The healing effect of unrestricted somatic stem cells loaded in collagen-modified nanofibrous PHBV scaffold on full-thickness skin defects

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Copyright © 2014 American Scientific PublishersAll rights reservedPrinted in the United States of America

Journal ofBiomaterials and Tissue Engineering

Vol. 4, 1–8, 2014

The Healing Effect of Unrestricted SomaticStem Cells Loaded in Nanofibrous Poly

Hydroxybutyrate-Co-Hydroxyvalerate Scaffoldon Full-Thickness Skin Defects

Esmaeil Biazar1�∗, Saeed Heidari Keshel2�3, Ali Sahebalzamani4,Mehran Hamidi5, and Maryam Ebrahimi2

1Department of Biomaterial Engineering, Tonekabon Branch, Islamic Azad University, Tonekabon, Iran2Faculty of Paramedical Sciences, Student Research Committee, Proteomics Research Center,

Shahid Beheshti University of Medical Sciences, Tehran, Iran3Tissue Engineering Department, School of Advanced Technologies in Medicine,

Tehran University of Medical Science, Tehran, Iran4Faculty of Biomedical Engineering, Science and Research Branch, Islamic Azad University, Tehran, Iran

5Department of Biomaterial Engineering, Science and Research Branch, Islamic Azad University, Yazd, Iran

Unrestricted somatic stem cells (USSCs) loaded in nanofibrous poly hydroxybutyrate-co-hydroxyvalerate (PHBV) scaffold can be used for skin regeneration when grafted into full thicknessskin defects of rats. Nanofibrous PHBV scaffolds were designed by electro spinning method. After-wards, the scaffolds were evaluated by scanning electron microscopy, physical and mechanicalassays. In this study; nanofibrous PHBV scaffolds loaded with and without USSCs were graftedinto the skin defects. The wounds were subsequently investigated at 21 days after grafting. Resultsof mechanical and physical analyses were showed good resilience and compliance to movementas a skin graft. In animal models; all study groups excluding the control group exhibited the mostpronounced effect on wound closure, with the statistically significant improvement in wound heal-ing being seen on post-operative day 21. Histological and immunostaining examinations of healedwounds from all groups, especially the groups treated with stem cells, were showed a thin epider-mis plus recovered skin appendages in the dermal layer. The graft of nanofibrous PHBV scaffoldloaded with USSC showed better results during the healing process of skin defects in rat models.

Keywords: Nanofibrous PHBV Scaffold, Unrestricted Somatic Stem Cells (USSCs), WoundHealing, Rat.

1. INTRODUCTIONThe final goal of tissue engineering should be the func-tional recovery of damaged tissue in vivo, and in vitroreconstruction of tissue architecture whilst realizingexquisite tissue-specific functions.1 The reconstructionof skin defects remains a major concern when thedefective area is widespread, severely contaminated bymicroorganisms, or poorly vascularized, as can be thecase with irradiation defects, congenital skin disorders,or extensive burns.1 Mesenchymal stem cells (MSCs)have the prospect of broad clinical application. MSCsare abundant and present in various tissues, such as the

∗Author to whom correspondence should be addressed.

bone marrow, periosteum, thymus, skin, adipose tissue,muscle, umbilical cord, and cord blood2–7 and as seedcells they are commonly used in tissue engineering.Umbilical cord blood contains haematopoietic as well asnon-haematopoietic mesenchymal stem cells, these latteralso named as CBEs (Cord Blood Embryonic-like stemcells).8 CBEs have been shown to differentiate into neural,hepatobiliary, pancreatic-like precursors and potentiallyothers.9 Human umbilical cord blood is a rich source ofhemopoietic stem cells for clinical application and may beone of the largest sources of stem cells with naive immunestatus.10 Cord lining-mesenchymal stem cells expressCD23, CD14 and low amounts of CD34 and CD35;they do not express endothelial marker CD31 and havegreater in vitro expansion than Wharton’s jelly-derived

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The Healing Effect of USSCs Loaded in Nanofibrous PHBV Scaffold on Full-Thickness Skin Defects Biazar et al.

