Method: A three-dimensional organotypic model for regenerating oral mucosa using immortalized epithelial progenitor cell line (OKF-6/TERT2), was replicated using hBM-MSCs. To obtain a optimal cell seeding density, the various cell numbers (1x106 cells/gel, 3x106 cells/gel, 6x106 cells/gel) of cultured hBM-MSCs (passage 2 to 3) were plated separately on the top of collagen/3T3 fibroblast gels, and then cultured in α-MEM + 15% FBS for 7 days. The gels were then cultured for an additional 15 days at the air-liquid interface with an “airlift culture medium” containing a 3:1 mix of DMEM and Ham’s F-12 supplemented with 5% FBS, hydrocortisone, T3, cholera toxin, adenine, insulin, and transferrin. To visualize histological structure, paraffin sections of the gel matrices were stained with H&E and viewed using light microscopy.
Result: Histological analysis indicated that 3x106 cultured MSCs (P2) per gel was optimal for MSC proliferation and the formation of multiple layers as compared to the other cell seeding densities. In comparison to native oral mucosa, the aggregation of hBM-MSCs did not exhibit the distinct layers of epithelium (i.e. stratum basale, spinosum, granulosum, corneum).
Conclusion: Human BM-MSCs can proliferate and form a multilayered cell-stratified structure using a three-dimensional organotypic model of the oral mucosa. Although the structure was stratified, MSCs had not been fully differentiated into epithelial cells as compared to the typical histological structure of native full thickness human oral mucosa, because the intermediate layers were missing. An appropriate combination of the differentiation culture conditions will be investigated for further inducing these multilayered MSCs to form into the native full thickness human oral mucosa.
Keywords: Bioengineering, Epithelium/epithelial and Tissue engineering