Comparison of biopolymer scaffolds for the fabrication of skin substitutes in a porcine wound model.

This study compared three acellular scaffolds as templates for the fabrication of skin substitutes. A collagen-glycosaminoglycan (C-GAG), a biodegradable polyurethane foam (PUR) and a hybrid combination (PUR/C-GAG) were investigated. Scaffolds were prepared for cell inoculation. Fibroblasts and keratinocytes were serially inoculated onto the scaffolds and co-cultured for 14 days before transplantation. Three pigs each received four full-thickness 8 cm × 8 cm surgical wounds, into which a biodegradable temporising matrix (BTM) was implanted. Surface seals were removed after integration (28 days), and three laboratory-generated skin analogues and a control split-thickness skin graft (STSG) were applied for 16 weeks. Punch biopsies confirmed engraftment and re-epithelialisation. Biophysical wound parameters were also measured and analysed. All wounds showed greater than 80% epithelialisation by day 14 post-transplantation. The control STSG displayed 44% contraction over the 16 weeks, and the test scaffolds, C-GAG 64%, Hybrid 66.7% and PUR 67.8%. Immunohistochemistry confirmed positive epidermal keratins and basement membrane components (Integrin alpha-6, collagens IV and VII). Collagen deposition and fibre organisation indicated the degree of fibrosis and scar produced for each graft. All scaffold substitutes re-epithelialised by 4 weeks. The percentage of original wound area for the Hybrid and PUR was significantly different than the STSG and C-GAG, indicating the importance of scaffold retainment within the first 3 months post-transplant. The PUR/C-GAG scaffolds reduced the polymer pore size, assisting cell retention and reducing the contraction of in vitro collagen. Further investigation is required to ensure reproducibility and scale-up feasibility.

A bioreactor for studying negative pressure wound therapy on skin grafts.

Negative pressure wound therapy (NPWT) has become the prevailing standard of care for treating complex soft tissue wounds and is now being considered for use in alternative applications including improving skin graft take. While it is generally agreed that negative pressure leads to improved wound healing, universal consensus on its optimal application is not supported in the literature. We describe the design and validation of a bioreactor to determine the prospective benefits of NPWT on skin grafts and engineered skin substitutes (ESS). Clinically relevant pressures were applied, and the native human skin was able to withstand greater negative pressures than the engineered substitutes. Both skin types were cultured under static, flow-only, and -75 mm Hg conditions for 3 days. While it remained intact, there was damage to the epidermal-dermal junction in the ESS after application of negative pressure. The normal skin remained viable under all culture conditions. The engineered skin underwent apoptosis in the flow-only group; however, the application of negative pressure reduced apoptosis. Vascular endothelial growth factor levels were significantly higher in the normal flow-only group, 152.0 ± 75.1 pg/mg protein, than the other culture conditions, 81.6 ± 35.5 pg/mg for the static and 103.6 ± pg/mg for the negative pressure conditions. The engineered skin had a similar trend but the differences were not significant. This bioreactor design can be used to evaluate the impacts of NPWT on the anatomy and physiology of skin to improve outcomes in wounds after grafting with normal or engineered skin.

Development of the mechanical properties of engineered skin substitutes after grafting to full-thickness wounds.

Engineered skin substitutes (ESSs) have been reported to close full-thickness burn wounds but are subject to loss from mechanical shear due to their deficiencies in tensile strength and elasticity. Hypothetically, if the mechanical properties of ESS matched those of native skin, losses due to shear or fracture could be reduced. To consider modifications of the composition of ESS to improve homology with native skin, biomechanical analyses of the current composition of ESS were performed. ESSs consist of a degradable biopolymer scaffold of type I collagen and chondroitin-sulfate (CGS) that is populated sequentially with cultured human dermal fibroblasts (hF) and epidermal keratinocytes (hK). In the current study, the hydrated biopolymer scaffold (CGS), the scaffold populated with hF dermal skin substitute (DSS), or the complete ESS were evaluated mechanically for linear stiffness (N/mm), ultimate tensile load at failure (N), maximum extension at failure (mm), and energy absorbed up to the point of failure (N-mm). These biomechanical end points were also used to evaluate ESS at six weeks after grafting to full-thickness skin wounds in athymic mice and compared to murine autograft or excised murine skin. The data showed statistically significant differences (p <0.05) between ESS in vitro and after grafting for all four structural properties. Grafted ESS differed statistically from murine autograft with respect to maximum extension at failure, and from intact murine skin with respect to linear stiffness and maximum extension. These results demonstrate rapid changes in mechanical properties of ESS after grafting that are comparable to murine autograft. These values provide instruction for improvement of the biomechanical properties of ESS in vitro that may reduce clinical morbidity from graft loss.