MSCs.11 CD14 inhibits T cells. Wharton’s jelly derivedMSCs do not express CD14 or CD23. Despite thosedescriptions, the cell markers of umbilical cord-derivedMSCs are under great debate.12�13 Numerous materialsfor skin regeneration, including temporary substitutes,such as porcine xenografts, synthetic membranes, andallogeneic substitutes or permanent skin substitutes, suchas cultured epidermis and dermal substitutes, have beeninvestigated.14 Among these materials, artificial dermalsubstitutes are structurally optimized to be incorporatedinto the surrounding tissue and to allow cell invasionby fibroblasts and capillaries for subsequent dermalremodeling.15 The mechanical properties of a scaffoldcan influence the resulting stem cell differentiation.16

One of the key factors of tissue engineering is to cre-ate a three-dimensional scaffold with suitable propertiesalso, degradation rate, high porosity, interconnected poresand etc. Typically, biodegradable polymeric scaffoldsare fabricated using different methods.17 Wide vari-ety of natural materials, such as collagen [Biobrane™,Integra®, Alloderm™], fibrin [Bioseed™], HA [Laserskin™]and GAGs [Integra®] etc. have been used in com-mercialized skin grafts.18�19 Nylon [Transcyte™] andbiodegradable polymers such as polyglactin [Dermgraft®],polycaprolactone (PCL)20 and PLGA21 were used forfabricating skin substitutes. PHA has been investigatedfor their applications as biodegradable and biocompat-ible materials, including applications in chondrocytesand bone tissue engineering.22 Among many reportedPHA, only poly-R-3-hydroxybutyrate (PHB) copoly-mers of R-3-hydroxybutyrate and R-3-hydroxyvalerate(PHBV), copolymers of R-3-hydroxybutyrate and4-hydroxybutyrate (P3HB4HB) and copolymers of R-3-hydroxybutyrate and R-3-hydroxyhexanoate (PHBHHx)are available in sufficient quantity for various biomedicalstudies along with the FDA approved polylactic acid(PLA).22 Poly (3-hydroxybutyrate-co-3-hydroxyvalerate)(PHBV) scaffold has shown good biocompatibility,biodegradable and also piezoelectric properties and usedfor skin regeneration.23�24 In natural tissues, cells aresurrounded by extracellular matrix, which has physi-cal structural features ranging from nanometer scale tomicrometer scale. Hence, a nano-structured porous andlarge surface area is needed as an alternate to natu-ral ECM. To mimic the natural ECM, many researchgroups tried to fabricate nanofibrous scaffold by dif-ferent methods also electro spinning. Nanofibers haveimproved the performance of biomaterials. Electro spin-ning is one of the most important methods for fabricationof nanofibrous scaffolds.25–33 In this study, nanofibrouspoly hydroxybutyrate-co-hydroxyvalerate (PHBV) scaf-fold were fabricated by electro spinning. The sampleswere evaluated by scanning electron microscope (SEM),physical and mechanical analysis and In-vitro assays thenloaded with to USSCs and implanted in damaged rat andinvestigated by different analyses.

2. MATERIALS AND METHODSPHBV (molecular weight of 680 KDa) was purchasedfrom Sigma-Aldrich (USA). 2,2,2-trifluoroethanol (TFE)was also purchased from Sigma-Aldrich and was used assolvent to prepare PHBV solutions. Both polymer and sol-vent were used without further purification.Electro-spinning apparatus used in this study prepared

from Fanavaran Nano-Meghyas Company (Iran). PHBVwas dissolved in TFE at a concentration of 2% w/v andthe resulting solution was poured into a glass syringe con-trolled by syringe pump. A positive high voltage sourcethrough a wire was applied at the tip of a syringe needleand a strong electric field (20 Kv) is generated betweenthe PHBV solution and the collector. As soon as the elec-tric field reached a critical value with increasing voltage,mutual charge repulsion overcame the surface tension ofthe polymer solution and an electrically charged jet wasejected from the tip of a conical shape as the Taylor cone.Ultrafine fibers are formed by narrowing the ejected jetfluid as it undergoes increasing surface charge densitydue to the evaporation of the solvent. An electro spunPHBV nanofibrous mat was carefully detached from thecollector and dried in vacuum for 2 days at room tem-perature to remove the solvent molecules completely. Thenanofibers fabricated with a pre-determined variables ofelectro-spinning (Syringe Size: 17 mm, Collector speed:1000 rpm, Injected speed: 2 ml/min, Syringe tip distanceto collector: 75 mm, Voltage: 20 Kv, Temperature: 30 �C,Time: 7 h). The electro-spinning set has been shown inFigure 1. The samples were examined by attenuated totalreflection Fourier transform infrared (ATR-FTIR; Nexus;Thermo Niocolet, Waltham, MA) before and after mod-ifying of PHBV surfaces, and then were put under theinstrument for investigation. The surface characteristics ofneural guides were studied by a scanning electron micro-scope (Cambridge Stereo-scan, S-360, Wetzlar, Germany)to analyze the changes in the surface morphology. Themats were first gold sputtered for two hours (Ion Sput-ter, JFC-1100-JOEL, Japan) to provide surface conductionbefore scanning. The sample surfaces static contact angleswere investigated by a contact angle measuring appara-tus (Krüss G10, Matthews, NC, Germany) according tothe sessile drop method. For mechanical investigations,

Fig. 1. The nanofibrous mat designed by electro spinning method.(a) Electro-spinning set, (b) the manofibrous film.

