The prognosis for extensive and deep skin injury is not satisfactory because of scar formation and the loss of normal function and skin appendages. Several novel therapies for skin repair and regeneration have emerged. Stem cell–based therapies are attractive candidates in regenerative medicine to treat skin injuries, such as chronic wounds and radiolesions. Stem cell therapy has emerged as a promising new approach in almost every medicine specialty. It has been demonstrated that mesenchymal stem cells (MSCs) play an effective role in promoting wound healing when injected into the skin defects, either alone or in combination with scaffold.1,2 Umbilical cord blood (CB) contains hematopoietic as well as nonhematopoietic MSCs, these latter also named as CB embryonic–like stem cells.3 Umbilical cord MSCs have become a unique, accessible, and noncontroversial source of regeneration in medicine.4,5 Cord blood embryonic–like stem cells have been shown to differentiate into neural, hepatobiliary, pancreatic-like precursors, and potentially to the other ones.3,6 Unrestricted somatic stem cells (USSCs) from umbilical cord blood (UCB) isolated by Jager and his colleagues and evaluated the differentiation capacity and cytokine production of these cells for transplantation. In fact, USSCs are one of the rare cell populations in UCB which are considered pluripotent. Human UCB-derived USSCs have previously been demonstrated to have a broad differentiation potential and regenerative beneficial effects when administered in animal models of multiple degenerative diseases.7 It was also suggested that stem cells from UCB are able to differentiate into epithelial cells under in vitro conditions and could therefore be used as a starting material for isolation and expansion of cells in large skin defects.8 Tissue regeneration is a process aided by cellular sciences, engineering, and medicine to repair defected tissues and restore their functions.9 One of the key factors of tissue engineering is to create a three-dimensional scaffold with suitable properties also, degradation rate, high porosity, interconnected pores, etc. Wide variety of natural materials, such as collagen (Biobrane, Integra, Alloderm), fibrin (Bioseed), HA (Laserskin), and GAGs (Integra), have been used in commercialized skin grafts.10–12 Nylon (Transcyte) and biodegradable polymers, such as polyglactin (Dermgraft), polycaprolactone,13 and poly(lactic-co-glycolic acid) (PLGA)14 were used for fabricating skin substitutes. Polyhydoxyalkanoates are polyesters produced by microorganisms under unbalanced growth conditions. Polyhydoxyalkanoates are generally biodegradable, with good biocompatibility, making them attractive as tissue engineering biomaterials.15–25 It is well-known that the chitosan is a natural biomaterial derived from the chitin. It is found in the shells of crustaceans, such as lobsters, crabs, and shrimp, and many other organisms, including insects and fungi. Chitosan is a linear polysaccharide composed of randomly distributed β-(1–4)-linked D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit). It has a number of commercial and possible biomedical applications especially for skin regeneration.26 Controlling the biomaterials surface properties is essential for the high performance of cell adhesion. Wettability is an important factor in the surface modification of biomaterials. Modification of hydrophobic polymer surfaces can be achieved by wet (acid, alkali), dry (plasma), radiation treatments (ultraviolet radiation and laser), and chemical methods like cross-linking.18,27,28
In this study, porous poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) scaffold was fabricated by freeze-drying and crosslink with chitosan by chemical method. The samples were evaluated by scanning electron microscope (SEM), physical and mechanical analysis, and in vitro assays; loaded with USSCs; implanted in damaged skin; and then investigated by different analyses.
Materials and Methods
Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) containing 12% by mole of 3-hydroxyvalerate, were supplied by Sigma-Aldrich (St. Louis, MO) and were used to fabricate polymeric scaffolds. For scaffold fabrication, PHBV powder was weighed accurately and poured into a beaker (50 ml). Then, an accurately measured amount of dichloromethane was added to the tube to make a solution with a desired PHBV concentration at 10% (w/v). To obtain a homogeneous PHBV solution, the mixture was kept at 50°C in a water bath and mixed thoroughly.
