Bilayer Skin Research Articles

Vital roles of stem cells and biomaterials in skin tissue engineering.

Tissue engineering essentially refers to technology for growing new human tissue and is distinct from regenerative medicine. Currently, pieces of skin are already being fabricated for clinical use and many other tissue types may be fabricated in the future. Tissue engineering was first defined in 1987 by the United States National Science Foundation which critically discussed the future targets of bioengineering research and its consequences. The principles of tissue engineering are to initiate cell cultures in vitro, grow them on scaffolds in situ and transplant the composite into a recipient in vivo. From the beginning, scaffolds have been necessary in tissue engineering applications. Regardless, the latest technology has redirected established approaches by omitting scaffolds. Currently, scientists from diverse research institutes are engineering skin without scaffolds. Due to their advantageous properties, stem cells have robustly transformed the tissue engineering field as part of an engineered bilayered skin substitute that will later be discussed in detail. Additionally, utilizing biomaterials or skin replacement products in skin tissue engineering as strategy to successfully direct cell proliferation and differentiation as well as to optimize the safety of handling during grafting is beneficial. This approach has also led to the cells' application in developing the novel skin substitute that will be briefly explained in this review.

In vivo assessment of printed microvasculature in a bilayer skin graft to treat full-thickness wounds.

Chronic wounds such as diabetic foot ulcers and venous leg ulcers are common problems in people suffering from type 2 diabetes. These can cause pain, and nerve damage, eventually leading to foot or leg amputation. These types of wounds are very difficult to treat and sometimes take months or even years to heal because of many possible complications during the process. Allogeneic skin grafting has been used to improve wound healing, but the majority of grafts do not survive several days after being implanted. We have been studying the behavior of fibroblasts and keratinocytes in engineered capillary-like endothelial networks. A dermo-epidermal graft has been implanted in an athymic nude mouse model to assess the integration with the host tissue as well as the wound healing process. To build these networks into a skin graft, a modified inkjet printer was used, which allowed the deposit of human microvascular endothelial cells. Neonatal human dermal fibroblast cells and neonatal human epidermal keratinocytes were manually mixed in the collagen matrix while endothelial cells printed. A full-thickness wound was created at the top of the back of athymic nude mice and the area was covered by the bilayered graft. Mice of the different groups were followed until completion of the specified experimental time line, at which time the animals were humanely euthanized and tissue samples were collected. Wound contraction improved by up to 10% when compared with the control groups. Histological analysis showed the neoskin having similar appearance to the normal skin. Both layers, dermis and epidermis, were present with thicknesses resembling normal skin. Immunohistochemistry analysis showed favorable results proving survival of the implanted cells, and confocal images showed the human cells' location in the samples that were collocated with the bilayer printed skin graft.

An in vitro priming step increases the expression of numerous epidermal growth and migration mediators in a tissue-engineering construct.

An FDA-approved, prototypic, living, bilayered skin construct (BSC) has been used for non-healing wounds. Using this particular construct as proof of principle, we hypothesized that an in vitro 'priming' step may enhance its repertoire of expression of key mediators and genes. The priming step used here was incubation in Dulbecco's modified Eagle's medium (DMEM) for 24 h at 37°C and 5% CO2 , with or without construct meshing. Microarray and ingenuity pathway analysis (IPA) showed that >1000 genes were overexpressed by the priming step, including interleukin 6 (IL-6), which plays important roles in wound healing. Genes highly overexpressed by priming were those involved in epidermal proliferation and migration. Quantitative real-time PCR (qRT-PCR), immunostaining and western blots verified the results. An epiboly assay (epidermal migration over dermis) showed that BSC epiboly was inhibited by IL-6 neutralizing antibody. Back wounds of nude mice were treated with primed or control BSCs for 3 days prior to harvesting; primed BSCs showed a significantly (p = 0.006) greater level of epidermal migration vs unprimed. Our study demonstrates that an in vitro priming step induces wound healing-related genes in the BSC, leading to a construct that could prove more effective in stimulating wound healing. Copyright © 2014 John Wiley & Sons, Ltd.

