1
Bone formation via endochondral
ossification within high-density
hMSC aggregates
Department of Biomedical Engineering,
Case Western Reserve University, Cleveland, Ohio
Abstract
The goal of this project is to study the effects of sequentially presenting chondrogenic
(cartilage tissue formation inducing) and osteogenic (bone tissue formation inducing) growth
factors over various well-defined timelines on high-density human bone marrow-derived stem
cells (hMSC) aggregates for tissue engineering bone. I hope to be able to elucidate key timebased patterns of this natural process for tissue engineering applications to heal critical-sized
bone defects. The timeline that best demonstrates endochondral ossification in this in vitro
system may be used to guide the development of a system for sequential delivery of TGF-1 and
BMP-2 for bone tissue engineering. This proposal provides the relevant background needed to
understand the relevance of this project to devise alternative methods to treat critical-sized bone
defects. It also includes an ideal timeline and information on the expected budget for the
completion of this project. The proposal also addresses my qualifications to ideally be able to
carry out this project successfully.
Bone formation via endochondral ossification within high-density hMSC aggregates
Neha Dwivedi
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Table of Contents
1. Project Description………………………………………………………………….................4
1.1. Overview…………………………………………………………………………………………..4
1.2. Literature Review………………………………………………………………………………….4

Conventional Tissue Grafting Techniques and Drawback………………………………..4

Human Mesenchymal Stem Cells: An attractive option for bone tissue engineering…….4

Endochondral vs Intramembranous ossification…………………………………………..5
1.3.Problem Statement………………………………………………………………………………...6
2. Research Plan and Schedule…………………………………………………………………..7
2.1.Specific Aim………………………………………………………………………………………7
2.2.Methods and Timeline of Project……….…………………………………………………………7
3. Qualifications of Researcher…………………………………………………………………..9
4. Budget…………………………………………………………………………………………9
5. Anticipated Audience Involvement……………………………………………………….....10
6. Bibliography…………………………………………………………………………………11
Bone formation via endochondral ossification within high-density hMSC aggregates
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1. Project Description
1.1. Overview
Bone is one of the most critical and complex organs in the body. It accounts for a variety of
extremely essential body functions that include support and structure, mineral storage, blood
production, pH regulation etc. Thus, any morphological, congenital defect in this tissue can have
severe consequences. Because of their size, ‘critical-sized defects’ are too large for bone to heal
by itself without adding a bone graft or a suitable substitute instead. Current treatment options
include Autogenic grafting, Allografting and Xenografting.
These procedures, although reasonable options, have shown to result in side effects due to the
risk of virus transmission from donor to host, infection and host rejection. (2)
The endochondral ossification approach to bone tissue engineering has a major advantage over
the intramembranous ossification pathway owing to its avascular onset. Many tissue engineered
constructs are limited by the lack of a vascular network. Thus, the endochondral ossification
approach is an attractive alternative to bone regeneration as issues of early vascularization may
be circumvented.
1.2.Literature Review

Conventional Tissue Grafting Techniques and Drawbacks
Autogenous grafting, the gold standard for treating bone defects, is a technique that involves
extracting bone tissue from a different site in the patient and using it to fill the defected site.
However, this option is limited by drawbacks such as pain, donor-site morbidity, and donor graft
availability. (1) Another common treatment is known as Allografting. This involves the removal
of bone tissue from a donor of the same species to fill in the defect in the patient. This procedure,
although a reasonable option has shown to result in side effects due to host-donor infections.
Another option that one may opt for is the Xenografting technique. This involves bone tissue
grafting from a non-human donor. Although it was considered as a perfectly viable option for
several years, it is now considered harmful due to the risk of virus transmission from donor to
host, infection and host rejection. (2)
The drawbacks of the treatments mentioned above have encouraged the development of tissue
engineering techniques that might succeed in eliminating the risk of diseases and infections and
the need for complicated surgical procedures. This project employs the tissue engineering
approach to form bone via endochondral ossification by culturing human bone marrow-derived
stem cells (hMSCs) in a suitable microenvironment that mimics the conditions in the body.
