Enhanced Diabetic Rat Wound Healing by Platelet-Rich Plasma Adhesion Zwitterionic Hydrogel.

BACKGROUND: The skin is the largest organ in the human body and serves as a barrier for protective, immune, and sensory functions. Continuous and permanent exposure to the external environment results in different levels of skin and extracellular matrix damage. During skin wound healing, the use of good dressings and addition of growth factors to the wound site can effectively modulate the rate of wound healing. A dressing containing bioactive substances can absorb wound exudates and reduce adhesion between the wound and dressing, whereas growth factors, cytokines, and signaling factors can promote cell motility and proliferation. AIM AND OBJECTIVES: We prepared a functional wound dressing by combining platelet-rich plasma (PRP) and zwitterionic hydrogels. Functional wound dressings are rich in various naturally occurring growth factors that can effectively promote the healing process in various types of tissues and absorb wound exudates to reduce adhesion between wounds and dressings. Furthermore, PRP-incorporated zwitterionic hydrogels have been used to repair full-thickness wounds in Sprague-Dawley rats with diabetes (DM SD). MATERIALS AND METHODS: Fibroblasts and keratinocytes were cultured with PRP, zwitterionic hydrogels, and PRP-incorporated zwitterionic hydrogels to assess cell proliferation and specific gene expression. Furthermore, PRP-incorporated zwitterionic hydrogels were used to repair full-thickness skin defects in DM SD rats. RESULTS: The swelling ratio of hydrogel, hydrogel + PRP1000 (108 platelets/mL), and hydrogel + PRP1000 (109 platelets/mL) groups were similar (~07.71% ± 1.396%, 700.17% ± 1.901%, 687.48% ± 4.661%, respectively) at 144 hours. The tensile strength and Young modulus of the hydrogel and hydrogel + PRP10000 groups were not significantly different. High concentrations of PRP (approximately 108 and 109 platelets/mL) effectively promoted the proliferation of fibroblasts and keratinocytes. The zwitterionic hydrogels were not cytotoxic to any cell type. High PRP concentration-incorporated zwitterionic hydrogels increased the rate of cell proliferation and significantly increased the expression of characteristic genes such as collagen, fibronectin, involucrin, and keratin. Subsequently, zwitterionic hydrogels with high PRP concentrations were used to repair full-thickness skin defects in DM SD rats, and a wound healing rate of more than 90% was recorded on day 12. CONCLUSIONS: PRP contains high concentrations of growth factors that promote cell viability, enhance specific gene expression, and have a high medical value in cell therapy. Zwitterionic hydrogels have a 3-dimensional interconnected microporous structure and can resist cell adhesion without causing cytotoxicity. Platelet-rich plasma-incorporated zwitterionic hydrogels further enhance the cellular properties and provide an effective therapeutic option for wound healing.

An In Vitro Study of the Effects of Mechanical and Enzymatic Isolation of Stromal Vascular Fraction on Wound Healing.

ABSTRACT: The adipose-derived stromal vascular fraction (SVF) is considered to be an attractive source of stem cells in cell therapy. Besides stem cells, it also contains functional cells, such as macrophages, precursor cells, somatic stem cells, and pericytes. Collagenase digestion is the most frequently used method to isolate SVF, but it is time-consuming and costly and has some problems, such as infectious agents and immune reactions. In this research, we compared the yield, cell population ratios, and cell viability when isolating SVF by the ultrasonic physics (U-SVF) method and traditional enzymatic method (E-SVF). Then, we isolated exosomes from U-SVF and E-SVF, respectively, and cocultured them with fibroblasts to investigate the potential of applying this cell secretion in wound repair. The results showed that there was no significant difference between the ultrasonic method and enzymatic method in terms of cell viability, cell numbers, or the expression of CD markers of stem cells. However, exosome analysis identified a greater number and smaller size of exosome particles obtained by U-SVF. In terms of cell proliferation efficiency, although the proliferation efficiency of U-SVF was lower than that of E-SVF. Trilineage differentiation experiments revealed that both E-SVF and U-SVF had good differentiation ability, owing to high stem cell content. Finally, E-SVF and U-SVF exosomes were cocultured with fibroblasts. The efficiency of fibroblast migration increased in the SVF exosome treated groups, and the expression of related genes (integrin α5ß1) was slightly upregulated; however, the expression of FAK, AKT, ERK, and RhoA was significantly upregulated at 24 hours. From the abovementioned experiments, we found that there was no significant difference in stem cell-related characteristics between SVF isolated by ultrasonic cavitation and SVF isolated by the enzymatic method. In addition, exosomes secreted by SVF may have excellent therapeutic effect on skin injuries, which provides a new viewpoint and therapeutic strategy for soft tissue repair.

