ray beam turned on with no sample in beam), and density
calibration phantom (hydroxyapatite, solid water, air) images were taken at the end
of each imaging session for correction of the x-ray projection data (dark- and brightfield normalization, ring artifact reduction, and CT attenuation to Hounsfield Unit
conversion during projection reconstruction). Projection images were preprocessed
and reconstructed into 3D volumes (45 lim resolution) on a 16PC reconstruction
cluster using a modified tent-FDK cone beam algorithm (GE reconstruction
software).
98
g
Each specimen was analyzed with a segmentation software developed in-house,
in which a 3D cylindrical "defect template" volume, 10 mm in diameter and 15 mm
in length size, was manually positioned to define the boundaries of the defect site
using the circular introitus and marks from the flat finishing drill on the opposite
cortex as fiduciary guides. Regions of new bone formation within the defect site
were identified based on calibrated Hounsfield Units (HU) within each specimen to
provide reproducible thresholding across all specimens. Each specimen was
visualized as a 3D volume using MicroView software (GE Healthcare, Milwaukee,
WI). A skilled operator who was blinded with respect to the material being assessed
used the native bone tissue outside of the defect site as a reference to define, for
each sample: 1) a lower bone threshold which excluded soft tissue, but was below
the density of native trabecular bone and any periosteal new bone formation evident
in the sample and 2) an upper density threshold, which excluded voxels with a
density that was too high to be considered as new bone formation and therefore
indicative of unresorbed TCP. This upper threshold was set as a level that excluded
all voxels that were at or above the density of native cortical bone, based on the
rationale that little of the new bone formation present in the defect could achieve
the mineral density of cortical bone by 4 weeks after grafting and that any voxel
with a density that was equal to or greater than the density of native cortical bone
must be assumed to represent residual TCP material.
The mean upper and lower thresholds for the two groups were not different: 1)
for the TCP group: 1935 +/- 141 HU and 1140 +/- 74 HU, respectively; 2) for the
tEGF-TCP group: 1907 +/- 96 HU and 1115 +/- 87 HU, respectively.
As described in previous studies (60,112), to eliminate the inclusion of
confounding sampling artifacts in the analysis, regions of the top, bottom, periphery
and center of the defect were excluded from analysis. The 3-mm regions from the
top and bottom of the defect were excluded to eliminate the potentially confounding
effects associated with tissue reaction at the cortical bone surface at the base of the
99
defects and tissue reaction at the opening of the defect sites. Data from the very
edge (4.75.-5.0 mm from the center) were excluded to avoid inclusion of existing
cortex at the edge of the defect in the analysis. The very center (0-0.25 mm) was
also excluded from analysis due to the vanishing small sampling volume that is
assessed as one approaches zero in the defect and the increased variability that this
creates in data from the very center. Specific regions of interest were defined for
analysis based on vertical position and radial position within the defect site (Figure
4-2). The pericortical (PC) region was defined as that region between 8 to 12 mm
from the bottom of the grafted site. The intramedullary (IM) region was defined as
that region between 3 to 7 mm from the bottom of the grafted site.
4.4.10
Histology Analysis
Each sample was processed and analyzed in the Bone Histomorphometry
Laboratory at the Mayo Clinic (Rochester, MN) using undecalcified processing.
After rehydration in a graded series of alcohols, and embedded in Methyl
Methacrylate without decalcification, a Leica RM 2265 microtome was used to cut 5
micron thick sections along the long bone axis, through the middle of the defect site.
Sections were stained with modified Gomori's Trichrome, then scanned using a
NanoZoomer Digital Pathology System (Hamamatsu, Bridgewater, NJ) and
analyzed using manual analysis with the OsteoMeasure system (OsteoMetrics,
Decatur, GA). In order to provide systematic and representative sampling, a
contiguous series of 20x magnification fields were examined extending in a
transaxial plane across the defect site through the mid-portion of the PC and IM
regions (across the entire defect width at 10 mm and 5 mm, respectively, from the
bottom of the defect). The center, middle, and outer regions are compared using the
same radial dimensions as in microCT analysis (60,112). Regions of cellular new
unmineralized bone (active osteoblasts), mineralized woven bone, acellular residual
MCA, fibrous marrow tissue, sinus/vascular space, hematopoietic marrow and void
100
"
space were identified and manually traced by a skilled technician. The relative
area for each of these parameters was expressed as a percentage of the total tissue
area in the defect site.
