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DOI: 10.3727/096368909X481782
E-ISSN 1555-3892
www.cognizantcommunication.com
Cell Transplantation, Vol. 19, pp. 399–408, 2010
Printed in the USA. All rights reserved.
Copyright  2010 Cognizant Comm. Corp.
Continuous Delivery of Stromal Cell-Derived Factor-1
From Alginate Scaffolds Accelerates Wound Healing
Sina Y. Rabbany,*†1 Joseph Pastore,‡1 Masaya Yamamoto,†§ Tim Miller,‡
Shahin Rafii,†¶ Rahul Aras,‡ and Marc Penn‡#
*Bioengineering Program, Hofstra University, Hempstead, NY, USA
†Department of Genetic Medicine, Howard Hughes Medical Institute, Weill Cornell Medical College, New York, NY, USA
‡Juventas Therapeutics, Inc., Cleveland, OH, USA
§Department of Biomaterials, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan
¶Howard Hughes Medical Institute, Weill Cornell Medical College, New York, NY, USA
#Department of Stem Cell Biology and Regenerative Medicine, Cleveland Clinic, Cleveland, OH, USA
Proper wound diagnosis and management is an increasingly important clinical challenge and is a large and
growing unmet need. Pressure ulcers, hard-to-heal wounds, and problematic surgical incisions are emerging
at increasing frequencies. At present, the wound-healing industry is experiencing a paradigm shift towards
innovative treatments that exploit nanotechnology, biomaterials, and biologics. Our study utilized an alginate
hydrogel patch to deliver stromal cell-derived factor-1 (SDF-1), a naturally occurring chemokine that is
rapidly overexpressed in response to tissue injury, to assess the potential effects SDF-1 therapy on wound
closure rates and scar formation. Alginate patches were loaded with either purified recombinant human SDF1 protein or plasmid expressing SDF-1 and the kinetics of SDF-1 release were measured both in vitro and
in vivo in mice. Our studies demonstrate that although SDF-1 plasmid- and protein-loaded patches were able
to release therapeutic product over hours to days, SDF-1 protein was released faster (in vivo Kd 0.55 days)
than SDF-1 plasmid (in vivo Kd 3.67 days). We hypothesized that chronic SDF-1 delivery would be more
effective in accelerating the rate of dermal wound closure in Yorkshire pigs with acute surgical wounds, a
model that closely mimics human wound healing. Wounds treated with SDF-1 protein (n = 10) and plasmid
(n = 6) loaded patches healed faster than sham (n = 4) or control (n = 4). At day 9, SDF-1-treated wounds
significantly accelerated wound closure (55.0 ± 14.3% healed) compared to nontreated controls (8.2 ± 6.0%,
p < 0.05). Furthermore, 38% of SDF-1-treated wounds were fully healed at day 9 (vs. none in controls) with
very little evidence of scarring. These data suggest that patch-mediated SDF-1 delivery may ultimately
provide a novel therapy for accelerating healing and reducing scarring in clinical wounds.
Key words: Dermal delivery; Tissue remodeling; Neovascularization; Tissue scarring; Hydrogels
INTRODUCTION
the principal cells involved in stimulating blood vessel
growth. In response to injury, these cells initiate the angiogenic process, which eventually leads to remodeled
tissue and a newly formed vasculature. There are many
therapies directed at accelerating wound healing, such
as the application of electric currents, living skin equivalents, and pharmacological manipulation of factors that
modulate the wound-healing process. However, few
treatments are able to combine the ability to improve
wound healing with ease of transport and efficacy in
a cost-effective manner. Furthermore, there are limited
Wound healing is a complex and coordinated series
of events involving inflammation, tissue deposition, remodeling, and scarring. In the epidermis, this is a dynamic process involving multiple skin layers, specifically the interaction of infiltrative cells (leukocytes,
progenitor cells) with the underlying stroma (keratinocytes, endothelial cells, epithelia, extracellular matrix)
(26). Angiogenesis is one of the important events during
wound healing, and microvascular endothelial cells are
Received April 30, 2009; final acceptance December 2, 2009. Online prepub date: December 8, 2009.