Scaffold preparation by particular leaching method (sample 1).
A small amount of salts (NaCl) 1% (w/v) were optionally added, and the solution was homogenized using a homogenizer (Diax 600, Heidolph, Germany) at high speeds. Then, the solution was poured into a filled 5 mm deep stainless to remove solvent after 48 hours. The produced foam was immerged in di-ionized water to removed salts. The scaffolds were stored in a vacuum desiccator at room temperature for storage and for further removal of any residual solvent.
Scaffold preparation by freeze-drying method (sample 2).
The produced PHBV solution was poured in glass mold, then was quenched in liquid nitrogen, then transferred to a freeze dryer apparatus (Gamma 2–16 LSC, Martin Christ, Germany) and kept at −50°C for 24 hours. The scaffolds were stored in a vacuum desiccator at room temperature for storage and for further removal of any residual solvent.
Chitosan-modified scaffold preparation by chemical and freeze-drying method (sample 3).
Chitosan (DA: 75–85%; medium molecular weight; Sigma-Aldrich) was immobilized on the PHBV surfaces based on the following protocol. A weighted amount of the chitosan was rinsed with acetic acid buffer solution (50 mM, pH = 5.0). Then, the scaffold (sample 2) was submerged into the 6 M NaOH solution for 15 minutes. The hydrolyzed scaffold mats were rinsed into 2-(N-morpholino)ethane sulfonic acid buffer (pH = 6.0) containing 10 mM 1-ethyl-3-(3-dimethylaminopropyl) and 10 mM sulfo-sulfo-N-hydroxysuccinimide to activate the carboxyl groups on the surfaces. Chitosan solution (15 mg/ml in acetic acid buffer solution, 50 mM, pH = 5.0) was injected into the previous solution and was shaken gently for 24 hours at 4°C. Cross-linked scaffold was quenched in liquid nitrogen, then transferred to a freeze dryer apparatus (Gamma 2–16 LSC, Martin Christ, Germany), and kept at −50°C for 24 hours. The scaffolds were stored in a vacuum desiccator at room temperature for storage and for further removal of any residual solvent. The preparation process is shown in Figure 1.
Characterization of Scaffolds
The samples were examined by attenuated total reflection Fourier transform infrared (ATR-FTIR; Nexus; Thermo Nicolet, Waltham, MA) before and after modifying scaffold with chitosan, and then were put under the instrument for investigation. The surface characteristics of the chitosan cross-linked and the uncross-linked scaffolds were studied by an SEM (Cambridge Stereoscan, S-360, Wetzlar, Germany) to analyze the changes in the surface morphology.
Static contact angles of the samples surface were investigated by a contact angle measuring apparatus (Krüss G10, Matthews, NC) according to the sessile drop method. Compressive mechanical properties of PHBV scaffolds were determined at room temperature using an Instron mechanical tester (Instron, Norwood, MA) with a 100 N load cell and at a crosshead speed of 0.5 mm/min. The compressive modulus was calculated from the initial linear region of stress-strain curves. The pore diameters and porosity were calculated using SEM micrographs and porosity analyzer.
Culture and isolation protocol of the USSC from fresh UCB was previously described by Kogler et al.29 and also by Heidari et al.30 After consent from mothers, their umbilical cords were obtained from the cord vein. Cell proliferation and viability in vitro were analyzed with the tetrazolium salt 3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyltetrazolium bromide (MTT). Briefly, 5,000 cells of USSCs were seeded on the PHBV scaffolds. For analysis, 20 µl of MTT (Sigma) solution (5 mg/ml stock solution in phosphate-buffered saline [PBS]) was added to each well, and the plates were returned to standard tissue incubator conditions for an additional 4 hours. Medium was then removed, the cells were solubilized in 100 µl of dimethyl sulfoxide, and colorimetric analysis was performed (wavelength, 570 nm RAYTO microplate reader). For electron microscopic investigations, the USSCs (0.5 ml) with an initial density of 4 × 105 cells/ml were seeded into each well of 24 well plates covered with PHBV scaffolds. The USSCs suspension was exposed to these substrates at 37°C in a humidified atmosphere of 5% CO2/95% air in an incubator for defined periods of time (48 hours) and washed by Hank’s Balanced Salt Solution twice to remove unattached cells. The cultured scaffolds with cells were washed by PBS, and then fixed by glutaraldehyde (2.5%) at 4°C for 2 hours. The samples were dehydrated by alcohols, and then kept with tetraoxide osmium vapors at 4°C for 2 hours. The samples were kept in desiccator, then coated with gold, and investigated by an SEM (Cambridge Stereoscan, S-360).
