Repair Of Full-thickness Skin Defects Research Articles

Three-dimensional structured PLCL/ADM bioactive aerogel for rapid repair of full-thickness skin defects.

The failure to treat deep skin wounds can result in significant complications, and the limitations of current clinical treatments highlight the pressing need for the development of new deep wound healing materials. In this study, a series of three-dimensional structured PLCL/ADM composite aerogels were fabricated by electrospinning and subsequently characterized for their microstructure, compression mechanics, exudate absorption, and hemostatic properties. Additionally, the growth of HSFs and HUVECs, which are involved in wound repair, was observed in the aerogels. The composite aerogel was subsequently employed in wound repair experiments on rat full-thickness skin with the objective of observing the wound healing rate and examining histological utilizing H&E, Masson, CD31, and COL-I staining. The findings indicated that the PLCL/ADM composite aerogel with a 10% concentration exhibited uniform pore size distribution, a good three-dimensional structure, and compression properties comparable to those of human skin, which could effectively absorb exudate and exert hemostatic effect. In vivo experiment results demonstrated that the aerogel exhibited superior efficacy to conventional oil-gauze overlay therapy and ADM aerogel in promoting wound healing and could facilitate rapid, high-quality in situ repair of deep wounds, thereby offering a novel approach for skin tissue engineering and clinical wound treatment.

Biocompatible and antibacterial Flammulina velutipes-based natural hybrid cryogel to treat noncompressible hemorrhages and skin defects.

Hemorrhage, infection, and frequent replacement of dressings bring great clinical challenges to wound healing. In this work, Flammulina velutipes extract (FV) and hydroxyethyl cellulose (HEC) were chemically cross-linked and freeze-dried to obtain novel HFV cryogels (named HFVn, with n = 10, 40, or 70 corresponding to the weight percentage of the FV content), which were constructed for wound hemostasis and full-thickness skin defect repair. Systematic characterization experiments were performed to assess the morphology, mechanical properties, hydrophilic properties, and degradation rate of the cryogels. The results indicated that HFV70 showed a loose interconnected-porous structure and exhibited the highest porosity (95%) and water uptake ratio (over 2,500%) with a desirable degradation rate and shape memory properties. In vitro cell culture and hemocompatibility experiments indicated that HFV70 showed improved cytocompatibility and hemocompatibility. It can effectively mimic the extracellular matrix microenvironment and support the adhesion and proliferation of L929 cells, and its hemolysis rate in vitro was less than 5%. Moreover, HFV70 effectively induced tube formation in HUVEC cells in vitro. The results of the bacteriostatic annulus confirmed that HFV70 significantly inhibited the growth of Gram-negative E. coli and Gram-positive S. aureus. In addition, HFV70 showed ideal antioxidant properties, with the DPPH scavenging rate in vitro reaching 74.55%. In vivo rat liver hemostasis experiments confirmed that HFV70 showed rapid and effective hemostasis, with effects comparable to those of commercial gelatin sponges. Furthermore, when applied to the repair of full-thickness skin defects in a rat model, HFV70 significantly promoted tissue regeneration. Histological analysis further confirmed the improved pro-angiogenic and anti-inflammatory activity of HFV70 in vivo. Collectively, our results demonstrated the potential of HFV70 in the treatment of full-thickness skin defects and rapid hemostasis.

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.

The research of artificial dermis in promotion of skin and its appendage regeneration

Objective To explore the function of artificial dermis in repair of full-thickness skin defects, and investigate its potential of inducing skin appendage regeneration. Methods A 3 cm×3 cm full-thickness skin defect was made on the dorsal skin of SD rats. In the experimental group, artificial dermis was used to cover the wound, and the control group was covered with silicon film only. The wound healing and hair regeneration were observed at 2 and 4 weeks after the operation. At these time points, HE and Masson staining were performed to detect the regeneration of skin appendages under the microscope. Simultaneously, BrdU (5-bromodeoxyuridine) incorporation test was used to verify whether the tissue of wound healing was derived from cell proliferation. Results The wounds in the experimental group was significantly smaller at 2 and 4 weeks after the operation than those in the control group. In the experimental group, hair growth was seen in the wound healing area at 4 weeks postoperatively. HE and Masson staining showed the regeneration of hair follicles and sebaceous glands in the newborn skin area. BrdU test indicated that there were a large number of BrdU positive cells in the epithelial tissue, hair follicles and sebaceous glands of the newborn skin area in the experimental group, which also showed that the cells in the above tissue were derived from division and proliferation. Conclusion Artificial dermis can play a good role in the repair of full-thickness skin defects. It not only can induce the regeneration of epithelial tissue, but also can guide the skin appendages stem/progenitor cell migration, proliferation and differentiation to promote the regeneration of skin appendages. The discovery will provide new ideas for the clinical and basic research of tissue repair and skin regeneration. Key words: Regeneration; Animals,laboratory; Artificial dermis; Full-thickness skin defects; Skin appendage

