Bilayer Skin Research Articles

Standalone single- and bi-layered human skin 3D models supported by recombinant silk feature native spatial organization

Physiologically relevant human skin models that include key skin cell types can be used forin vitrodrug testing, skin pathology studies, or clinical applications such as skin grafts. However, there is still no golden standard for such a model. We investigated the potential of a recombinant functionalized spider silk protein, FN-silk, for the construction of a dermal, an epidermal, and a bilayered skin equivalent (BSE). Specifically, two formats of FN-silk (i.e. 3D network and nanomembrane) were evaluated. The 3D network was used as an elastic ECM-like support for the dermis, and the thin, permeable nanomembrane was used as a basement membrane to support the epidermal epithelium. Immunofluorescence microscopy and spatially resolved transcriptomics analysis demonstrated the secretion of key ECM components and the formation of microvascular-like structures. Furthermore, the epidermal layer exhibited clear stratification and the formation of a cornified layer, resulting in a tight physiologic epithelial barrier. Our findings indicate that the presented FN-silk-based skin models can be proposed as physiologically relevant standalone epidermal or dermal models, as well as a combined BSE.

Arginine-Biofunctionalized Ternary Hydrogel Scaffolds of Carboxymethyl Cellulose–Chitosan–Polyvinyl Alcohol to Deliver Cell Therapy for Wound Healing

Wound healing is important for skin after deep injuries or burns, which can lead to hospitalization, long-term morbidity, and mortality. In this field, tissue-engineered skin substitutes have therapy potential to assist in the treatment of acute and chronic skin wounds, where many requirements are still unmet. Hence, in this study, a novel type of biocompatible ternary polymer hybrid hydrogel scaffold was designed and produced through an entirely eco-friendly aqueous process composed of carboxymethyl cellulose, chitosan, and polyvinyl alcohol and chemically cross-linked by citric acid, forming three-dimensional (3D) matrices, which were biofunctionalized with L-arginine (L-Arg) to enhance cellular adhesion. They were applied as bilayer skin biomimetic substitutes based on human-derived cell cultures of fibroblasts and keratinocytes were seeded and grown into their 3D porous structures, producing cell-based bio-responsive hybrid hydrogel scaffolds to assist the wound healing process. The results demonstrated that hydrophilic hybrid cross-linked networks were formed via esterification reactions with the 3D porous microarchitecture promoted by foam templating and freeze-drying. These hybrids presented chemical stability, physicochemical properties, high moisture adsorption capacity, surface properties, and a highly interconnected 3D porous structure well suited for use as a skin substitute in wound healing. Additionally, the surface biofunctionalization of these 3D hydrogel scaffolds with L-arginine through amide bonds had significantly enhanced cellular attachment and proliferation of fibroblast and keratinocyte cultures. Hence, the in vivo results using Hairless mouse models (an immunocompromised strain) confirmed that these responsive bio-hybrid hydrogel scaffolds possess hemocompatibility, bioadhesion, biocompatibility, adhesiveness, biodegradability, and non-inflammatory behavior and are capable of assisting the skin wound healing process.

Three-dimensional bioprinted cell-adaptive hydrogel with anisotropic micropores for enhancing skin wound healing

Engineered matrices with aligned microarchitectures are pivotal in regulating the fibroblast-to-myofibroblast transition, a critical process for wound healing and scar reduction. However, developing a three-dimensional (3D) aligned matrix capable of effectively controlling this transition remains challenging. Herein, we developed a cell-adaptive hydrogel with highly oriented microporous structures, fabricated through bioprinting of thermo/ion/photo-crosslinked gelatin methacrylate/sodium alginate (GelMA/SA) incorporating shear-oriented polyethylene oxide (PEO) filler. The synergistic interactions among GelMA, PEO, and SA yield a homogeneous mixture conducive to the printing of biomimetic 3D constructs with anisotropic micropores. These anisotropic micropores, along with the biochemical cues provided by the GelMA/PEO/SA scaffolds, enhance the oriented spreading and organization of fibroblasts. The resultant spread and aligned cellular morphologies promote the transition of fibroblasts into myofibroblasts. By co-culturing human keratinocytes on the engineered dermal layer, we successfully create a bilayer skin construct, wherein the keratinocytes establish tight junctions accompanied by elevated expression of cytokeratin-14, while the fibroblasts display a highly spread morphology with increased fibronectin expression. The printed hydrogels accelerate full-thickness wound closure by establishing a bioactive microenvironment that mitigate inflammation and stimulate angiogenesis, myofibroblast transition, and extracellular matrix remodeling. This anisotropic hydrogel demonstrates substantial promise for applications in skin tissue engineering.

