Treatment Of Full-thickness Skin Defects Research Articles

Autologous Skin Cell Suspension for Full-Thickness Skin Defect Reconstruction: Current Evidence and Health Economic Expectations.

Despite differing etiologies, acute thermal burn injuries and full-thickness (FT) skin defects are associated with similar therapeutic challenges. When not amenable to primary or secondary closure, the conventional standard of care (SoC) treatment for these wound types is split-thickness skin grafting (STSG). This invasive procedure requires adequate availability of donor skin and is associated with donor site morbidity, high healthcare resource use (HCRU), and costs related to prolonged hospitalization. As such, treatment options that can facilitate effective healing and donor skin sparing have been highly anticipated. The RECELL® Autologous Cell Harvesting Device facilitates preparation of an autologous skin cell suspension (ASCS) for the treatment of acute thermal burns and FT skin defects. In initial clinical trials, the approach showed superior donor skin-sparing benefits and comparable wound healing to SoC STSG among patients with acute thermal burn injuries. These findings led to approval of RECELL for this indication by the US Food and Drug Administration (FDA) in 2018. Subsequent clinical evaluation in non-thermal FT skin wounds showed that RECELL, when used in combination with widely meshed STSG, provides donor skin-sparing advantages and comparable healing outcomes compared with SoC STSG. As a result, the device received FDA approval in June of 2023 for treatment of FT skin defects caused by traumatic avulsion or surgical excision or resection. Given that health economic advantages have been demonstrated for RECELL ± STSG versus STSG alone when used for burn therapy, it is prudent to examine similarities in the burn and FT skin defect treatment pathways to forecast the potential health economic advantages for RECELL when used in FT skin defects. This article discusses the parallels between the two indications, the clinical outcomes reported for RECELL, and the HCRU and cost benefits that may be anticipated with use of the device for non-thermal FT skin defects.

A bioactive composite hydrogel dressing that promotes healing of both acute and chronic diabetic skin wounds

Mesenchymal stem cell derived exosomes (MSC-Exos) demonstrate beneficial effects on wound healing via anti-inflammatory and angiogenic properties. Chitosan (CS) exhibits excellent biocompatibility and accelerates cellular migration, adhesion, and proliferation. The ions released from bioactive glass (BG) and titanium dioxide (TiO2) nanoparticles exhibit sustained angiogenic and antibacterial potency. In this study, CMCS-CEBT hydrogel was synthesized from exosomes encapsulated carboxymethyl chitosan (CMCS), chitosan nanoparticles (CS-NPs), BG, and TiO2 nanoparticles for a preliminary evaluation of its impacts on the treatment of full-thickness skin defects, diabetic wounds, and burn skin injury due to burns. In vitro analysis indicated that the hydrogel exhibits excellent cell compatibility, stimulates endothelial cell adhesion and proliferation, and presents anti-inflammatory, angiogenic, and antibacterial activities. In vivo, the composite hydrogel dressing accelerated a wound healing acceleration effect, stimulated angiogenesis, and increased collagen deposition and the expression of anti-inflammatory factors. This innovative composite hydrogel dressing as a potential clinical therapy, utilizing bioactive materials, holds promise as a potential clinical therapy that aims to facilitate the regeneration of acute and chronically damaged skin tissue.

Application of a meshed artificial dermal scaffold and negative-pressure wound therapy in the treatment of full-thickness skin defects: a prospective in vivo study.