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the neural guides were subjected to stress–strain analysisusing a universal testing machine under an extension rateof 5 mm/min and 100 N load cell. The specific surfacearea of guides was determined by the surface area and poresize analyzer (BEL Japan).

2.1. Cell Culture and Surgical ProceduresCulture and isolation protocol of the USSC from freshumbilical cord blood was previously described by Kogler34

and also Heidari et al.35 Briefly, after consent from moth-ers, their umbilical cords were obtained from the cord vein.Red blood cells were then induced to settle using ammo-nium chloride (NH4Cl) (Sigma, Chemical Co, St. Louis),and the plasma was removed from the upper layer of thesample. The marrow sample was centrifuged at 400 gfor 20 min at room temperature in 1.077 g/ml Ficoll-Hypaque (Amersham Biosciences, Sweden) with a den-sity gradient. The enriched cells were collected from theinter-phase, re-suspended in basal medium and stimula-tory supplements (GIBCO, Grand Island, N.Y., USA) inthe presence of 10% fetal bovine serum (GIBCO, GrandIsland, N.Y., USA) and antibiotics (100 IU/ml penicillinG and 100 �g/ml streptomycin), and then transferredto tissue culture flasks (T25 flask) at densities of about1× 106 cell/ml. Cultures were maintained at 37 �C in ahumidified atmosphere of 5% CO2/95% air in an incu-bator (BINDER, Germany). The medium was changedafter 48 h and then every 2–3 days. Primary cultureswere usually maintained for 8–10 days, during whichtime the non-adherent hematopoietic cell fraction wasdepleted. After the cells had grown to near confluence,they were passaged 2–3 times by digestion with 0.25%trypsin and 0.02% EDTA (GIBCO, Grand Island, N.Y.,USA). Cell proliferation and viability in vitro was ana-lyzed with the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT). Briefly, 5000cells of unrestricted somatic stem cells were seeded on thenanofibrous PHBV scaffolds. For analysis, 20 �l of MTT(sigma) solution (5 mg/ml stock solution in phosphate-buffered saline [PBS]) was added to each well, and theplates were returned to standard tissue incubator condi-tions for an additional 4 hours. Medium was then removed,the cells were solubilized in 100 �l of dimethyl sulfox-ide, and colorimetric analysis was performed (wavelength,570 nm RAYTO micropleat reader). For electron micro-scopic investigations, the USSCs (0.5 ml) with an initialdensity of 4×105 cells/ml were seeded into each well of24-well plates covered with nanofibrous PHBV scaffolds.The USSCs suspension was exposed to these substrates at37 �C in a humidified atmosphere of 5% CO2/95% air inan incubator for defined periods of time (72 h) and washedby HBSS twice to remove unattached cells.Thirty male wistar white rats aged approximately 4–8

weeks and weighing 180–220 g at the beginning of theexperiment were divided into five groups. The protocol forthe experiment was approved by the institutional animal

care and use committee of Shahid Beheshti University ofMedical Sciences (Iran). Animals were handled accordingto the guidelines established for animal care at the center.Each rat had free access to both sterile water and standardrodent soft chow ad libitum. Animals were anesthetized by80 mg/kg ketamine; 10 mg/kg xylazin injection and theirback were shaved and swabbed with povidine-iodine fol-lowed by 70% ethanol. The scrubbing was repeated twomore times. Then a sterile template measuring 1.5 cm×1.5 cm was placed on their skin and the outline was tracedusing a sterile fine felt-tipped pen. The medial border ofthe template was oriented parallel to the sagittal axis of theanimal. Full-thickness wounds of 1.5 cm× 1.5 cm weremade by excising the skin within the confines of the squaredown to the level of subcutaneous panniculuscarnosus. Thewound cavity was covered with the designated scaffold andwas sutured using 10–0 nylon at 0.75-cm intervals. Among30 of the skin defects, ten defects were grafted withnanofibrous scaffolds without unrestricted somatic stemcells (USSCs) (group A), ten defects were grafted withnanofibrous scaffolds loaded with about 2× 106 USSCs(group B), and then ten defects were control group withoutany treatment (group C). The wounds were covered witha standard wet compress to prevent scaffold detachmentand desiccation, and then each animal was housed in itsown cage to avoid damage to the wound. Every day, theanimals received a subcutaneous injection of immunosup-pressive drug (cyclosporine (10 mg/kg)) and checked anypost-surgery pain, distress or complications after surgery.The process of reconstruction of defected skin with USSCsand nanofibrous scaffold has been shown in Figure 2.