4.4.11
Statistical Analysis
Statistical Analysis was performed using JMP® 10.0.1 software (SAS@, Cary,
NC). An ANOVA model was used to test for differences between scaffolds (TCP and
tEGF-TCP groups), sites (proximal or distal), and depth regions (PC or IM). The
effect of canine (each dog) was included as a random factor. All two-way interactions
were included in the model. The results are expressed as mean
Statistical significance was reached if p < 0.05 (60,112).
101
standard error.
Chapter 5
Conclusion
5.1 Summary
This thesis demonstrates the novel use of a beta-tricalcium phosphate
binding peptide-EGF fusion protein (BP-EGF) for tethering EGF onto BTCP
materials. Beta-TCP-containing materials often serve as bone graft substrates in
the clinic. In order to test the bioactivity of these tEGF protein fusions, a
combination of novel methods was developed for analysis of tEGF-induced MSC and
CTP phenotypes in vitro before moving onto more traditional in vivo methods of
assessment of bioactive graft performance. These included the fabrication and
characterization of 2D-like BTCP coverslips for rigorous analysis of CTP colony
formation and BTCP scaffold demineralization technique that allowed for early time
points assessment of MSC attachment to grafts.
In chapter 1, efforts were concerned with demonstration of the work leading
to the characterization of a novel BTCP binding peptide (BP) discovered in our
laboratory and the design and production of fusion proteins containing this peptide
fused to EGF. Other aspects pertained to characterization of binding of the final BPEGF fusion protein to BTCP substrates. Results showed that this fusion provided for
sufficient theoretical tEGF surface densities needed to stimulate proliferation of
cells based on previous works (74,76,82). Upon testing with human MSCs, the BPEGF fusion proteins retained bioactivity equivalent to wild-type EGF. Hence, fusion
of the N terminus of the EGF domain to the C terminus of the BP domain did not
affect EGF's folding or its ability to bind and activate the EGF receptor. The EGF
domain in BP-EGF also retained bioactivity upon tethering as seen by stimulation
of MSC proliferation on BTCP substrates. Most of the EGF initially tethered onto
102
these scaffolds stayed bound for over a week's period of time, a time span that will
allow for interaction of tEGF with surrounding transplanted cells during the
inflammatory stage and the beginning of the proliferative stage in bone wounds.
The data presented in chapter 2 shows that CTP colony formation is
enhanced under tethered EGF conditions on BTCP substrates. The custom-made
BTCP substrates showed to be properly foster osteogenic colony formation to a
similar extent as glass substrates, as their CFEs were qualitatively comparable.
The increase in colony forming efficiency under tEGF was observed in 8 out of 8
patient samples from plated bone marrow and trabecular surface, showing the
robust nature of EGF signaling in human cells across patients. More importantly,
tethered EGF did not affect the osteogenic differentiation potential of CTPs, as
assessed by alkaline phosphatase expression. No inflammatory response towards
recombinant BP-EGF was observed in these experiments.
These results showing tEGF-mediated enhancement of osteoprogenitor
proliferation and colony formation without loss of differentiation potential
motivated the testing of EGF-tethered BTCP grafts in a relevant, pre-clinical bone
defect model (Chapter 3). Canines, although human companions and considered
man 's best friend, offer one of the best resemblances to human bone physiology
while being relatively easy to take care of (99). The defect model used for graft
evaluation is known as the canine femoral multi-defect model (CFMD). The CFMD
provides a 1.Oxl.5 cm load-bearing cylindrical defect that provided up to N=2 for
both controls and tEGF conditions per animal (4 dogs, N=8 total per condition). BMflushed, BTCP bone void filler scaffolds with and without tEGF were loaded onto the
defects and bone formation was later assessed (4 weeks). From analysis of bone
formation through microCT and histological analysis it was concluded that both
control BTCP scaffolds and EGF-tethered BTCP scaffolds performed to the level of
mineralized cancellous allografts tested previously under the same animal model
(60,112). Hence, there is a possibility that we observed a ceiling effect. Using
103
histological analysis as a higher-resolution guide as to what was going on within the
defects, we observed trends toward lower residual scaffold with what seemed to be a
healthier hematopoietic microenvironment within the intramedullary regions of the
defects loaded with EGF-tethered BTCP scaffolds. These could indicate that defects
treated with tEGF are at a more advanced stage of wound healing. No signs of
inflammatory response were observed through histology.