Bone formation via endochondral ossification within high-density hMSC aggregates
Neha Dwivedi
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Hence, the problems that arise with rejection of grafts from Autografts, Allografts and
Xenografts are circumvented. (3)

Human Mesenchymal Stem Cells: An attractive option for bone tissue engineering
Human Mesenchymal Stem Cells are an attractive cell source because they are easily accessible
and abundant and have the ability to differentiate into various cell lineages including osteoblasts,
chondrocytes and adipocytes under appropriate conditions. (3)
These cells continuously replicate themselves, while a portion of these cells become committed
to mesenchymal cell lineages such as bone, cartilage, tendon, ligament and muscle. The
differentiation of these cells, within each lineage, is a complex multistep pathway involving
discrete cellular transitions much like that which occurs during hematopoiesis. Progression from
one stage to the next depends on the presence of specific bioactive factors, nutrients, and other
environmental cues. (4)

Endochondral vs Intramembranous Ossification
For critical-sized bone defects, endochondral ossification, the approach of forming bone via a
cartilaginous intermediate, may circumvent issues faced by the intramembranous ossification
approach with initially supplying oxygen and nutrients to cells because chondrocytes are
equipped to survive in hypoxic conditions. (5)
During fetal development, bone formation occurs via two processes, namely intramembranous
ossification and endochondral ossification. Intramembranous ossification, which is involved in
skull formation, is characterized by the direct differentiation of mesenchymal stem cells into
bone-forming cells, osteoblasts. Endochondral ossification, which is involved in the
development of most other bones, including long bones, has been often overlooked in the context
of bone engineering.(4) The process of Endochondral Ossification starts off by the recruitment of
MSCs and their subsequent condensation and differentiation into chondrocytes. The
chondrocytes form an unmineralized, avascular cartilage model. The chondrocytes gradually
begin to increase in volume and differentiate into hypertrophic chondrocytes. These hypertrophic
chondrocytes facilitate calcification of the cartilage matrix, which lead to chondrocyte death due
to nutrients getting cut off, and secrete signals such as matrix metalloproteinases (MMPs), and
vascular endothelial growth factor (VEGF) to prepare the extracellular matrix (ECM) for and to
stimulate vascular invasion, respectively for stimulation. The blood vessels bring in perivascular
cells such as osteoprogenitor cells and undifferentiated MSCs into the lacunae that resulted from
chondrocyte death. These perivascular cells then develop bone and marrow to replace the
cartilage tissue. (6)
Vascularization is the process of the development of a network of blood vessels around the
newly formed tissue. The blood vessels carry the minerals and nutrients required for the
Bone formation via endochondral ossification within high-density hMSC aggregates
Neha Dwivedi
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nourishment and growth of the tissue. The endochondral ossification approach to bone tissue
engineering has a major advantage over the intramembranous ossification pathway owing to its
avascular onset. This simply implies that the intermediate formed doesn’t require a welldeveloped vascular network for its maintenance. Cartilage, which is formed first in the
endochondral ossification pathway, is an avascular tissue so chondrocytes are able to survive in
poor environmental conditions. Therefore, tissue engineering bone via endochondral ossification
may circumvent issues of early vascularization to supply oxygen and nutrients to cells within the
construct. One of the biggest challenges in bone tissue engineering is early formation of a
vascular network within the constructs. (7) Early vascularization is an essential step in bone
tissue engineering via the intramembranous ossification approach until proper vasculature has
been established, the constructs has to rely on diffusion for oxygen and nutrient supply. For
bigger constructs, this is a huge problem as oxygen and nutrients cannot diffuse into the interior
of the construct due to diffusion limitations, which can result in cell death. Many tissue
engineered constructs are limited by the lack of a vascular network. (8) Thus, the endochondral
ossification approach is an attractive alternative to bone regeneration as issues of early
vascularization may be circumvented.
