Formation of Organized Periodontal Tissues Using Scaffold-Free Tissue Engineering

Objectives:
A major challenge in regenerating periodontal tissues is emulating its complex structure containing both mineralized tissues and ligament. The goal of this study was to determine if a multi-tissue construct resembling periodontium could be formed from human periodontal ligament stem/progenitor cells (PDLCs) using scaffold-free tissue engineering. Scaffold-free tissue engineering allows cells to generate and organize a 3D structure without exogenous scaffold material. Previously, scaffold-free tissue engineering has facilitated the formation of spatially organized multi-tissue constructs. Here we hypothesized that scaffold-free tissues engineered from PDLCs would result in a multi-tissue construct containing multiple organized tissues of the periodontium.

Methods: Scaffold-free 3D tissues were engineered from human PDLCs isolated from human third molars. PDLCs were cultured in osteogenic differentiation media. After the cells became confluent, they contracted their tissue monolayer around two constraint points and formed a scaffold-free cylindrical tissue construct. The resulting constructs were sectioned and histologically characterized using hematoxylin and eosin (H&E) staining, alizarin red staining to detect mineralization, and immunostaining against bone sialoprotein (BSP) as a marker of osteogenic cell differentiation and scleraxis as a marker of ligament formation.
Results: H&E staining showed that the constructs formed from PDLCs were solid and cellular. Positive alizarin red staining and the expression of BSP was localized to the center of the construct indicating the formation of mineralized tissue. Strong scleraxis expression was detected on the periphery of the construct demonstrating formation of ligament.
Conclusions: Scaffold-free tissue engineering using PDLCs resulted in spatially organized multi-tissue constructs with a mineralized bone/cementum core and a periodontal ligament periphery. These results are significant because the generated constructs can be used as a regenerative therapy to treat periodontal disease or serve as a functional model to study mechanisms of tissue patterning in an engineered tissue.