The high amounts of bone formation in both the PC and IM regions of control
and tEGF scaffolds give motive for evaluation of these bioactive grafts in a more
compromised model, where bone defects are critically-sized and soft-tissue
surrounding the graft is compromised, allowing for greater distinction between the
differential performance of unmodified scaffolds vs. tEGF scaffolds. A cronic caprine
tibial defect (CCTD) model with similar defect characteristics to the ones described
above is currently being developed and could represent the next step forward in
testing these bioactive grafts (203). Overall, the efforts in this thesis demonstrate
the capability of tEGF to improve synthetic bone graft performance by enhancing
upstream biological processes (attachment, survival, proliferation, migration, colony
formation) related to local and transplanted osteoprogenitor- mediated bone wound
healing.
5.2 Future Perspectives
Future work should be aimed at testing the performance of EGF-tethered
BTCP scaffolds in more compromised, pre-clinical bone defect models (203). These
results will ultimately determine how effective tEGF can be as a therapeutic.
Aspects to improve upon efforts shown in this thesis should be focused on achieving
the proper tEGF dose to stimulate the desired osteoprogenitor phenotypes. The
optimal in vitro tEGF dose for MSC proliferation might not translate to the best
dose for CTP colony formation/growth or for bone formation in vivo. Hence, efforts
at deciphering which doses are optimal for in vivo use should be a priority and
104
should be conducted in multiple animal models, as dose requirements might change
depending on the nature of the defect. Tracing what happens to the tethered protein
in vivo should also be investigated. The numerous proteases and variety of cell
populations within bone wound may cause early release or degradation of tethered
EGF that might not be properly predicted by our current in vitro models. This can
lead to reduced bioactivity or loss of tEGF-induced cell phenotypes not seen under
soluble EGF delivery (i.e. enhanced cell attachment, survival, colony formation, etc).
If undesirable early release/degradation is observed, mutagenesis of the
recombinant protein can be used to eliminate protease-sensitive regions or to
improve binding peptide affinity under the microenvironment of the bone wound.
Enhancing CTP retention and concentration in BTCP scaffolds through
tethered factors should also be explored, as it can easily be incorporated to current
clinical procedures. Our results using the CFMD model revealed that tEGF has no
significant effect on CTP concentration and retention through use of Selective Cell
Retention (SCR). Addition of other adhesion factors or other bioactive ligands
within BTCP scaffolds that can further improve CTP retention and concentration
through SCR are of value. For example, fusing hyaluronic acid binding proteins
(HAbp) to our binding peptide could allow for enhanced selection of CTPs from
marrow, as observed previously (60). The heterogeneity of the cell populations and
surface markers associated with CTPs makes it hard to find other factors like
HAbps that would improve their selective retention within scaffolds, but as we gain
more knowledge about them other markers will arise. On the other hand, MSCs are
better characterized with respect to surface markers (31) (REF-position paper) and
-
adhesion receptors (204,205) (Semon/Prockop 2010, integrin paper; Docheva 2007
stem cell Review series), which provides additional (potential) candidates to
improve cell retention and concentration within scaffolds. However, MSC isolation
and culture expansion can be lengthy and requires specialized equipment, which
impedes same-day intraoperative use.
105
All the prior literature and the data presented in this thesis regarding tEGFinduced osteoprogenitor phenotypes seem to indicate that if tEGF is presented and
dosed properly within bone grafts it might be capable of enhancing bone formation
without the need for addition of other exogenous factors. However, in the case
where tEGF falls short of enhancing bone formation as we move into larger preclinical models, the BTCP binding peptide presented here can be readily used to
tether or deliver other ligands, which might complement or synergize with tEGF to
stimulate osteoprogenitors or other specialized cells that might be present within
the bone wound. These ligands may be other growth factors that have been
demonstrated to be capable of signaling from the solid phase (i.e. FGF-2 (78), VEGF
(117), and Insulin (77)) as well as a controlled delivery method for other ligands,
which would only be active upon release. If activity is only achieved upon release,
then the affinity of the binding peptide can be tuned to release the ligand at a
particular time window during the healing process.
In theory, combinatorial delivery of factors that can complement each other
or synergize to enhance cell attachment, survival, colony formation, migration, etc.,
should have therapeutic value. The facile tethering approach presented here allows
for combinatorial delivery of ligands that target upstream processes in bone
regeneration (i.e. attachment, migration, proliferation/colony formation) as well as
downstream differentiation like BMPs. Release of these proteins at particular
stages of bone wound healing can allow for induction of temporal phenotypes that
can aid osteoprogenitor survival and performance. By timing the release rate of
each ligand, one can potentially initially deliver pro-survival and proliferation cues
followed by osteogenic differentiation factors, avoiding differentiation of the stem
cell compartment prior to entry into the proliferative stage of wound healing but
also allowing for enhanced differentiation of the osteoprogenitors that make it past
that stage.
106
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