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3D biomaterial matrix to support long term, full thickness, immuno-competent human skin equivalents with nervous system components.

ABSTRACT: Current commercially available human skin equivalents (HSEs) are used for relatively short term studies (?1 week) due in part to the time-dependent contraction of the collagen gel-based matrix and the limited cell types and skin tissue components utilized. In contrast, here we describe a new matrix consisting of a silk-collagen composite system that provides long term, stable cultivation with reduced contraction and degradation over time. This matrix supports full thickness skin equivalents which include nerves. The unique silk-collagen composite system preserves cell-binding domains of collagen while maintaining the stability and mechanics of the skin system for long-term culture with silk. The utility of this new composite protein-based biomaterial was demonstrated by bioengineering full thickness human skin systems using primary cells, including nerves and immune cells to establish an HSE with a neuro-immuno-cutaneous system. The HSEs with neurons and hypodermis, compared to in vitro skin-only HSEs controls, demonstrated higher secretion of pro-inflammatory cytokines. Proteomics analysis confirmed the presence of several proteins associated with inflammation across all sample groups, but HSEs with neurons had the highest amount of detected protein due to the complexity of the model. This improved, in vitro full thickness HSE model system utilizes cross-linked silk-collagen as the biomaterial and allows reduced reliance on animal models and provides a new in vitro tissue system for the assessment of chronic responses related to skin diseases and drug discovery.

SUBMITTER: Vidal SEL

PROVIDER: S-EPMC6200656 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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3D biomaterial matrix to support long term, full thickness, immuno-competent human skin equivalents with nervous system components.

Vidal Sarah E Lightfoot SEL Tamamoto Kasey A KA Nguyen Hanh H Abbott Rosalyn D RD Cairns Dana M DM Kaplan David L DL

Biomaterials 20180424

Current commercially available human skin equivalents (HSEs) are used for relatively short term studies (∼1 week) due in part to the time-dependent contraction of the collagen gel-based matrix and the limited cell types and skin tissue components utilized. In contrast, here we describe a new matrix consisting of a silk-collagen composite system that provides long term, stable cultivation with reduced contraction and degradation over time. This matrix supports full thickness skin equivalents whic ...[more]

PMID: 29709325

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Project description:Over 1 million burn injuries are treated annually in the United States, and current tissue engineered skin fails to meet the need for full-thickness replacement. Bioprinting technology has allowed fabrication of full-thickness skin and has demonstrated the ability to close full-thickness wounds. However, analysis of collagen remodeling in wounds treated with bioprinted skin has not been reported. The purpose of this study is to demonstrate the utility of bioprinted skin for epidermal barrier formation and normal collagen remodeling in full-thickness wounds. Human keratinocytes, melanocytes, fibroblasts, dermal microvascular endothelial cells, follicle dermal papilla cells, and adipocytes were suspended in fibrinogen bioink and bioprinted to form a tri-layer skin structure. Bioprinted skin was implanted onto 2.5 × 2.5 cm full-thickness excisional wounds on athymic mice, compared with wounds treated with hydrogel only or untreated wounds. Total wound closure, epithelialization, and contraction were quantified, and skin samples were harvested at 21 days for histology. Picrosirius red staining was used to quantify collagen fiber orientation, length, and width. Immunohistochemical (IHC) staining was performed to confirm epidermal barrier formation, dermal maturation, vascularity, and human cell integration. All bioprinted skin treated wounds closed by day 21, compared with open control wounds. Wound closure in bioprinted skin treated wounds was primarily due to epithelialization. In contrast, control hydrogel and untreated groups had sparse wound coverage and incomplete closure driven primarily by contraction. Picrosirius red staining confirmed a normal basket weave collagen organization in bioprinted skin-treated wounds compared with parallel collagen fibers in hydrogel only and untreated wounds. IHC staining at day 21 demonstrated the presence of human cells in the regenerated dermis, the formation of a stratified epidermis, dermal maturation, and blood vessel formation in bioprinted skin, none of which was present in control hydrogel treated wounds. Bioprinted skin accelerated full-thickness wound closure by promoting epidermal barrier formation, without increasing contraction. This healing process is associated with human cells from the bioprinted skin laying down a healthy, basket-weave collagen network. The remodeled skin is phenotypically similar to human skin and composed of a composite of graft and infiltrating host cells. Impact statement We have demonstrated the ability of bioprinted skin to enhance closure of full-thickness wounds through epithelialization and normal collagen remodeling. To our knowledge, this article is the first to quantify collagen remodeling by bioprinted skin in full-thickness wounds. Our methods and results can be used to guide further investigation of collagen remodeling by tissue engineered skin products to improve ongoing and future bioprinting skin studies. Ultimately, our skin bioprinting technology could translate into a new treatment for full-thickness wounds in human patients with the ability to recapitulate normal collagen remodeling in full-thickness wounds.

Project description:BackgroundFull thickness burn wounds are lack of angiogenesis, cell migration, epithelialisation and finally scar tissue formation. Tissue engineered composite graft can provide sustained release of growth factor and promote the wound healing by cell migration, early angiogenesis and proliferation of extracellular matrix and wound remodeling. The objective of this study was to evaluate the gene embedded (pDNA-platelet-derived growth factor, PDGF-B) porcine acellular urinary bladder matrix with transfected mesenchymal stem cells (rBMSC) on healing of full thickness burn wound in rat model.MethodsFull thickness burn wound of 2 × 2 cm size was created in dorsum of rat model under general anesthesia. Burn wounds were treated with silver sulfadiazine; porcine acellular urinary bladder matrix (PAUBM); PAUBM transfected with pDNA-PDGF-B; PAUBM seeded with rBMSC; PAUBM seeded with rBMSC transfected with pDNA-PDGF-B in groups A, B, C, D and E respectively. The wound healing was assessed based on clinical, macroscopically, immunologically, histopathological and RT-qPCR parameters.ResultsWound was significantly healed in group E and group D with early extracellular matrix deposition, enhanced granulation tissue formation and early angiogenesis compared to all other groups. The immunologic response against porcine acellular matrix showed that PDGF-B gene activated matrix along with stem cell group showed less antibody titer against acellular matrix than other groups in all intervals. PDGF gene activated matrix releasing the PDGF-B and promote the healing of full thickness burn wound with neovascularization and neo tissue formation. PDGF gene also enhances secretion of other growth factors results in PDGF mediated regenerative activities. This was confirmed in RT-qPCR at various time intervals.ConclusionGene activated matrix encoded for PDGF-B protein transfected stem cells have been clinically proven for early acceleration of angiogenesis and tissue regeneration in burn wounds in rat models. Evaluation of PDGF-B gene-activated acellular matrix and mesenchymal stem cell in full thickness skin burn wound in rat.

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3D biomaterial matrix to support long term, full thickness, immuno-competent human skin equivalents with nervous system components.

Publications

3D biomaterial matrix to support long term, full thickness, immuno-competent human skin equivalents with nervous system components.

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