A potential bilayer skin substitute based on electrospun silk-elastin-like protein nanofiber membrane covered with bacterial cellulose

Skin substitutes are designed to promote wound healing by replacing extracellular matrix. Silk-elastin-like protein is a renewable extracellular matrix-like material that integrated the advantages of silk and elastin-like protein. In this study, electrospun silk-elastin-like protein (SELP) nanofiber membrane covered with bacterial cellulose (BC) was created as a potential skin substitute to mimic gradient structure of epidermis and dermis of skin. The two layers were glued together using adhesive SELP containing 3,4-dihydroxyphenylalanine (DOPA) converted from tyrosine by tyrosinase. Skin topical drugs commonly used in clinical practice can penetrate through the SELP/BC barrier, and the rate of penetration is proportional to drug concentration. BC with dense fibrous structure can act as a barrier to preserve the inner SELP layer and prevent bacterial invasion, with a blocking permeation efficiency over 99% against four species of bacteria. Cell experiments demonstrated that the reticular fibers of SELP could provide an appropriate growth environment for skin cells proliferation and adhesion, which is considered to promote tissue repair and regeneration. The promising results support this strategy to fabricate a silk-elastin-like protein-based biomaterial for skin substitutes in the clinical treatment of full skin injuries and ulcers.

Radial matrix constraint influences tissue contraction and promotes maturation of bi-layered skin equivalents

Human skin equivalents (HSEs) serve as important tools for mechanistic studies with human skin cells, drug discovery, pre-clinical applications in the field of tissue engineering and for skin transplantation on skin defects. Besides the cellular and extracellular matrix (ECM) components used for HSEs, physical constraints applied on the scaffold during HSEs maturation influence tissue organization, functionality, and homogeneity.In this study, we introduce a 3D-printed culture insert that exposes bi-layered HSEs to a static radial constraint through matrix adhesion. We examine the effect of various diameters of the ring-shaped culture insert on the HSE's characteristics and compare them to state-of-the-art unconstrained and planar constrained HSEs.We show that radial matrix constraint of HSEs regulates tissue contraction, promotes fibroblast and matrix organization that is similar to human skin in vivo and improves keratinocyte differentiation, epidermal stratification, and basement membrane formation depending on the culture insert diameter.Together, these data demonstrate that the degree of HSE's contraction is an important design consideration in skin tissue engineering. Therefore, this study can help to mimic various in vivo skin conditions and to increase the control of relevant tissue properties.

Angiogenesis, hemocompatibility and bactericidal effect of bioactive natural polymer-based bilayer adhesive skin substitute for infected burned wound healing.

Thermal wounds are complex and lethal with irregular shapes, risk of infection, slow healing, and large surface area. The mortality rate in patients with infected burns is twice that of non-infected burns. Developing multifunctional skin substitutes to augment the healing rate of infected burns is vital. Herein, we 3D printed a hydrogel scaffold comprising carboxymethyl chitosan (CMCs) and oxidized alginate grafted catechol (O-AlgCat) on a hydrophobic electrospun layer, forming a bilayer skin substitute (BSS). The functional layer (FL) was fabricated by physiochemical crosslinking to ensure favorable biodegradability. The gallium-containing hydrophobic electrospun layer or backing layer (BL) could mimic the epidermis of skin, avoiding fluid penetration and offering antibacterial activity. 3D printed FL contains catechol, gallium, and biologically active platelet rich fibrin (PRF) to adhere to both tissue and BL, show antibacterial activity, encourage angiogenesis, cell growth, and migration. The fabricated bioactive BSS exhibited noticeable adhesive properties (P≤0.05), significant antibacterial activity (P≤0.05), faster clot formation, and the potential to promote proliferation (P≤0.05) and migration (P≤0.05) of L929cells. Furthermore, the angiogenesis was significantly higher (P≤0.05) when evaluated in vivo and in ovo. The BSS-covered wounds healed faster due to low inflammation and high collagen density. Based on the obtained results, the fabricated bioactive BSS could be an effective treatment for infected burn wounds.

