Cell Transplantation, Vol. 26, pp. 103–113, 2017
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DOI: https://doi.org/10.3727/096368916X692249
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Improved Transplanted Stem Cell Survival in a Polymer Gel
Supplemented With Tenascin C Accelerates Healing and Reduces
Scarring of Murine Skin Wounds
Cecelia C. Yates,*†‡§1 Austin Nuschke,*‡1 Melanie Rodrigues,¶ Diana Whaley,*§ Jason J. Dechant,†
Donald P. Taylor,‡#**†† and Alan Wells*‡§††
*Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
†Department of Health Promotion and Development, University of Pittsburgh School of Nursing, Pittsburgh, PA, USA
‡McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA, USA
§Veterans Affairs Medical Center, Pittsburgh, PA, USA
¶Department of Plastic Surgery, Stanford University, CA, USA
#Department of Biomedical Informatics, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
**Department of Plastic Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
††Department of Bioengineering, University of Pittsburgh Swanson School of Engineering, Pittsburgh, PA, USA
Mesenchymal stem cells (MSCs) remain of great interest in regenerative medicine because of their ability
to home to sites of injury, differentiate into a variety of relevant lineages, and modulate inflammation and
angiogenesis through paracrine activity. Many studies have found that despite the promise of MSC therapy,
cell survival upon implant is highly limited and greatly reduces the therapeutic utility of MSCs. The matrikine
tenascin C, a protein expressed often at the edges of a healing wound, contains unique EGF-like repeats that
are able to bind EGFR at low affinities and induce downstream prosurvival signaling without inducing receptor internalization. In this study, we utilized tenascin C in a collagen/GAG-based polymer (TPolymer) that
has been shown to be beneficial for skin wound healing, incorporating human MSCs into the polymer prior to
application to mouse punch biopsy wound beds. We found that the TPolymer was able to promote MSC survival for 21 days in vivo, leading to associated improvements in wound healing such as dermal maturation and
collagen content. This was most marked in a model of hypertrophic scarring, in which the scar formation was
limited. This approach also reduced the inflammatory response in the wound bed, limiting CD3e+ cell invasion
by approximately 50% in the early wound-healing process, while increasing the numbers of endothelial cells
during the first week of wound healing as well. Ultimately, this matrikine-based approach to improving MSC
survival may be of great use across a variety of cell therapies utilizing matrices as delivery vehicles for cells.
Key words: Cellular therapy; PEG-polymer; Mesenchymal stem cells (MSCs); Extracellular matrix;
Collagen; Tenascin C; Dermal wound healing
INTRODUCTION
Mesenchymal stem cells/multipotent stem cells (MSCs)
possess strong potential to positively influence the wound
microenvironment and therefore are considered attractive
candidates for regenerative therapy. MSCs have shown
beneficial effects in preclinical models of wound healing
by accelerating wound closure, reducing scar formation,
and even improving neovascularization1–3. However, the
exact mechanism for MSC-mediated improvements in
these models of tissue repair is still poorly understood,
as the cells do not contribute to the repaired tissue, due
to limited survival upon implant, typically less than a
week after application4. Thus, the beneficial effects are
deemed to be due to paracrine signaling through soluble
and matrix factors5–7. Unfortunately, countering this beneficial aspect, the rapid death of the implanted MSC creates a nonspecific inflammation response dampening the
success of these preclinical studies.
Received February 4, 2016; final acceptance October 25, 2016. Online prepub date: July 22, 2016.
1
These authors provided equal contribution to this work.