Thirty male Wistar white rats aged approximately 4–8 weeks and weighing 180–220 g at the beginning of the experiment were divided into five groups. The protocol for the experiment was approved by the institutional animal care and use committee of Shahid Beheshti University of Medical Sciences (Iran). Animals were handled according to the guidelines established for animal care at the center. Each rat had free access to both sterile water and standard rodent soft chow ad libitum. Animals were anesthetized by 80 mg/kg ketamine; 10 mg/kg xylazine injection and their back were shaved and swabbed with povidone-iodine followed by 70% ethanol. The scrubbing was repeated two more times. Then, a sterile template measuring 1.5 cm × 1.5 cm was placed on their skin, and the outline was traced using a sterile fine felt-tipped pen. The medial border of the template was oriented parallel to the sagittal axis of the animal. Full-thickness wounds of 1.5 cm × 1.5 cm were made by excising the skin within the confines of the square down to the level of subcutaneous panniculus carnosus. The wound was covered with the designed scaffold and sutured using 10–0 nylon at 0.75 cm intervals. Among 30 of the skin defects, 10 defects were grafted with chitosan cross-linked scaffolds without stem cells, 10 defects were grafted with chitosan cross-linked scaffolds loaded with about 2 × 106 USSCs, and then 10 defects were grafted with control group. The wounds were covered with a standard wet compress to prevent scaffold detachment and desiccation, and then each animal was housed in its own cage to avoid damage to the wound. Every day, the animals received a subcutaneous injection of immunosuppressive drug (cyclosporine [10 mg/kg]) and checked any postsurgery pain, distress, or complications after surgery. The process of reconstruction of defected skin with USSCs and scaffold has been shown in Figure 2.
For in vivo detection and tracking of stem cells, USSCs were labeled with the nuclear stain 4′,6 diamidino-2-phenylindole dihydrochloride (DAPI; Sigma-Aldrich). Briefly, DAPI was added to the culture medium for 2 hours when the cells were 80% confluent. Then, the cells were harvested using trypsinization and prepared for seeding on the PHBV scaffold for 72 hours and transplantation into the rat skin defect. The skins were harvested 21 days after grafting. The skin specimens were cut and fixed in formalin 10%. 4′,6 diamidino-2-phenylindole dihydrochloride fluorescence was analyzed using a fluorescence microscope.
The wounds were harvested 21 days after grafting, then stained and investigated for histological assessments. The reconstituted skin was cut to the control depth determined as the excision down to the level of panniculus carnosus (15 mm wide, 15 mm long, and a depth of 5 mm), and fixed in 10% formalin at 4°C for 5 days, dehydrated, and then paraffin-embedded. Serial 2 µm paraffin sections were cut with a Senior Precision Rotary Microtome (Model-RMT-30, Radical Instruments, Haryana, India) and stained with hematoxylin and eosin according to the routine histology protocol. The wounds were evaluated for the presence of smooth muscle, number of sebaceous units and hair follicle, epithelialization, and vascular hyperplasia, etc., by optical microscope (Nikon, Mellville, NY). The results of each group were statistically analyzed using analysis of variance. The quantitative data analyses were carried out with the statistical procedures of Excel program.