The combination of cigarette smoke and solar rays causes effects similar to skin aging in a bilayer skin model

Skin aging is a multifactorial process influenced by internal and external factors. The contribution of different environmental factors has been well established individually in the last few years. On the one hand, man is rarely exposed to a single factor, and on the other hand, there is very little knowledge about how these extrinsic factors may interact with each other or even how the skin may react to chronic exposure. This study aimed to evaluate the effect on skin aging of a chronic co-exposure of tissue-engineered skin substitutes to cigarette smoke extract (CSE) and solar simulator light (SSL). Skin substitutes were reconstructed according to the self-assembly method and then exposed to CSE followed by irradiation with SSL simultaneously transmitting UVA1, visible light and infrared. When skin substitutes were chronically exposed to CSE and SSL, a significant decrease in procollagen I synthesis and the inhibition of Smad2 phosphorylation of the TGF-β signaling pathway were observed. A 6.7-fold increase in MMP-1 activity was also observed when CSE was combined with SSL, resulting in a decrease in collagen III and collagen IV protein expression. The secretory profile resulting from the toxic synergy was investigated and several alterations were observed, notably an increase in the quantities of pro-inflammatory cytokines. The results also revealed the activation of the ERK1/2 (3.4-fold) and JNK (3.3-fold) pathways. Taken together, the results showed that a synergy between the two environmental factors could provoke premature skin aging.

Mutable Collagenous Tissue Isolated from Echinoderms Leads to the Production of a Dermal Template That Is Biocompatible and Effective for Wound Healing in Rats.

The mutable collagenous tissue (MCT) of echinoderms possesses biological peculiarities that facilitate native collagen extraction and employment for biomedical applications such as regenerative purposes for the treatment of skin wounds. Strategies for skin regeneration have been developed and dermal substitutes have been used to cover the lesion to facilitate cell proliferation, although very little is known about the application of novel matrix obtained from marine collagen. From food waste we isolated eco-friendly collagen, naturally enriched with glycosaminoglycans, to produce an innovative marine-derived biomaterial assembled as a novel bi-layered skin substitute (Marine Collagen Dermal Template or MCDT). The present work carried out a preliminary experimental in vivo comparative analysis between the MCDT and Integra, one of the most widely used dermal templates for wound management, in a rat model of full-thickness skin wounds. Clinical, histological, and molecular evaluations showed that the MCDT might be a valuable tool in promoting and supporting skin wound healing: it is biocompatible, as no adverse reactions were observed, along with stimulating angiogenesis and the deposition of mature collagen. Therefore, the two dermal templates used in this study displayed similar biocompatibility and outcome with focus on full-thickness skin wounds, although a peculiar cellular behavior involving the angiogenesis process was observed for the MCDT.

Fabrication and evaluation of a bi-layered gelatin based scaffold with arrayed micro-pits for full-thickness skin construct

There is an unmet need for a reliable and reproducible method for incorporating hair follicle derived stem cells in tissue engineered skin models to reconstitute hair follicles. This study discloses a novel method for introducing hair follicle derived stem cells in microneedle embossed micro-pits of a bilayer skin equivalent fabricated from a gelatin based scaffold. The microneedles are hard and strong enough to penetrate the upper layer of the bilayer gelatin based scaffold that corresponds to the epidermis and permeates down to lower layer that corresponds to dermal layer. This strategic location will mimic the natural niche of hair follicle stem cells for picking up signals from both the epidermis and dermis. Hair follicle stem cells are trapped in to these micro-pits by vacuum assisted cell seeding. The bilayer system consists of two distinct electrospun layers in a single processing step, representing outer epidermal layer and inner dermal layer with hair follicle stem cells in embedded pits, resulting in the formation of a closed representation of a complete skin.

Bilayered skin substitute incorporating rutin nanoparticles for antioxidant, anti-inflammatory, and anti-fibrotic effect

Hypertrophic scarring in large burns and delayed healing in chronic wounds are consequences of prolonged and aggravated inflammation, sustained infiltration of immune cells, free radical generation, and abundance of inflammatory mediators. Therefore, it is imperative to curb hyperinflammation to expedite wound healing. In this study, rutin nanoparticles (RNPs) were synthesized without an encapsulant and incorporated into eggshell membrane powder-crosslinked gelatin-chitosan cryogels to impart antioxidant and anti-inflammatory properties for treating hyperinflammation. The resultant nanoparticles were found to be 17.53 ± 4.03 nm in size and were stable at room temperature for a month with no visible sedimentation. RNPs were found to be non-cytotoxic and exhibited anti-inflammatory (by increasing IL-10 levels) and antioxidant properties (by controlling the generation of reactive oxygen species and enhancing catalase production in human macrophages). Additionally, RNPs were found to reduce α-SMA expression in fibroblasts, thereby demonstrating their anti-scarring effect. In vivo studies with a bilayered skin substitute constituting an RNP-incorporated cryogel proved that it is biocompatible, does not induce renal toxicity, aids wound healing, and induces better re-epithelialization than the control groups at the initial stages. Thus, RNP-incorporated cryogels containing bilayered skin substitutes are an advanced and novel alternative to commercial dermo-epidermal substitutes that lack anti-inflammatory or anti-scarring properties.