Address correspondence to Alan Wells, M.D., D.M.Sc., Department of Pathology, University of Pittsburgh, 3550 Terrace Street, Scaife Hall, S-713,
Pittsburgh, PA 15261, USA. Tel: 412-647-7813; Fax: 412-647-8567; E-mail: [email protected] or Cecelia C. Yates, Ph.D., University of Pittsburgh School
of Nursing, 3500 Victoria Street, Victoria Building, 458A, Pittsburgh, PA 15261, USA. Tel: 412-624-5728; Fax: 412-624-8521; E-mail: [email protected]
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Efforts to optimize the therapeutic effects of MSCs
now focus on the secretion of proregenerative factors
to stimulate host recovery and regeneration8. Several
well-documented studies suggest that the multiple antiinflammatory, angiogenic, neurotrophic, immunomodulatory, and antifibrotic factors are derived from the transplanted MSCs, suggesting that the release of these factors
may depend on the specific contextual demands of the
type and stage of injury9. This allows for the consideration of MSCs to be viewed as a “smart,” site-specific
multidrug release system.
Although MSCs are shown to demonstrate clear therapeutic potential, current paradigms for their use are
clearly not optimal10. Among these obstacles are delivery, safety, survival, dosing, immune response, and
costly infusion procedures. Current injury models have
shown that MSCs are cleared within days11 in the heart,
kidney, and liver with the delivery of MSCs to cutaneous wound sites demonstrating even lower persistence12.
Engineering-based strategies have suggested that the use
of biomaterial-based therapeutics for cell delivery may
not only enhance cell viability but also contribute to
MSC fate determination13. We have found earlier that cell
surface-restricted activation of epidermal growth factor
(EGF) receptor (EGFR), accomplished by tethering of
the EFGR ligands themselves, promotes MSC survival12
while allowing for expansion of the MSCs and maintaining differentiation capacity2. One way to prolong survival
of MSCs is using the matrikine tenascin C (TNC); this
matricellular protein contains cryptic low-affinity ligands
for the EGFR14. Incorporation of TNC into a matrix protects MSCs from killing by death cytokines15,16.
We have proposed that to improve pathologic healing,
the cellular delivery system must promote survival and
allow the cells to be responsive to environmental cues6.
Delivery systems such as hydrogels and acellular dermal
matrices lack the beneficial structural, mechanical, and
chemical properties needed to provide microenvironmentresponsive regeneration. As such, we have developed a
gel that shapes the space to be replaced by tissue containing matrikines to enhance survival16. Our biodegradable
3,4-dihydroxyphenylalanine (DOPA)-cross-linked gel is
built from polyethylene glycol (PEG) and lysine diisocyanate (LDI). Incorporated into this gel are bioactive
agents collagen type I to provide hemostasis and TNC
to promote engraftment efficiency and survival17,18. The
antimicrobial and hemostatic agents incorporated into
this polymer-based wound gel bode well as a cell delivery system, as these components would reduce innate
inflammatory responses and reduce subsequent scarring.
Herein we examined the beneficial effect of this polymer
for xenogeneic MSC delivery using our recently developed wound-scarring model based on the absence of
wound resolution through the chemokine receptor CXCR3
YATES ET AL.
signaling axis. This model yields mice that develop sterile chronic inflammation and hypertrophic scars19,20.
MATERIALS AND METHODS
Reagents
For all cell culture, a-minimum essential medium
(MEM) (15-012-CV) was obtained from Mediatech
(Manassas, VA, USA), fetal bovine serum (FBS)
(S11550H) was obtained from Atlanta Biologicals (Flowery Branch, GA, USA), and L-glutamine (25030-081)
was obtained from Thermo Fisher Scientific (Gibco,
Grand Island, NY, USA). CM-DiI cell tracker (C7000)
used in culture was obtained from Life Technologies
(Grand Island, NY, USA), and an infrared cell tracker
was also obtained from Life Technologies (D12731). For
plate coating, rat tail collagen I (354236) was obtained
from BD Biosciences (San Jose, CA, USA) at 4.41
mg/ml concentration, and TNC (CC065) was obtained
from EMD Millipore (Billerica, MA, USA) at 100 µg/
ml. Na2B4O7 (S9640-25G) for prepolymer solvent was
obtained from Sigma-Aldrich (St. Louis, MO, USA),
and AgNO3 and (NH4)2S2O8 for prepolymer catalyst solutions were obtained directly from the polymer manufacturer CM-Tec (Newark, DE, USA). For plug digestion
and cell processing for flow, Liberase TL (0540102001)
was obtained from Roche (Indianapolis, IN, USA), and
40-µm filters (352340) for cell filtration were obtained
from BD Biosciences.