1These two authors contributed equally.
Address correspondence to Marc Penn, Department of Stem Cell Biology and Regenerative Medicine, Cleveland Clinic, Cleveland, OH, 44195,
USA. Tel: (216) 444-6697; E-mail: pennm@ccf.org or Sina Y. Rabbany, Department of Genetic Medicine, Weill Cornell Medical College, New
York, NY 10021, USA. Tel: (212) 746-7014; E-mail: sir2007@med.cornell.edu
399
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studies that directly compare gene- and protein-based biological delivery in order to define optimal delivery
strategies.
Dermal Delivery of Biologic Material
Alginate hydrogels have proven to be useful scaffolds
for tissue engineering and delivery vehicles for drug
treatments. Hydrogels can be loaded with a variety of
growth factors for controlled release during different
wound-healing stages and in light of wound severity.
Furthermore, alginate gels and patches are extremely
versatile and can be tailored to account for the stiffness,
swelling, cell attachment, or degradation of bioreactive
polymers (3). Improving the rate of dermal wound healing has been identified as a target for demonstrating the
therapeutic potential of growth factor delivery. A number of factors have been identified that mediate wound
healing and promote proliferation and differentiation of
skin cells, such as platelet-derived growth factor
(PDGF) and epidermal growth factor (EGF) (20). Our
study focused on stromal cell-derived factor-1 (SDF-1),
a naturally occurring chemokine that is rapidly overexpressed in response to tissue injury, particularly during
ischemia, to expedite wound closure and decrease scar
formation (2,15). SDF-1 plays an important role in the
migration, recruitment, and retention of endothelial progenitor cells (EPCs) to the site of injury, hence contributing to neovascularization. Findings from multiple independent studies suggest that inducing SDF-1 expression
at a wound site has therapeutic potential to promote
rapid repair of hard-to-heal wounds, such as deep incisions, ulcerations, and burns (5,6,10).
Therapeutic Applications of SDF-1
A unique aspect of wound healing in adult tissue is
that regeneration is generally driven by environmental
stimuli and is often initiated by local release of chemokines. SDF-1 treatment has been shown to reduce wound
size by recruiting progenitor cells to the site of injury,
promoting full reepithelialization of the wound and enhancing revascularization of the damaged tissue. A
prominent feature of this treatment is the decreased amount
of tissue scarring, a particularly important feature that
will increase the health, mobility, and well-being of patients. The beneficial effects of SDF-1 treatment are well
characterized in cardiovascular models of ischemia and
hypoxic stress (7,30). Similarly, dermal wounds may be
a particularly relevant therapeutic target for SDF-1 therapy due to the ability of SDF-1 to recruit EPCs and
epithelial cells and thus accelerate natural wound repair.
Importantly, SDF-1 treatment has been shown to accelerate wound healing. In two studies, direct injection of
SDF-1 protein or lentivirus significantly reduced wound
size after 1 week in diabetic mice (5,11). We therefore
RABBANY ET AL.
hypothesized that slow-release delivery of either SDF-1
protein or plasmid expressing SDF-1 would increase the
rate of wound closure in a porcine model of full-thickness dermal wound healing. We employed a clinically
relevant delivery system, an alginate scaffold, to deliver
SDF-1 plasmid or protein over time to acute surgical
wounds. In this article, we characterized SDF-1 plasmid
or protein dermal delivery using alginate scaffolds in
vitro and demonstrate the potential for therapeutic benefit in vivo by using the scaffolds to deliver the SDF-1
protein and plasmid to acute surgical wounds.
MATERIALS AND METHODS
Construction of Honeycomb Alginate Patches
Chemicals were obtained from Nacalai Tesque. Inc.
