Perfusion of medium improves growth of human oral neomucosal tissue constructs

Tissue engineering of the oral mucosa may be useful in congenital cleft palate repairs, defects following extirpative oncologic surgery, and periodontal disease. One of the limitations of in vitro growth of oral mucosal constructs is central necrosis of 3-dimensional tissues. We tested the hypothesis that medium perfusion would enhance oral mucosal histogenesis in vitro. Normal human oral keratinocytes were obtained from young to middle-aged adults. Porous 3-dimensional matrices were prepared from collagen and chondroitin sulfate with some crosslinked with glutaraldehyde. Each device was seeded with 5.0 x 10(5) human oral keratinocytes. The seeded matrices were cultured with or without perfusion of medium at 1.3 ml/min. Histologic analysis of samples cultured for 3, 7, or 14 days showed superior viability and proliferation when perfused. At day 7, the average number of cell layers of the neoepithelium of sponges in the perfused culture system (9.4 +/- 1.0) was 88% greater than for the nonperfused culture system (5.0 +/- 0.9, p<0.005). Glutaraldehyde crosslinking did not influence cellular proliferation or the extent of matrix's shrinkage in either culture system. This study shows that medium perfusion enhanced cell viability and proliferation of human oral keratinocytes cultured in porous 3-dimensional matrices.