Construction and Characterization of Human Oral Mucosal Equivalent Using Amniotic Membrane as a Matrix
Fangfang Qi, Tetsu Shimane, Hitoshi Aizawa, Yinghui Li, Hiroshi Kurita
Department of Oral and Maxillofacial Surgery, Shinshu University School of Medicine
Statement of the problem
A clinical need exists for an immunologically compatible surgical patch for the use of soft tissue replacement, body wall repair, and as a wound dressing. Amniotic membrane is a thin light-weight and elastic material and it can promote cell attachment, proliferation and differentiation1. Hyper-dry amniotic membrane as a natural extracellular matrix is a novel dried amniotic, can be preserved at room temperature2and has been used to culture epithelial cell for transplantation. In this study, we made human oral mucosal equivalent using hyper-dry amniotic membrane as a matrix. The cultured oral mucosal equivalent was transplanted in mouse full-thickness skin defect, the wound contraction and characterization of the grafted material was evaluated.
Materials and methods
Gingival mucosa was obtained from wisdom teeth extraction patient. Keratinocytes extracted from gingival mucosa and developed in a serum-free culture system without a feeder layer. Oral keratinocytes were seeded and cultured on hyper-dry amniotic membrane (HAM, Toyama University) for two weeks. Full-thickness skin wounds (1 cm x 1 cm) were made on athymic mice (BALB/C) and covered with the oral mucosal equivalent cultured on hyper-dry AM (OME on HAM), with the oral mucosal equivalent cultured on artificial membrane (MillcellR culture plate insert) , and with no material offers as control. 3 weeks after grafting, constriction of wound and characterization of grafted material were examined with hematoxylin and eosin and immunohistochemcial staining for ck10, ck1, and involucrin.
Results
By two weeks that Keratinocytes cultured on HAM revealed 4 to 5-layered well-organized stratified epithelium on the surface. Presence of ck10, ck1 in all suprabasal layers , While involucrin was presence in all cell layers.
By three weeks of mice grafting with OME on HAM, the regenerated oral mucosal showing a good morphology which is similar to gingival mucosa and the epithelial cells expressed ck10, ck1 and involucrin in supra-basal layers is similar to that of gingival mucosa.
Concerning wound contraction, OME on HAM showed a smallest contraction rate (87.97 ± 2.39 % with OME on HAM, 89.11±1.66%with OME on MillcellR, 91.23±2.1% in control).
Conclusions
Hyperdry AM have sufficient elasticity and can be cut in a suitable shape easily. This study demonstrated that use Hyper-dry AM as a matrix developed oral mucosal equivalent revealed a good morphology and contribute to a stable wound healing.
References
1. Lujun Yang, Yuji Shirakata, Sho Tokumara. Living skin equivalents constructed using human amnions as a matrix. Journal of Dermatological Science 56, (2009) 188-195
2. Ayaka Toda, Motonori Okabe, Toshiko Yoshida. The potential of amniotic membrane/amnion-derived cells for regeneration of Various Tissues. Journal of Pharmacological sciences. 105, (2007) 215-228