Beneficial Effects of Astragaloside IV-Treated and 3-Dimensional-Cultured Endothelial Progenitor Cells on Angiogenesis and Wound Healing.

INTRODUCTION: Astragaloside IV (AS-IV) is a natural herb extract and a popular compound used in traditional Chinese medicine because of its effect on multiple biological processes, such as promotion of cell proliferation, improvement in cardiopulmonary and vascular function, and promotion of angiogenesis around wounds, leading to accelerated wound healing. Vascular regeneration primarily results from the differentiation of endothelial progenitor cells (EPCs). Biomedical acceleration of angiogenesis and differentiation of EPCs around the wound remain challenging. MATERIALS AND METHODS: In this study, we treated human umbilical cord blood-derived EPCs with AS-IV and cultured them on 2-dimensional (tissue culture polystyrene) and 3-dimensional culture plates (3DPs). These cultured cells were then combined with human blood plasma gel and applied on the skin of nude mice in an attempt to repair full-thickness skin defects. RESULTS: The results show that using 3DP culture could increase vascular-related gene expression in EPCs. Furthermore, 12.5 µg/mL AS-IV-treaded EPCs were combined with plasma gels (P-3DP-EPC12.5) and showed enhanced vascular-related protein expression levels after 3 days of culture. Finally, P-3DP-EPC12.5s were used to repair full-thickness skin defects in nude mice, and we could register a wound healing rate greater than 90% by day 14. CONCLUSIONS: Based on these results, we concluded that we have developed a potential therapeutic approach for wound healing using plasma gel containing 3-dimensional surface-cultured AS-IV-treated EPCs.

Complete zwitterionic double network hydrogels with great toughness and resistance against foreign body reaction and thrombus.

Conventional tough hydrogels offer enhanced mechanical properties for load-bearing implants; however, their application is still hindered by a lack of biocompatibility. In this study, we demonstrate a new methodology for developing biocompatible double network (DN) hydrogels by using a responsive amphoteric polymer as a first framework. Tough DN hydrogels were formed by penetrating zwitterionic poly(sulfobetaine acrylamide) (PSBAA) into a swollen poly(lysine acrylamide) (PLysAA) network in an acidic or alkaline solution, and polymerizing under UV irradiation. The DN hydrogels were able to become zwitterionic entirely under physiological conditions, and possess excellent mechanical strength, comparable to conventional DN hydrogels with permanently charged polyelectrolyte frameworks. Additionally, in vitro studies including biofouling, cytotoxicity and hemolysis were conducted to show the superior biocompatibility of the complete zwitterionic DN hydrogels. After the circulation of human blood in tubular DN hydrogels, the zwitterionic DN gels displayed negligible thrombus formation. Furthermore, PLysAA/PSBAA hydrogels were implanted subcutaneously, showing excellent resistance against inflammatory response and long-term capsule formation. This work has presented a new strategy for synthesizing a biocompatible tough DN hydrogel to effectively mitigate the foreign body reaction to render great benefit for the development of biomedical implants.

Evaluation of Polycaprolactone/Gelatin/Chitosan Electrospun Membrane for Peritoneal Adhesion Reduction.

In this study, a novel antiadhesion membrane made of polycaprolactone, gelatin, and chitosan was fabricated using the electrospinning technique. A series of polycaprolactone/gelatin/chitosan (PGC) electrospun membranes with different amounts of chitosan (0%, 0.5%, 1%, and 2% in weight percentage) was synthesized. The physicochemical properties and biocompatibility of the fabricated membranes were examined and compared with the aim to select an effective antiadhesion membrane. Scanning electron microscopy showed that these 4 electrospun membranes had similar fiber diameter and pore area, with no statistical differences between them. Furthermore, the contact angle decreased with increased chitosan content, indicating that chitosan may contribute to increased hydrophilic properties. The in vitro degradation test revealed that the higher chitosan content corresponded to a lower degradation rate in PGC membranes within 7 days. All PGC membranes exhibited similar cell proliferation; however, cell proliferation was lower than tissue culture polystyrene (P < 0.05). To compare antiadhesion ability, the adhesion between the cecum and abdominal wall was created in a rat model. Assessment after implantation of electrospun membranes revealed that PGCs with higher chitosan content (PGC2) had better antiadhesion effects, as evaluated by an adhesion score at day 14 postsurgery. Thus, PGC2 was effective in reducing the formation of tissue adhesion. Therefore, PGC electrospun membrane may provide a potential peritoneal antiadhesion barrier for clinical use.

