Sensory Innervation follows Angiogenesis in Grafted Tissue-Engineered Oral Mucosa
Sam S. Bae, Joseph P. Long, Igor Makovey, Shiuhyang Kuo, Stephen E. Feinberg
Statement of the Problem
Partial or complete loss of the lips from trauma or surgery debilitates individuals from carrying out daily functional activities and social gestures. Reconstruction of the lips is particularly difficult and often limited due to the intricate relationship 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 NeuN were used during the first three months as a marker 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 in these distances helped determine whether 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 will help highlight clinical applications, limitations, and expectations of its usage. Reconstructed tissue that achieves anatomical and functional, including sensory, restorability will greatly influence the success of the graft and the overall quality of life. This study may also allow discovery of methods that could possibly alter the rate of innervation and progression. By better understanding the process of re-establishing sensory innervation to engineered tissues in the oral and maxillofacial region, it can help guide reconstructive processes for each individual patient in future clinical application.
References
1. Peramo, A., Marcelo, C.L., Feinberg, S.E. Tissue engineering of lips and muco-cutaneous junctions: in vitro development of tissue engineered constructs of oral mucosa and skin for lip reconstruction. J of Tissue Engineering 18, 4, 2012.
2. Izumi, K., Song, J., and Feinberg, S.E. Development of a tissue engineered human oral mucosa: from the bench to the bed side. Cells Tissues Organs 176, 134, 2004.