Keloid-derived keratinocytes exhibit an abnormal gene expression profile consistent with a distinct causal role in keloid pathology.

Keloids are disfiguring scars that extend beyond the original wound borders and resist treatment. Keloids exhibit excessive extracellular matrix deposition, although the underlying mechanisms remain unclear. To better understand the molecular basis of keloid scarring, here we define the genomic profiles of keloid fibroblasts and keratinocytes. In both cell types, keloid-derived cells exhibit differential expression of genes encompassing a diverse set of functional categories. Strikingly, keloid keratinocytes exhibited decreased expression of a set of transcription factor, cell adhesion, and intermediate filament genes essential for normal epidermal morphology. Conversely, they exhibit elevated expression of genes associated with wound healing, cellular motility, and vascular development. A substantial number of genes involved in epithelial-mesenchymal transition were also up-regulated in keloid keratinocytes, implicating this process in keloid pathology. Furthermore, keloid keratinocytes displayed significantly higher migration rates than normal keratinocytes in vitro and reduced expression of desmosomal proteins in vivo. Previous studies suggested that keratinocytes contribute to keloid scarring by regulating extracellular matrix production in fibroblasts. Our current results show fundamental abnormalities in keloid keratinocytes, suggesting they have a profoundly more direct role in keloid scarring than previously appreciated. Therefore, development of novel therapies should target both fibroblast and keratinocyte populations for increased efficacy.

Composition and Performance of Autologous Engineered Skin Substitutes for Repair or Regeneration of Excised, Full-Thickness Burns.

Prompt and permanent wound closure after burn injuries remains a requirement for patient recovery. Historically, split-thickness skin autograft (STAG) has served as the prevailing standard of care for closure of extensive, deep burns. Because STAG availability may be insufficient in life-threatening burns, alternatives have been evaluated for safety and efficacy of wound closure. Since the 1970s, alternatives consisting of cultured epidermal keratinocytes, and/or acellular dermal substitutes were studied and translated into services and devices that facilitated wound closure, survival, and recovery after major burns. Cultured epithelial autografts (CEA) promoted epidermal closure of wounds but were not stable during long-term recovery. An acellular dermal substitute consisting of collagen and glycosaminoglycans (C-GAG) provided more uniform dermal repair, and reduced needs for epidermal harvesting but was subject to loss from microbial contamination. More recently, an autologous engineered skin substitute (ESS) has been reported and includes a C-GAG polymer populated with fibroblasts and keratinocytes which form basement membrane. ESS can be applied clinically over a vascularized dermal substitute and generates stable wound closure that is smooth, soft, and strong. Despite these advances, no current alternatives for permanent wound closure restore the anatomy and physiology of uninjured skin. Current alternatives act by mechanisms of wound healing, not by developmental biology by which skin forms in utero with pigment, hair, sweat and sebaceous glands, microvasculature, and nerve. Until full-thickness burns are restored with all of the normal structures and functions of uninjured skin, regenerative medicine of skin will remain an ambitious aspiration for future researchers and engineers to achieve.

Morphogenesis of chimeric hair follicles in engineered skin substitutes with human keratinocytes and murine dermal papilla cells.

Engineered skin substitutes (ESS) have been used successfully to treat life-threatening burns, but lack cutaneous appendages. To address this deficiency, dermal constructs were prepared using collagen-glycosaminoglycan scaffolds populated with murine dermal papilla cells expressing green fluorescent protein (mDPC-GFP), human dermal papilla cells (hDPC) and/or human fibroblasts (hF). Subsequently, human epidermal keratinocytes (hK) or hK genetically modified to overexpress stabilized ß-catenin (hK') were used to prepare ESS epithelium. After 10 days incubation at air-liquid interface, ESS were grafted to athymic mice and were evaluated for 6 weeks. Neofollicles were observed in ESS containing mDPC-GFP, but not hDPC or hF, independent of whether or not the hK were genetically modified. Based on detection of GFP fluorescence, mDPC were localized to the dermal papillae of the well-defined follicular structures of grafted ESS. In addition, statistically significant increases in LEF1, WNT10A and WNT10B were found in ESS with neofollicles. These results demonstrate a model for generation of chimeric hair in ESS.