A gelatin/collagen/polycaprolactone scaffold for skin regeneration.

BACKGROUND: A tissue-engineered skin substitute, based on gelatin ("G"), collagen ("C"), and poly(ε-caprolactone) (PCL; "P"), was developed. METHOD: G/C/P biocomposites were fabricated by impregnation of lyophilized gelatin/collagen (GC) mats with PCL solutions, followed by solvent evaporation. Two different GC:PCL ratios (1:8 and 1:20) were used. RESULTS: Differential scanning calorimetry revealed that all G/C/P biocomposites had characteristic melting point of PCL at around 60 °C. Scanning electron microscopy showed that all biocomposites had similar fibrous structures. Good cytocompatibility was present in all G/C/P biocomposites when incubated with primary human epidermal keratinocytes (PHEK), human dermal fibroblasts (PHDF) and human adipose-derived stem cells (ASCs) in vitro. All G/C/P biocomposites exhibited similar cell growth and mechanical characteristics in comparison with C/P biocomposites. G/C/P biocomposites with a lower collagen content showed better cell proliferation than those with a higher collagen content in vitro. Due to reasonable mechanical strength and biocompatibility in vitro, G/C/P with a lower content of collagen and a higher content of PCL (GCLPH) was selected for animal wound healing studies. According to our data, a significant promotion in wound healing and skin regeneration could be observed in GCLPH seeded with adipose-derived stem cells by Gomori's trichrome staining. CONCLUSION: This study may provide an effective and low-cost wound dressings to assist skin regeneration for clinical use.

Melanocyte Differentiation From Induced Pluripotent Stem Cells Derived From Human Adipose-Derived Stem Cells.

The pigment melanin is produced by melanocytes, is primarily responsible for skin color, and protects it against ultraviolet rays that can cause the destruction of genetic material within the keratinocytes. To elucidate the mechanisms of many diseases associated with melanocytes, such as melanoma and albinism, or burns with uneven pigment distribution, the disease model needs to be established first. In this study, we aimed to construct the melanocyte model from patients in a short period.Sandai virus vector containing 4 stemness genes (Oct4, Sox2, Klf4, c-Myc) was transfected into human adipose-derived stem cells to produce induced pluripotent stem cells (iPSCs). Immunofluorescence staining was used to confirm the expression of specific proteins for iPSCs, including Tra-1-60, Tra-1-81, Oct-4, Sox-2, and Nango. polymerase chain reaction results also showed that specific genes of iPSCs with the ability to cause the differentiation of cells into the 3 germ layers were expressed. In our in vivo experiments, iPSCs were subcutaneously injected into nude mice to induce teratoma formation for 2 months. The morphology of the 3 germ layers was confirmed by hematoxylin and eosin staining. Furthermore, melanocytes were purified by serial induction medium, and their presence was confirmed by flow cytometry and the expression of different markers for melanocytes.

Evaluation and characterization of waterborne biodegradable polyurethane films for the prevention of tendon postoperative adhesion.

BACKGROUND: Tendon adhesion is a serious problem and it affects tendon gliding and joint motion. Although recent studies have yielded promising results in developing anti-adhesion materials, there are still many problems. Polycaprolactone (PCL)-based polyurethane (PU) has good mechanical properties and biocompatibility, and it has a potential in anti-adhesion applications. MATERIALS AND METHODS: In this study, a series of waterborne biodegradable polyurethane (WBPU) films with different ratios of ionic groups were synthesized. In order to select an effective anti-adhesion film, the WBPU films were cast and characterized for physicochemical properties and biocompatibility. RESULTS: All WBPU films were non-cytotoxic in the cell viability test and had suitable physicochemical and mechanical properties based on the measurement of zeta potential, water contact angle, mechanical properties, water absorption, thickness change, and gelatin test. To evaluate the anti-adhesion effect, severely injured tendons of rabbits were sutured with the modified Kessler core suture technique and WBPU films were then wrapped around the tendon. Implantation in rabbits showed that the WBPU film had better anti-adhesion effect than PCL films and the untreated control, and demonstrated no significant difference in the anti-adhesion performance from the commercial product Seprafilm based on gross evaluation, histological analysis, and biomechanical assessment. CONCLUSION: Compared to Seprafilm and PCL applied in the tendon anti-adhesion, WBPU had better mechanical properties, low inflammatory reaction, and a proper degradation interval.