833 Ex vivo gene therapy of recessive dystrophic epidermolysis bullosa skin cells leads to dermo-epidermal adhesion strength restoration in autologous bilayered self-assembled skin substitutes

833 Ex vivo gene therapy of recessive dystrophic epidermolysis bullosa skin cells leads to dermo-epidermal adhesion strength restoration in autologous bilayered self-assembled skin substitutes

Artificial Skin Therapies; Strategy for Product Development.

Significance: Tissue-engineered artificial skin for clinical reconstruction can be regarded as an established practice. Bi-layered skin equivalents are available as established allogenic or autologous therapy, and various acellular skin replacements can support tissue repair. Moreover, there is considerable commonality between the skin and other soft tissue reconstruction products. This article presents an attempt to create a comprehensive global landscape review of advanced replacement materials and associated strategies for skin and soft tissue reconstruction. Recent Advances: There has been rapid growth in the number of commercial and pre-commercial products over the past decade. In this survey, 263 base products for advanced skin therapy have been identified, across 8 therapeutic categories, giving over 350 products in total. The largest market is in the United States, followed by the E.U. zone. However, despite these advances, and the investment of resources in each product development, there are key issues concerning the clinical efficacy, cost-benefit of products, and clinical impact. Each therapeutic strategy has relative merits and limitations. Critical Issues: A critical consideration in developing and evaluating products is the therapeutic modality, associated regulatory processes, and the potential for clinical adoption geographically, determined by regulatory territory, intellectual property, and commercial distribution factors. The survey identifies an opportunity for developments that improve basic efficacy or cost-benefit. Future Directions: The economic pressures on health care systems, compounded by the demands of our increasingly ageing population, and the imperative to distribute effective health care, create an urgent global need for effective and affordable products.

A single-stage bilayered skin reconstruction using Glyaderm® as an acellular dermal regeneration template results in improved scar quality: an intra-individual randomized controlled trial

Absence of almost the entire reticular dermal layer is inherent to the use of autologous split-thickness skin grafting (STSG) to close full-thickness wounds, often resulting in hypertrophic scars and contractures. Many dermal substitutes have been developed, but unfortunately most have varying results in terms of cosmetic and/or functional improvement as well as patient satisfaction, in addition to high costs. Bilayered skin reconstruction using the human-derived glycerolized acellular dermis (Glyaderm®) has been reported to result in significantly improved scar quality using a two-step procedure. Unlike the necessary two-step procedure for most commercially available dermal substitutes, in this study we aimed to investigate the use of Glyaderm® in a more cost-effective single-stage engrafting. This is a method which, if autografts are available, is preferred by the majority of surgeons given the reduction in costs, hospitalization time and infection rate. A prospective, randomized, controlled, intra-individual, single-blinded study was performed, investigating the simultaneous application of Glyaderm® and STSG vs. STSG alone in full-thickness burns or comparable deep skin defects. During the acute phase, bacterial load, graft take and time to wound closure were assessed and were the primary outcomes. Aesthetic and functional results (secondary outcomes) were evaluated at 3, 6, 9 and 12months follow-up using subjective and objective scar measurement tools. Biopsies for histological analysis were taken at 3 and 12months. A total of 66 patients representing 82 wound comparisons were included. Graft take (>95%), pain management and healing time were comparable in both groups. At 1year follow-up, the overall Patient and Observer Scar Assessment Scale assessed by the patient was significantly in favour of sites where Glyaderm® was used. Not infrequently, patients attributed this difference to improved skin sensation. Histological analysis showed the presence of a well-formed neodermis, with donor elastin present for up to 12months. A single-stage bilayered reconstruction with Glyaderm® and STSG results in optimal graft take without loss of Glyaderm® nor the overlaying autografts due to infection. The presence of elastin in the neodermis was demonstrated during long-term follow-up in all but one patient, which is a crucial factor contributing to the significantly improved overall scar quality as evaluated by the blinded patients. The trial was registered on clinicaltrials.gov and received the following registration code: NCT01033604.

