Characterization of heterogeneous skin constructs for full thickness skin regeneration in murine wound models.

An autologous heterogeneous skin construct (AHSC) has been developed and used clinically as an alternative to traditional skin grafting techniques for treatment of cutaneous defects. AHSC is manufactured from a small piece of healthy skin in a manner that preserves endogenous regenerative cellular populations. To date however, specific cellular and non-cellular contributions of AHSC to the epidermal and dermal layers of closed wounds have not been well characterized given limited clinical opportunity for graft biopsy following wound closure. To address this limitation, a three-part mouse full-thickness excisional wound model was developed for histologic and macroscopic graft tracing. First, fluorescent mouse-derived AHSC (mHSC) was allografted onto non-fluorescent recipient mice to enable macroscopic and histologic time course evaluation of wound closure. Next, mHSC-derived from haired pigmented mice was allografted onto gender- and major histocompatibility complex (MHC)-mismatched athymic nude mouse recipients. Resulting grafts were distinguished from recipient murine skin via immunohistochemistry. Finally, human-derived AHSC (hHSC) was xenografted onto athymic nude mice to evaluate engraftment and hHSC contribution to wound closure. Experiments demonstrated that mHSC and hHSC facilitated wound closure through production of viable, proliferative cellular material and promoted full-thickness skin regeneration, including hair follicles and glands in dermal compartments. This combined macroscopic and histologic approach to tracing AHSC-treated wounds from engraftment to closure enabled robust profiling of regenerated architecture and further understanding of processes underlying AHSC mechanism of action. These models may be applied to a variety of wound care investigations, including those requiring longitudinal assessments of healing and targeted identification of donor and recipient tissue contributions.

Evaluation in a porcine wound model and long-term clinical assessment of an autologous heterogeneous skin construct used to close full-thickness wounds.

Acute and chronic wounds involving deeper layers of the skin are often not adequately healed by dressings alone and require therapies such as skin grafting, skin substitutes, or growth factors. Here we report the development of an autologous heterogeneous skin construct (AHSC) that aids wound closure. AHSC is manufactured from a piece of healthy full-thickness skin. The manufacturing process creates multicellular segments, which contain endogenous skin cell populations present within hair follicles. These segments are physically optimized for engraftment within the wound bed. The ability of AHSC to facilitate closure of full thickness wounds of the skin was evaluated in a swine model and clinically in 4 patients with wounds of different etiologies. Transcriptional analysis demonstrated high concordance of gene expression between AHSC and native tissues for extracellular matrix and stem cell gene expression panels. Swine wounds demonstrated complete wound epithelialization and mature stable skin by 4 months, with hair follicle development in AHSC-treated wounds evident by 15 weeks. Biomechanical, histomorphological, and compositional analysis of the resultant swine and human skin wound biopsies demonstrated the presence of epidermal and dermal architecture with follicular and glandular structures that are similar to native skin. These data suggest that treatment with AHSC can facilitate wound closure.

Physical characterization of swine and human skin: Correlations between Raman spectroscopy, Tensile testing, Atomic force microscopy (AFM), Scanning electron microscopy (SEM), and Multiphoton microscopy (MPM).

BACKGROUND: Swine dorsum is commonly utilized as a model for studying skin wounds and assessment of dermatological and cosmetic medicaments. The human abdomen is a common location for dermatological intervention. OBJECTIVE: This study provides a correlation between spectral, mechanical, and structural characterization techniques, utilized for evaluating human abdominal skin and swine dorsum. METHODS: Raman spectroscopy (RS), tensile testing, ballistometry, AFM, SEM, and MPM were utilized to characterize and compare full-thickness skin properties in swine and human model. RESULTS: RS of both species' skin types revealed a similar assignment of vibrations in the fingerprint and the high wavenumber spectral regions. Structural imaging and mechanical characterization using ballistometry and tensile testing displayed differences in the inherent functional properties of human and swine skin. These differences correlated with variations in the Raman peak ratios, collagen intensity measured using SEM and MPM and collagen density measured using AFM. CONCLUSION: A comprehensive evaluation of swine skin as a suitable substitute for human skin for mechanical and structural comparisons was performed. This data should be considered for better understanding the swine skin model for cutaneous drug delivery and wound applications. Additionally, correlation between RS, tensile testing, AFM, SEM, and MPM was performed as skin characterization tools.