Development of a Novel Pre-Vascularized Three-Dimensional Skin Substitute Using Blood Plasma Gel.

Skin substitutes with existing vascularization are in great demand for the repair of full-thickness skin defects. In the present study, we hypothesized that a pre-vascularized skin substitute can potentially promote wound healing. Novel three-dimensional (3D) skin substitutes were prepared by seeding a mixture of human endothelial progenitor cells (EPCs) and fibroblasts into a human plasma/calcium chloride formed gel scaffold, and seeding keratinocytes onto the surface of the plasma gel. The capacity of the EPCs to differentiate into a vascular-like tubular structure was evaluated using immunohistochemistry analysis and WST-8 assay. Experimental studies in mouse full-thickness skin wound models showed that the pre-vascularized gel scaffold significantly accelerated wound healing 7 days after surgery, and resembled normal skin structures after 14 days post-surgery. Histological analysis revealed that pre-vascularized gel scaffolds were well integrated in the host skin, resulting in the vascularization of both the epidermis and dermis in the wound area. Moreover, mechanical strength analysis demonstrated that the healed wound following the implantation of the pre-vascularized gel scaffolds exhibited good tensile strength. Taken together, this novel pre-vascularized human plasma gel scaffold has great potential in skin tissue engineering.

High correlation between skin color based on CIELAB color space, epidermal melanocyte ratio, and melanocyte melanin content.

BACKGROUND: To treat skin color disorders, such as vitiligo or burns, melanocytes are transplanted for tissue regeneration. However, melanocyte distribution in the human body varies with age and location, making it difficult to select the optimal donor skin to achieve a desired color match. Determining the correlations with the desired skin color measurement based on CIELAB color, epidermal melanocyte numbers, and melanin content of individual melanocytes is critical for clinical application. METHOD: Fifteen foreskin samples from Asian young adults were analyzed for skin color, melanocyte ratio (melanocyte proportion in the epidermis), and melanin concentration. Furthermore, an equation was developed based on CIELAB color with melanocyte ratio, melanin concentration, and the product of melanocyte ratio and melanin concentration. The equation was validated by seeding different ratios of keratinocytes and melanocytes in tissue-engineered skin substitutes, and the degree of fitness in expected skin color was confirmed. RESULTS: Linear regression analysis revealed a significant strong negative correlation (r =  - 0.847, R2 = 0.717) between CIELAB L* value and the product of the epidermal melanocyte ratio and cell-based melanin concentration. Furthermore, the results showed that an optimal skin color match was achieved by the formula. DISCUSSION: We found that L* value was correlated with the value obtained from multiplying the epidermal melanocyte ratio (R) and melanin content (M) and that this correlation was more significant than either L* vs M or L* vs R. This suggests that more accurate prediction of skin color can be achieved by considering both R and M. Therefore, precise skin color match in treating vitiligo or burn patients would be potentially achievable based on extensive collection of skin data from people of Asian descent.

Efficacy of Lyophilised Platelet-Rich Plasma Powder on Healing Rate in Patients With Deep Second Degree Burn Injury: A Prospective Double-Blind Randomized Clinical Trial.