Assessment of replication rates of human keratinocytes in engineered skin substitutes grafted to athymic mice.

Stable closure of skin wounds with engineered skin substitutes (ESS) requires indefinite mitotic capacity to generate the epidermis. To evaluate whether keratinocytes in ESS exhibit the stem cell phenotype of label retention, ESS (n = 6-9/group) were pulsed with 5-bromo-2'-deoxyuridine (BrdU) in vitro, and after grafting to athymic mice (n = 3-6/group). Pulse and immediate chase in vitro labeled virtually all basal keratinocytes at day 8, with label uptake decreasing until day 22. Label retention in serial chase decreased more rapidly from day 8 to day 22, with a reorganization of BrdU-positive cells into clusters. Similarly, serial chase of labeled basal keratinocytes in vivo decreased sharply from day 20 to day 48 after grafting. Label uptake was assessed by immediate chases of basal keratinocytes, and decreased gradually to day 126, while total labeled cells remained relatively unchanged. These results demonstrate differential rates of label uptake and retention in basal keratinocytes of ESS in vitro and in vivo, and a proliferative phenotype with potential for long-term replication in the absence of hair follicles. Regulation of a proliferative phenotype in keratinocytes of ESS may improve the biological homology of tissue-engineered skin to natural skin, and contribute to more rapid and stable wound healing.

Advances in Skin Tissue Bioengineering and the Challenges of Clinical Translation.

Skin tissue bioengineering is an emerging field that brings together interdisciplinary teams to promote successful translation to clinical care. Extensive deep tissue injuries, such as large burns and other major skin loss conditions, are medical indications where bioengineered skin substitutes (that restore both dermal and epidermal tissues) are being studied as alternatives. These may not only reduce mortality but also lessen morbidity to improve quality of life and functional outcome compared with the current standards of care. A common objective of dermal-epidermal therapies is to reduce the time required to accomplish stable closure of wounds with minimal scar in patients with insufficient donor sites for autologous split-thickness skin grafts. However, no commercially-available product has yet fully satisfied this objective. Tissue engineered skin may include cells, biopolymer scaffolds and drugs, and requires regulatory review to demonstrate safety and efficacy. They must be scalable for manufacturing and distribution. The advancement of technology and the introduction of bioreactors and bio-printing for skin tissue engineering may facilitate clinical products' availability. This mini-review elucidates the reasons for the few available commercial skin substitutes. In addition, it provides insights into the challenges faced by surgeons and scientists to develop new therapies and deliver the results of translational research to improve patient care.

Direct comparison of reproducibility and reliability in quantitative assessments of burn scar properties.

INTRODUCTION: Determining the efficacy of anti-scar technologies can be difficult as qualitative, subjective assessments are often utilized instead of systematic, objective measures. Perceptions regarding the reliability of instruments for quantitative measurements along with their high cost and increased data collection time may discourage their use, leading to use of scar scales which are relatively quick and low-cost. To directly evaluate the reliability of instruments for quantitative measurements of scar properties, instruments and two qualitative scales were compared by assessing a variety of cutaneous scars. METHODS: Scar height and surface texture were evaluated using a 3D scanner and a mold/cast technique. Scar color was evaluated by using a spectroscopy-based tool, the Mexameter®, and digital photography with image analysis. Scar biomechanics were evaluated using the BTC-2000™, Dermal Torque Meter (DTM®), and ballistometer®. The Vancouver Scar Scale (VSS) and Patient and Observer Scar Assessment Scale (POSAS) were used to qualitatively evaluate the same scar properties. Intraclass correlation coefficients (ICC) were used to determine inter- and intra-user reliability (poor, moderate, good, excellent) with all instruments and the kappa reliability statistic was used to asses inter-user reliability (poor, fair, moderate, good, very good) for VSS and POSAS. Time for measurement collection and after collection analysis was also recorded. RESULTS: The Mexameter® was the most reliable method for evaluating erythema and pigmentation compared to digital photography and image processing, POSAS and VSS. Digital photography and analysis was more reliable than POSAS and VSS. Assessment of scar height was significantly more reliable when using a 3D scanner versus VSS and POSAS. The 3D scanner and mold-cast techniques also offered an additional benefit of providing an absolute value of scar height relative to the surrounding tissue. Intra-user reliability for all mechanical tests was moderate to good. Inter-user reliability was greater when using the BTC-2000™ and ballistometer® versus the DTM®. All quantitative measurements took less than 90 s for collection, with the exception of the mold/cast technique. CONCLUSION: Non-invasive instruments allow scar properties to be quantitatively assessed with high sensitivity and as a function of time and/or treatment without the need for biopsy collection. Overall, the reliability of scar assessments was significantly improved when quantitative instruments were utilized versus scar scales. Quantitative assessment of color and biomechanics were swift, requiring less than 90 s per measurement while assessments of texture and height required additional analysis time after collection. With proper training of clinical staff and well-defined protocols for measurement collection, reliable, quantitative assessments of scar properties can be collected with little disruption to the clinical workflow.