Synthesis of Polymers
The PEG-(HDI-DOPA)2 (DHP) prepolymer-based gel
was generated by CM-Tec in the manner described previously17. In brief, a PEG-DOPA/Col I prepolymer was
dissolved in 0.1 M Na2B4O7 aqueous solution (0.3 g
prepolymer/ml solution). This underwent polymerization initiated by the redox initiation system AgNO3/
(NH4)2S2O8 (7 µl/ml gel), yielding 20 µg of Ag/gm polymer gel wet weight. This formulated the PEG-polymer.
Specifics of polymer stability and silver diffusion have
been published previously17. TNC incorporation into the
PEG-polymer was accomplished by coating tissue culture dishes for 16 h at 37°C with 1 µg/cm2 Col I and 1 µg/
cm2 TNC diluted in phosphate-buffered saline (PBS).
PBS was aspirated, and the coated surfaces were placed
under UV light for 10 min before cell seeding. Human
bone marrow-derived MSCs were seeded on the dish for
24 h before harvesting the matrix and cells (if present)
by rubber policeman and mixing with the PEG-polymer
(referred to hereafter as PEG-TPolymer).
Animals
C57BL/6J and FVB wild-type (WT) mice and CXCR3
expression-abrogated mice [obtained from Dr. Wayne
Hancock and Dr. Bao Lu (Millennium Pharmaceutical
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IMPROVED TRANSPLANTED STEM CELL SURVIVAL IN A POLYMER GEL ACCELERATES HEALING OF SKIN WOUNDS
Inc., Cambridge, MA, USA and Perlmutter Laboratory,
Children’s Hospital, Boston, MA, USA)] were bred inhouse. All of the offspring were genotyped by polymerase
chain reaction (PCR) before use. Control, age-matched
C57BL/6J mice were obtained from Jackson Laboratories
(Bar Harbor, ME, USA). Serological analyses did not
detect blood-borne pathogens or evidence of infection.
Mice were housed in individual cages after wound induction and maintained under a 12-h light/dark cycle and
room temperature (20 ± 2°C). Both male and female
mice were used, as earlier studies did not reveal any gender differences in healing among the mice at the ages
used. All studies on these animals were performed after
approval by, and in compliance with, the Institutional
Animal Care and Use Committee (IACUC) of the Veteran’s
Administration and University of Pittsburgh; these animals were housed in a facility of the Veteran’s Affair
Medical Center (Pittsburgh, PA, USA) accredited by
the Association for the Assessment and Accreditation of
Laboratory Animal Care.
Wounding
Male and female mice [7 to 8 weeks (considered as
young) weighing approximately 25 g] were anesthetized
with an intraperitoneal (IP) injection containing ketamine
(75 mg/kg; Phoenix Scientific Inc., Solana Beach, CA)
and xylazine (5 mg/kg; VEDCO, Saint Joseph, MO,
USA). No less than eight mice from each group were
used in each experiment. The backs were cleaned, shaved,
and sterilized with betadine solution. For full-thickness
wounds, a 6-mm-diameter punch biopsy through the epidermis and dermis was made on one side of the dorsal
midline (to minimize wound contraction), and the contralateral uninjured skin served as a control. Wounds
were either left untreated or treated with TPolymer or
TPolymer-MSCs. Thirty minutes after the treatments,
we covered the wounds with Tegaderm (3M, St. Paul,
MN, USA) in all five groups to maintain uniformity and
prevent the mice from removing the treatments. Wounds
were examined at 3, 7, 14, 21, 60, or 90 days postwounding. This was done to capture the transitions from inflammatory to regenerative and regenerative to resolving
phases of wound healing. Animals were euthanized by
CO2 inhalation, as approved by the American Veterinary
Medical Association (AVMA), and the wounds harvested
for assessment. Each wound was measured and then
removed from the animal, with unwounded skin taken
from the contralateral dorsum as a control.
