Comparison of Autologous Full-Thickness Gingiva and Skin Substitutes for Wound Healing

Abstract

Ideally tissue-engineered products should maintain the characteristics of the original tissue. For example, skin represents orthokeratinized epithelium and oral gingiva represents parakeratinized epithelium. The aim of this study was to develop an autologous full-thickness gingiva substitute suitable for clinical applications and to compare it with our autologous full-thickness skin substitute that is routinely used for healing chronic wounds. Autologous full-thickness skin and gingiva substitutes were constructed under identical culture conditions from 3-mm punch biopsies isolated from the upper leg or gingiva tissue, respectively. Both consisted of reconstructed epithelia on acellular dermis repopulated with fibroblasts. To compare the characteristics of the original and reconstructed tissue, differential morphological observations and expression of differentiation markers (keratins 6, 10, and 17 and stratum corneum precursors involucrin, loricrin, and SKALP) were determined. Skin and gingiva substitutes were transplanted onto therapy-resistant leg ulcers or tooth extraction sites in order to determine their effects on wound healing. The tissue-engineered constructs maintained many of the differential histological and immunohistochemical characteristics of the original tissues from which they were derived. The skin substitute was orthokeratinized, and the gingiva substitute was parakeratinized. Transplantation of skin (n = 19) and gingiva substitutes (n = 3) resulted in accelerated wound healing with no adverse effects. As identical culture systems were used to generate both the skin and gingiva substitutes, the differences observed in tissue (immuno)histology can be attributed to intrinsic properties of the tissues rather than to environmental factors (e.g., air or saliva). This study emphasizes the importance of closely matching donor sites with the area to be transplanted. Our results represent a large step forward in the area of clinical applications in oral tissue engineering, which have until now greatly lagged behind skin tissue engineering.

Comparison of advanced therapy medicinal product gingiva and skin substitutes and their in vitro wound healing potentials

“…SS and GS were constructed essentially as described previously (Gibbs et al, 2006; Vriens et al, 2008). For this study, tissue from seven gingiva donors and seven skin donors was received in the culture facility.…”

“…Immunohistochemical staining was performed essentially as described previously (Vriens et al, 2008). For staining of keratin 6, 10, 13, 16, 17, Ki‐67 and vimentin, antigen retrieval was performed by immersion of the slides in 0.01  m citrate buffer (pH 6.0) for 15 min at 100°C, followed by slow cooling to room temperature for 3 h. For loricrin and involucrin, endogenous peroxidase was inhibited by 20 min incubation in 0.3% H 2 O 2 in methanol solution.…”

“…Previously, we described a method for producing an autologous bi‐layered skin (Gibbs et al, 2006) and oral mucosa (gingiva) (Vriens et al, 2008) substitute from very small 3 mm punch biopsies during just a 3 week culture period. The unique method of constructing skin substitutes (SS) and gingival substitutes (GS) involves isolation of the intact epithelial sheet from the biopsy and fibroblasts from the dermis or lamina propria, respectively, followed by proliferation and migration of keratinocytes and melanocytes out of the epithelial sheet over acellular human dermis, and migration of fibroblasts into the dermis (patent no.…”

“…The SS consists of a multilayered orthokeratinized epithelium, whereas the GS consists of a parakeratinized epithelium in which the terminally differentiated cells in the upper layers retained remnants of nuclei. Several keratins, as well as loricrin and involucrin expression, were tissue specific, representative of the original biopsy tissue (Gibbs et al, 2006; Vriens et al, 2008). …”

“…The GS was implemented in a proof‐of‐concept pilot study, closing three gingiva lesions (Vriens et al, 2008). Excellent safety data and promising efficacy data were obtained in both studies.…”