Artificial dermal scaffolds (ADSs) have great value in repairing deep skin defects. However, problems such as unsatisfactory angiogenesis and local dropsy or empyema often occur, resulting in delayed or even failed wound healing. Negative pressure wound therapy (NPWT) is an effective therapy to promote wound healing or shorten wound bed preparation time. Studies on whether it can improve the effects of ADSs have never been interrupted, and no consensus has been reached. In this study, an improved ADS was prepared by mesh technology, physicochemical experiments were conducted, cell adhesion and proliferation were assessed with the meshed ADS, and in vivo experiments were conducted to investigate the effects of meshed ADS or ADS combined with NPWT in repairing full-thickness skin defects. The results showed that the meshed ADS showed through-layer channels arranged in parallel longitudinal and transverse intersections. The cell experiments confirmed the good cytocompatibility. The in vivo experiments showed that there were no differences in the take rate or contraction of grafted skin among all experiment groups. The meshed ADS exhibited good histocompatibility, and there were no differences in tissue inflammation, dermal angiogenesis, or degradation among all groups. In addition, necrosis, dropsy, or empyema of the dermal scaffold were found in all experiment groups except for the meshed ADS + NPWT group, which showed better wound repair results, including fewer scaffold-related complications and satisfactory skin graft survival and wound contraction. In conclusion, this novel meshed ADS, which has a regular through-layer mesh structure and possesses stable physicochemical properties and good biocompatibility, combined with NPWT can ensure adequate subdermal drainage and reduce the risk of scaffold-related complications, thereby improving the quality and efficiency of wound repair, promoting a broader application of biomaterials, and helping physicians and readers implement more effective wound management.

Physical dual-network photothermal antibacterial multifunctional hydrogel adhesive for wound healing of drug-resistant bacterial infections synthesized from natural polysaccharides

Wound-healing of drug-resistant bacterial infections has always been a clinical challenge. The design and development of effective and economically safe wound dressings with antimicrobial activity and healing-promoting properties is highly desirable, especially in the context of wound-infections. Herein, we designed a physical dual-network multifunctional hydrogel adhesive based on polysaccharide material for the treatment of full-thickness skin defects infected with multidrug-resistant bacteria. The hydrogel utilized ureido-pyrimidinone (UPy)-modified Bletilla striata polysaccharide (BSP) as the first physical interpenetrating network for providing some brittleness and rigidity; and then branched macromolecules formed after cross-linking Fe3+ with dopamine-conjugated di-aldehyde-hyaluronic acid as the second physical interpenetrating network for providing some flexibility and elasticity. In this system, BSP and hyaluronic acid (HA) are used as synthetic matrix materials to provide strong biocompatibility and wound-healing ability. In addition, ligand cross-linking of catechol-Fe3+ and quadrupole hydrogen-bonding cross-linking of UPy-dimer can form a highly dynamic physical dual-network structure, which imparts good rapid self-healing, injectability, shape-adaptation, NIR/pH responsiveness, high tissue-adhesion and mechanical properties of this hydrogel. Meanwhile, bioactivity experiments demonstrated that the hydrogel also possesses powerful antioxidant, hemostatic, photothermal-antibacterial and wound-healing effects. In conclusion, this functionalized hydrogel is a promising candidate for clinical treatment of full-thickness bacteria-stained wound dressing materials.

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.

Fabrication of SA/Gel/C scaffold with 3D bioprinting to generate micro-nano porosity structure for skin wound healing: a detailed animal in vivo study

Bioprinting has exhibited remarkable promises for the fabrication of functional skin substitutes. However, there are some significant challenges for the treatment of full-thickness skin defects in clinical practice. It is necessary to determine bioinks with suitable mechanical properties and desirable biocompatibilities. Additionally, the key for printing skin is to design the skin structure optimally, enabling the function of the skin. In this study, the full-thickness skin scaffolds were prepared with a gradient pore structure constructing the dense layer, epidermis, and dermis by different ratios of bioinks. We hypothesized that the dense layer protects the wound surface and maintains a moist environment on the wound surface. By developing a suitable hydrogel bioink formulation (sodium alginate/gelatin/collagen), to simulate the physiological structure of the skin via 3D printing, the proportion of hydrogels was optimized corresponding to each layer. These results reveal that the scaffold has interconnected macroscopic channels, and sodium alginate/gelatin/collagen scaffolds accelerated wound healing, reduced skin wound contraction, and re-epithelialization in vivo. It is expected to provide a rapid and economical production method of skin scaffolds for future clinical applications.