Histological Analysis
Wound bed biopsies surrounded by a margin of nonwounded skin were collected after euthanasia at 3, 7,
14, 21, 60, or 90 days postwounding. Wound biopsies
were fixed in 10% buffered formalin, processed, and
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embedded in paraffin blocks using standard protocols.
Tissue sections (5 µm) were stained with hematoxylin
and eosin (H&E) (Sigma-Aldrich), Masson’s trichrome
(Sigma-Aldrich), and PicroSirius Red (Sigma-Aldrich)
and analyzed for general tissue and cellular morphology
and analyzed using the MetaMorph software (Molecular
Devices LLC, Sunnyvale, CA, USA). All images were
collected at original magnification 10´ and 40´.
Epidermal and Dermal Maturation
Histopathological examination of mouse tissue was performed blinded by a trained histopathologist. Qualitative
assessments were made concerning aspects of dermal and
epidermal maturation as previously described20. In brief,
the samples were scored on a scale of 0 to 4 for epidermal
healing (0, no migration; 1, partial migration; 2, complete
migration with partial keratinization; 3, complete keratinization; and 4, normal epidermis) and dermal healing
(0, no healing; 1, inflammatory infiltrate; 2, granulation
tissue present-fibroplasias and angiogenesis; 3, collagen
deposition replacing granulation tissue >50%; and 4,
complete healing).
Flow Cytometry
Twelve-millimeter punch biopsies of the wound and
12-mm punch biopsies of unwounded regions were isolated for quantification of MSC engraftment in wounded
skin at days 3, 7, 14, and 21. Tissue was minced and
incubated in 0.5 mg/ml Liberase TL (Roche) for 1 h at
37°C. Tissue was filtered; cells were washed and labeled
for analysis. Human leukocyte antigen (HLA) markers
were used to identify positive human MSCs in the wound
bed. Appropriate isotype controls, unstained cells, and
untreated wounds were used as controls. Flow cytometry
was performed on an LSR II Cytometer (BD Biosciences)
and subsequently analyzed using FlowJo digital FACS
software (Tree Star, Inc., Ashland, OR, USA).
Cell Culture and MSC Labeling
Primary bone marrow-derived MSCs were obtained
from the National Institutes of Health (NIH)-funded
MSC repository at Texas A&M under the direction of
Dr. Darwin Prockop and primary human mesenchymal
stem cells (prhMSCs) immortalized by human telomerase reverse transcriptase (hTERT) (imhMSCs) were
obtained from Junya Toguchida’s laboratory (Kyoto
University, Japan). Both imhMSCs and primary bone
marrow-derived cells are considered both established
cell lines and commercially available primary cells,
and as such are IRB exempted 4e at the University of
Pittsburgh (IRB No. PRO13080262). These cells are
certified and analyzed for appropriate surface marker
expression/differentiation potential by the repository
prior to shipment. All primary MSCs were cultured in
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a-MEM supplemented with 17.5% triple-dialyzed FBS
and L-glutamine. Passage 1 (P1) MSCs were expanded at
37°C to approximately 70% confluence, when they were
passaged until P3 to allow for sufficient cell numbers
for implant.
For cell tracking, CM-DiI (C7000) (Thermo Fisher)
was resuspended at 1 mg/ml in dimethyl sulfoxide
(DMSO) (Sigma-Aldrich) as a stock solution, and then
diluted to 1 µM in warm PBS, where it was left on cells
at 37°C for 5 min followed by 15 min at 4°C. The flask
was then washed and incubated in proliferation media
overnight. Fluorescent signal from MSCs in culture
was confirmed prior to use in experiments. For infrared
tracking of implanted cells, 1,1¢-dioctadecyl-3,3,3¢,3¢tetramethylindotricarbocyanine iodide (D12731) (Life
Technologies) was added directly to culture media at
1 µM from a stock solution of 1 mg/ml. Labeling solution was incubated on the adherent MSCs for 30 min, followed by replacement with normal proliferation media.
