Evaluation of Transplanted Tissue-Engineered Oral Mucosa Equivalents in Severe Combined Immunodeficient Mice

Abstract

The aim of this study was to determine the optimal stage of development at which transplant human ex vivo-produced oral mucosa equivalents (EVPOMEs) in vivo. EVPOMEs were generated in a serum-free culture system, without the use of an irradiated xenogeneic feeder layer, by seeding human oral keratinocytes onto a human cadaveric dermal equivalent, AlloDerm. EVPOMEs were cultured for 4 days submerged and then for 7 or 14 days at an air-liquid interface to initiate stratification before transplantation into SCID mice. AlloDerm, without epithelium, was used as a control. Mice were killed on days 3, 10, and 21 posttransplantation. Epithelium of the transplanted EVPOMEs was evaluated with the differentiation marker keratin 10/13. Dermal microvessel ingrowth was determined by immunohistochemistry with a mouse vascular marker, lectin binding from Triticum vulgaris. The presence and stratification of the epithelium were correlated with revascularization of the underlying dermis. The microvessel density of AlloDerm without epithelium was less than that of EVPOMEs with an epithelial layer. Microvessel density of the dermis varied directly with the degree of epithelial stratification of the EVPOMEs. The EVPOMEs cultured at an air-liquid interface for 7 days had the optimal balance of neoangiogenesis and epithelial differentiation necessary for in vivo grafting. 163

Ex Vivo Produced Oral Mucosa Equivalent by Using the Direct Explant Cell Culture Technique

“…At the end of this study, histological findings of "EVPOME"s development showed the same epithelial architecture as the previous studies performed by Izumi et al (9,25). Also, the epithelial architecture of "EVPOME"s resembled that of normal oral mucosa.…”

Tissue Engineering of Lips and Muco-Cutaneous Junctions: In Vitro Development of Tissue Engineered Constructs of Oral Mucosa and Skin for Lip Reconstruction

“…13 In addition ex vivo produced oral mucosal equivalents have been fabricated 25 and grafted into humans. 26 However, they have been separately implemented and the oral constructs have only been used for intraoral grafting procedures.…”

“…35 Other dermal substitutes for development of nonkeratinized oral mucosa constructs have been reported using collagen-based membranes. 36 The testing of these 3D constructs in vivo could initially be done by grafting the constructs into athymic (nude) rats to allow integration with the underlying striated muscular bed as previously done with mice 25 to evaluate vascular ingrowth. It has been shown in clinical trials that use of the Alloderm Ò as a dermal substitute without the presence of fibroblasts in keratinized oral mucosa repair is successful on transplantation.…”

Biochemical Indicators of Implantation Success of Tissue-Engineered Oral Mucosa

“…Surgical procedures in mice were detailed in a previous article (Izumi et al 2003). Briefly, each batch of control and stressed EVPOMEs was grafted into 7-to 8-wk-old SCID mice (Charles River Laboratories, Wilmington, MA) for 1 wk.…”