Engineering Osteogenesis with Hypertrophic Chondrocytes in Tissue-engineered Cartilaginous Template for Rudimentary Bone Formation

Description

The general objective of this project is to implement and study postnatal osteogenesis by natural hypertrophic chondrocytes (HCCs) associated with osteogenic progenitor cells (OPCs) in a tissue-engineered cartilaginous template for rudimentary bone formation. The ultimate goal is to develop viable and vascularized bone grafts for regenerative purpose. As the functional cells, HCCs are hypothesized and attempted to play a central role executing osteogenesis with or without involving OPCs. As an artificial osteogenic niche, a novel decellularized, tissue-engineered cartilaginous template that is made of pure hyaline cartilaginous extracellular matrices (ECMs), named dLhCG, will be produced and recellularized with the functional cells to implement postnatal osteogenesis via a pathway similar to endochondral ossification during the primary development. Accordingly, the Specific Objectives are listed as follows. The Objective 1 is to achieve relatively pure and natural population of HCCs from primary chondrocytes via a microfluidic approach. Rather than inducing or producing HCCs from chondrocytes or OPCs via deliberate biochemical treatments such as exposure to particular cytokines or hormones, which result in unnatural and/or already apoptotic HCC-like cells, relatively pure and natural population of HCCs will be sourced from primary chondrocytes harvested from native articular cartilage. The primary chondrocytes will be settled into Passage 0 without further subculture, from which smaller-sized regular (non-hypertrophic) chondrocytes (PCCs) and greater-sized HCCs are critically isolated from each other via a size exclusion cell sorting through an inertial spiral microfluidic approach. The Objective 2 is to achieve rudimentary osteogenesis from the tissue-engineered osteogenic niche made of functional cell-laden cartilaginous template in vitro. The functional cell populations that will be employed to re-cellularize dLhCG template are, respectively: HCCs, marrow derived stromal cells (MSCs) as the OPC, and co-culture of HCCs and MSCs; the Controls are, respectively: PCCs and co-culture of PCCs and MSCs. The Objective 3 is to achieve vascularized rudimentary bone formation from the re-cellularized dLhCG Samples using the chorioallantoic membrane (CAM) of fertilized chicken eggs as a natural bioreactor. To fulfil this Objective and reveal the mechanism, as hypothesized and proposed, the central roles played by HCCs will be investigated with emphasis, including execution of direct osteogenic trans-differentiation, initiation of the co-cultured OPCs’ osteogenesis, induction of vascularization via angiogenesis in situ, and successive degradation of the cartilaginous template – the dLhCG scaffold. In summary, the study in this project can fulfil dual missions of understanding osteogenic biology and developing live bone grafts for regenerative applications.