Polymer Characterization Analyses
For in vitro assessment of the polymer, degradation
assays and cell incorporation assays were performed.
Degradation was allowed to proceed with 0.4 g of formed
polymer in 2 ml of PBS at 37°C over 10 days, with a
daily scoring of remaining polymer area as a means of
analyzing degradation. Cells tracked with CM-DiI label
were visualized via fluorescence microscopy within the
polymer immediately after incorporation.
Statistical Analyses
For wound-healing outcomes (wound maturation,
dermal maturation, and collagen content), a two-way
Student’s t-test was performed for direct comparisons of
treatments, with values of p < 0.05 considered statistically
significant. For inflammatory infiltrate, one-way analysis
of variance (ANOVA) of each mouse model group was
performed for all three conditions, with p < 0.05 considered significant. Posteriori test [Tukey’s honestly significant difference (HSD)] was used to confirm where the
differences occurred between groups on all groups that
showed an overall significant difference. Data are presented as mean and SEM. GraphPad software was used.
RESULTS
MSC-Incorporated PEG-Polymer Is Biodegradable
To assess the polymer as a biodegradable MSC delivery system, we evaluated its degradation and cell survival
properties. PEG-TPolymer degradation was tested in vitro
by placing 100 mg of polymer in phosphate buffer solution and changing the buffer daily. Approximately 60%
of the PEG-TPolymer was still intact 10 days after formulation compared to PEG-Polymer alone being almost
absent by this time (Fig. 1A and B). Thus, the addition
YATES ET AL.
of TNC promoted gel retention. Imaging of the PEGTPolymer with and without MSC incorporation indicated
that the gel maintained viable MSCs during the studied
time period (Fig. 1C). The polymer with TNC and MSCs
quickly embedded within a mouse wound (Fig. 1D) and
remained intact for days (data not shown).
PEG-TPolymer/MSC Survival and Engraftment
In Vivo Capabilities
To determine whether the MSCs survive in the wound
bed, we labeled the cells with an infrared tracking dye
and imaged them over a weeklong period. Qualitatively,
there was a decrease in the xenotransplanted human
MSCs (Fig. 2A). To quantitate this effect, we assessed
MSC survival after 3, 7, or 21 days, using flow cytometric detection of cells stained for human HLA (Fig. 2B).
Three days after treatment with PEG-TPolymer + MSCs,
50% of the transplanted cells were still present in the
wound bed. The MSCs survived in the gel over 21 days,
but declined in cell number, as expected in immunocompetent animals. This survival was noted in both WT
mice and CXCR3−/− [knockout (KO)] mice. This demonstrated that not only does the polymer have superior
biocompositional properties but that it also enhances the
persistence of the human MSCs.
PEG-TPolymer/MSC Effect on Improved Scarring
With the enhanced MSC survival, we next determined
whether this would improve the healing of wounds and
normalize the scar phenotype. Full-thickness wounds were
created on the WT and CXCR3−/− (KO) mice, and followed
for up to 90 days to track the full healing or scarring as noted
in our earlier publications18. Wound maturation was measured to account for a composite of epidermal thickness,
keratinization, chronic inflammation, fibroblast cellularity,
vascularity, and collagen modeling. Quantification of these
wounds revealed that wound repair and dermal maturation
in mice that were treated with PEG-TPolymer + MSCs
showed significant improvement in comparison with
untreated mice in the first 60 days postwounding (Fig. 3A
and B). The PEG-TPolymer itself, without MSCs, does not
alter the wound healing significantly.