Exogenous Keratinocyte Growth Factor Is Not Required for Pigmentation of Skin Substitutes with Three Isogeneic Cell Types.

Engineered skin substitutes (ESS) containing human fibroblasts (hF) and human keratinocytes (hK) provide significant medical benefits for treatment of acute and chronic skin wounds, including, but not limited to, burns, burn scars, congenital skin lesions, and cutaneous ulcers. However, anatomic deficiencies, such as lack of pigment, can contribute to long-term morbidity, including hypopigmentation and reduced solar protection. To address the deficiency of hypopigmentation, ESS were populated sequentially with cultured hF, human melanocytes (hM), and hK to generate ESS with pigment (ESS-P). Constructs were incubated in media containing 0.0, 1.5, or 5.0 ng/mL keratinocyte growth factor (KGF), which promotes survival and differentiation of hM in ESS-P, and had media changed at 24 or 48 h intervals. ESS-P were evaluated in vitro for surface hydration, surface color, and distribution of hM. Proliferation was assessed by measuring incorporation of 5-bromo-2'-deoxyuridine into replicating DNA in basal epidermal cells. ESS-P from test conditions were grafted to immunodeficient mice, and were assessed over 12 weeks for pigmented area, pigment density, and distribution of hM in healed human grafts. The in vitro data showed differences among test groups, including increase in hydration of the epidermal surface with higher KGF, increase of surface pigmentation with 24 h media changes, increase of hM density with higher KGF and 24 h media changes, and time-dependent decrease of proliferation. At 12 weeks after grafting, differences among groups were found for pigment density, but not for distribution of hM or percentage of pigmented area. These differences demonstrate that a higher concentration of KGF (5 ng/mL) in the maturation medium of ESS-P and more frequent media changes (24 h interval) promote higher viability and hM differentiation of ESS-P before grafting, but are not required for full pigmentation (pigmented area, pigment density, hM distribution) of grafted wounds. Based on these results, reductions of the concentration of KGF (i.e., 1.5 ng/mL) in the maturation medium, and of the frequency of medium changes (48 h intervals) would be expected to support survival, continued replication, and restoration of skin color by hM in therapeutic transplantation of ESS-P. Impact Statement Restoration of skin color after traumatic injury affects personal identity and provides protection from exposure to solar radiation. Keratinocyte growth factor (KGF) and nutrient supply are known to regulate survival of melanocytes before transplantation in engineered skin substitutes with pigment (ESS-P). This report demonstrates that exogenous KGF is not required to restore skin color and that replacement of the nutrient medium at lower frequency (48 versus 24 h) does not inhibit development of skin color after melanocyte transplantation. These results offer new alternatives to conserve resources in fabrication of ESS-P and to maintain efficacy for restoration of skin color.

Light or Dark Pigmentation of Engineered Skin Substitutes Containing Melanocytes Protects Against Ultraviolet Light-Induced DNA Damage In Vivo.