Characterization of Scaffolds
The results, from the ATR-FTIR spectrum of the unmodified scaffold PHBV sample, the chitosan film, and the chitosan-modified linked scaffold sample, are shown in Figure 3. In these figures, the strong bands in 1,720 cm−1 and 1,450 cm−1 as related to the C=O and C-O groups. The stretching band in 1,180 cm−1 is shown to be related to the -C-O-C- group, and the stretching band in 2,800–3,000 cm−1 is shown to be related to the C-H methyl group. The spectra shows that the strong bands in 3,318 cm−1 and 3,746 cm−1 are related to the OH and NH groups of chitosan structure. While for the chitosan-modified PHBV scaffold, besides the characteristic adsorption peaks of PHBV, the characteristic peaks of chitosan were also exhibited, the amide I at 1,650 cm−1 and the amide II at 1,540 cm−1 caused by C=O stretching vibrations and the combination of N-H in plane bending and C-N stretching vibrations, respectively. The stretching bands in 3,429 cm−1 and 3,783 cm−1 are related to the OH and NH groups because of the presence of chitosan.
Figure 4A and B shows scaffold samples produced through particular leaching method. Porosity of PHBV scaffolds was calculated to be 75%. The pore size of foams was 20–50 µm and open–close cells and noninterconnect. Figure 4C and D shows scaffold samples produced through freeze-drying method. The pore structure became uniform and small, with pore sizes ranging from 20 µm and high porosity. Interconnected structures of the microporous foam were observed. Figure 4E and F shows chitosan-modified scaffold samples produced through chemical method with to freeze-drying. Porosity of modified scaffold was calculated 80%, and their pore size also measured as 15 µm. The porosity and pore size was decreased with to coating scaffold by chitosan, but contact angle increased than other samples. Compressive properties of PHBV scaffolds decreased with increasing porosity (Table 1). The scaffolds from particular leaching and freeze-drying methods had a compressive modulus of 6.5 and 4.1 MPa, respectively, whereas chitosan-modified scaffolds had a compressive modulus of 4.8 MPa.
Cell Culture Results
Table 2 shows the MTT assay for the control or tissue culture polystyrene, the chitosan cross-linked scaffold, and unmodified samples. The results showed a high viability for chitosan cross-linked scaffold samples, which caused more cell proliferation. Figure 5 shows images of the cell culture on the chitosan cross-linked, unmodified scaffolds, and control samples. Image A is related to the control sample and images B and C are related to the unmodified and cross-linked scaffolds. Cellular images showed good growth in the vicinity of scaffolds, especially for chitosan cross-linked samples. Figure 6 shows the SEM images of cultured stem cells on the samples. Figures show suitable cell attachment on the chitosan-modified scaffold surfaces.
The skin repair efficacy in different states by means of chitosan-modified scaffold, with and without USSCs transplanted modalities, is compared in Figure 8. Figure 8A and B show fluorescence-tracked DAPI-labeled USSCs in skin. Blue dots are DAPI-labeled USSCs, which are well discernible in the figures. The chitosan-modified scaffolds without any cells and with cells have been displayed in Figure 8A and B, respectively. As it is seen, the chitosan-modified scaffold combined with the USSCs and many of DAPI-labeled cells have been distributed in derma, and a few labeled cells can be seen in epidermal.
Histological images related to the control, the grafted, and the normal skin groups have been displayed in Figure 9. Histological examination of wounds in group D, 21 days after grafting, exhibited the well-recovered epidermal and dermal layers. All the study groups well showed the formation of epidermal layer, but skin appendages were not formed in the dermal section in all groups. Formation of epidermal and dermal layers and especially, formation of sebaceous gland was well seen in the chitosan-modified PHBV scaffold groups with stem cell and normal skin. However, a thickened epidermis with immature epithelial cells and recovered skin appendages and also hair follicles were formed in the dermal layer in the modified scaffold group with stem cell similar to the histological features of the normal group. The formation of hair bulbs was well seen in the modified scaffold and normal groups. The control group showed slightly thickened epidermis with the fibrosis tissue. In addition, skin appendages were partially recovered in modified scaffold group with stem cell (Figure 9).