Glyaderm® dermal substitute: Clinical application and long-term results in 55 patients

IntroductionGlycerol preserved acellular dermis (Glyaderm®) consists of collagen and elastin fibers and is the first non-profit dermal substitute derived from glycerol-preserved, human allogeneic skin. It is indicated for bi-layered skin reconstruction of full thickness wounds. MethodsA protocol for clinical application and optimal interval before autografting with split thickness skin graft (STSG) was developed in a pilot study.A phase III randomized, controlled, paired, intra-individual study compared full thickness defects engrafted with Glyaderm® and STSG versus STSG alone.Outcome measures included percentage of Glyaderm® take, STSG take, and scar quality assessment. ResultsPilot study (27 patients): Mean take rates equaled 91.55% for Glyaderm® and 96.67% for STSG. The optimal autografting interval was 6 days (±1 day).Randomized trial (28 patients): Mean Glyaderm® take rate was 88.17%. STSG take rates were comparable for both research groups (p=0.588). One year after wound closure, Glyaderm®+STSG was significantly more elastic (p=0.003) than STSG alone. Blinded observers scored Glyaderm® treated wounds better in terms of scar quality. DiscussionThe efficacy of Glyaderm® as a suitable dermal substitute for full thickness wounds is attested. Currently a procedure for simultaneous application of Glyaderm® and STSG is adopted, allowing for further widespread use of Glyaderm®.

Full-Thickness Skin Wound Healing Using Autologous Keratinocytes and Dermal Fibroblasts with Fibrin

Split-skin grafting (SSG) is the gold standard treatment for full-thickness skin defects. For certain patients, however, an extensive skin lesion resulted in inadequacies of the donor site. Tissue engineering offers an alternative approach by using a very small portion of an individual's skin to harvest cells for propagation and biomaterials to support the cells for implantation. The objective of this study was to determine the effectiveness of autologous bilayered tissue-engineered skin (BTES) and single-layer tissue-engineered skin composed of only keratinocytes (SLTES-K) or fibroblasts (SLTES-F) as alternatives for full-thickness wound healing in a sheep model. Full-thickness skin biopsies were harvested from adult sheep. Isolated fibroblasts were cultured using medium Ham's F12: Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum, whereas the keratinocytes were cultured using Define Keratinocytes Serum Free Medium. The BTES, SLTES-K, and SLTES-F were constructed using autologous fibrin as a biomaterial. Eight full-thickness wounds were created on the dorsum of the body of the sheep. On 4 wounds, polyvinyl chloride rings were used as chambers to prevent cell migration at the edge. The wounds were observed at days 7, 14, and 21. After 3 weeks of implantation, the sheep were euthanized and the skins were harvested. The excised tissues were fixed in formalin for histological examination via hematoxylin-eosin, Masson trichrome, and elastin van Gieson staining. The results showed that BTES, SLTES-K, and SLTES-F promote wound healing in nonchambered and chambered wounds, and BTES demonstrated the best healing potential. In conclusion, BTES proved to be an effective tissue-engineered construct that can promote the healing of full-thickness skin lesions. With the support of further clinical trials, this procedure could be an alternative to SSG for patients with partial- and full-thickness burns.

The Use of a Pure Native Collagen Dressing for Wound Bed Preparation Prior to Use of a Living Bi-layered Skin Substitute

Management of chronic wounds in the outpatient setting is quite challenging. The extensive co-morbid medical problems of the chronically ill patient along with the complexities of the wound bed and its biochemical environment has led to a plethora of patients with poor wound healing. This ever increasing population is a challenge for the wound care practitioner and cost to the health care system and patient. Increased wound chronicity has promulgated the use of advanced wound care products, including Living Skin Substitutes (LSS), in an attempt to obtain wound closure, and ultimately both physiological and functional healing.1–3 In the outpatient setting, it is evident that the efficacy of the LSS varies widely depending on the patient type with some patients responding quite favorably while others who do not achieve healing despite repeated applications of LSS. This case series demonstrates that a systematic method of wound bed preparation prior to the application of LSS improved healing outcomes. The entire wound bed preparation protocol included autolytic, non-selective, and sharp-selective debridement, if deemed appropriate, followed by the weekly application of a pure native collagen. The wound bed preparation protocol was completed prior to LSS application. This case series presents evidence supporting the application of a 100% native collagen dressing to wound bed prior to the final step of LSS utilization.

