Capturing the Regenerative Potential of Periodontal Ligament Fibroblasts

The cell population within the periodontal ligament (PDL) tissue is remarkably heterogeneous1. Fibroblasts, a mixed population of cells, are the main cellular component of the PDL and the cell type most often studied for periodontal regeneration. Osteoblasts and osteoclasts are found on the bone side, while fibroblasts, macrophages, undifferentiated adult/mesenchymal stem cells, neural elements, and endothelial cells are found throughout the PDL. Epithelial rests of Malassez cells and cementoblasts are focused near the root surface. PDL tissue also includes loose connective tissue between dense fiber bundles that contain branches of the periodontal blood vessels and nerves2. The complexity of the PDL tissue, with its various cell types and cell progenitor components, explains the challenges involved in therapies to restore tissue following periodontal disease. Cementoblasts, osteoblasts, and endothelial cells must migrate, differentiate, and coordinately interact with a variety of soluble mediators to regenerate the periodontium3. Stem cells located in the PDL tissue are key contributors to this process4. Stem cells in the PDL are important not only for formation and maintenance of the tissue but also for repair, remodeling, and regeneration of adjacent alveolar bone and cementum5. Our laboratory has shown that progenitor cells isolated from PDL tissue by selection with cell surface markers STRO-1+ and CD146+ are capable of differentiating into chondrogenic, osteogenic, and adipogenic phenotypes under appropriate culture conditions6. Immortalized cell lines are genetically modified human cells that proliferate beyond the point at which their original in vivo primary culture counterparts become senescent7. Immortalized PDL cells have been genetically altered, with unknown effects on cellular processes. Also, many immortalized cell lines harbor intraspecies and interspecies cross-contamination. Authentication tests to confirm the identity of immortalized cell lines is now a requirement from funding agencies8. In order to study the regenerative potential of PDL fibroblasts, our laboratory uses primary cells for in vitro studies. Primary PDL cell cultures used at early passages have the advantage of maintaining the rich phenotypic and functional heterogeneity of fibroblasts in the original tissue. Here, we present a detailed method for culturing PDL fibroblasts, based on over 10 years of experience of culturing PDL cells from primary cultures, which has been effective in maintaining the progenitor phenotype of PDL stem cells.