Platelet-rich plasma (PRP) is a kind of plasma that is rich in platelets after processing. It includes various growth factors and cytokines, which speed up the process of wound healing and hemostasis. The PRP solution used in this study is diluted from lyophilized PRP powder, which decreased the possibility of contamination, facilitated the storage, and prolonged the storage life. From in vitro fibroblast proliferation testing, the numbers of PRP supplement were performed for 1, 4, and 7 times by continuous replacement of culture medium each day. Four times of lyophilized PRP supplement was selected for clinical study due to sufficient promotion of fibroblast proliferation. Next, 27 patients of deep second-degree burn wound were included in this study. Patients were assigned to two groups: PRP group (n = 15) and control group (n = 12). A concentration of 1.0 × 10 platelets/cm (wound area) according to wound size was sprayed on the wound evenly. Function was mainly assessed by the percentage of wound closure and bacteria picking out rate in 2 and 3 weeks. The wound closure at 3 weeks showed a significant difference in PRP group (P < 0.05). The healing rate of PRP group reached nearly 80% and made a breakthrough of 90% in 3 weeks, showing a significant difference compared with the control group (P < 0.05). Lyophilized PRP can be considered as an effective treatment to increase healing rate in patients with deep second-degree burn injury.

Restoration of skin pigmentation after deep partial or full-thickness burn injury.

Significant skin pigmentation changes occur when patients suffer deep burn injuries. These pigmentation disorders may cause not only cosmetic and psychological issues, but more importantly it increases the risk of skin cancer or photoaging. Severe burns significantly effect on the process of repigmentation as the pigmentation is tightly regulated by cell proliferation and differentiation of melanocytes and melanocyte stem cells which are housing in the epidermis and hair follicles of the skin. In the present review, we discuss the possible mechanisms to replenish the melanocytes from the healthy epidermis and hair follicles surrounding burn wounds. The molecular mechanisms of skin repigmentation following healing of burn injuries includes the differentiation of melanoblasts into melanocytes, the distribution and responses of melanocytes and melanocyte stem cells after burn injury, and the regulation of melanin production. We also reviewed advanced therapeutic strategies to treat pigmentation disorders, such as convectional surgery, laser, UV treatment and emerging concepts in skin tissue-engineering.

A Biodegradable Hemostatic Gelatin/Polycaprolactone Composite for Surgical Hemostasis.

Massive bleeding is the leading cause of battlefield-related deaths and the second leading cause of deaths in civilian trauma centers. One of the challenges of managing severe wounds is the need to promote hemostasis as quickly as possible, which can be achieved by using hemostatic dressings. In this study, we fabricated 2 kinds of gelatin/polycaprolactone composites with 2 ratios of gelatin/polycaprolactone, 1:1 and 2:1 (GP11 and GP21, respectively). Scanning electron microscopy revealed that the GP11 composite exhibited rougher and more porous structure than the GP21 composite did. Furthermore, both composites showed similar biocompatibility as that of tissue culture polystyrene. Moreover, both GP composites tended to show a gradual decrease in contact angle to zero within 40 minutes. The in vitro blood plasma coagulation assay revealed that the prothrombin time was significantly longer for the GP composites than it was for the Quikclot composite, whereas the activated partial thromboplastin time of the GP11 composite was significantly shorter than that of the gauze. Furthermore, the GP11 had the largest platelet adsorption of all the composites. The in vivo coagulation test showed an obvious shortening of the bleeding time with the Quikclot and GP21 compared with gauze sample. In conclusion, the GP composites showed superior biocompatibility and hemostasis to the gauze and comparable effects with the Qickclot composite. Therefore, the GP composites have the potential for development as biodegradable surgical hemostatic agents.

Water-based polyurethane 3D printed scaffolds with controlled release function for customized cartilage tissue engineering.

Conventional 3D printing may not readily incorporate bioactive ingredients for controlled release because the process often involves the use of heat, organic solvent, or crosslinkers that reduce the bioactivity of the ingredients. Water-based 3D printing materials with controlled bioactivity for customized cartilage tissue engineering is developed in this study. The printing ink contains the water dispersion of synthetic biodegradable polyurethane (PU) elastic nanoparticles, hyaluronan, and bioactive ingredients TGFß3 or a small molecule drug Y27632 to replace TGFß3. Compliant scaffolds are printed from the ink at low temperature. These scaffolds promote the self-aggregation of mesenchymal stem cells (MSCs) and, with timely release of the bioactive ingredients, induce the chondrogenic differentiation of MSCs and produce matrix for cartilage repair. Moreover, the growth factor-free controlled release design may prevent cartilage hypertrophy. Rabbit knee implantation supports the potential of the novel 3D printing scaffolds in cartilage regeneration. We consider that the 3D printing composite scaffolds with controlled release bioactivity may have potential in customized tissue engineering.