Three-dimensional bioprinting of a full-thickness functional skin model using acellular dermal matrix and gelatin methacrylamide bioink

Treatment of full-thickness skin defects still presents a significant challenge in clinical practice. Three-dimensional (3D) bioprinting technique offers a promising approach for fabricating skin substitutes. However, it is necessary to identify bioinks that have both sufficient mechanical properties and desirable biocompatibilities. In this study, we successfully fabricated acellular dermal matrix (ADM) and gelatin methacrylamide (GelMA) bioinks. The results demonstrated that ADM preserved the main extracellular matrix (ECM) components of the skin and GelMA had tunable mechanical properties. Both bioinks with shear-thinning properties were suitable for 3D bioprinting and GelMA bioink exhibited high printability. Additionally, the results revealed that 20% GelMA with sufficient mechanical properties was suitable to engineer epidermis, 1.5% ADM and 10% GelMA displayed relatively good cytocompatibilities. Here, we proposed a new 3D structure to simulate natural full-thickness skin, which included 20% GelMA with HaCaTs as an epidermal layer, 1.5% ADM with fibroblasts as the dermis, and 10% GelMA mesh with human umbilical vein endothelial cells (HUVECs) as the vascular network and framework. We demonstrated that this 3D bioprinting functional skin model (FSM) could not only promote cell viability and proliferation, but also support epidermis reconstruction in vitro. When transplanted in vivo, the FSM could maintain cell viability for at least 1 week. Furthermore, the FSM promoted wound healing and re-epithelization, stimulated dermal ECM secretion and angiogenesis, and improved wound healing quality. The FSM may provide viable functional skin substitutes for future clinical applications. Statement of significanceWe propose a new 3D structure to simulate natural full-thickness skin, which included 20% GelMA with HaCaTs as an epidermal layer, 1.5% ADM with fibroblasts as the dermis, and 10% GelMA mesh with HUVECs as the vascular network. It could not only maintain a moist microenvironment and barrier function, but also recreate the natural skin microenvironment to promote cell viability and proliferation. This may provide viable functional skin substitutes for future clinical applications.

Experimental evaluation of the effectiveness of adipose mesenchymal stem cells in full-thickness skin wounds

BACKGROUND: Currently, numerous techniques or medical devices that allow complete or partial restoration of the lost skin within a short time remain as subjects of development. Many studies have shown the effectiveness of using stem cells in the treatment of full-thickness skin defects, but their use remains very limited. At present, there is no consensus among researchers about the advisability of the use of stem cells in the treatment of burns as well as about the method of their introduction.
 AIM: This study aimed to examine the possibility of accelerating the reparative histogenesis of tissues in the zone of deep skin burns using cultures of adipogenic mesenchymal cells, as well as to evaluate the effectiveness of various methods of introducing cultures of these cells into the defect zone.
 MATERIALS AND METHODS: An experimental study was carried out on small laboratory animals (rats). After simulating a grade III burn, mesenchymal stem cells were transplanted and superficially applied to the wound surface or injected under the fascia. In the control group, no wound treatment was performed. To assess the effectiveness of the wound-healing preparations, the appearance of wounds was assessed daily, the nature of the discharge and presence and type of granulations were noted, and the timing of scab rejection and wound healing was recorded. The wound area was assessed using a planimetric method. A histological examination of wound biopsies was carried out on days 7, 14, 21, and 28 of observation.
 RESULTS: The application of adipogenic mesenchymal stem cells demonstrated the greatest efficiency on the developed burn model. Subfascial administration was less effective, but this method achieved a significant acceleration of wound healing in comparison with the control group. An increase in the healing index by 56.6% demonstrates the highest intensity of reparative regeneration in animals applied with adipogenic mesenchymal stem cells.
 CONCLUSIONS: The preliminary results show that the application of adipogenic mesenchymal stem cells on the skin defect is more effective than subfascial administration based on the healing index. The use of adipogenic mesenchymal stem cells may substantially increase the effectiveness of the treatment of full-thickness skin defects.

Three-Dimensional Bioprinting of a Full-Thickness Functional Skin Model Using Acellular Dermal Matrix and Gelatine Methacrylamide Bioink