More strikingly, wounds treated with PEG-TPolymer +
MSCs resulted in significantly more collagen earlier in
the healing process than untreated at days 7 and 21 postinjury; this was determined by Masson’s trichrome staining of histological sections and subsequent MetaMorph
analysis for quantitation (Fig. 3C and D). PicroSirius Red
staining, which detects appropriately aligned collagen
bundles, shows that the PEG-TPolymer + MSCs promoted
the replacement of immature collagen fibers that fail to
provide strength in the KO scarring mice to more mature
aligned fibers (Fig. 4). Of interest, and more strikingly
in KO mice, PEG-TPolymer (even without the added
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IMPROVED TRANSPLANTED STEM CELL SURVIVAL IN A POLYMER GEL ACCELERATES HEALING OF SKIN WOUNDS
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Figure 1. The PEG-collagen polymer degrades gradually in vitro and integrates into mouse wounds. The PEG-collagen polymer was
assessed in vitro for degradation in PBS over 10 days as a function of preparation state (prepolymer, polymer alone, or polymer + TNC)
(A), with percent degradation assessed as a size of the original polymer aggregate (B; day 8 shown). (A) Gel polymerizes with a tissuelike consistency in vitro. For MSC incorporation testing, cell presence was monitored via CM-DiI cell tracker on MSCs prior to addition
to the polymer (C; day 8 shown). 10´ images, 200 mM. For polymer application, the functional polymer showed adequate integration into
wound beds after 40–60 min (D). Shown is one of at least two experiments with similar outcomes. Scale bars: 400 µm.
MSCs) improved the collagen alignment. This indicates a
dual benefit of the therapy, in part coming from the polymer gel and in part from the cellular products.
PEG-TPolymer/MSC Effect on Immune Response
To assess potential paracrine activity from MSCs surviving in the wound bed, we assessed CD31+ cell infiltration as a metric of angiogenic signaling and CD3e+
infiltration as an indicator of inflammatory response
in relation to T-cell invasion (Fig. 5). Flow cytometric
analysis suggested that the inflammatory infiltrate early
in the wound-healing response was blunted with less
CD3+ cell invasion and at both day 3 and day 7 during
the peak inflammatory response (Fig. 5A). Additionally,
we assessed CD11B, a leukocyte adhesion and migration
marker, to mediate the inflammatory response in general,
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108
YATES ET AL.
Figure 2. Mesenchymal stem cells survive in the polymer in vivo over 21 days in the presence of TNC. MSCs were tracked prior
to polymer application with an infrared membrane label, and wound areas were imaged directly on wild-type mice using a LiCOR
Odyssey imaging system to detect the IR label. Shown here are three representative images over 18 days (A). To quantify the MSC
survival, cells were extracted from mouse wounds via collagenase digestion and probed with a human-specific HLA antibody to detect
human MSCs via flow cytometry (B). MSC survival in the wound bed was assessed for both wild-type and CXCR3−/− mice. Percent
positive for HLA (left) normalized to PEG-TPolymer and absolute numbers (right) were determined via flow cytometry analysis.
Background was subtracted. n = 3–5 for flow cytometry analyses; n = 1 for IR tracking confirmation.
and results did now show a consistent change as a result
of including MSCs in the PEG-TPolymer for CD3+ cell
results, although in the presence of the MSCs, the CD3+
and CD11B+ cell infiltration tended to be less.
CD31+ cell infiltration, as a method for assessing angiogenic response in the wound bed, did show marked
changes as a result of PEG-TPolymer treatment (Fig. 5B).