Evaluation of Wound Healing Efficacy of an Antimicrobial Spray Dressing at Skin Donor Sites.

INTRODUCTION: Autologous skin transplantation is a common treatment for patients with full-thickness burns. Postoperative wound care is essential for skin graft donor and recipient sites, but traditional wound dressings such as cotton and gauze do not form an effective barrier to bacteria, and patients can feel uncomfortable when replacing dressings. MATERIALS AND METHODS: The goal of this study was to evaluate the use of an antimicrobial spray dressing (JUC Spray Dressing, NMS Technologies Co Ltd, Nanjing, China), with respect to its antimicrobial efficiency and the degree of pain experienced by patients. RESULTS: The authors found the antimicrobial spray can reduce pain during the recovery period, while providing equivalent antibacterial protection to the control treatment (AQUACEL Hydrofiber Wound Dressing, ConvaTec, Bridgewater, NJ) based on skin culture tests. The spray did not adversely affect the wound site recovery. No significant side effects were present during the treatment period. CONCLUSION: This antimicrobial spray could potentially be used in wound dressing applications.

Fast isolation and expansion of multipotent cells from adipose tissue based on chitosan-selected primary culture.

Adipose-derived adult stem cells (ASCs) have gained much attention because of their multipotency and easy access. Here we describe a novel chitosan-based selection (CS) system instead of the conventional plastic adherence (PA) to obtain the primary ASCs. The minimal amount of adipose tissue for consistent isolation of ASCs is reduced from 10 mL to 5 mL. The selection is based on the specific interaction between cells and chitosan materials, which separate ASCs by forming spheroids during primary culture. The primary culture period was reduced from 4 days to one day and more ASCs (ten-fold expansion) were achieved in a week. The average duration for obtaining 1 × 10(7) cells takes about seven days from 5 mL of adipose tissue, compared to 14 days using the conventional PA method from 10 mL of adipose tissue. The replicative senescence of CS-ASCs is not evident until the fifteenth passage (vs. eighth for the PA-ASCs). The obtained ASCs (CS-ASCs) have less doubling time for the same passage of cells and show greater stemness than those obtained from the conventional PA method (PA-ASCs). Moreover, CS-ASCs undergo trilineage differentiation more effectively than PA-ASCs. The greater differentiation potential of CS-ASCs may be associated with the enrichment and maintenance of CD271 positive cells by chitosan selection of primary culture.

Histochemical and functional improvement of adipose-derived stem cell-based tissue-engineered cartilage by hyperbaric oxygen/air treatment in a rabbit articular defect model.

Cartilage is exposed to compression forces during joint loading. Therefore, exogenous stimuli are frequently used in cartilage tissue engineering strategies to enhance chondrocyte differentiation and extracellular matrix (ECM) secretion. In this study, human adipose-derived stem cells were seeded on a gelatin/polycaprolactone scaffold to evaluate the histochemical and functional improvement of tissue-engineered cartilage after hyperbaric oxygen/air treatment in a rabbit articular defect model. Behavior tests showed beneficial effects on weight-bearing and rear leg-supporting capacities after treatment of tissue-engineered cartilage with 2.5 ATA oxygen or air. Moreover, positron emission tomography images and immunohistochemistry staining demonstrated hydroxyapatite formation and increased ECM synthesis, respectively, at the tissue-engineered cartilage graft site after high pressure oxygen/air treatment. Based on these results, we concluded that hyperbaric oxygen and air treatment can improve the quality of tissue-engineered cartilage in vivo by increasing the synthesis of ECM.

The effects of microenvironment on wound healing by keratinocytes derived from mesenchymal stem cells.