(Kyoto, Japan), Calbiochem (La Jolla, CA, USA), BD
Biosciences (Franklin Lakes, NJ, USA), and Sigma
Chemical Co. (St. Louis, MO, USA) and used without
further purification. Alginate gels were prepared according to the method described previously (8). Briefly, sodium alginate (MW, 110k, Wako Pure Chemical Industry,
Osaka, Japan) was dissolved at different concentrations in
distilled water. The inner wall of glass beakers was
coated with a thin layer of sodium alginate and dried at
110°C for 0.5 h. An aqueous solution of sodium alginate
was poured into the alginate-coated beaker and calcium
chloride solution was sprayed until a thin alginate gel
layer was formed on the top of the sodium alginate solution. Then, calcium chloride solution was added on the
thin gel layer, followed by overnight incubation at room
temperature to complete gelation. This ionotropic gelation generated parallel channel-like pores in alginate
gels, which were subsequently washed with deionized
water to remove free calcium ions. The resulting gels
were frozen at −80°C, lyophilized under vacuum at
room temperature for 2 days to form a honeycomb structure, cut into 5 mm diameter/2 mm thick disc-shaped
units, and subjected to further chemical modifications.
Alginate discs were immersed in 2-(N-morpholino)
ethanesulfonic acid (MES) buffer (0.2 M, pH 6.0) containing ethylenediamine (ED, 5.17 mM), NHS (114
mM), and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, 305 mM) and centrifuged at 1500 rpm for
5 min to eliminate air bubbles. After 24 h of cross-linking, the resulting gels were washed with phosphatebuffered saline (PBS) three times.
Radioiodination of SDF-1 Protein
Human recombinant SDF-1 was radioiodinated according to the method of Greenwood et al. (12). Briefly,
10 µl of Na125I solution was added to 40 µl of 0.1 mg/
ml SDF-1 solution, followed by the addition of 0.2 mg/
ml of chloramine-T potassium phosphate-buffered solution (0.5 M, pH 7.5) containing 0.5 M sodium chloride
ALGINATE SCAFFOLDS AS DELIVERY VEHICLE FOR SDF-1 IN INJURY
(100 µl). After agitation at room temperature for 2 min,
100 µl of PBS solution (pH 7.5) containing 0.4 mg of
sodium metabisulfate was added to the reaction solution
to stop the radioiodination. The reaction mixture was
passed through an anionic-exchange column to remove
the uncoupled, free 125I molecules from the 125I-labeled
SDF-1 protein.
Radioiodination of SDF-1 Plasmid DNA
PBS containing 1.5 mg/ml of SDF-1 plasmid DNA
(1.33 ml) was added to the 125I-Bolton-Hunter reagent
nitrogen dried. The resulting solution was kept at room
temperature for 6 h to introduce 125I residue into the
amino groups of SDF-1 plasmid DNA. Noncoupled, free
125
I-labeled reagent was separated from 125I-labeled SDF1 plasmid DNA by gel filtration with a PD-10 column
(Amersham Pharmacia Biotech K.K, Tokyo, Japan).
Preparation of Alginate Patches Incorporating
I-Labeled SDF-1 Protein or 125I-Labeled
SDF-1 Plasmid DNA
Cross-linked alginate patches were immersed into
0.015 M spermine chloride/MES buffer solution containing NHS (114 mM) and EDC (305 mM), and stored
at room temperature overnight. Unbound spermine chloride was removed by washing with PBS and distilled
water. After lyophilization, the spermine-immobilized
patch was sterilized with 70% ethanol and air dried in
an aseptic tissue culture hood. An aqueous solution (20
µl) of 125I-labeled SDF-1 protein (1.0 ng/µl) containing
sodium heparin (1 U/µl) was dropped onto the sterilized
scaffolds and incubated at 4°C overnight to complete
protein incorporation. Similarly, 15 µl of an aqueous solution of 125I-labeled SDF-1 plasmid (1.0 mg/ml) was
dropped onto the sterilized scaffolds and incubated at
4°C overnight to complete plasmid DNA incorporation.
125
In Vitro Evaluation of SDF-1 Protein and Plasmid
DNA Release From Alginate Patches
An alginate patch saturated with 125I-labeled SDF-1
protein or plasmid DNA was placed in 1 ml of PBS
containing bovine serum albumin fraction V (BSA, 0.1
mg/ml), followed by incubating at 37°C. The supernatant was periodically sampled to measure the radioactivity of released 125I-labeled SDF-1 protein or plasmid
DNA by a gamma counter (ARC-380, Aloka Co., Ltd.,
Japan). Fresh BSA solution of the same volume was
added to continue the release test.