Both WT and KO mice demonstrated an approximately
twofold increased presence of CD31+ cells after 3 days,
with relatively comparable levels to the untreated mice at
day 21. Day 7 results also showed a trend toward increased
CD31+ cell invasion in both models, although the KO mice
PEG-TPolymer + MSC condition demonstrated statistical
significance. However, the WT model reverted to a lower
level of invasion at day 7. Again, there did not appear to
be a consistent change as a result of MSC presence in the
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IMPROVED TRANSPLANTED STEM CELL SURVIVAL IN A POLYMER GEL ACCELERATES HEALING OF SKIN WOUNDS
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Figure 3. MSC-based polymer yields improved wound-healing outcomes. Full-thickness sections of mouse wounds across 60 days
were assessed via H&E stain (A; day 7 shown) and quantified for whole-wound maturation and dermal maturation (B; day 7 shown)
by a veterinary pathologist in a double-blinded analysis. Both wild-type and CXCR3−/− mice were assessed. Wound tissue was also sectioned and treated with a trichrome stain to assess matrix content and organization at the same time points (C; day 60 shown). Total percent collagen content was analyzed through a MetaMorph analysis normalized to unwounded tissue (D). n = 3. Scale bars: 200 µm.
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YATES ET AL.
Figure 4. Collagen alignment and maturation improve using the MSC-based polymer. Full-thickness mouse skin sections from wound
beds were excised over 60 days and probed with PicroSirius Red (PS red) stain to assess collagen outcomes in the wounds (A; day
21 shown; scale bar: 200 µm) for both wild-type and CXCR3−/− mice. PS red was scored for collagen alignment in a double-blinded
fashion by a veterinary pathologist for wild-type (B) and knockout (C) models across 90 days of wound healing. n = 3. Full-thickness
mouse skin sections from wound beds were excised over 60 days and probed with PS red stain to assess collagen outcomes in the
wounds (A; day 21 shown) for both wild-type and CXCR3−/− mice. n = 3. *p < 0.05.
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IMPROVED TRANSPLANTED STEM CELL SURVIVAL IN A POLYMER GEL ACCELERATES HEALING OF SKIN WOUNDS
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Figure 5. MSC-based polymer augments murine cell infiltrate in wound beds. Cell extracts from mouse wounds in both wild-type and
CXCR3−/− mice were probed for the presence of CD3e+ and CD11b+ (A) and CD31+ (B) cells to assess inflammatory and angiogenic
infiltrate over the first 21 days of wound repair. Treatment conditions analyzed for murine infiltrate were untreated mice, mice treated
with a TNC-based polymer only, and mice treated with the TNC-based polymer combined with primary human MSCs. n = 3–5.
wound bed, suggesting the PEG-TPolymer itself acted as a
paracrine attractant for the invading murine cells.
DISCUSSION
In this study, we utilized a PEG-collagen-based polymer
to effectively deliver xenogeneic MSCs into the wound
and appropriately alter the pathological state of the wound.
Herein we have shown that our polymer-based approach
enhances the survival of these cells while retaining stem cell
markers, resulting in a microenvironment projected toward
a more regenerative healing. Compared to hydrogel and
injection delivery systems, the PEG-collagen delivers the
MSCs in a protective scaffold that biodegrades17,21,22 over
time to allow for a reduction in the otherwise robust and
damaging immune influx, and increased angiogenesis with
a subsequent improvement in healing.
Matrix-based approaches to wound healing are not a
particularly novel innovation, and many commercially
available treatments for chronic wound dressings utilize
fabricated or existing matrices to that effect. This has
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previously consisted of collagen-based dressings, including decellularized collagen matrices in concert with
glycosaminoglycans23, bilayered matrices incorporating
fibroblasts and keratinocytes to jump-start the healing
process24, and others. While these approaches provide
the host tissue with space for native cells to migrate and
engraft into the new wound matrix, they ultimately fail to
address many of the key pathologies for chronic wound
healing, such as extended or persistent inflammation
or reduced angiogenesis25–28. Thus, novel treatments to
address these factors have become necessary in the next
generation of wound-healing strategies. We have investigated MSCs for this purpose because of their widely
acknowledged role in normal wound healing, as well as
their capacity to modulate the chronic inflammation and
avascularity that plague such wounds1,7,29–32.