Endochondral ossification has been partially recreated in vitro by culturing hMSCs in
chondrogenic media followed by osteogenic media in several studies. However, no studies have
reported to compare the effects of different timelines of sequentially presenting chondrogenic
and osteogenic signals to hMSC aggregates on endochondral ossification. Transforming growth
factor-1 (TGF- β1) and bone morphogenetic protein-2 (BMP-2) are two growth factors that
regulate this process. TGF-1 is a strong inducer of hMSC chondrogenesis whereas BMP-2 is a
potent inducer of osteogenesis and has been shown to stimulate chondrocyte hypertrophy during
endochondral ossification. (9)
1.3. Problem Statement
There are nearly 6,000 disorders associated with the bone tissue that, taken together, affect
approximately 25 million Americans. One in every 10 individuals in this country has received a
diagnosis of a bone tissue disease. As explained above, there are numerous drawbacks associated
with the current options to treat bone defects. These drawbacks have encouraged the
development of tissue engineering techniques that might succeed in eliminating the risk of
diseases and infections and the need for complicated surgical procedures.
This project employs the tissue engineering approach to form bone via endochondral ossification
by culturing human bone marrow-derived stem cells (hMSCs) in a suitable microenvironment
that mimics the conditions in the body. If funded sufficiently, this project has the potential to
create an alternative method to treat bone defects that affect a major proportion of the population
today. Not only is the method being deciphered non-invasive and less harmful to the body, it
circumvents issues of time and finding donors for bone tissue implants.
Bone formation via endochondral ossification within high-density hMSC aggregates
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2. Research Plan and Schedule
2.1.Specific Aims
The proposed project aims at recreating the process of bone formation, in vitro, to study the
effects of sequentially presenting exogenous chondrogenic (TGF-β1) and osteogenic (BMP-2)
growth factors on aggregates. The understanding of the time dependent process of bone
regeneration is expected to help guide the development of a growth factor delivery system that
will ideally be tested in vivo once the in vitro studies show positive results. Ideally, this system
will be optimized to induce maximum bone tissue regeneration.
2.2.Methods and Timeline of Project
This project will start off with the isolation of hMSCs (human mesenchyme stem cells) from
bone marrow aspirates of healthy donors. These hMSCs will then be expanded in monolayer
culture and then stored in liquid nitrogen until use. These cells will be thawed and cultured on
tissue culture plastic, as and when needed. Once the cells have expanded and reached a
confluence of around 90%, they will be trypsinized. Trypsin will help detach the cells from the
surface of the tissue culture plastic. Next, a hemacytometer will be used to count the number of
cells in the suspension containing detached cells. This suspension will be diluted or concentrated
by resuspending in a known volume of Basal Pellet Media (BPM). Next, 200 ul aliquots of this
suspension will be added to each well of a V-bottom polypropylene plate. This plate needs to be
autoclaved and sterilized before use. To obtain free floating hMSC aggregates the plate will be
centrifuged. The aggregates thus formed will then be cultured in different conditions to proceed
with the study of endochondral ossification. Table 1 gives an overview of the timeline involved
with exogenously introducing the growth factors into the system created. Exogenous introduction
implies introducing growth factors required directly into the media that the aggregates are being
cultured in.
Timeline Planned for hMSC Culture
Chondrogenic Media + TGF-B1 (1 week), Osteogenic Media + BMP-2 (4 weeks)
Chondrogenic Media + TGF-B1 (2 weeks), Osteogenic Media + BMP-2 (3 weeks)
Chondrogenic Media + TGF-B1 (3 weeks), Osteogenic Media + BMP-2 (2 weeks)
Table 1: Timeline for exogenously introducing growth factors into bone tissue aggregates
Bone formation via endochondral ossification within high-density hMSC aggregates
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To study and understand the temporal patterns of endochondral ossification, these aggregates
will be harvested at two different time points. Week 2 and week 5 are two time points that have
shown to work best in understanding the patterns in bone formation. Harvests from these two
time points will then be analyzed biochemically and histologically.