Stem cell therapy may help in the treatment of epithelial level burn injury: clinical truth is that epithelial (first degree) burns usually heal well without any special forms of treatment. Dai et al.31 studied investigation of the potential to isolate MSCs from human UCB and differentiate them into epithelial cells in mouse skin tissues. Mononuclear cells (MNCs) from UCB (UCB–MNCs) isolated and induced to MSCs in culture. The results demonstrate that UCB-derived MSCs contribute to skin tissue regeneration in vivo and may be an ideal cell source for therapy of skin epithelial tissue injury, including burns.31 Liao et al.7 demonstrated that USSCs could be induced to express genes which hallmark keratinocyte differentiation. They also demonstrated that USSCs express type VII collagen, a protein that is absent or defective in patients with an inherited skin disease, recessive dystrophic epidermolysis bullosa. In mice with full-thickness excisional wounds, a single intradermal injection of USSCs at a 1 cm distance to the wound edge resulted in significantly accelerated wound healing. Unrestricted somatic stem cell–treated wounds displayed a higher density of CD31+ cells, and the wounds healed with a significant increase in skin appendages. These beneficial effects demonstrated without apparent differentiation of the injected USSCs into keratinocytes or endothelial cells. These data suggest that CB-derived USSCs could significantly contribute to wound repair and be potentially used in the cell therapy.7 The combination of scaffold with stem cells considered being useful as a method for bone regeneration. Our previous studies20,21 revealed the great efficacy of different nanofibrous scaffolds (NFSs) loaded with USSCs as skin grafts for treating the acute full-thickness skin wounds in a rat model. The combined use of nanofibrous mat and USSCs for repairing the acute full-thickness wound of 1.5 cm ×1.5 cm gave rise to a successful wound healing and skin regeneration in rats. On the postoperative day 21, the reconstructed skin in the NFSs loaded with USSCs demonstrated an intact epithelium together with the formation of new hair follicles and sebaceous glands and the collagen deposition, which is reminiscent of the structures of the natural skin.20,21 Here, we confirm previous results and demonstrate that our designed porous scaffold with stem cells does significantly promote skin regeneration. In the full-thickness wounding model, we demonstrated that USSCs with porous scaffold facilitated the rate of wound healing by promoting re-epithelialization and dermal layers. Improved skin histology with USSC/scaffold-treated wounds, when compared with the other samples, was observed at all the time points that were analyzed, as determined by the progression of epithelialization and formation of epidermis. One interesting observation is the presence of skin appendages in the regenerated wound of the USSC/scaffold-treated rat. It has previously been shown that transplantation of MSCs together with NFS in the wounding animal model, resulted in formation of hair follicles and sebaceous glands.21,22,32 In this current study, we demonstrated that chitosan-modified PHBV scaffold loaded with CB-derived USSCs also resulted in the formation of skin appendages at the wounds. These observations suggested that the porous PHBV scaffold can be used as a potential candidate material for stem cells especially in skin regeneration.
The combined application of scaffolds and stem cells to the acute full-thickness wound of 15 mm × 15 mm in a rat model achieved successful wound healing and skin regeneration by day 21. On day 21, the reconstructed skin in this grafted groups specially porous scaffold with USSCs demonstrated an intact epithelium with the formation of new hair follicles and sebaceous glands, which were reminiscent of the structures of normal skin. This study demonstrated a great efficacy of scaffold/USSCs as skin grafts in the treatment of acute full-thickness skin wounds in a rat model. Biodegradable porous scaffold accelerated wound healing by providing a provisional matrix for cellular invasion and adhesion, collagen synthesis, and deposition also USSCs had a specific effect on wound healing. Taken together, these findings suggest a great potential of the scaffold/USSCs as efficient skin grafts for the treatment of acute full-thickness skin wounds.
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