Treatment of full-thickness skin defects still presents a significant challenge in clinical practice. There is a need for accurate patterning of living cells in pre-defined spatial positions, and the three-dimensional (3D) bioprinting technique offers a promising approach for fabricating skin substitutes. However, it is necessary to identify bioinks that have both sufficient mechanical properties to simulate the complex skin structure, and can provide a suitable microenvironment to support cell growth. In this study, we found that acellular dermal matrix (ADM) bioink preserved the main extracellular matrix (ECM) components of the skin to promote cell growth, and gelatine methacrylamide (GelMA) bioink with tunable and sufficient mechanical properties was strong and elastic for skin tissues. Here, we propose a new 3D structure to simulate natural full-thickness skin, which included 20% GelMA with HaCaTs as an epidermal layer, 1.5% ADM with fibroblasts as the dermis, and 10% GelMA mesh with human umbilical vein endothelial cells (HUVECs) as the vascular network. We demonstrated that this 3D bioprinting functional skin model (FSM) could not only maintain a moist microenvironment and barrier function, but also recreate the natural skin microenvironment to promote cell viability and proliferation in vitro. When transplanted in vivo, our FSM could maintain cell viability for at least 1 week. Furthermore, the FSM promoted wound healing and re-epithelization, stimulated dermal ECM secretion and angiogenesis, and enhanced wound healing quality. Therefore, the FSM may provide viable functional skin substitutes for future clinical applications.

Human decellularized dermal matrix seeded with adipose-derived stem cells enhances wound healing in a murine model: Experimental study.

ObjectiveFull-thickness cutaneous wounds treated with split-thickness skin grafts often result in unaesthetic and hypertrophic scars. Dermal substitutes are currently used together with skin grafts in a single treatment to reconstruct the dermal layer of the skin, resulting in improved quality of scars. Adipose-derived stem cells (ASCs) have been described to enhance wound healing through structural and humoral mechanisms. In this study, we investigate the compatibility of xenogen-free isolated human ASCs seeded on human acellular dermal matrix (Glyaderm®) in a murine immunodeficient wound model.MethodsAdipose tissue was obtained from abdominal liposuction, and stromal cells were isolated mechanically and cultured xenogen-free in autologous plasma-supplemented medium. Glyaderm® discs were seeded with EGFP-transduced ASCs, and implanted on 8 mm full-thickness dorsal wounds in an immunodeficient murine model, in comparison to standard Glyaderm® discs. Re-epithelialization rate, granulation thickness and vascularity were assessed by histology on days 3, 7 and 12. Statistical analysis was conducted using the Wilcoxon signed-rank test. EGFP-staining allowed for tracking of the ASCs in vivo. Hypoxic culture of the ASCs was performed to evaluate cytokine production.ResultsASCs were characterized with flowcytometric analysis and differentiation assay. EGFP-tranduction resulted in 95% positive cells after sorting. Re-epithelialization in the ASC-seeded Glyaderm® side was significantly increased, resulting in complete wound healing in 12 days. Granulation thickness and vascularization were significantly increased during early wound healing. EGFP-ASCs could be retrieved by immunohistochemistry in the granulation tissue in early wound healing, and lining vascular structures in later stages.ConclusionGlyaderm® is an effective carrier to deliver ASCs in full-thickness wounds. ASC-seeded Glyaderm® significantly enhances wound healing compared to standard Glyaderm®. The results of this study encourage clinical trials for treatment of full-thickness skin defects. Furthermore, xenogen-free isolation and autologous plasma-augmented culture expansion of ASCs, combined with the existing clinical experience with Glyaderm®, aid in simplifying the necessary procedures in a GMP-laboratory setting.

Silver nanoparticle loaded collagen/chitosan scaffolds promote wound healing via regulating fibroblast migration and macrophage activation

Treatment of full-thickness skin defects poses significant clinical challenges including risk of infection and severe scaring. Silver nanoparticle (NAg), an effective antimicrobial agent, has provided a promising therapeutic method for burn wounds. However, the detailed mechanism remains unknown. Hence, we constructed a metallic nanosilver particles-collagen/chitosan hybrid scaffold (NAg-CCS) and investigated its potential effects on wound healing. In vitro scratch assay, immunofluorescence staining and antibacterial activity of the scaffold were all studied. In vivo NAg-CCS was applied in full-thickness skin defects in Sprague-Dawley (SD) rats and the therapeutic effects of treatment were evaluated. The results showed that NAg at a concentration of 10 ppm accelerated the migration of fibroblasts with an increase in expression of α-smooth muscle actin (α-SMA). Furthermore, in vivo studies showed increased levels of pro-inflammatory and scar-related factors as well as α-SMA, while markers for macrophage activation were up-regulated. On day 60 post transplantation of ultra-thin skin graft, the regenerated skin by NAg-CCS had a similar structure to normal skin. In summary, we demonstrated that NAg-CCS was bactericidal, anti-inflammatory and promoted wound healing potentially by regulating fibroblast migration and macrophage activation, making it an ideal dermal substitute for wound regeneration.