2.2. Histological AssessmentThe wounds were harvested 21 days after grafting, thenstained and investigated for histological assessment. Thereconstituted skin was cut to the control depth determinedas the excision down to the level of panniculus carnosus(15 mm wide, 15 mm long, and a depth of 5 mm), andfixed in 10% formalin at 4 �C for 5 days, dehydratedand then paraffin-embedded. Serial 2 �m paraffin sectionswere cut with a rotating microtome (Microm) and stainedwith hematoxylin and eosin according to the routine histol-ogy protocol. The wounds were evaluated for the presenceof smooth muscle, number of sebaceous units and hairfollicle, epithelialization, fibroplasias and vascular hyper-plasia etc. by optical microscopy (Nikon; Japan).

2.3. Immunostaining AnalysisFor in vivo detection and tracing of Stem Cells, USSCswere labeled with the nuclear stain 4-6 diamidino-2-phenylindole dihydrochloride (DAPI; Sigma-Aldrich).Briefly, DAPI was added to the culture medium for 2 hourswhen the cells were 80% confluent. Then, the cells wereharvested using trypsinization and prepared for seeding on

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Fig. 2. The grafting process of scaffolds loaded with stem cells on damaged skin.

PHBV scaffold for 72 hours and transplanted in to the ratskin defect. The skins were harvested 21 days after graft-ing. The skin specimens were cut and fixed in formalin10%. DAPI fluorescence was analyzed using a fluorescencemicroscope. Keratinocytes from the rat skin equivalentswere analyzed for their specific expression of cytokeratin10 (K10) by means of immunohistochemical analysis. Theresults of each group were statistically analyzed using theANOVA analysis. The quantitative data analyses were car-ried out with the statistical procedures of excel program.

3. RESULTS AND DISCUSSION3.1. Characterizations of Nanofibrous ScaffoldThe nanofibrous PHBV scaffolds were designed by electrospinning method. Figure 3 shows the SEM images of thenanofibrous mats at different magnifications. The smoothand homologous nanofibers have clearly been shown inthis figure. The average size for the nanofibers was about100 nm.The mechanical and physical properties of nanofibrous

PHBV scaffolds have been presented in Table I. The con-tact angle of 105� was measured for the nanofibrous PHBVscaffolds. The porosity of nanofibrous PHBV scaffold wascalculated 91.62% and their pore size also measured as

Fig. 3. SEM images of the designed PHBV nanofibers in different magnifications. (A) 1000×; (B) 5000×; (C) 20000×.

Table I. The mechanical and physical properties of nanofibrous PHBVmats.

Contact Porosity Tensile Ultimate tensileSample angle (deg)� (%) modulus (MPa) stress (MPa)

Nanofibrous 105±3�2� 91 110±18 5.9±0.5PHBV mat

Note: The data are presented as the mean values S.D, p < 0�05.

0�45± 0�25 �m. Specific surface area for the electrospunnanofibrous scaffolds was about 138 m2/g. It is discerniblethat the electrospun nanofibrous mat has high porosity andhigh level of specific surface area, as well. The tensilemodulus of the nanofibrous scaffolds is equal to that ofthe human skin. Moreover, the ultimate tensile stress valueof the nanofibrous PHBV scaffolds is suitable comparedto that of the human skin, (Tensile modulus of the humanskin is 15–150 (MPa) and its Ultimate tensile stress is5–30 (MPa)).Table II shows the MTT assay for the control or tissue

culture polystyrene (TCPS), and the nanofibrous scaffold.The results showed a high viability for the nanofibrousscaffold sample, which were caused more cell prolifera-tion. Figure 4 shows images of the cell culture on thenanofibrous scaffold and control sample. Image A and B


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Table II. Results of MTT assay for the nanofibrous PHBV mat.