Engineered skin substitutes (ESS) containing autologous fibroblasts and keratinocytes provide stable wound closure in patients with large, full-thickness burns, but are limited by hypopigmentation due to absence of added melanocytes. DNA damage caused by ultraviolet radiation (UV) increases risk for skin cancer development. In human skin, melanocytes provide pigmentation that protects skin from UV-induced DNA damage. This study investigated whether inclusion of human melanocytes (hM) affects the response of ESS to UV in vivo. Specifically, pigmentation and formation of cyclobutane pyrimidine dimers (CPDs), the most prevalent UV-induced DNA photoproduct, were analyzed. Three groups of ESS were prepared with fibroblasts and keratinocytes, ± melanocytes, and grafted orthotopically to immunodeficient mice: ESS without melanocytes (ESS-hM), ESS with light skin-derived (Caucasian) melanocytes (ESS+hM-L), and ESS with dark skin-derived (African-American) melanocytes (ESS+hM-D). Pigmentation of ESS+hM-L and ESS+hM-D increased significantly after grafting; pigmentation levels were significantly different among groups. Mean melanocyte densities in ESS+hM-L and ESS+hM-D were similar to each other and to densities in normal human skin. After 8 weeks in vivo, grafts were irradiated with 135 mJ/cm2 UV; non-UV-treated mice served as controls. UV modestly increased pigmentation in the ESS+hM groups. UV significantly increased CPD levels in ESS-hM, and levels in ESS-hM were significantly greater than in ESS+hM-L or ESS+hM-D. The results demonstrate that light or dark melanocytes in ESS decreased UV-induced DNA damage. Therefore, melanocytes in ESS play a photoprotective role. Protection against UV-induced DNA damage is expected to reduce skin cancer risk in patients grafted with ESS containing autologous melanocytes.

Wound healing on athymic mice with engineered skin substitutes fabricated with keratinocytes harvested from an automated bioreactor.

The Kerator is a computer controlled bioreactor for the automated culture and harvest of keratinocytes that can reduce labor and materials involved in the fabrication of engineered skin substitutes (ESS). Previous studies have shown that the Kerator is comparable to tissue culture flasks by keratinocyte confluence during culture, clonogenic potential of harvested keratinocytes and microanatomy, cell viability, and surface hydration of ESS fabricated with the harvested keratinocytes. In this study, the Kerator and tissue culture flasks were further compared by keratinocyte proliferation in vitro and wound healing after transplantation of ESS to athymic mice. The number of bromodeoxyuridine-positive keratinocytes in ESS fabricated with keratinocytes harvested from Kerator after 2 wk of in vitro maturation was 34 +/- 3 per high power field (hpf) (mean +/- SEM), which was not significantly different from that fabricated with keratinocytes harvested from flasks (34 +/- 1.5 per hpf). Percentage original wound area 6 wk after surgery of ESS fabricated with keratinocytes from the Kerator was 36% +/- 3.3%, which was not significantly different from that of ESS fabricated with keratinocytes from flasks (30% +/- 4.3%). In both cases, 78% (7 of 9) mice transplanted were positive for engraftment of human keratinocytes by direct immunofluorescence for HLA-ABC antigens. These results further confirm that the ESS fabricated with keratinocytes harvested from Kerator and flasks are equivalent in vitro and in vivo. Therefore, use of Kerator for large scale production of ESS can lead to increased availability at reduced cost while maintaining ESS quality for grafting.

Fabrication of Chimeric Hair Follicles for Skin Tissue Engineering.

Fabrication of engineered skin substitutes provides an alternative approach for the treatment of full-thickness burns and other skin injuries. Improving the functionality of current skin substitute models requires incorporation of skin appendages, including hair follicles, sebaceous glands, and sweat glands. In this chapter, methods for generating skin substitutes incorporating chimeric hair follicles are described. Isolation of human keratinocytes, human fibroblasts, and murine dermal papilla cells is first outlined. These cell types are then combined with collagen-glycosaminoglycan (GAG) scaffolds to generate human-murine chimeric grafts which are then grafted to full-thickness surgical wounds in immunodeficient mice. The methods described allow for the generation of a human-mouse follicular structure.

Identification of Merkel cells associated with neurons in engineered skin substitutes after grafting to full thickness wounds.

Engineered skin substitutes (ESS), prepared using primary human fibroblasts and keratinocytes with a biopolymer scaffold, were shown to provide stable closure of excised burns, but relatively little is known about innervation of ESS after grafting. This study investigated innervation of ESS and, specifically, whether Merkel cells are present in healed grafts. Merkel cells are specialized neuroendocrine cells required for fine touch sensation in skin. We discovered cells positive for keratin 20 (KRT20), a general marker for Merkel cells, in the basal epidermis of ESS after transplantation to mice, suggesting the presence of Merkel cells. Cells expressing KRT20 were not observed in ESS in vitro. However, widely separated KRT20-positive cells were observed in basal epidermis of ESS by 2 weeks after grafting. By 4 weeks, these cells increased in number and expressed keratins 18 and 19, additional Merkel cells markers. Putative Merkel cell numbers increased further between weeks 6 and 14; their densities varied widely and no specific pattern of organization was observed, similar to Merkel cell localization in human skin. KRT20-positive cells co-expressed epidermal markers E-cadherin and keratin 15, suggesting derivation from the epidermal lineage, and neuroendocrine markers synaptophysin and chromogranin A, consistent with their identification as Merkel cells. By 4 weeks after grafting, some Merkel cells in engineered skin were associated with immature afferents expressing neurofilament-medium. By 8 weeks, Merkel cells were complexed with more mature neurons expressing neurofilament-heavy. Positive staining for human leukocyte antigen demonstrated that the Merkel cells in ESS were derived from grafted human cells. The results identify, for the first time, Merkel cell-neurite complexes in engineered skin in vivo. This suggests that fine touch sensation may be restored in ESS after grafting, although this must be confirmed with future functional studies.