Poly(hydroxybutyrate‐co‐hydroxyvalerate) Bilayer Skin Tissue Engineering Constructs with Improved Epidermal Rearrangement

Bilayer skin substitutes constitute an attractive strategy towards improved skin wound healing. Therefore, solvent casting and freeze-drying methodologies are used to produce polyhydroxybutyrate-co-hydroxyvalerate (PHBV) thin nanoporous membranes and 3D porous scaffolds that are combined in bilayer structures to recreate the epidermal and dermal layers, respectively. The combination of these methodologies allow attaining a bilayer structure with a high water retention capability and adequate mechanical properties, susceptible to enzymes degradative action. Cultures established with human keratinocytes (hKC) and dermal fibroblasts (hDFb) confirm the suitability of the PHBV structures to support cell adhesion and proliferation. Nonetheless, when co-cultured under defined conditions, hKC are able to grow and rearrange in a multilayer structure with proliferative cells in the basal layer, and cells expressing a terminal differentiation marker in the upper layer. Therefore, PHBV bilayer structures demonstrate properties that favor skin cells performance, thus representing a promising strategy to improve wound healing.

Kynurenine Increases Matrix Metalloproteinase-1 and -3 Expression in Cultured Dermal Fibroblasts and Improves Scarring In Vivo

We previously demonstrated that the formation of hypertrophic scarring on the wounds of a rabbit ear fibrotic model was significantly reduced by grafting a bilayer skin substitute expressing indoleamine 2,3-dioxygenase (IDO). Here, we hypothesize that the improved healing quality is due to extracellular matrix modulatory effect of IDO-mediated tryptophan metabolites. To test this hypothesis, a series of in vitro and in vivo experiments were conducted and the findings revealed a significant increase in the expression of matrix metalloproteinase 1 (MMP-1) in fibroblasts either transduced with human IDO gene or cultured with conditioned media obtained from IDO-expressing cells. Consistent with this finding, kynurenine (Kyn) treatment markedly increased the levels of MMP-1 and MMP-3 expression through activation of the MEK (mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase)-ERK1/2 MAPK signaling pathway. On the other hand, Kyn significantly suppressed the expression of type I collagen in fibroblasts as compared with that of control. To test the anti-fibrogenic effect of Kyn in an in vivo model, rabbit ear fibrotic wounds were topically treated with cream containing 50 μg Kyn per l00 μl of cream per wound. The result showed a marked improvement in scar formation relative to the controls. These findings collectively suggest that Kyn can potentially be used as an anti-fibrogenic agent for treating hypertrophic scarring.

Differential Response of Human Adipose Tissue-Derived Mesenchymal Stem Cells, Dermal Fibroblasts, and Keratinocytes to Burn Wound Exudates: Potential Role of Skin-Specific Chemokine CCL27