(I)
Histological Analysis:
Aggregates harvested were stained with various different kinds of stains as explained further
ahead. Once stained, these were sent in for histological analysis. The three major kinds of
stainings performed were H&E, Alizarin Red S Staining and Aggregates will be stained with
cartilage (glycoscaminoglycan (GAG), type II collagen, and type X collagen) and bone (type I
collagen and bone sialoprotein (BSP)) markers. GAG is a polysaccharide that is abundantly
found in hyaline articular cartilage extracellular matrix (ECM). Type II collagen is also
predominant in articular cartilage ECM and is secreted by chondrocytes. Type X collagen is
secreted by hypertrophic chondrocytes. Staining for type X cartilage will allow us to verify the
occurrence of chondrocyte hypertrophy, a key process in Endochondral ossification. Type I
collagen is predominantly present in the ECM of bone and bone sialoprotein is a late osteogenic
marker that constitutes mineralized tissue such as bone and calcified cartilage. It is a major
component of bone ECM. (8) Calcium staining will be performed to analyze the extent of tissue
mineralization. Histological analysis will also allow us to examine cell and tissue morphology
within the aggregates.
(II)
Biochemical Analysis:
To support the results from the histological assays, biochemical analysis will be performed.
These will be performed to quantify the amount of DNA to measure cell viability, GAG to
ensure articular cartilage formation, ALP (Alkaline Phosphatase) to verify osteogenic activity
and Calcium content to determine the extent of mineralization in the constructs at these time
points. Once, this entire procedure is carried out for the first donor, it will be repeated with two
other donors to reinforce the results obtained. Figure 1 contains pictures obtained from
histological analysis of week 2 and week 5 aggregates exposed to exogenous treatment.
However, these pictures are from a previous but similar study on bone marrow obtained from a
different donor.
Bone formation via endochondral ossification within high-density hMSC aggregates
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Figure 1: Depicts three different groups of hMSC aggregates cultured in different conditions.
Column 1 shows H&E Staining, Colum 2 shows Safranin O and Column 3 shows Alizarin Red S
Staining of aggregates at 5 week timepoint
3. Qualifications of Researcher
I am a third year undergraduate student at Case Western Reserve University, majoring in
Biomedical Engineering (BME) with a specialty in Tissue Engineering. The courses I have taken
so far, as BME core classes, have helped me develop the level of understanding in the field of
basic biological and engineering principles necessary. Moreover, working in the Alsberg Stem
Cell Engineering and Novel Therapeutics (ASCENT) Lab, as an undergraduate researcher, for
over 10 months has helped me achieve a strong grasp of the engineering technologies and ideas
associated with bone tissue regeneration. Working in the ASCENT Lab and collaborating with
other graduate level students working on similar projects revolving around tissue engineering has
essentially helped me develop a research oriented mindset that encourages scientific inquiry. It
has also help me familiarize myself with laboratory work ethics. I believe that these factors along
with my technical knowledge in the field will ideally help me complete the proposed project
successfully.
4. Budget
Stem cell engineering is a relatively novel method to approach treatment of bone defects.
Furthermore, research in this area involves using high level technology not generally available at
economical costs. The funding that the ASCENT Lab receives to conduct all the experiments
that are carried out on a daily basis is only enough to cover the basic costs of in-lab facilities.
However, materials such as bone marrow harvests from donors, required for this project, can be
Bone formation via endochondral ossification within high-density hMSC aggregates
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highly expensive. Table 2 shows a breakdown of the total amount that will be spent on basic
amenities that will be needed to complete this project. Essentially, the two areas that will most
need funding are project supplies and hours of research invested by researcher.