Collagen VII Expression Is Required in Both Keratinocytes and Fibroblasts for Anchoring Fibril Formation in Bilayer Engineered Skin Substitutes.

The blistering disease recessive dystrophic epidermolysis bullosa (RDEB) is caused by mutations in the gene encoding collagen VII (COL7), which forms anchoring fibrils that attach the epidermis to the dermis. Cutaneous gene therapy to restore COL7 expression in RDEB patient cells has been proposed, and cultured epithelial autograft containing COL7-modified keratinocytes was previously tested in clinical trials. Because COL7 in normal skin is expressed in both fibroblasts and keratinocytes, cutaneous gene therapy using a bilayer skin substitute may enable faster restoration of anchoring fibrils. Hypothetically, COL7 expression in either dermal fibroblasts or epidermal keratinocytes might be sufficient for functional anchoring fibril formation in a bilayer skin substitute. To test this, engineered skin substitutes (ESS) were prepared using four combinations of normal + RDEB cells: (1) RDEB fibroblasts + RDEB keratinocytes; (2) RDEB fibroblasts + normal keratinocytes; (3) normal fibroblasts + RDEB keratinocytes; and (4) normal fibroblasts + normal keratinocytes. ESS were incubated in vitro for 2 weeks prior to grafting to full-thickness wounds in immunodeficient mice. Biopsies were analyzed in vitro and at 1, 2, or 3 weeks after grafting. COL7 was undetectable in ESS prepared using all RDEB cells (group 1), and macroscopic blistering was observed by 2 weeks after grafting in ESS containing RDEB cells. COL7 was expressed, in vitro and in vivo, in ESS prepared using combinations of normal + RDEB cells (groups 2 and 3) or all normal cells (group 4). However, transmission electron microscopy revealed structurally normal anchoring fibrils, in vitro and by week 2 in vivo, only in ESS prepared using all normal cells (group 4). The results suggest that although COL7 protein is produced in engineered skin when cells in only one layer express the COL7 gene, formation of structurally normal anchoring fibrils appears to require expression of COL7 in both dermal fibroblasts and epidermal keratinocytes.

Influence of electrospun collagen on wound contraction of engineered skin substitutes.

The treatment of massive full-thickness burns with engineered skin substitutes has shown promise in clinical trials. The majority of skin substitutes are comprised of fibroblasts and/or keratinocytes on collagen scaffolds, commonly generated by freeze drying which can generate significant structural heterogeneity. Electrospinning may generate collagen scaffolds with greater homogeneity. Skin substitutes were fabricated using either freeze-dried (FD) or electrospun (ES) collagen scaffolds. Cell distribution, proliferation, organization, and maturation were assessed on each scaffold type in vitro, and engraftment and healing of full thickness wounds in athymic mice were tested. In vitro evaluation of freeze-dried collagen skin substitutes (FCSS) and electrospun collagen skin substitutes (ECSS) revealed no significant differences in cell proliferation, surface hydration, or cellular organization between the ECSS and FCSS groups. Both groups exhibited excellent stratification with a continuous layer of basal keratinocytes present at the dermal-epidermal junction. After grafting to full thickness wounds in athymic mice, both skin substitutes had high rates of engraftment: 87.5% in the FCSS group and 100% in the ECSS group. Histological evaluation of wounds revealed that bovine collagen persisted in the wound at week 8 in the FCSS group while no bovine collagen was seen in the ECSS group. At 8 weeks post-grafting, the ECSS grafts were 61.3+/-7.9% original graft area whereas the FCSS grafts were 39.2+/-8.8% original area (p<0.01). These results indicate that ES scaffolds can be used to fabricate skin substitutes with optimal cellular organization and can potentially reduce wound contraction compared to FD scaffolds. These advantages may lead to reduced morbidity in patients treated with skin substitutes fabricated from ES collagen.