Fabrication and characterization of the porous duck’s feet collagen sponge for wound healing applications

There are several artificial dermis commonly use to cover the wound and promote healing. The major goal of wound management is fast and scarless healing. However, there is no ideal skin substitute, that is effective to accelerate wound healing without scar formation. Artificial dermis substitute also has some drawbacks, such as high cost, insufficient available period and donor pathogen infection. To overcome these problems, we developed duck’s feet collagen (DFC) sponge as artificial dermal substitutes for the treatment of full-thickness skin defects. We measured these DFC sponge’s comparative characteristics and performances with an artificial dermis Colladerm by carried out SEM-EDX analyze, water-binding abilities and porosity test. Biocompatibility test was also performed using CCK-8 cytotoxicity assay. We also evaluated its wound healing effects for a full-thickness skin wound and compared with Colladerm in a rat model. Histological studies were carried via hematoxylin and eosin and Masson’s Trichrome staining. Although the wound healing effect of the DFC sponge was almost similar to that of Colladerm, the DFC sponge did not induce scar formation and wound contracture like Colladerm. We suggest that DFC sponge can be used as an ideal dermal substitute to the treatment of full-thickness skin wound.

Evaluation of cultured human dermal- and dermo-epidermal substitutes focusing on extracellular matrix components: Comparison of protein and RNA analysis

Treatment of full-thickness skin defects with split-thickness skin grafts is generally associated with contraction and scar formation and cellular skin substitutes have been developed to improve skin regeneration. The evaluation of cultured skin substitutes is generally based on qualitative parameters focusing on histology. In this study we focused on quantitative evaluation to provide a template for comparison of human bio-engineered skin substitutes between clinical and/or research centers, and to supplement histological data. We focused on extracellular matrix proteins since these components play an important role in skin regeneration. As a model we analyzed the human dermal substitute denovoDerm and the dermo-epidermal skin substitute denovoSkin.The quantification of the extracellular matrix proteins type III collagen and laminin 5 in tissue homogenates using western blotting analysis and ELISA was not successful. The same was true for assaying lysyl oxidase, an enzyme involved in crosslinking of matrix molecules. As an alternative, gene expression levels were measured using qPCR. Various RNA isolation procedures were probed. The gene expression profile for specific dermal and epidermal genes could be measured reliably and reproducibly. Differences caused by changes in the cell culture conditions could easily be detected. The number of cells in the skin substitutes was measured using the PicoGreen dsDNA assay, which was found highly quantitative and reproducible. The (dis) advantages of assays used for quantitative evaluation of skin substitutes are discussed.

Adipose Tissue-Derived Microvascular Fragments Improve Vascularization, Lymphangiogenesis, and Integration of Dermal Skin Substitutes

Full-thickness skin defects can be covered with dermal skin substitutes in combination with split-thickness skin grafts. However, slow vascularization of the matrices bears the risk of wound infection and extends the length of hospitalization. To overcome these problems, we describe a promising vascularization strategy. Green fluorescent protein+ adipose tissue-derived microvascular fragments (ad-MVF) were isolated from epididymal fat pads of C57BL/6-Tg(CAG-EGFP)1Osb/J mice. ad-MVF were seeded on collagen-glycosaminoglycan matrices, which were implanted into full-thickness skin defects in the dorsal skinfold chamber of wild-type C57BL/6 mice. Nonseeded matrices served as controls. Vascularization, lymphangiogenesis, and integration of the implants were studied by using intravital fluorescence microscopy, histology, and immunohistochemistry over 14 days. ad-MVF rapidly reassembled into microvascular networks within the implants, which developed interconnections to the host microvasculature. Accordingly, vascularization of the implants was markedly accelerated, as indicated by a significantly higher microvessel density when compared with controls. Moreover, dense lymphatic networks originating from the green fluorescent protein+ ad-MVF developed within the implants. This was associated with an improved implant integration. Hence, seeding ad-MVF on collagen-glycosaminoglycan matrices represents a potential strategy to reduce morbidity and hospitalization of patients undergoing the treatment of full-thickness skin defects.

