Assessing Chondrogenesis of Chondroprogenitors and Chondrocytes on Trilaminar Electrospun Scaffolds

Abstract

between the soft tissue components and the neurovascular bundle that accompany them. Functional movements rely on mechanical and sensory innervation. This is a challenging process especially in engineered tissue. The hypothesis of this study is that sensory innervation will follow the path developed by neoangiogenesis of the grafted engineered oral mucosal tissue; ex-vivo produced oral mucosa equivalents (EVPOME). EVPOMEs have already been shown to develop neoangiogenesis when implanted to SCID (severely compromised immunodeficient) mice. Reconstructed tissue that achieves anatomical and functional, including sensory, restorability will greatly influence the success of the graft and the overall quality of life. Materials and Methods: Thirty mice were used for subcutaneous implantation of the grafts (18 with cellular EVPOMEs and 12 with AlloDerm for control). Every month for a period of six months five mice (3 with cellular EVPOMEs, 2 with AlloDerm as control) were harvested and samples of EVPOME taken for immunohistochemistry (IHC) and routine histology. Antibodies to NeuNwereused duringthefirstthreemonthsasamarker of early innervation process. The last three months we used antibodies to Neurofilament as an indication of terminal neuronal differentiation. Throughout all six months antibody to CD31 was used simultaneously, which is specific to angiogenesis to show that innervation follows angiogenesis. Methods of Data Analysis: IHC assays of the EVPOMEs were analyzed using a data analysis program specifically written for our study, which looked at the images of slides prepared by the histology core. The program was designed to count the number of cell signals that reach a specific threshold and to analyze the quality of cell signals from the above-listed antibodies. Distances between blood vessel and nerve cells were measured, looking for significance compared to a random disbursement of the signals throughout the slide. Measurement of significance inthesedistanceshelpeddeterminewhether or not the nerve growth followed a similar path as prior angiogenesis into the grafted tissue. Results: Preliminary studies have shown detection of early stage neuronal precursor cell marker, NeuN, from as early as 4 weeks post-implant, suggesting the development of nerve growth in concert with microvascular ingrowth into the grafted EVPOMEs. Conclusions: This study will provide insight into the development and relationship between two processes ‐ angiogenesis and sensory innervation ‐ in full-thickness oral mucosa grafts. A better understanding of the engineered tissue willhelphighlight clinicalapplications, limitations, and expectations of its usage. Reconstructed tissue that achieves anatomical and functional, including sensory, restorability will greatly influence the success of the graftand the overallqualityof life.This studymayalso