Embryonic stem cells (ESCs) are pluripotent cells that can differentiate into various cell types, including keratinocyte-like cells, within suitable microniches. In this study, we aimed to investigate the effects of culture media, cell coculture, and a tissue-engineering biocomposite on the differentiation of mouse ESCs (MESCs) into keratinocyte-like cells and applied these cells to a surgical skin wound model. MESCs from BALB/c mice (ESC26GJ), which were transfected using pCX-EGFP expressing green fluorescence, were used to track MESC-derived keratinocytes. Weak expression of the keratinocyte early marker Cytokeratin 14 (CK-14) was observed up to 12 days when MESCs were cultured in a keratinocyte culture medium on tissue culture plastic and on a gelatin/collagen/polycaprolactone (GCP) biocomposite. MESCs cocultured with human keratinocyte cells (HKCs) also expressed CK-14, but did not express CK-14 when cocultured with human fibroblast cells (HFCs). Furthermore, CK-14 expression was observed when MESCs were cocultured by seeding HKCs or HFCs on the same or opposite side of the GCP biocomposite. The highest CK-14 expression was observed by seeding MESCs and HKCs on the same side of the GCP composite and with HFCs on the opposite side. To verify the effectiveness of wound healing in vivo, adipose-derived stem cells were applied to treat surgical wounds in nude mice. An obvious epidermis multilayer and better collagen deposition during wound healing were observed, as assessed by Masson staining. This study demonstrated the potential of keratinocyte-like differentiation from mesenchymal stem cells for use in promoting wound closure and skin regeneration.

Sciatic nerve regeneration by cocultured Schwann cells and stem cells on microporous nerve conduits.

Cell transplantation is a useful therapy for treating peripheral nerve injuries. The clinical use of Schwann cells (SCs), however, is limited because of their limited availability. An emerging solution to promote nerve regeneration is to apply injured nerves with stem cells derived from various tissues. In this study, different types of allogeneic cells including SCs, adipose-derived adult stem cells (ASCs), dental pulp stem cells (DPSCs), and the combination of SCs with ASCs or DPSCs were seeded on nerve conduits to test their efficacy in repairing a 15-mm-long critical gap defect of rat sciatic nerve. The regeneration capacity and functional recovery were evaluated by the histological staining, electrophysiology, walking track, and functional gait analysis after 8 weeks of implantation. An in vitro study was also performed to verify if the combination of cells led to synergistic neurotrophic effects (NGF, BDNF, and GDNF). Experimental rats receiving conduits seeded with a combination of SCs and ASCs had the greatest functional recovery, as evaluated by the walking track, functional gait, nerve conduction velocity (NCV), and histological analysis. Conduits seeded with cells were always superior to the blank conduits without cells. Regarding NCV and the number of blood vessels, conduits seeded with SCs and DPSCs exhibited better values than those seeded with DPSCs only. Results from the in vitro study confirmed the synergistic NGF production from the coculture of SCs and ASCs. It was concluded that coculture of SCs with ASCs or DPSCs in a conduit promoted peripheral nerve regeneration over a critical gap defect.

Spheroid formation of mesenchymal stem cells on chitosan and chitosan-hyaluronan membranes.

Stem cells can lose their primitive properties during in vitro culture. The culture substrate may affect the behavior of stem cells as a result of cell-substrate interaction. The maintenance of self-renewal for adult human mesenchymal stem cells (MSCs) by a biomaterial substrate, however, has not been reported in literature. In this study, MSCs isolated from human adipose (hADAS) and placenta (hPDMC) were cultured on chitosan membranes and those further modified by hyaluronan (chitosan-HA). It was observed that the MSCs of either origin formed three-dimensional spheroids that kept attached on the membranes. Spheroid formation was associated with the increased MMP-2 expression. Cells on chitosan-HA formed spheroids more quickly and the size of spheroids were larger than on chitosan alone. The expression of stemness marker genes (Oct4, Sox2, and Nanog) for MSCs on the materials was analyzed by the real-time RT-PCR. It was found that formation of spheroids on chitosan and chitosan-HA membranes helped to maintain the expression of stemness marker genes of MSCs compared to culturing cells on polystyrene dish. The maintenance of stemness marker gene expression was especially remarkable in hPDMC spheroids (vs. hADAS spheroids). Blocking CD44 by antibodies prevented the spheroid formation and decreased the stemness gene expression moderately; while treatment by Y-27632 compound inhibited the spheroid formation and significantly decreased the stemness gene expression. Upon chondrogenic induction, the MSC spheroids showed higher levels of Sox9, aggrecan, and collagen type II gene expression and were stained positive for glycosaminoglycan and collagen type II. hPDMC had better chondrogenic differentiation potential than hADAS upon induction. Our study suggested that the formation of adhered spheroids on chitosan and chitosan-HA membranes may sustain the expression of stemness marker genes of MSCs and increase their chondrogenic differentiation capacity. The Rho/Rho-associated kinase (ROCK) signaling pathway may be involved in spheroid formation.