A two-component pre-seeded dermal–epidermal scaffold

We have developed a bilayered dermal–epidermal scaffold for application in the treatment of full-thickness skin defects. The dermal component gels in situ and adapts to the lesion shape, delivering human dermal fibroblasts in a matrix of fibrin and cross-linked hyaluronic acid modified with a cell adhesion-promoting peptide. Fibroblasts were able to form a tridimensional matrix due to material features such as tailored mechanical properties, presence of protease-degradable elements and cell-binding ligands. The epidermal component is a robust membrane containing cross-linked hyaluronic acid and poly-l-lysine, on which keratinocytes were able to attach and to form a monolayer. Amine–aldehyde bonding at the interface between the two components allows the formation of a tightly bound composite scaffold. Both parts of the scaffold were designed to provide cell-type-specific cues to allow for cell proliferation and form a construct that mimics the skin environment.

Spray-assisted layer-by-layer assembly on hyaluronic acid scaffolds for skin tissue engineering

Tissue engineering approaches for the development of a single epidermal-dermal scaffold to treat full-thickness skin defects have been limited by difficulties in the fabrication of a bilayer scaffold combining the specific properties of the epidermis and the dermis. Here we present an innovative approach to developing a scaffold that holds promise for skin tissue engineering. We utilize the spray-assisted layer-by-layer assembly technique to deposit a polyelectrolyte multilayer film composed of hyaluronic acid and poly-L-lysine (the epidermal component) on a porous hyaluronic acid scaffold (the dermal component), in a rapid and controlled manner. The multilayer film promotes cell adhesion, contributing to regeneration of the epidermal barrier functions of skin. While human keratinocytes attached and proliferated on the coated porous scaffolds, they did not invade the porous dermal component, thus leaving room for seeding of relevant fibroblast cell types in this scaffold. This scaffold therefore holds promise for co-culture of different cells, which may be useful for treatment of full-thickness skin defects as well as other tissue engineering applications.

Nanofibrous structured biomimetic strategies for skin tissue regeneration

Mimicking porous topography of natural extracellular matrix is advantageous for successful regeneration of damaged tissues or organs. Nanotechnology being one of the most promising and growing technology today shows an extremely huge potential in the field of tissue engineering. Nanofibrous structures that mimic the native extracellular matrix and promote the adhesion of various cells are being developed as tissue-engineered scaffolds for skin, bone, vasculature, heart, cornea, nervous system, and other tissues. A range of novel biocomposite materials has been developed to enhance the bioactive or therapeutic properties of these nanofibrous scaffolds via surface modifications, including the immobilization of functional cell-adhesive ligands and bioactive molecules such as drugs, enzymes, and cytokines. In skin tissue engineering, usage of allogeneic skin is avoided to reestablish physiological continuity and also to address the challenge of curing acute and chronic wounds, which remains as the area of exploration with various biomimetic approaches. Two-dimensional, three-dimensional scaffolds and stem cells are presently used as dermal regeneration templates for the treatment of full-thickness skin defects resulting from injuries and severe burns. The present review elaborates specifically on the fabrication of nanofibrous structured strategies for wound dressings, wound healing, and controlled release of growth factors for skin tissue regeneration.