Construction of tissue-engineered skin by culture of human amniotic epithelial cells on human de-epidermized dermis for the repair of skin defects in nude mice: an experimental study

Objective To evaluate the feasibility of repair of full-thickness skin defects in nude mice with tissue-engineered skin which was constructed by culture of human amniotic epithelial cells (hAECs) and fibroblasts on human de-epidermized dermis (DED) . Methods Healthy human amniotic tissues were treated with trypsin at a low concentration in multi-steps to prepare hAECs, and a two-step collagenase digestion was used to treat healthy children′s prepuce tissues to prepare fibroblast suspensions. When fibroblasts were cultured in vitro up to passage 3-5 and hAECs up to passage 2, they were seeded on the reticular dermal surface and basement membrane surface of the DED respectively to construct the tissue-engineered skin. A total of 20 healthy male nude mice aged 3-4 weeks were enrolled into this experiment, and full-thickness skin defects were made on the middle of the back of mice. Then, these mice were randomly divided into 2 groups by using a lottery method, and reconstructed full-thickness tissue-engineered skin grafts and vaseline oil gauze were used to cover the wounds in the tissue-engineered skin group and control group respectively. The whole body and transplantation sites of the nude mice were observed on day 7, 14, 21 and 28 after transplantation, the wound healing time and rate were compared between the above two groups, and skin tissues at the transplantation site were harvested at 4 weeks after transplantation and subjected to histological examination. Results HAECs had stem-cell characteristics and expressed octamer-binding protein-4 (OCT-4) and embryonic marker stage-specific embryonic antigen-4 (SSEA-4) . After 2-week organ culture, the in vitro reconstructed tissue-engineered skin showed 4-9 continuous layers of stratified epithelium, and the histological structure of the epidermis was similar to that of the normal human skin. Compared with the control group, the tissue-engineered skin group showed significantly higher wound healing rates on day 7, 14 and 21 after transplantation (57.49% ± 6.11% vs. 22.93% ± 4.26%, 92.80% ± 3.10% vs. 54.57% ± 7.94%, 98.83% ± 0.25% vs. 91.16% ± 4.79%, respectively; n= 10, t= 27.36, 32.23, 11.80, respectively, all P < 0.001) , shorter wound healing time[ (21.51 ± 1.51) d vs. (28.80 ± 1.14) d, n= 10, t= 42.23, P < 0.001], with the color of skin grafts closer to that of autologous skin on day 28 after transplantation. Histological examination revealed distinct stratification of the epithelium, obvious keratinization and favorable growth of cells in the dermis in the tissue-engineered skin group, but thin epithelium with some defects, indistinct stratification of the dermis, and inflammatory cell infiltration in the control group. Conclusion Tissue-engineered skin constructed by the culture of hAECs and fibroblasts on human DED can survive in nude mice after transplantation, resulting in a more favorable healing of wounds, and is expected to serve as a kind of ideal tissue-engineered skin. Key words: Tissue engineering; Epithelial cells; Dermis; Skin transplantation; Wound healing; De-epidermized dermis; Human amniotic epithelial cells

Growth Differentiation Factor 5 Accelerates Wound Closure and Improves Skin Quality During Repair of Full-Thickness Skin Defects.

A fast and stable wound closure is important, especially for extended and unstable wounds found after burn injuries. Growth can regulate a variety of cellular processes, including those involved in wound healing. Growth differentiation factor 5 (GDF-5) can accelerate fibroblast cell migration, cell proliferation, and collagen synthesis, which are essential for wound healing. Nevertheless, no standardized evaluation of the effect of GDF-5 on the healing of full-thickness wounds has been published to date. Five full-thickness skin defects were created on the backs of 6 minipigs. Three wounds were treated with GDF-5 in different concentrations with the help of a gelatin-collagen carrier, and 2 wounds served as control group. The first was treated with the gelatin carrier and an Opsite film (Smith & Nephew, Fort Worth, Texas), and the other was treated solely with an Opsite film that was placed above all wounds and renewed every second day. Growth differentiation factor 5 accelerates wound closure (10.91 [SD, 0.99] days) compared with treatment with the carrier alone (11.3 [SD, 1.49] days) and control wounds (13.3 [SD, 0.94] days). Epidermal cell count of wounds treated with GDF-5 revealed a higher number of cells compared with the control group. In addition, mean epidermal thickness was significantly increased in GDF-5-treated wounds compared with the control wounds. Because of its ability to improve skin quality, GDF-5 should be considered when developing composite biomaterials for wound healing.