Conductive biomimetic bilayer fibrous scaffold for skin regeneration

The treatment of severely damaged wounds is a serious challenge as these wounds have poor self-healing capacity, take longer to heal, and are prone to becoming chronic wounds. To solve this problem, PCL/gelatin electrospinning nanofiber membranes were combined with rGO-loaded chitosan non-woven fabrics to prepare biomimetic bilayer skin scaffolds. Among them, the PCL/gelatin nanofiber membrane was used to simulate the epidermis layer with a dense structure, and the chitosan non-woven fabric was used to simulate the dermis layer with a loose structure. When the concentration of the spinning solution was 7 %, and the average fiber diameter was 241 nm, HaCaTs had the best cell proliferation and adhesion, and the re-epithelialization process was fast. To enhance the induction effect of scaffolds on dermal cells, the electroactive substance rGO was used to give the scaffold electrical conductivity. Considering the biocompatibility, the concentration of GO dispersion was controlled at 1 mg/mL, and the obtained CHI@ 1rGO scaffolds had high conductivity, as well as the improved mitochondrial membrane potential of HFF-1 s which increased cell viability. So the induction of HFF-1 s migration was obvious. After cross-linking composite scaffolds with glutaraldehyde, the scaffolds still showed good cytocompatibility, at the same time, the peel strength was increased from 7.21 cN to 350 cN, and the structural stability of the nanofiber membrane layer in the liquid environment was also enhanced. Both the moisture absorption and moisture permeability of composite scaffolds were improved, which could maintain the moist environment of the wound to a certain extent. Altogether, results showed that electroactive biomimetic bilayer fibrous scaffolds have great application potential in promoting skin tissue regeneration.

Properties of Collagen/Sodium Alginate Hydrogels for Bioprinting of Skin Models

3D printing technology has great potential for the reconstruction of human skin. However, the reconstructed skin has some differences from natural skin, largely because the hydrogel used does not have the appropriate biological and physical properties to allow healing and regeneration. This study examines the swelling, degradability, microstructure and biological properties of Collagen/Sodium Alginate (Col/SA) hydrogels of differing compositions for the purposes of skin printing. Increasing the content of sodium alginate causes the hydrogel to exhibit stronger mechanical and swelling properties, a faster degradation ratio, smaller pore size, and less favorable biological properties. An optimal 1% collagen hydrogel was used to print bi-layer skin in which fibroblasts and keratinocytes showed improved spreading and proliferation as compared to other developed formulations. The Col/SA hydrogels presented suitable tunability and properties to be used as a bioink for bioprinting of skin aiming at finding applications as 3D models for wound healing research.

A bioprinted vascularized skin substitute with fibroblasts, keratinocytes, and endothelial progenitor cells for skin wound healing

Skin substitutes are highly demanded by patients with extensive burns and full-thickness skin wounds. Bioprinting offers a promising technology to fabricate customized cell-laden skin substitutes. In this study, a hydrogel of biodegradable polyurethane (PU)-gelatin (4:1) laden with human fibroblasts, endothelial progenitor cells (EPCs), and keratinocytes was used as the bioink for building a bi-layer dermo-epidermal skin substitute. The seven-layer cell-laden constructs with stack thickness of 1.4 mm were precisely deposited through a 210 μm nozzle with an air pressure of 0.055–0.175 MPa and nozzle temperature of 19 °C. When grown in vitro, three types of cells in the bioprinted constructs showed good cell viability (>99%) in 24 h and reached a high proliferation rate (>220%) in 14 days. Significantly, EPCs were successfully differentiated into endothelial-like cells in the constructs and expressed the vasculogenesis-related proteins (CD31+ and eNOS+). When implanted in vivo, the bi-layer constructs attained ∼90% wound healing ratio and ∼76% re-epithelialization after 28 days in the nude mice model. Histological analyses revealed that skin wounds treated with the bi-layer constructs achieved high degrees of tissue integration and collagen production after 28 days. Vasculogenesis and angiogenesis of the wound treated with bi-layer constructs was significantly greater (∼300%) than those of the untreated wounds. The in vitro and in vivo findings indicate that the bioprinted skin substitutes with fibroblasts, EPCs, and keratinocytes embedded in PU-gelatin hydrogel may offer a promising strategy for clinical wound treatment and development of bioprinted skin.

Single unit functionally graded bioresorbable electrospun scaffold for scar-free full-thickness skin wound healing.