Sample Viability %

TCPS 100Nanofibrous mats 112

Note: The data are presented as the mean values S.D, p < 0�05.

Nanofibrous mat

Stem cellsStem cells

A B

Fig. 4. Cell culture on the nanofibrous, and the control samples.(A) The control, and (B) the nanofibrous PHBV mat.

are related to the control sample and nanofibrous scaffold,respectively. Cellular images showed good growth in thevicinity of nanofibrous scaffolds.

3.2. Wound-Size MeasurementAny inflammation symptoms were not seen in the woundsurface of study groups. The study groups A and B showedthe most pronounced effect on wound closure, with sta-tistically significant improvements in wound healing beingseen at 21 day after grafting (Fig. 5). The earliest differ-ence was observed between the groups A and B, when theydemonstrated 70% and 80% wound closure, respectively(Fig. 6). By contrast, the study group C, demonstrated≤ 20% wound closure and was still poorly healed (P <0�05). Although a slight trend towards improved woundhealing was noticeable in the group A compared to thegroup B, no statistically significant differences were foundat any time period between the two groups.

3.3. Histological ResultsHistological images related to the control, the grafted andthe normal skin groups have been displayed in Figure 7.

A B C

Fig. 5. Wound healing at the day 21 after grafting. (A) The nanofibrous scaffold without USSCs, (B) The nanofibrous scaffold with USSCs, (C) Thecontrol.

Fig. 6. Percentage of original wound area after 21 days for the control,the nanofibrous scaffold without USSCs, the nanofibrous scaffold withUSSCs.

Histological examination of wounds in group B, 21 daysafter grafting exhibited the well-recovered epidermal anddermal layers. The epidermis was thin and showed amature differentiation, and the dermal layer showed athick collagen bundle deposition and well-recovered skinappendages at this time point. Both the study groups Aand B well showed the formation of epidermal layer, butskin appendages were not formed in the dermal section inboth groups. Skin appendages such hair follicles and seba-ceous units, were formed in the dermal layer in the groupB, similar to the histological features of the normal group.The formation of hair bulbs was well seen in the groupsA and B, especially in the group B. The Group C showedslightly thickened epidermis with the fibrosis tissue.

3.4. Immunostaining ResultsThe morphology of DAPI-labeled unrestricted somaticstem cells (USSCs) has been depicted in Figure 8. Themicroscopic image of USSCs on control (TCPS) at pas-sage 3 have been shown in Figure 8(A). After expansion tothe third passage, a monolayer of adherent, fibroblast-like


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A B

D

EP

SB

EP

SB

VS

EP

VS

HB

HF

SB

HBHF

FD

C

Fig. 7. Histology of wounds by H&E staining in the different groupson post-operative day 21. (A) The nanofibrous PHBV scaffold withoutUSSCs (group A); (B) The nanofibrous PHBV scaffold with USSCs(group B); (C) The normal skin; (D) control. (Abbreviations: FD-fibrinous debris; EP—epithelialization; HF—hair follicle; HB—hair bulb;SB—sebaceous gland; VS—vascularized section).

cells was labeled with DAPI. Figures 8(B) and, (C) showdapi labeling of USSCs culture on the scaffold and controlsamples, respectively.The skin repair efficacy in different states by means

of nanofibrous scaffold, with and without unrestrictedsomatic stem cells (USSCs) transplanted modalities hascompared in Figure 9. Figures 8(A) and (B) show flu-orescence tracked 4′,6-diamidino-2-phenylindole (DAPI)-labeled USSCs in skin. Blue dots are DAPI-labeled USSCswhich are well discernible in the Figure 9(A). Thenanofibrous scaffolds with cells have been displayed inFigure 9(B). As it is seen, the nanofibrous scaffold com-bined with the USSCs and many of DAPI-labeled cellshave been distributed in derma and a few labeled cells canbe seen in epidermal.The identification of keratinocytes of the skin equiv-

alents was performed by the immunodetection of

C

Fig. 8. Morphologic analysis of DAPI-labeled unrestricted somatic stem cells (USSCs). (A) Phase contrast microscopy of USSCs in the cell culturedish at passage 3 with stretched fibroblastic phenotype. (B) Fluorescence microscopy of adhesion of USSCs labeled with DAPI culture on TCPS.(C) Dapi labeling of USSCs on the scaffold. Original magnification: 150×, Scale bars: 70 �m.