In Vivo Evaluation of SDF-1 Protein and SDF-1
Plasmid DNA Release From Alginate Patches
in Rodents
Alginate patches incorporating 125I-labeled SDF-1
protein or plasmid DNA were implanted subcutaneously
401
into the backs of 6-week-old female ddY mice (Shimizu
Laboratory Supply Inc., Japan) at the central position 15
mm away from their tail root. At different time intervals,
the mouse skin including the implanted site of the
sponge (3 × 5 cm) was excised and the corresponding
fascia was thoroughly wiped off with a filter paper to
absorb 125I-labeled SDF-1 protein or plasmid DNA. The
radioactivity of the patch, the excised skin strip, and the
filter paper were measured on a gamma counter (ARC380) to assess the time profile of in vivo SDF-1 protein
and plasmid DNA release. The experimental group was
composed of three mice unless otherwise mentioned.
The use of animals in this study was approved by MPI’s
Research Institutional Animal Care and Use Committee
(IACUC) prior to starting, and was based on current International Conference on Harmonisation (ICH) Tripartite Guidelines.
Porcine Surgical Wound Healing Model
and Antemortem Follow-up
A standard model of acute surgical wound healing
was performed in domestic Yorkshire pigs. A cuffed endotracheal tube was placed and general anesthesia was
maintained with isoflurane delivered in oxygen through
a rebreathing system with ventilator assist. Each animal
received 12 full-thickness 5-cm incisions (six on each
side of the spine) spaced approximately 7.5 cm apart.
Each incision was made perpendicular to the spine, starting 7.5 cm from the spine and cutting toward the abdomen. Gauze was placed in the incision until the bleeding
stopped. The gauze was removed, and the incision was
sutured closed.
Alginate patches (1 × 6 cm) incorporating SDF-1
protein, plasmid DNA, or PBS were applied to wounds.
Scaffolds were prepared with a solution of plasmid encoding human SDF-1 in a pcDNA3.1 backbone (Invitrogen Corporation, Carlsbad, CA, USA) by mixing
3.5 mg of the SDF-1 plasmid in 2.33 ml PBS to create
a 1.5 mg/ml solution. For the SDF-1 protein scaffolds,
a solution was prepared by mixing 10 µg of carrier-free
SDF-1 protein (R&D Systems, Minneapolis, MN, USA)
with 5 ml PBS and 3 ml of 1000 IU/ml injection heparin
(Baxter Healthcare Corporation, Deerfield, IL, USA) to
create a 1.5 µg/ml solution. PBS scaffolds served as a
negative control and were prepared by mixing 1.35 ml
PBS and 0.45 ml of 1000 IU/ml injection heparin. On
each scaffold, the loading solution was pipetted under
sterile conditions onto the scaffold in six equally spaced
60-µl drops (360 µl total) so that each drop covered a
1-cm2 area of the scaffold. All loaded scaffolds were
stored at 4°C for 12 h prior to wound application.
Following wound closure, the scaffold was placed
next to the wound and photographed. The scaffold
placement order was randomized with the following dis-
402
tribution: SDF-1 protein (n = 10), SDF-1 plasmid (n =
6), PBS (n = 4), or sham (no scaffold, n = 4) wounds.
The scaffold was placed over the wound (except in the
sham group), and each wound was dressed with a Tegaderm patch. To determine the effect of SDF-1 on the
rate of wound healing, wound length was measured by
the same veterinarian at day 0 (prior to scaffold placement) and prior to sacrifice. Wound length was converted to percent healing by the following equation: (initial wound length − final wound length)/(initial wound
length) × 100%. To monitor the time course of the effects of SDF-1 on wound healing, the first pig was sacrificed at 4 days and the second pig was sacrificed at 9
days.
RABBANY ET AL.
formed using Microsoft Office Excel 2007 (Microsoft,
Redmond, WA, USA).
