Collaborative Research: 3D-Biofabrication of hASC-based Biomimetic Osteochondral Tissue and the Role of Extracellular Calcium Receptor

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PIs: Loboa, Elizabeth/Shirwaiker, RohanProposals: 1702841/1703466Osteoarthritis (OA)is a degenerative joint disease that limits mobility of the affected joint due to gradual degradation of the cartilage. It currently affects over 25 million people in the US, and these numbers are only expected to rise. Current clinical approaches to cartilage and osteochondral tissue repair are limited by donor site morbidity, tissue scarcity, and poor long term functional outcomes. Engineered tissues can potentially serve as more effective alternatives if they can be designed to mimic native tissue characteristics. This project investigates a new osteochondral tissue engineering strategy using human adipose-derived (fat-derived) stem cells (hASCs) and 3D biofabrication techniques. A 3D construct will be engineered to mimic the structural and biological characteristics of native osteochondral tissue. This construct will be 3D-bioprinted with physiologically-inspired materials that will produce site-specific chondrogenesis and osteogenesis of hASCs. The mechanistic role of the extracellular calcium sensing receptor in regulating hASC differentiation, control that is critical for such an engineered construct, will be also be studied. The characteristics of the engineered construct will be assessed using in vitro methods as well as a proof-of-concept study using a miniature pig model. The ability to create such a hASC-based biomimetic tissue transplant can lead to a paramount clinical advancement for osteoarthritis treatment. The knowledge about how the calcium sensing receptor(CaR)influences stem cell differentiation can be utilized to develop engineered tissue alternatives for other musculoskeletal tissues and regenerative medicine applications. This project will also directly support the multidisciplinary education of a post-doctoral scholar, and graduate and undergraduate students at the partnering institutions. The PIs will continue their focus and efforts in recruiting students from groups underrepresented in STEM. The learnings and outcomes from the studies will be incorporated in multiple undergraduate and graduate courses, and used during outreach activities including university open houses and summer camps. Important findings will be disseminated through scholarly journal publications and at local, national and international meetings.The project focuses on investigating a new 3D tissue engineering approach with possible translation to OA treatment. Specifically, a novel 3D bioprinted quadriphasic construct will be engineered using physiologically inspired chemical cues to induce site-specific differentiation of human adipose-derived stem cells in a manner that recapitulates the depth dependent compositional and architectural heterogeneity of osteochondral tissue varying from the depth dependent compositional and architectural heterogeneity of osteochondral tissue varying from the dense surface layer of cartilage to the porous subchondral one, which is crucial to its function during load bearing. The mechanistic role of the extracellular calcium sensing receptor in regulating the osteogenic and chondrogenic lineage specification of hASC, controlling of which is critical for this engineered construct is also being investigated. To enable the advancement of fundamental science while ensuring future clinical relevance, a synergistic approach utilizing in vitro methods and an in vivo model will be used to achieve project goals. The primary hypothesis is that gradients of the bioprinted beta tricalcium phosphate (TCP) and decellularized articular cartilage extracellular matrix (dECM) hydrogel with optimized stand-pore architecture will induce site-specific hASC osteogenesis and chondrogenisis, with the CaR playing a central role in this lineage specification. This hypothesis will be tested by completing the following specific aims: 1) Investigate and characterize the effects of TCP and cartilage dECM hydrogel gradients (in polycaprolactone (PCL) based composites) and their 3D-bioplotted strand-pore geometries on site specific osteogenesis and chondrogenesis, respectively, of hASC, 2) Determine the effects of inactivation (siRNA and MPS2143, calcilytic) and activation (Cinacalcet, calcimimetic) of the CaR on hASC response to extracellular Ca2+. In particular, evaluate the ability of the CaR to regulate hASC osteogenesis as well as chondrogenesis, 3) Assess in vivo site-specific hASC osteo- and chondrogenic differentiation and integration of the optimized full-thickness quadriphasic construct using a porcine osteochondral defect model and surgical approaches and tools used in human surgeries (proof-of-concept pilot study). The planned approach overcomes many of the limitations of current clinical approaches that fail to mimic native tissue heterogeneity. The project uses multidisciplinary methods from manufacturing, biomedical engineering and clinical sciences to develop an understanding of the role of extracellular Ca2+ and the CaR in the differentiation of hASC, and represents a new approach to creating a multiphasic biomimetic tissue with the potential for clinical translation. From a tissue design and manufacturing perspective, the project will provide new knowledge about the material-process-structure interactions in the 3D bioplottig of PCL-TCP/dECM composite constructs. From a clinical science perspective, understanding and elucidating the role of the CaR in regulation of the effects of extracellular Ca2+ on hASC lineage specification will lead to new understanding of how pharmacological inhibition of activation of the CaR could be used to optimize hASC based approaches to engineer bone and/or cartilage gradients at desired tissue interfaces (e.g. cartilage, ligament.). Successful osteochondral TE using hASC will allow for autologous tissue transplantation using a patient's abundant ASC. The ability to place an hASC-induced transplant that replicates full thickness osteochondral tissue will be a paramount clinical advancement for chondral and osteochondral defect repair. Understanding the mechanistic role of the CaR in hASC lineage specification will provide critical knowledge for utilization of hASC in other musculoskeletal TE and regenerative medicine applications.