455. Genetically Engineered Adult Stem Cells and Hybrid Scaffolds as a Platform for Intervertebral Disc Regeneration

Abstract

Current therapeutic solutions for intervertebral disc (IVD) degeneration include prosthetic implants, which fail in the long term. Adult mesenchymal stem cells (MSCs), progenitor cells that give rise to chondroblasts, tenocytes, and osteoblasts, are excellent candidates for tissue engineering of skeletal tissues. We have previously shown that MSCs overexpressing the Brachyury or the Smad8/BMP-2 genes induced cartilage and tendon formation respectively. Therefore, we hypothesized that genetically engineered MSCs combined with a hybrid of polymeric scaffolds could be used to replace a degenerative IVD. A hybrid of fibrin gel and a filamentous Poly Lactide-Glycolide acid (PLGA) scaffold were molded into the shape of a rat IVD (1a). 1X106 MSCs over expressing the Brachyury transcription factor were suspended in the fibrin gel, forming the center part of the construct. 3X106 MSCs expressing the Smad8/BMP-2 genes were seeded on the PLGA, forming the outer ring of the construct. Grafts were implanted subcutaneously in C3H/HeN mice or used to replace caudal discs of Nude rats. The cells were also labeled with Luciferase or GFP gene, which enabled non- invasive monitoring of cell survival using the Roper Chemiluminescence1s Imaging System, or minimally invasively using the Fibered confocal microscopy system (CellVisio). MRI was utilized to compare the engineered disc water content to a native one. Grafts were harvested after three weeks, fixed in 4% formalin and paraffin/plastic embedded. Histological sections were subjected to immunohistology against Collagen I, II, and VI. Our results indicated that an IVD-like tissue was formed subcutaneously and in the IVD space. Smad8/BMP-2 expressing MSCs formed an annulus fibrosus (AF)-like tissue in the outer portion of the graft (1b). MSCs expressing the Brachyury gene gave raise to cartilaginous nucleus pulposus (NP)-like tissue in the center of the construct, positively stained against Collagen II and VI (1c). The cells survived at least two weeks post implantation as was demonstrated by the bioluminescence and the high-resolution fluorescence imaging systems (1d-e). MRI analysis indicated that similar water content was present in the engineered IVD compared to adjacent intact controls. This is the first report demonstrating the use of genetically engineered MSCs for IVD tissue engineering. We conclude, that engineered biological disc composed of genetically engineered MSCs seeded on an IVD-shaped hybrid biodegradable scaffolds can serve as a platform for IVD regeneration. (See Figure 1).