RESULTS
SDF-1 Protein and Plasmid Release Characteristics
Release characteristics of radioiodinated SDF-1 protein and plasmid from alginate scaffolds are shown in
Figure 1. SDF-1 protein and plasmid release in vitro was
measured at multiple time points over 150 h (Fig. 1A).
The scaffolds released approximately 40–60% of plasmid or protein SDF-1 within the first day, with Kd of
24.5 and 15.8 h, respectively. Thereafter, SDF-1 plasmid
continued to be released, whereas SDF-1 protein release
was minimal after 24 h. Because neither the SDF-1 plas-
Postmortem Follow-up
Following sacrifice, one section from the middle of
each wound site was excised for histopathological and
immunohistochemical analysis. Standard hematoxylin
and eosin (H&E) stain was used to assess extent of fibroplasia, inflammation, and necrosis at day 4 and necrosis, fibrosis, and granulomatous inflammation at day
9. Each parameter was graded on a qualitative scale by a
histopathologist blinded to randomization as either: none
(not present), minimal, mild, moderate, or severe. Immunohistochemical staining was performed on the same
tissue section.
The effect of SDF-1 on fibroblast infiltration into the
wound was detected by vimentin staining. Vimentin is a
type III intermediate filament often expressed in epithelial cells undergoing mesenchymal transition. The effect
of SDF-1 on blood vessel formation was determined by
CD31 and the presence of smooth muscle was detected
by smooth muscle actin staining. CD31, also known as
platelet endothelial cell adhesion molecule 1 (PECAM1),
is a type I integral membrane glycoprotein and a member of the immunoglobulin superfamily of cell surface
receptors. It is constitutively expressed on the surface of
endothelial cells and concentrated at the junction between them. CD31 has been used to measure angiogenesis and can be used as a marker for myeloid progenitor
cells. Smooth muscle α-actin is used because it is one
of a few genes whose expression is relatively restricted
to vascular smooth muscle cells. The amount of each
stain per sample was graded by the same pathologist
using the same qualitative scale as above.
Statistical Analysis
All data are presented as mean ± SEM. Comparisons
between groups were performed using a Student’s t-test.
A value of p < 0.05 was considered statistically significant. For SDF-1 release data, a logarithmic curve was
fit to each data set. All statistical analyses were per-
Figure 1. Characterization of SDF-1 release from alginate
scaffolds in vitro and in vivo. Scaffolds were loaded with radioiodinated protein (squares) or plasmid (diamonds) and release was measured in vitro (A) by quantifying radioactivity
from the supernatant at multiple time points or in vivo (B)
from filter paper wiped along the fascia of scaffolds implanted
subcutaneously at the base of the neck in mice. Dotted lines
indicate the time points at which 50% of the plasmid or protein
was dissociated (Kd) from the scaffold.
ALGINATE SCAFFOLDS AS DELIVERY VEHICLE FOR SDF-1 IN INJURY
403
mid nor protein reached 100% release at 1 week, we
ensured the patches retained the originally loaded SDF1 by detaching the remaining protein or plasmid from
the patch by soaking in a high salt solution (data not
shown), suggesting that release may have reached equilibrium with the supernatant. This indicated that subcutaneous scaffold application in vivo may sustain plasmid
and protein release over time, and may be effective release of therapeutic vectors or proteins.
To test this hypothesis in vivo, SDF-1 plasmid and
protein scaffolds were implanted subcutaneously into
the backs of 6-week-old ddY female mice and evaluated
for release over 2 weeks (Fig. 1B). The in vivo profile
of SDF-1 protein release was similar to the in vitro profile, as the majority of the SDF-1 protein was released
within the first day (Kd, 0.55 days) compared to the in
vivo release of SDF-1 plasmid, which demonstrated a
slower release profile (Kd 3.67 days). Dissociation values are presented in Table 1. The total amount of SDF1 plasmid released initially was significantly lower than
that of protein for the first 6 days, while the amount
remaining after 2 weeks was similar. Based on these
release characteristics, we tested the effects of SDF-1
treatment in a well-characterized porcine model of dermal wound healing.