Many cell-based regenerative medicine strategies toward tissue-engineered constructs are currently being explored. Cell-cell interactions and interactions with different biomaterials are extensively investigated, whereas very few studies address how cultured cells will interact with soluble wound-healing mediators that are present within the wound bed after transplantation. The aim of this study was to determine how adipose tissue-derived mesenchymal stem cells (ASC), dermal fibroblasts, and keratinocytes will react when they come in contact with the deep cutaneous burn wound bed. Burn wound exudates isolated from deep burn wounds were found to contain many cytokines, including chemokines and growth factors related to inflammation and wound healing. Seventeen mediators were identified by ELISA (concentration range 0.0006-9 ng/mg total protein), including the skin-specific chemokine CCL27. Burn wound exudates activated both ASC and dermal fibroblasts, but not keratinocytes, to increase secretion of CXCL1, CXCL8, CCL2, and CCL20. Notably, ASC but not fibroblasts or keratinocytes showed significant increased secretion of vascular endothelial growth factor (5-fold) and interleukin-6 (253-fold), although when the cells were incorporated in bi-layered skin substitute (SS) these differences were less pronounced. A similar discrepancy between ASC and dermal fibroblast mono-cultures was observed when recombinant human-CCL27 was used instead of burn wound exudates. Although CCL27 did not stimulate the secretion of any of the wound-healing mediators by keratinocytes, these cells, in contrast to ASC or dermal fibroblasts, showed increased proliferation and migration. Taken together, these results indicate that on transplantation, keratinocytes are primarily activated to promote wound closure. In contrast, dermal fibroblasts and, in particular, ASC respond vigorously to factors present in the wound bed, leading to increased secretion of angiogenesis/granulation tissue formation factors. Our findings have implications for the choice of cell type (ASC or dermal fibroblast) to be used in regenerative medicine strategies and indicate the importance of taking into account interactions with the wound bed when developing advanced therapies for difficult-to-close cutaneous wounds.

Micropatterned dermal–epidermal regeneration matrices create functional niches that enhance epidermal morphogenesis

Although tissue engineered skin substitutes have demonstrated some clinical success for the treatment of chronic wounds such as diabetic and venous ulcers, persistent graft take and stability remain concerns. Current bilayered skin substitutes lack the characteristic microtopography of the dermal–epidermal junction that gives skin enhanced mechanical stability and creates cellular microniches that differentially promote keratinocyte function to form skin appendages and enhance wound healing. We developed a novel micropatterned dermal–epidermal regeneration matrix (μDERM) which incorporates this complex topography and substantially enhances epidermal morphology. Here, we describe the use of this three-dimensional (3-D) in vitro culture model to systematically evaluate different topographical geometries and to determine their relationship to keratinocyte function. We identified three distinct keratinocyte functional niches: the proliferative niche (narrow geometries), the basement membrane protein synthesis niche (wide geometries) and the putative keratinocyte stem cell niche (narrow geometries and corners). Specifically, epidermal thickness and keratinocyte proliferation is significantly (p<0.05) increased in 50 and 100μm channels while laminin-332 deposition is significantly (p<0.05) increased in 400μm channels compared to flat controls. Additionally, β1brip63+ keratinocytes, putative keratinocyte stem cells, preferentially cluster in channel geometries (similar to clustering observed in native skin) compared to a random distribution on flats. This study identifies specific target geometries to enhance skin regeneration and graft performance. Furthermore, these results suggest the importance of μDERM microtopography in designing the next generation of skin substitutes. Finally, we anticipate that 3-D organotypic cultures on μDERMS will provide a novel tissue engineered skin substitute for in vitro investigations of skin morphogenesis, wound healing and pathology.

Ease of Use, Safety, and Efficacy of Integra Bilayer Wound Matrix in the Treatment of Diabetic Foot Ulcers in an Outpatient Clinical Setting

Integra bilayer wound matrix (IBWM) is a bilayer skin replacement system composed of a dermal regeneration layer and a temporary epidermal layer. It is used to treat various types of deep, large wounds via an inpatient procedure in an operating room. We sought to determine ease of use and effectiveness of IBWM in an outpatient clinical setting when treating diabetic foot ulcers. In addition, no epidermal autografting was performed in conjunction with the IBWM after silicone release, as is common in the inpatient setting. This 12-week, single-arm, prospective pilot study was conducted in three outpatient clinics. Weekly evaluations included monitoring the wound for signs of infection during the 12-week follow-up phase. Eleven patients with diabetic foot ulcers who met the inclusion and exclusion criteria were enrolled. One patient was discontinued from the study owing to noncompliance leading to a serious adverse event. Therefore, ten patients who received the study intervention were included in the per-protocol population reported herein. The mean patient age was 60.6 years, with an average 11-year history of diabetes mellitus. Each ulcer was located on the plantar aspect of the foot. No infection was reported during the study. Patients treated with IBWM showed progressive wound healing over time: the greatest mean wound reduction was approximately 95% in week 12. Seven of ten patients (70%) achieved complete wound closure by week 12. No recurrent ulcers were reported during follow-up. These results are consistent with the hypothesis that IBWM is easy to use, safe, and effective when used on diabetic foot ulcers in an outpatient clinical setting without the secondary procedure of autografting for closure.