Full-thickness wounds are difficult to heal spontaneously. Scaffolds, meant for treating full-thickness wounds, should ensure proper tissue regeneration, both structurally and functionally. An ideal scaffold should mimic the physical, mechanical and biochemical properties of natural skin. However, available mono- or bi-layer skin scaffolds lack in the precise architecture and functionality, thus, failing to provide scar-free regeneration of full-thickness skin wounds. These unmet challenges of scar-free skin regeneration have been addressed in the present study for the first time. This research deals with the synthesis of a low-cost, structurally and functionally graded single unit biodegradable polymeric scaffold. The functional gradient in this scaffold was achieved by varying polymer concentration and electrospinning parameters. This gradient in the scaffold provided the required microenvironment for proper functional and structural reconstruction of all the layers of natural skin. The mechanical property of the scaffold matched that of the natural skin. Besides, the degradation kinetics of the scaffold was in coordination with the regeneration time for the full-thickness wound. The porosity and hydrophilicity gradients of the scaffold helped it mimic the in vivo hypodermal, dermal and epidermal microenvironments of the skin, simultaneously. Co-culturing PCS-201 (dermal fibroblasts) and HaCaT (keratinocytes) on the scaffold resulted in successful regeneration through cellular proliferation, differentiation and organization of the skin tissue. The scaffold also displayed better wound healing in vivo, in terms of speedy wound closure and proper tissue regeneration, in comparison to the standard treatment. Altogether, this study successfully established a simple, one-step synthesis process of a functionally graded, bioresorbable scaffold for scar-free, native-like, structural and functional regeneration of full-thickness skin wounds. Due to cost-effectiveness, easy synthesis process and microarchitectural features, the designed scaffold possesses a potential of translation to a good commercial wound healing product.

492 Bi-layered skin construct with gene modified keratinocytes reduces UVR DNA damage

Ultraviolet radiation (UVR) is among major contributors to skin cancer in humans. Production of melanin serves as a main defense mechanisms against UVR damage. This pigment, which gives color to hair, skin and eyes, protects the underlying tissue from UVR damage. However, in some patient populations, this protection mechanism is not effective, leaving patients highly susceptible to cancer. Similarly, mycosporine like amino acids (MAAs), which are commonly expressed in marine algae, have been shown to serve a protective role in UVR-induced DNA damage.

Polymerizable Skin Hydrogel for Full Thickness Wound Healing.

The skin is the largest organ in the human body, comprising the main barrier against the environment. When the skin loses its integrity, it is critical to replace it to prevent water loss and the proliferation of opportunistic infections. For more than 40 years, tissue-engineered skin grafts have been based on the in vitro culture of keratinocytes over different scaffolds, requiring between 3 to 4 weeks of tissue culture before being used clinically. In this study, we describe the development of a polymerizable skin hydrogel consisting of keratinocytes and fibroblast entrapped within a fibrin scaffold. We histologically characterized the construct and evaluated its use on an in vivo wound healing model of skin damage. Our results indicate that the proposed methodology can be used to effectively regenerate skin wounds, avoiding the secondary in vitro culture steps and thus, shortening the time needed until transplantation in comparison with other bilayer skin models. This is achievable due to the instant polymerization of the keratinocytes and fibroblast combination that allows a direct application on the wound. We suggest that the polymerizable skin hydrogel is an inexpensive, easy and rapid treatment that could be transferred into clinical practice in order to improve the treatment of skin wounds.

3D bioprinting of an implantable xeno-free vascularized human skin graft.

Bioengineered tissues or organs produced using matrix proteins or components derived from xenogeneic sources pose risks of allergic responses, immune rejection, or even autoimmunity. Here, we report successful xeno-free isolation, expansion, and cryopreservation of human endothelial cells (EC), fibroblasts (FBs), pericytes (PCs), and keratinocytes (KCs). We further demonstrate the bioprinting of a human skin substitute with a dermal layer containing xeno-free cultured human EC, FBs, and PCs in a xeno-free bioink containing human collagen type I and fibronectin layered in a biocompatible polyglycolic acid mesh and subsequently seeded with xeno-free human KCs to form an epidermal layer. Following implantation of such bilayered skin grafts on the dorsum of immunodeficient mice, KCs form a mature stratified epidermis with rete ridge-like structures. The ECs and PCs form human EC-lined perfused microvessels within 2 weeks after implantation, preventing graft necrosis, and eliciting further perfusion of the graft by angiogenic host microvessels. As proof-of-concept, we generated 12 individual grafts using a single donor of all four cell types. In summary, we describe the fabrication of a bioprinted vascularized bilayered skin substitute under completely xeno-free culture conditions demonstrating feasibility of a xeno-free approach to complex tissue engineering.