While MSCs undoubtedly hold great promise for
improving on existing matrix-based approaches to wound
healing, a fundamental constraint in all MSC therapies has
been limited cell survival upon implant due to the harsh
nature of the wound environments. Because of the matrixbased character of the wound-healing polymer (and other
cited treatments for wound healing), a logical additive to
our treatment were proteins that might assist with improving survival of implanted cells. We utilized the EGF-like
repeats of TNC here for this purpose, which are able to
bind EGFR at low affinity but high avidity to induce prosurvival signaling constitutively. While we showed here
that this approach can in fact promote weeks-long survival
of implanted cells, this treatment strategy also affords any
migrating resident cells the opportunity to survive much
longer than expected in the wound bed. Indeed, a major
factor contributing to the chronic nature of diabetic ulcers
and other comparable conditions is the lack of healthy
fibroblasts and/or keratinocytes lining the wound bed,
which are subjected to the same conditions that implanted
MSCs face in vivo33–35. By incorporating an abundance
of TNC, it is probable that these cells (fibroblasts, keratinocytes, CD31+ cells, and others) will have the ability to
invade and engraft in the same matrix, ideally surviving
on the same time scale and thus allowing a more complete
healing response. By the same token, invading inflammatory cells might also be able to exploit the TNC infrastructure to resist anti-inflammatory strategies, reinforcing the
importance of maintaining the immunomodulatory MSC
population at high quality in the long term.
A limitation of this study is the well-acknowledged
drawbacks of murine skin wound healing. As can be seen
even in our data in this study, mouse wounds rarely fail to
heal and, in fact, mostly heal at a rapid pace, entering the
remodeling phase within a few weeks at most. Long-term
outcomes that are analyzed such as our day 60 or day
90 time points here provide little insight beyond potential
scarring implications, as seen in the CXCR3−/− model as
YATES ET AL.
characterized previously18,20. As such, while we do show
an improved rate of healing and collagen maturation in
mouse wounds treated with our polymer, we have not
here delineated effects in a truly human relevant “chronic
wound” model or one with a delayed healing response in
general. We have also here only examined bone marrowderived MSCs, whereas many groups have proposed
adipose-derived MSCs for skin wound therapy in order to
ease the burden of translation to humans and potential differences in paracrine profiles of the two subpopulations.
Future studies examining the optimal conditions for MSC
success in this wound-healing polymer must include an
exploration of the MSC subtype most suited for the TNC
approach to extending survival while also leading to the
most desirable wound-healing outcomes.
In addition to the functional importance of MSCs in our
treatment strategy, the delivery polymer must also encapsulate the other necessary aspects of complete wound healing. Previously, we have shown that this PEG polymer
is able to promote wound healing in comparable mouse
models21, also leveraging an AgNO3/(NH4)2S2O8 catalyst
system for polymerization for this purpose. Silver has a
well-documented antimicrobial role36,37, and as such we
originally applied this as the rationale for a silver nitratebased redox catalysis method. Silver ions in solution as
part of the catalysis process are ultimately able to act
in a dual fashion as both a catalyst and an antimicrobial agent, which we showed was functional in previous
studies with this polymer17,21. In concert with the woundhealing matrix and MSC-based therapeutic for reducing
inflammation and inducing a more robust wound-healing
response, this polymer as a whole appears to encapsulate
the entire wound-healing process and holds great promise
for scaling to chronic wound therapies in the future.
ACKNOWLEDGMENTS: The authors thank the members of the
Wells laboratory for suggestions and discussions. The authors
also thank the members of the Yates laboratory for help in performing experiments and data analysis. These studies were supported by grants from the National Institute of General Medical
Science of the National Institutes of Health (USA) (GM063569).
Services in kind were provided by the Pittsburgh VA Medical
Center and the University of Pittsburgh School of Nursing.
C.C.Y., D.P.T., and A.W. have a patent position on the TPolymer
that is assigned to the University of Pittsburgh; this patent is
neither licensed nor under investigation.
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