Immunoprotective Role of Indoleamine 2,3-Dioxygenase in Engraftment of Allogenic Skin Substitute in Wound Healing

Delayed wound healing can significantly impact survival of patients who suffer from severe thermal injury. In general, the use of a wound coverage, particularly with those of bilayer skin substitute, would be ideal to promote healing and prevent infection and fluid loss. Although the use of an autologous skin substitute is desirable, its preparation is time consuming and its immediate availability is impossible. To overcome this difficulty, the authors have previously demonstrated that the expression of indoleamine 2,3 dioxygenase (IDO) could function as a local immune suppressive factor in protecting allogenic fibroblasts and keratinocytes without using any immunosuppressive medication in a wound healing animal model. IDO, which is naturally expressed in the placenta by trophoblast cells during pregnancy, plays an essential role in maternal tolerance toward the fetus. The potent and selective local immunosuppressive function of IDO makes this enzyme a very promising tool for engineering a nonrejectable skin allograft. Here, the authors reviewed and discussed how the expression of IDO by the primary cells of our skin substitute can serve as a source of IDO enzyme activity and generate a tryptophan-deficient environment. Under this condition, only skin cells but not immune cells (CD4(+) and CD8(+) cells) would survive and protect engraftment of this engineered and shelf-ready skin substitute to be used not only as wound coverage but also as a rich source of wound healing promoting factors. Therefore, this review summarizes the body of work on immunoprotective role of IDO in engraftment of allogenic skin substitute in wound healing, which has recently been reported by the authors' research group and others.

Prediction of Percutaneous Absorption in Human Using Three-Dimensional Human Cultured Epidermis LabCyte EPI-MODEL

The objective of this study is to establish a relationship of the skin penetration parameters between the three-dimensional cultured human epidermis LabCyte EPI-MODEL (LabCyte) and hairless mouse (HLM) skin penetration in vitro and to predict the skin penetration and plasma concentration profile in human. The skin penetration experiments through LabCyte and HLM skin were investigated using 19 drugs that have a different molecular weight and lipophilicity. The penetration flux for LabCyte reached 30 times larger at maximum than that for HLM skin. The human data can be estimated from the in silico approach with the diffusion coefficient (D), the partition coefficient (K) and the skin surface concentration (C) of drugs by assuming the bi-layer skin model for both LabCyte and HLM skin. The human skin penetration of β-estradiol, prednisolone, testosterone and ethynylestradiol was well agreed between the simulated profiles and in vitro experimental data. Plasma concentration profiles of β-estradiol in human were also simulated and well agreed with the clinical data. The present alternative method may decrease human or animal skin experiment for in vitro skin penetration.

Evaluation of the Biocompatibility of a Bilayer Chitosan Skin Regenerating Template, Human Skin Allograft, and Integra Implants in Rats

Introduction. Chitosan is a nontoxic, biocompatible, and biodegradable polymer obtained from chitin by N-deacetylation using strong alkali. Chitosan in a form of a bilayer skin regenerating template can act as a scaffold for regeneration. Integra is a two-layer skin regeneration system, constructed of a matrix of crosslinked fibers that acts as a scaffold for regenerating dermal skin cells. Human skin allografts (HSAs) are the “gold standard” for temporary coverage of clean burn wounds. Objectives. The objective of this study was to conduct in-vivo, preclinical biocompatibility evaluations of Integra, HSA, and Chitosan skin regenerating template (SRT). Methods. Paravertebral subcutaneous pockets were created for the implantation of test materials. Implants were retrieved after 4, 7, 14, 21, and 28 days. Slides of sections through the implants were examined to determine biocompatibility. Results. Chitosan SRT and Integra showed similar inflammatory patterns. HSA showed a higher inflammatory reaction initially which then reduced to levels similar to Integra and Chitosan SRT. Chitosan SRT and Integra also shared similar angiogenesis levels. Towards the end, all implants were degraded with decreased tissue response. Conclusion. Integra, Chitosan SRT, and HSA have been shown to be biocompatible. Integra and Chitosan SRT seem to illicit similar tissue responses.