SDF-1-Releasing Scaffold Accelerates Wound Healing
Recent work from several laboratories suggests that
SDF-1 has therapeutic potential for treating hard-to-heal
wounds complicated by diabetes or burns (4,5,10,19,29).
We hypothesized that SDF-1 treatment would increase
the rate of healing in acute dermal wounds, such as those
received after surgery. Therefore, 5-cm-long full-thickness incisions were made on the backs of Yorkshire pigs
and covered with Tegaderm alone or scaffolds soaked
with PBS, SDF-1 plasmid, or SDF-1 protein to assess
the effect of SDF-1 treatment on the rate of dermal
wound closure.
The impact of SDF-1-releasing scaffolds on wound
healing is shown in Figure 2. Wounds treated with either
SDF-1 plasmid or protein scaffolds (Fig. 2A, solid
markers and lines) trended towards accelerated wound
healing by day 4 and demonstrated a significantly higher
rate of wound closure compared to control wounds at
day 9 post-scaffold application (Fig. 2A, dotted lines).
Grouped data (Fig. 2B) demonstrate that SDF-1 treat-
Table 1. Kd Values for SDF-1 Patch Release
In vitro
In vivo
Plasmid
Protein
24.5 h
3.67 days
15.8 h
0.55 days
Figure 2. SDF-1 treatment accelerates healing of acute surgical wounds. (A) Percent wound healing over time shown for
SDF-1 plasmid (squares) or protein (diamonds) treated
wounds and nontreated saline (triangles) or sham (crosses) in
a porcine model of acute dermal wound closure. (B) SDF-1treated groups (n = 8) healed to a much greater extent compared to nontreated controls (n = 4). *p < 0.05.
ment significantly accelerated wound closure (55.05 ±
14.36%) compared to controls (8.14 ± 5.96%, p < 0.05).
Representative examples of wounds treated with PBS
(Fig. 3A, D, G), SDF-1 protein (Fig. 3B, E, H), or SDF1 plasmid (Fig. 3C, F, I) scaffolds at day 0 (top panel)
and day 9 (bottom panel) are shown. At day 9, the
wound treated with the control scaffold was still apparent, whereas there was no visible evidence of the SDF-1
protein- and SDF-1 plasmid-treated wounds (Fig. 3G–I).
Notably, one of three SDF-1 plasmid-treated wounds
and two of five SDF-1 protein-treated wounds are 100%
healed at 9 days (Fig. 3J).
SDF-1 has previously been shown to cause neovascularization (21,30) and promote homing of progenitor
cells (11,28). It is recently appreciated that SDF-1 may
also be important in regulating the wound-healing response in skin (23,26,27). Interestingly, macroscopic
wound evaluation by a pathologist blinded to treatment
group indicated that scar formation varied between
wounds. Therefore, we investigated the impact of SDF-1
404
RABBANY ET AL.
Figure 3. SDF-1 treatment reduces scar formation in dermal wounds and increases wound closure. Representative images of
wounds that received scaffolds containing PBS control (A, D), SDF-1 protein (B, E), or plasmid (C, F) at day 0 prior to scaffold
placement or on day 9 after scaffold removal. All full-thickness wounds are 5.0 ± 0.1 cm. (G–I, 2×) Wounds that received SDF-1
treatment demonstrated less visible scarring compared to controls and had a higher percentage of fully healed wounds (J).
ALGINATE SCAFFOLDS AS DELIVERY VEHICLE FOR SDF-1 IN INJURY
on fibroblast infiltration, smooth muscle, and new blood
vessel formation, using immunohistochemical staining
for vimentin (Fig. 4A–H), smooth muscle actin (Fig.
4I–L), and PECAM-1 (CD31) (Fig. 4K–O), respectively. A slight increase in vimentin staining was observed in PBS-treated compared to SDF-1-treated
wounds, which may reflect differences observed in scar
formation. There were no substantial differences in the
amount of CD31 or smooth muscle actin staining compared between treatment groups. H&E analysis showed
a slight decrease in fibrosis in the SDF-1 protein- and
plasmid-treated wounds compared to control or sham
wounds (Fig. 5A). Similarly, a slight increase in granulotamous inflammation was observed in control groups,
405
but no difference was observed in subacute/chronic inflammation or necrosis between groups (Fig. 5B–D).