A B

Dermis

Epidermis

Fig. 9. Fluorescence tracked 4′,6-diamidino-2-phenylindole (DAPI)-labeled USSCs in skin. (A). Blue dots are DAPI-labeled USSCs inthe nanofibrous sample without USSC. (B) The nanofibrous samplewith USSC. All figures are 10× 10 magnification. Scale bars represent100 mm.

cytokeratin, which are specific markers for the epithelialdifferentiation. On the other hand, supra basal cell layersof the skin equivalents expressed cytokeratin 10, a markerfor terminal differentiation, which were seen at the day 21.Figure 10 shows the well formation of epithelial layer anddetection or presentation of keratinocytes for the normalskin and the study groups A–C. In contrast to the groupswithout cells, the groups with the USSCs demonstrated anincreased amount of epidermis formation (brown dots) andpresence of keratinocytes.It was also suggested that stem cells from umbilical cord

blood are able to differentiate into epithelial cells underin vitro conditions and could therefore be uses as a start-ing material for isolation and expansion of cells in largeskin defects.36 These observations indicated that bone mar-row MSCs can differentiate into epidermal cells and repairskin defects. In addition, bone marrow and umbilical cord-derived MSCs can be induced to differentiate in vitro intoskin fibroblast cells. Progress has also been made in theculture and differentiation of hair follicle, sweat gland,and sebaceous gland appendage cells from MSCs. Shenget al.37 successfully induced the phenotypic transformationto sweat gland cells from bone marrow MSCs by direct co-culturing of bone marrow MSCs with normal sweat gland


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A B C

Fig. 10. Immunostaining or epidermal markers expression (cytokeratin-10) on day 21. (A) The nanofibrous scaffold without USSCs, (B) The nanofi-brous scaffold with USSCs, (C) The normal skin.

cells. Duffy et al. showed that MSCs growing in scaf-folds can be induced to differentiate into vascular endothe-lial cells in vitro.38 Some investigators have comparedthe proliferative and pluripotent potentials between humanMSCs derived from the umbilical cord and bone mar-row and found that human umbilical cord MSCs (HUCM-SCs) are more applicable for research and development.39

Liao et al.40 demonstrated that USSCs could be induced toexpress genes which hallmark keratinocyte differentiation.They also demonstrated that USSCs express type VII col-lagen (C7), a protein that is absent or defective in patientswith an inherited skin disease, recessive dystrophic epider-molysis bullosa (RDEB). In mice with full-thickness exci-sional wounds, a single intradermal injection of USSCsat a 1 cm distance to the wound edge resulted in signif-icantly accelerated wound healing. USSC-treated woundsdisplayed a higher density of CD31+ cells and the woundshealed with a significant increase in skin appendages.These beneficial effects demonstrated without apparentdifferentiation of the injected USSCs into keratinocytes orendothelial cells. In vivo bioluminescent imaging (BLI)revealed specific migration of USSCs modified with aluciferase reporter gene, from a distant intradermal injec-tion site to the wound, as well as following systemic injec-tion of USSCs. In view of this, HUCMSCs are an ideal cellchoice for skin tissue engineering, as they are a class ofself-renewable proliferating, and pluripotent stem cells andhave clear advantages over MSCs from other sources.40

4. CONCLUSIONThis study reveals the great efficacy of nanofibrous Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) scaffoldloaded with unrestricted somatic stem cells (USSCs) asskin grafts for treating the acute full-thickness skin woundsin a rat model. The combined use of nanofibers andUSSCs for repairing the acute full-thickness wound of1.5 cm× 1.5 cm gave rise to a successful wound heal-ing and skin regeneration in rats. On the post-operativeday 21, the reconstructed skin in the nanofibrous scaf-folds, especially ones loaded with USSCs, demonstratedan intact epithelium together with the formation of new

hair follicles and sebaceous glands, which were reminis-cent of the structures of the natural skin. The results ofimmunostainning with cytokeratin showed that implantingthe cell-laden scaffolds lead to the formation of a thickerepidermis layer due to the differentiation of stem cells intokeratinocytes and fibroblasts. The USSCs exhibited a spe-cific effect on wound healing, especially on regenerationof skin appendages. Taken together, these findings suggesta great potential of the nanofibrous scaffolds loaded withUSSCs as efficient skin grafts for the treatment of acutefull-thickness skin wounds.

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tation DOI:10.3727/096368913X663569 (2013).

Received: xx xxxx xxxx. Accepted: xx xxxx xxxx.


The healing effect of unrestricted somatic stem cells loaded in collagen-modified nanofibrous PHBV scaffold on full-thickness skin defects

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