Differential Keratin Expression During Epiboly in a Wound Model of Bioengineered Skin and in Human Chronic Wounds

Epiboly represents the process by which keratinocytes migrate to envelop a surface. The authors have been investigating a living bilayered skin construct (BSC) that is used in the treatment of lower extremity wounds due to venous insufficiency and diabetes. The construct demonstrates epiboly after injury and incubation in vitro, and this model may be useful for studying epidermal migration and the process of skin maturation. Punch biopsies of the construct in vitro were cultured and immunostained for specific keratins at baseline and at 24 to 72 hours. For comparison, skin biopsy specimens from human chronic venous ulcers and acute healing wounds were similarly processed. The authors found that K1 and K10 were fully expressed in the epidermis of the fully epibolized surface on BSC. K1 was also present in the migrating edge of specimens, whereas K10 was not detectable. K16 and K6 were evident in normal skin and the epibolized area of the construct; K6 expression was very prominent in the migrating edge. Importantly, K17 was distinctly limited to the epibolized surface and the migrating edge, and its expression was very similar to that observed in healing human wounds. In conclusion, differential expression of keratins in this epiboly model closely reflects in vivo studies and supports keratin specificity in the processes of migration and differentiation of new epidermis. Therefore, these findings provide further and important validity for the study of epithelialization and the hope of developing prognostic markers for venous ulcer healing.

Local expression of indoleamine 2,3-dioxygenase suppresses T-cell-mediated rejection of an engineered bilayer skin substitute

Engineered skin substitutes (ESSs) comprising both keratinocytes and fibroblasts can afford many advantages over the use of autologous keratinocyte grafts for the treatment of full-thickness and partial-thickness burns. In this study, we investigated the efficacy of a novel ESS containing both genetically altered fibroblasts that express the immunosuppressive factor indoleamine 2,3-dioxygenase (IDO) and primary keratinocytes from a nonautologous source to confer immune protection of xenogeneic cells cultured in a bilayer ESS. The results show that engraftment of IDO expressing skin substitutes on the back of rats significantly improves healing progression over 7 days compared with both nontreated and non-IDO-expressing skin substitutes (p<0.001). Immuno-staining of CD3 and CD31 suggests that IDO-expressing skin substitutes significantly suppress T cell infiltration (p<0.001) and improve neovascularization by four-fold (12.6±1.2 vs. 3.0±1.0 vessel-like structure/high power field), respectively. In conclusion, we found that IDO expression can improve the efficacy of nonautologous ESS for the purpose of wound healing by mitigating T-cell infiltration as well as promoting vascularization of the graft.

Cultured Bilayered Skin Allograft for Vaginal Construction

Objective This is the first report of live human cultured bilayered skin allograft (taken from another person) (LHCBSA) to line a dissected space to create a vagina. Case A 19-year-old with Mayer-Rokitansky-Küster-Hauser syndrome (MRKH syndrome) of vaginal and uterine agenesis had a space dissected and lined with LHCBSA. Although the lining devitalized within 2 weeks, there was a rapid ingrowth of vaginal mucosal cells from the vaginal dimple with an excellent long-term result. Conclusion This is the first report that LHCBSA is able to stimulate vaginal mucosal cell growth for a neovagina. It is possible that it might stimulate other surface tissue lining to cover adjacent raw areas such as bladder or esophagus.