DISCUSSION
Wound dressings capable of delivering proangiogenic
factors play a vital role in the early healing process, by
leaving wounds from being undisturbed and keeping
wounds intact at body temperature, giving the body a
chance to heal by recruiting the much needed blood supply. In this study, we demonstrated that a novel wound
dressing, an alginate scaffold releasing the proangiogenic molecule SDF-1, accelerated the wound-healing
process in a clinically relevant animal model of acute
surgical wound healing. Furthermore, we characterized
Figure 4. Immunhistochemical analysis of wounds treated with SDF-1 or PBS scaffolds. Representative images of immunohistochemical staining demonstrating that SDF-1 treatment decreased vimentin staining (A–D, 40×; E–H, 2×) in the wound scar. The
smooth muscle actin (I–K, 40×) illustrates lower level of remodeling in both SDF-1 protein and plasmid group in comparison with
PBS control. CD31 staining (L–P, 40×) also shows less vascularization in the SDF-1 groups compared with the PBS control.
406
RABBANY ET AL.
Figure 5. Scaffold application does not adversely affect wound healing. Microscopic evaluation
of dermal tissue at wound site 9 days after scaffold application demonstrates no significant difference in fibrosis (A), granulotamous inflammation (B), subacute/chronic inflammation (C), or necrosis (D) between controls (n = 4) and SDF-1-treated wounds (n = 8).
SDF-1 release kinetics from the scaffold for both the
SDF-1 plasmid and the SDF-1 protein. Interestingly, the
time course of the in vivo release kinetics differed between plasmid and protein; however, the SDF-1-induced
wound-healing benefit was observed independent of the
delivery modality.
Several investigators have employed model proteins
such as albumin and IgG to examine protein release
from carriers. Some used recombinant proteins, but very
few have shown in vivo release profiles. As shown in
Figures 1 and 2, we found that the difference between
the in vivo and in vitro profiles for both the SDF-1 protein and plasmid may be due to the displacement of the
SDF-1 protein with other heparin-binding proteins or to
the biological responses to the implanted vehicles. We
did not check the stability of SDF-1 protein and plasmid,
but rather the remaining radioactivity of radiolabeled
SDF-1 protein and plasmid.
Protein and plasmid can be cleaved by proteinases
and DNases in the body, respectively. Thus, some mechanisms that allow SDF-1 protein and plasmid to exhibit
their bioactivities by protecting them from enzymatic digestion should be considered to explain the accelerated
wound healing observed after therapeutic application of
SDF-1 protein and plasmid. It is well known that many
growth factors are stored in the body by binding to extracellular matrices, such as heparin sulfate proteogly-
cans and growth factor binding proteins, which protect
the growth factors from enzymatic digestion and regulate certain in vivo biological activities. In regenerating
tissues and organs, the binding of extracellular matrix
proteins and proteoglycans to growth factors is mitigated
by enzymes secreted by the healing tissue, leading to the
release of growth factors in a biologically active form.
It has been previously reported that the stability or resistance of growth factor proteins and plasmids against
proteinases or DNases is increased in biodegradable gelatin hydrogels due to their binding with gelatin molecules (14,24). Based on this mechanism, we mimicked
the naturally occurring release system for growth factor
proteins or plasmids and extended their biological activity in vivo by controlling their release from gelatin hydrogels.
As shown in Figure 2, we found that the alginate
patches prolonged the in vivo retention of both SDF-1
protein and plasmid over 2 weeks. Because heparin and
spermine introduced into the presented alginate scaffolds can bind to SDF-1 protein and plasmid, respectively, this result strongly suggests that SDF-1 protein
and plasmid binds to patches and consequently achieves
the prolonged in vivo retention in a biologically active
form.
As described above, the release of the plasmid was
markedly slower compared to the protein in vivo (Fig.