Use of Amniotic Microparticles Coated With Fibroblasts Overexpressing SDF-1α to Create an Environment Conducive to Neovascularization for Repair of Full-Thickness Skin Defects.

As angiogenesis and vasculogenesis involve the complex network structures of various types of cells, extracellular matrix components, and cytokines, it is still difficult to exactly mimic the microenvironment of vascularization in vivo. In our study, we constructed a complex containing highly proliferative fibroblasts that can secrete extracellular matrix components and growth factors to chemotaxize endothelial progenitor cells (EPCs) in an attempt to create an ideal microenvironment for quick vascularization. Amniotic membrane microparticles (mAM) rich in type IV collagen (COL IV) and laminin (LN) were prepared, and human dermal fibroblasts (HDF) were infected with lentivirus (LV) of overexpression of SDF-1α to construct SDF-1α(ov)HDF. Using the rotary cell culture system (RCCS), mAM was loaded with HDF or SDF-1α(ov)HDF to construct HDF-mAM and SDF-1α(ov)HDF-mAM complexes. The complexes were able to secrete various types of active peptides (IL-6, IL-8, TGF-β, and bFGF) during in vitro culture. In addition, SDF-1α(ov)HDF-mAM complex highly expressed SDF-1α. Transwell assay showed SDF-1α(ov)HDF-mAM complex had an apparent chemotactic effect on EPCs. Transplantation of complexes onto full-thickness skin defects of C57BL mice further demonstrated that SDF-1α expression and the number of peripheral EPCs at days 3, 5, and 7 in the SDF-1α(ov)HDF-mAM group were significantly higher than that in other groups (p < 0.01). The local microvascular density at day 10 of transplantation showed that the microvascular density in the SDF-1α(ov)HDF-mAM group was significantly higher than that in HDF-mAM group (p < 0.01). In conclusion, HDF-mAM had a strong proliferative activity and could be used to create a sound microenvironment for quick vascularization by secreting multiple cytokines and extracellular matrix components. Overexpression of SDF-1α could chemotaxize EPCs to reach local wounds, thus further accelerating angiogenesis in the transplant site. The technique described may prove to be a new model for accelerating vascularization of tissue and organ transplants and chronic ischemic wounds.

Polyurethane membrane/knitted mesh-reinforced collagen–chitosan bilayer dermal substitute for the repair of full-thickness skin defects via a two-step procedure

The advent of dermal substitutes provides a revolutionary strategy for the repair and reconstruction of deep skin defects. Dermal substitutes form a regenerative template that provides the porous structure and mechanical support necessary to guide cell migration, deposition of the extracellular matrix (ECM) and angiogenesis. Commercially available dermal substitutes, particularly collagen-based dermal scaffolds, are widely used in clinical practice. However, the poor mechanical properties of collagen-based dermal scaffolds compromise their biological effects, as well as the repair outcomes. Here, we describe a bilayer dermal substitute prepared by integrating a hybrid dermal scaffold with a polyurethane (PU) membrane to obtain a PU membrane/knitted mesh-reinforced collagen–chitosan bilayer dermal substitute (PU-PLGAm/CCS). The morphology of PU-PLGAm/CCS was investigated and, to characterize the effects of PU-PLGAm/CCS on tissue regeneration, dermal substitutes were transplanted to repair full-thickness skin wounds in Sprague-Dawley rats using a two-step surgical procedure. These results were then compared with those obtained using the PELNAC™ Artificial Dermis. In the weeks after the first operation, wound changes were analysed based on macroscopic observations, and tissue specimens were harvested for histology, immunohistochemistry, immunofluorescence real-time quantitative PCR, and Western blotting analysis. Following the second operation (i.e., transplantation of split-thickness skin grafts), the repair outcomes were investigated based on the mechanical strength and ECM expression. PU-PLGAm/CCS significantly inhibited wound contracture, promoted angiogenesis, and facilitated the ordered arrangement of neotissue, such that the repair outcomes were improved in the PU-PLGAm/CCS group compared with the PELNAC™ group. In conclusion, the favourable microstructure and structural stability of dermal substitutes facilitated tissue regeneration. PU-PLGAm/CCS achieved a balance between porous structure, biocompatibility and mechanical properties for dermal regeneration by integrating the advantages of biological and synthetic biomaterials, which demonstrates its potential for skin tissue engineering.