Functional Bilayered Skin Substitute Constructed by Tissue-Engineered Extracellular Matrix and Microsphere-Incorporated Gelatin Hydrogel for Wound Repair

Our aim in this study was to develop a new methodology for constructing a bilayer skin equivalent to create a clinically compliant skin graft composite for the treatment of various skin defects. We utilized tissue-engineered extracellular matrix (TEECM) that maintains the native dermal components, as analogical dermal layer, and gelatin hydrogel containing epidermal growth factor (EGF)-loaded microspheres, as epidermis layer upon TEECM. The bilayer skin equivalent (GG-EGF/TEECM) could be synthesized appropriately and has been hypothesized to provide an enhanced effect on re-epithelization, apart from dermal reconstruction improved by TEECM. Morphological properties, EGF release efficiency, cytotoxicity, and effects on wound repair of this composite were investigated in this study. It was shown that the two layers adhered firmly to each other, and the percentage of cumulative release was approximately 80% by day 14. Moreover, as a biocompatible equivalent, GG-EGF/TEECM improved the healing of the wound area as indicated by significantly earlier re-epithelialization and dermal maturation. These results suggest that GG-EGF/TEECM can provide an optimal microenvironment and proper template for skin tissue engineering.

Controlled vacuum seeding as a means of generating uniform cellular distribution in electrospun polycaprolactone (PCL) scaffolds.

A major challenge encountered in using electrospun scaffolds for tissue engineering is the non-uniform cellular distribution in the scaffold with increasing depth under normal passive seeding conditions. Because of the small surface pores, typically few microns in diameter, cells tend to congregate and proliferate on the surface much faster compared to penetrating the scaffold interior. In order to overcome this problem, we used a vacuum seeding technique on polycaprolactone electrospun scaffolds while using NIH 3T3 fibroblasts as the model cell system. This serves as a precursor to the bilayer skin model where the fibroblasts would be residing at an intermediate layer and the keratinocytes would be on the top. Vacuum seeding was used in this study to enhance fibroblasts seeding and proliferation at different depths. Our results show that the kinetics of cell attachment and proliferation were a function of varying vacuum pressure as well as fiber diameter. Cell attachment reached a maxima somewhere between 2-8 in. Hg vacuum pressure and fell for lower vacuum pressures presumably because of cell loss through the filtration process. Cell proliferation and collagen secretion over five days indicated that vacuum pressure did not affect cellular function adversely. We also compared the combined impact of scaffold architecture (400 nm versus 1100 nm average diameter fiber scaffolds) and vacuum pressure. At a given pressure, more cells were retained in the 400 nm scaffolds compared to 1100 nm scaffolds. In addition, the cell intensity profile shows cell intensity peak shift from the top to the inner layers of the scaffold by lowering the vacuum pressure from 0 in. Hg to 20 in. Hg. For a given vacuum pressure the cells were seeded deeper within the 1100 nm scaffold. The results indicate that cells can be seeded in electrospun scaffolds at various depths in a controlled manner using a simple vacuum seeding technique. The depth of seeding is a function of pressure and scaffold fiber diameter.

Human single-donor composite skin substitutes based on collagen and polycaprolactone copolymer

The development and characterization of an enhanced composite skin substitute based on collagen and poly(ε-caprolactone) are reported. Considering the features of excellent biocompatibility, easy-manipulated property and exempt from cross-linking related toxicity observed in the 1:20 biocomposites, skin substitutes were developed by seeding human single-donor keratinocytes and fibroblasts alone on both sides of the 1:20 biocomposite to allow for separation of two cell types and preserving cell signals transmission via micro-pores with a porosity of 28.8±16.1μm. The bi-layered skin substitute exhibited both differentiated epidermis and fibrous dermis in vitro. Less Keratinocyte Growth Factor production was measured in the co-cultured skin model compared to fibroblast alone condition indicating a favorable microenvironment for epidermal homeostasis. Moreover, fast wound closure, epidermal differentiation, and abundant dermal collagen deposition were observed in composite skin in vivo. In summary, the beneficial characteristics of the new skin substitutes exploited the potential for pharmaceutical screening and clinical application.