•
•
Cost of project supplies:
Bone Marrow Samples
Tissue Culture Flasks
Expansion Media
Homogenizer
Spectrometer
Multiplate Reader
Stipend for hours invested in the project
15-20 hours/week, $11/hour
TOTAL
Table 2: Tentative Budget Expected
$2500
$1100
$3,600
5. Future Considerations
Ideally, this study will lead into in vivo studies to understand the process of bone formation
inside a real biological system. This will essentially help us in testing whether or not our system
functions in vivo, which is extremely important since in vivo and in vitro conditions differ
significantly. Another advantage of growing our constructs in vivo will be that angiogenesis,
which is the process of formation of blood vessels will be induced naturally.
Another important consideration for the future will be the use of multilayer microspheres. The
layers on these microspheres will be coated with different growth factors. Based on where these
growth factors are coated on to the microsphere, the growth factors will be released into the
construct. Ideally, the microspheres that we will use will have the inner layer coated with BMP-2
and the outer layer coated with VEGF. Addition of VEGF will induce angiogenesis. This will
help recruit blood vessel invasion in our constructs.
6. Anticipated Audience Involvement
This proposal is intended to raise awareness regarding the dire need for alternative options to
treat bone defects in order to circumvent the drawbacks associated with current treatment options
and understand the significance of stem cell research in this area. However, in order to conduct
stem cell research, it is extremely essential to understand the need for funding since stem cell
research can amount up to be extremely expensive. Since this kind of research is still in its novel
stages, there are a lot of principles involved that might be hard to decipher. With this proposal I
Bone formation via endochondral ossification within high-density hMSC aggregates
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would like to respectfully extend invitation for any kind of feedback that might help me guide
my project towards completion.
I believe that this project has the potential to guide further studies in the area of treatment of
bone defects using high density stem cell aggregates and hence, can prove extremely useful to
future researchers in labs similar to the ASCENT Lab. In context of a much wider audience, this
research project can essentially prove as a time-saving, safer and less-invasive technique to treat
bone defects, which affect a major proportion of the current population in the form of congenital
and morphological defects.
7. Bibliography
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
J.R. Porter, T.T. Ruckh, K.C. Popat, “Bone Tissue Engineering: A Review in Bone
Biomimetics and Drug Delivery Strategies”, in Biotechnol. Prog, Vol. 25, pp.1539-1540,
2009.
B. Schmitt, J. Ringe, Thomas Ha¨upl, M. Notter, R. Manz, G. Burmester, M. Sittinger,
Christian, “BMP2 initiates chondrogenic lineage development of adult human
mesenchymal stem cells in high-density culture”, in Differentiation, pp. 1-2, 2003.
D. Marolt, M. Knezevic and G.V Novakovic,” Bone tissue engineering with human stem
cells”, in Marolt et al. Stem Cell Research & Therapy, pp. 1-2, 2010.
S. Provot, E. Schipani, “Molecular mechanisms of endochondral bone development”, in
Biochemical and Biophysical Research Communications, pp. 658–665, 2005.
L.C. Cerstenfeld and F.D. Shapiro, “Expression of Bone-Specific Genes by Hypertrophic
Chondrocytes: Implications of the Complex Functions of the Hypertrophic Chondrocyte
during Endochondral Bone Development”, in Journal of Cellular Biochemistry 62, pp. 19, 1996.
Studer D, Millan C, Öztürk E, Maniura-Weber K, Zenobi-Wong M. Molecular and
biophysical mechanisms regulating hypertrophic differentiation in chondrocytes and
mesenchymal stem cells”, in Eur Cell Mater, pp. 24-35, 2012.
Jeroen Rouwkema, Peter E. Westerweel, Jan de Boer, Marianne C. Verhaar, and
Clemens A. van Blitterswijk, “The Use of Endothelial Progenitor Cells for
Prevascularized Bone Tissue Engineering”, in ROUWKEMA ET AL. , 2005-2006.
Craig A. Simmons, Eben Alsberg, Susan Hsiong, Woo J. Kim, and David J. Mooney,
“Dual growth factor delivery and controlled scaffold degradation enhance in vivo bone
formation by transplanted bone marrow stromal cells”, in Bone 35, pp. 1-2, 2004.
Bone formation via endochondral ossification within high-density hMSC aggregates
Neha Dwivedi