Expression of genes encoding antimicrobial proteins and members of the toll-like receptor/nuclear factor-kappaB pathways in engineered human skin.

Skin functions as a first line of defense against microbial invasion. Tissue-engineered cultured skin substitutes (CSS) are used to aid wound closure in massively burned patients, and have been used to facilitate safe and effective wound closure in adult patients with chronic wounds. Although they contain only two cell types at grafting, they can potentially contribute to innate defense against pathogens and stimulation of adaptive immunity. Gene microarrays were used to identify expression in cultured skin of genes involved in innate and adaptive immune responses, and to evaluate the effects of cytokine stimulation on expression levels. Cultured skin expressed multiple antimicrobial protein genes, including human beta defensins 1 and 2 and S100A12. In addition, the antiviral gene APOBEC3G, which was not previously identified in skin, was expressed in CSS and up-regulated by interleukin-1alpha and tumor necrosis factor alpha. Cathelicidin was not expressed in unstimulated CSS, but was induced by cytokine treatment. Further, genes encoding several proinflammatory cytokines and members of the toll-like receptor and nuclear factor kappa B pathways were expressed in CSS, suggesting that cells in CSS can mediate activation of inflammatory responses. The observed expression patterns indicate that engineered human skin utilizes innate defense mechanisms similar to those reported for native skin. Therefore, regulation of these pathways by cytokine stimulation may offer a mechanism for increasing innate immunity in CSS to combat wound infection after grafting onto patients.

Improved barrier function observed in cultured skin substitutes developed under anchored conditions.

BACKGROUND/PURPOSE: The current method of producing cultured skin substitutes (CSS) is focused on providing treatments for severe skin wounds/burns. We have developed a modified growth method to make them more suitable for in vitro product-testing/toxicity-testing purposes. METHOD: CSS grown in Petri dishes were either transferred to Franz diffusion cells on day 5 (modified method) or left in the Petri dish (standard method) and maintained in these environments for the remainder of the growth phase. Mitochondrial metabolism (MTT assay) was measured on days 5, 10 and 14 and histology was studied on days 5, 10 and 14. Barrier function for all tissues was evaluated by transferring them to Franz cells (standard method) and measuring transepidermal water loss (TEWL), 3H2O penetration and 14C-niacinamide permeability on days 7, 14 and 21. RESULTS: CSS grown by the standard and modified methods showed comparable cell viability and tissue morphology. Barrier function, however, was markedly improved in CSS grown by the modified method. The average improvement at days 7 and 14 was 1.3-fold for TEWL, 2.1-fold for 3H2O penetration and 6.4-fold for 14C-niacinamide permeability. The barrier function of CSS grown by the modified method was still significantly lower than that of human cadaver skin tested by the same methods. CONCLUSIONS: CSS developed using the anchored multi-cell system showed similar cell viability and morphology and improved barrier function compared with CSS produced by the standard Petri dish method, thereby improving its potential as an in vitro skin permeability and toxicity model.

Tissue engineering of skin and regenerative medicine for wound care.

Engineering of biologic skin substitutes has progressed over time from individual applications of skin cells, or biopolymer scaffolds, to combinations of cells and scaffolds for treatment, healing, and closure of acute and chronic skin wounds. Skin substitutes may be categorized into three groups: acellular scaffolds, temporary substitutes containing allogeneic skin cells, and permanent substitutes containing autologous skin cells. Combined use of acellular dermal substitutes with permanent skin substitutes containing autologous cells has been shown to provide definitive wound closure in burns involving greater than 90% of the total body surface area. These advances have contributed to reduced morbidity and mortality from both acute and chronic wounds but, to date, have failed to replace all of the structures and functions of the skin. Among the remaining deficiencies in cellular or biologic skin substitutes are hypopigmentation, absence of stable vascular and lymphatic networks, absence of hair follicles, sebaceous and sweat glands, and incomplete innervation. Correction of these deficiencies depends on regulation of biologic pathways of embryonic and fetal development to restore the full anatomy and physiology of uninjured skin. Elucidation and integration of developmental biology into future models of biologic skin substitutes promises to restore complete anatomy and physiology, and further reduce morbidity from skin wounds and scar. This article offers a review of recent advances in skin cell thrapies and discusses the future prospects in cutaneous regeneration.