Preparation of Collagen/Gelatin Sponge Scaffold for Sustained Release of bFGF

Artificial dermis (AD) has been used to regenerate dermis-like tissues in the treatment of full-thickness skin defects, but it takes 2 or 3 weeks to complete dermal regeneration. Our previous study demonstrated that injection of basic fibroblast growth factor (bFGF)-impregnated gelatin microspheres (MS) into the AD accelerates the regeneration of dermis-like tissue. However, injection of gelatin MS before clinical use is complicated and time consuming. This study investigated a new scaffold, in which collagen and gelatin are integrated, and which is capable of sustained bFGF release. We produced collagen/gelatin sponges with a gelatin concentration of 0wt%, 10wt%, 30wt%, and 50wt%. The mean pore size in each sponge decreased with the gelatin concentration. In an in vitro study, proliferation of fibroblasts in each sponge was not significantly different over 7 days of culture. As for in vivo sustained release of bFGF, a radioisotope study demonstrated that retention of bFGF in gelatin 10wt% and 30wt% sponges was significantly larger than that in gelatin 0wt% sponge. The collagen/gelatin sponges were grafted on full-thickness skin defects created on a rabbit ear, and we evaluated regeneration of dermis-like tissue by measuring the amount of hemoglobin and size of dermis-like tissue on histological sections. Seven days after implantation, the amount of hemoglobin in dermis-like tissue in gelatin 10wt% sponge was significantly larger than those in control and gelatin 50wt% sponge. Twenty-eight days after implantation, the area of dermis-like tissue in gelatin 10wt% sponge was significantly larger than those in the other specimens. We conclude that the collagen sponge integrated with 10wt% gelatin has the most potential for sustained release of bFGF and that the combination of collagen/gelatin 10wt% sponge and bFGF is a promising therapeutic modality for the treatment of full-thickness skin defects.

Endothelial network formed with human dermal microvascular endothelial cells in autologous multicellular skin substitutes

A human skin equivalent from a single skin biopsy harboring keratinocytes and melanocytes in the epidermal compartment, and fibroblasts and microvascular dermal endothelial cells in the dermal compartment was developed. The results of the study revealed that the nature of the extracellular matrix of the dermal compartments plays an important role in establishment of endothelial network in vitro. With rat-tail type I collagen matrices only lateral but not vertical expansion of endothelial networks was observed. In contrast, the presence of extracellular matrix of entirely human origin facilitated proper spatial organization of the endothelial network. Namely, when human dermal fibroblasts and microvascular endothelial cells were seeded on the bottom of an inert filter and subsequently epidermal cells were seeded on top of it, fibroblasts produced extracellular matrix throughout which numerous branched tubes were spreading three-dimensionally. Fibroblasts also facilitated the formation of basement membrane at the epidermal/matrix interface. Under all culture conditions, fully differentiated epidermis was formed with numerous melanocytes present in the basal epidermal cell layer. The results of the competitive RT-PCR revealed that both keratinocytes and fibroblasts expressed VEGF-A, -B, -C, aFGF and bFGF mRNA, whereas fibroblasts also expressed VEGF-D mRNA. At protein level, keratinocytes produced 10 times higher amounts of VEGF-A than fibroblasts did. The generation of multicellular skin equivalent from a single human skin biopsy will stimulate further developments for its application in the treatment of full-thickness skin defects. The potential development of biodegradable, biocompatible material suitable for these purposes is a great challenge for future research.

Static and dynamic fibroblast seeding and cultivation in porous PEO/PBT scaffolds.

The present study aims at optimizing dermal fibroblast seeding and cultivation in Polyactive scaffolds in order to limit the biopsy size needed for autologous treatment of full-thickness skin defects and chronic wounds. Three methods for seeding and cultivation of fibroblasts in porous scaffolds were compared: dynamic seeding followed by static cultivation (DS), static seeding followed by static cultivation (SS) and dynamic seeding followed by dynamic cultivation (DD). Human dermal fibroblasts isolated from cultured explants were seeded in porous PEO/PBT (Polyactive) scaffolds. Samples were taken from 6 h to 21 days post-seeding for both histological analysis (cell distribution and extracellular matrix (ECM) formation), and quantitative cell number assay. The seeding efficiency 24 h post-seeding was 76% (+/-3.6%) for dynamically seeded matrices, whereas it was only 30% (+/-5%) for statically seeded matrices (p<0.001). ECM formation was abundant in DS samples already at day 10, while even after 21 days ECM formation was less pronounced in SS samples. Surprisingly, cells detached from DD samples as aggregates, starting from day 10. Cell numbers as assayed quantitatively correlated with the histological results. At all timepoints cell numbers found for DS samples were significantly higher as compared to SS samples. At day 21, DS samples contained approximately twofold more cells as compared to SS and DD samples and comprised ECM consisting of collagen types I and III. Our results indicate that the combination of dynamic seeding and static cultivation assures efficient utilization of isolated fibroblasts and improved neodermis formation, thereby allowing a reduction in the skin biopsy size needed for the engineering of living skin substitute.