ALGINATE SCAFFOLDS AS DELIVERY VEHICLE FOR SDF-1 IN INJURY
1). However, both provided similar acceleration of the
healing of surgical wounds (Figs. 2 and 3). There are a
number of potential explanations. SDF-1’s wound-healing benefit may be relatively time independent, and delivery of SDF-1 within a certain number of days following wound creation is sufficient. In contrast to the
kinetics of SDF-1 protein, which releases quickly in
vivo, only 20% of SDF-1 plasmid is released 1 day following delivery and is likely not significantly expressed
until at least 24 h after delivery, but 75% is released
after 6 days (Fig. 1). Although the release characteristics
differed, both SDF-1 protein and plasmid treatment accelerated wound healing (Fig. 2). A second explanation
might be that SDF-1 must be delivered close to the time
of wound creation. However, in this study an exceptionally high dose of SDF-1 was delivered, so that even 20%
of total plasmid release/60% of protein release after 1
day is sufficient to provide benefit. This is supported by
previous work demonstrating that the application of
SDF-1 within 2 days postwounding is effective in accelerating the healing of diabetic wounds (11). Furthermore, under different experimental conditions, stem cell
homing has been induced with lower concentrations of
SDF-1 protein and plasmid than those used in this study
(22,25).
Histological analysis of the wound measured by
PECAM-1 (CD31) on day 9 revealed that the SDF-1
release decreased vascularization of the wound. Similarly, smooth muscle α-actin staining confirms a lower
level of remodeling. Staining of the PBS control group
is shown for comparison (Fig. 4).
The beneficial effect of SDF-1 on wound healing in
this large-animal model (Figs. 2 and 3) is novel but consistent with recent reports identifying an important role
for SDF-1 in wound healing. Not only has SDF-1 been
shown to be preferentially expressed at the wound margin
where healing occurs, two independent rodent studies
show that administration of SDF-1 protein or lentivirus
encoding SDF-1 improved wound healing in hard-to-heal
diabetic wounds by increasing homing of endothelial progenitor cells (EPCs) to the wound site (5,6,11,23,26). The
same mechanism has been implicated in patients with
thermal wounds (i.e., burns) where, following injury,
SDF-1 levels are increased proportionally to the extent
of EPC mobilization (10). Finally, these findings in
wound healing are supported by a large body of literature demonstrating that SDF-1 recruits progenitor cells,
induces angiogenesis, and promotes healing when delivered to ischemically damaged cardiac, peripheral vascular, or renal tissue (2,18,28,30).
One particularly clinically relevant finding in this
study is that SDF-1-treated wounds that fully closed
showed no scarring at the wound site (Fig. 3, vimentin
2× images in Fig. 4). Dermal wounds created during fe-
407
tal development heal without remnants of a scar (16,17).
One of the important molecular pathways responsible
for regulating fetal development in a number of organ
systems is the sonic hedgehog–Gli pathway (9), which
has been implicated in healing ischemic injury as well.
Delivery of a sonic hedgehog (Shh) plasmid to cardiac
ischemic tissue has improved cardiac function (9,13). In
both this myocardial injury model and in wound-healing
models, treatment with Shh has been shown to upregulate SDF-1 and SDF-1’s enhancement of EPC homing
to the site of injury (1,13). Therefore, our data suggest
that Shh-mediated SDF-1 upregulation may be the
mechanism by which there is decreased scarring following fetal surgery. If correct, our data further suggest that
SDF-1 overexpression in adult would healing may be a
strategy to decrease scar formation in adulthood. Interestingly, our histology does not demonstrate a significant increase in vascular density in those animals that
received SDF-1 protein or plasmid. This lack of angiogenesis suggests that the benefits associated with SDF1 treatment in our study may be a mechanism other than
EPC homing.
In conclusion, continuous delivery of the proangiogenic modulator, SDF-1 plasmid or protein, through an
alginate scaffold significantly accelerates wound healing
and may reduce scarring in a clinically relevant acute
surgical wound model.
ACKNOWLEDGMENTS: We thank Matthew Kiedrowski for
plasmid production and Amanda Finan for scaffold loading.
We thank MPI Research (Kalamazoo, MI) for the execution of
this study.
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