The roles of knitted mesh-reinforced collagen–chitosan hybrid scaffold in the one-step repair of full-thickness skin defects in rats

Full-thickness skin defects represent a significant and urgent clinical problem. Dermal substitutes serving as a regenerative template to induce dermal reconstruction provide a promising method to treat serious skin defects. Although collagen–chitosan dermal scaffolds display good biocompatibility and a suitable porous structure for angiogenesis and tissue regeneration, their poor mechanical properties compromise their application. To develop a well-supported dermal substitute, a poly(l-lactide-co-glycolide) (PLGA) knitted mesh was fabricated and integrated with collagen–chitosan scaffold (CCS) to obtain a PLGA knitted mesh-reinforced CCS (PLGAm/CCS). The morphology of this PLGAm/CCS was investigated in vitro. To characterize the tissue response, specifically angiogenesis and tissue regeneration, the PLGAm/CCS was transplanted in combination with thin split-thickness autografts to repair full-thickness skin wounds using a one-step surgical procedure in Sprague–Dawley rats. These results were then compared with CCSs. At weeks 2, 4 and 8 after the operation, the healing wounds were imaged to analyse wound changes, and tissue specimens were harvested for histology, immunohistochemistry, real-time quantitative polymerase chain reaction and Western blot analysis. The results demonstrated that collagen–chitosan sponge in the PLGAm/CCS remained porous, interconnected and occupied the openings of PLGA mesh, and the incorporation of the PLGA knitted mesh into CCS improved the mechanical strength with little influence on its mean pore size and porosity. Following transplantation, PLGAm/CCS inhibited wound contraction, and effectively promoted neotissue formation and blood vessel ingrowth. In conclusion, the mechanical strength of the scaffolds plays an important role in the process of tissue regeneration and vascularization. The ability of PLGAm/CCS to promote angiogenesis and induce in situ tissue regeneration demonstrates its potential in skin tissue engineering.

Promotion of angiogenesis by sustained release of rhGM‐CSF from heparinized collagen/chitosan scaffolds

A novel dermal substitute of combining recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) with a porous heparinized collagen/chitosan scaffolds was developed, considering the inadequate angiogenesis during repair of full-thickness skin defects. The physicochemical properties of heparinized collagen/chitosan scaffolds were examined and in vitro release pattern of rhGM-CSF from scaffolds was measured by ELISA. Four groups of composite scaffolds (heparinized or unheparinized scaffolds loaded with or without rhGM-CSF) were fabricated for subcutaneous implantation in young adult male Sprague-Dawley (SD) rats. Tissue specimens were harvested at different time points after implantation for histopathological, immunohistochemical observation, and Western blotting analysis. The heparinized scaffolds (H(1)E) showed slower biodegradation and sustained release of rhGM-CSF in vitro, although no significantly different release pattern was observed between the H(1)E and unheparinized scaffolds (H(0)E). In vivo investigation revealed that the heparinized scaffolds loaded with rhGM-CSF (H(1)E/rhGM-CSF) had the best cellular adhesion and migration, new vessel formation, and highest expression of VEGF and TGF-β1, indicating promoted angiogenesis. This study demonstrated that composite dermal substitute of combining rhGM-CSF with a porous heparinized collagen/chitosan scaffolds could be a potential therapeutic agent for full-thickness skin defects because of its sustained delivery of rhGM-CSF.

An epidermal stem cells niche microenvironment created by engineered human amniotic membrane

How to amplify epidermal stem cells (ESCs) rapidly is a challenging crux in skin tissue engineering research. The present study describes the preparation of 3D micronized (300–600 μm) amniotic membrane (mAM) by means of repeated freeze-thawing cycles to deplete cell components and homogenized with a macrohomogenizer in liquid nitrogen. This newly prepared mAM not only possessed the characteristics of a microcarrier but completely retained the basement membrane structure and abundant active substances such as NGF, HGF, KGF, bFGF, TGF-β1 and EGF in the AM matrix. The result showed that mAM combined with rotary cell culture system (RCCS) was able to amplify ESCs quickly. The relative cell viability at day 7 and 14 was significantly higher than that of the conventional 2D plate culture (326 ± 28% and 535 ± 47% versus 232 ± 21% and 307 ± 32%, P < 0.05). In addition, the new method was able to prevent cell differentiation effectively and retain the characteristics of stem cells. When mAM loaded with ESCs (ESC-mAM) was further transplanted to full-thickness skin defects in nude mice, ESCs survived well and formed a new epidermis. Four weeks after transplantation, papilla-like structures were observed, and collagen fibers were well and regularly arranged in the newly formed dermal layer. In conclusion, the mAM as a novel natural microcarrier possesses an intact basement membrane structure and bioactivities. It not only provides the microenvironment similar to the stem cell niche within the human body favorable for ex vivo culture and amplification of ESCs but can be used as the dermal scaffold in constructing a skin substitute containing ESCs for the repair of full-thickness skin defects.