(680b) Three-Dimensional in Vitro Culture Model of Growth Plate Cartilage Using Alginate Hydrogel Scaffolds

The growth plate cartilage of long bones acts as a master regulator of bone growth and ossification during skeletal development. The coordination of growth and mineralization depends on both long- and short-range signals that collectively regulate the rate of chondrocyte maturation. The net result of these regulatory interactions is the formation of a unique tissue architecture that features morphologically and functionally distinct zones of chondrocytes. From least to most mature, these zones are composed of resting, proliferative, prehypertrophic and hypertrophic chondrocytes. Injury or disease that disrupts this architecture often leads to growth defects for which therapeutic interventions are generally not available. A better understanding of the regulatory network that coordinates chondrocyte maturation could greatly expand treatment options. In vitro tissue models are useful tools for elucidating the structure and function of regulatory networks. However, traditional two dimensional (2-D) cell culture models have failed to recapitulate the multi-zonal properties of the growth plate, and these 2-D cultured chondrocytes have shown a propensity to hypertrophy. Therefore, a more accurate model of growth plate cartilage is needed in order to study the development and cellular processes of growth plate chondrocyte maturation. Alginate hydrogels have been utilized in many tissue engineering applications over the past few decades because of biocompatibility, mild polymerization conditions and tunable mechanical properties. In this project alginate hydrogels are used in the design of a novel, three-dimensional (3-D) in vitro culture system to study the development and biology of growth plate cartilage.  In addition to providing a supportive extracellular matrix, this systems allow presentation of specific signaling factors, including PTH/PTHrP signaling and cell matrix attachment ligands, which are two factors identified in genetic studies that inhibit hypertrophy and promote normal cartilage architecture. Therefore, we used our alginate in vitro culture system to test the hypothesis that activation of PTH/PTHrP signaling and presentation of RGD cell attachment ligands reduce hypertrophy and maintain the resting and proliferative zones in culture. We show that mouse growth plate chondrocytes encapsulated in alginate rapidly enter hypertrophy as determined by quantitative PCR (qPCR) analysis of collagen X expression. Individually, treatment with PTH or presentation of RGD cell attachment peptides conjugated to the alginate matrix significantly delays hypertrophy of encapsulated chondrocytes. Together, these data suggest that optimization of PTH signaling and RGD-mediated cell adhesion within alginate cultures will result in an in vitro model of the proliferative and hypertrophic zones of growth plate cartilage.  Future studies will identify other matrix and soluble factors, as well as mechanical and chemical gradients that can result in mimics of other growth plate zones, as steps toward our long-term goal to recapitulate the zonal arrangement and columnar architecture of growth plate cartilage in vitro.