The overall scientific mission of the Cartilage Biology and Orthopaedics Branch is the study of the biology of cartilage tissues and the application of such knowledge to musculoskeletal and orthopaedic medicine. This project focuses on cartilage development, functional cartilage tissue engineering, the cellular basis of orthopaedic implant stability, physical influences on skeletal tissue function, and the biology of skeletal tissue injury repair. There are five interrelated parts to the project:? ? (1) Cellular and Molecular Mechanisms of Chondrogenesis. Using embryonic skeletal mesenchymal cells and multipotent embryonic mesenchymal cell lines, we have analyzed the role of cell-cell and cell-matrix interactions, growth factors and other signaling molecules, and gene expression and regulation events during chondrogenesis. Our studies have analyzed cell membrane, matrix, and signaling molecules, such as N-cadherin, fibronectin, integrins, connexin 43, transforming growth factor beta and bone morphogenetic proteins, fibroblast growth factors, and Wnts. We have also focused on nuclear components that regulate gene transcription, including AP-1, LEF, beta-catenin, and LEF. Results from these studies point to the initial cellular condensation event, and the cross-talk of multiple signaling pathways, as key regulatory events of developmental chondrogenesis. ? ? (2) Adult Mesenchymal Stem Cell Biology. The information gained from these developmental studies are also being applied to the study of adult tissue derived mesenchymal stem cells (MSCs), a highly promising candidate cell type for cartilage tissue engineering and regeneration. Multipotent MSCs are isolated from bone marrow stroma and from adult trabecular bone and studied in vitro for their ability to undergo multi-lineage differentiation along the osteogenic, chondrogenic, and adipogenic pathways. The mechanisms of action of growth factors (e.g. TGF-beta superfamily members), signaling molecules (e.g. Wnts), and hormonal regulators (e.g. glucocorticoids) in the maintenance of their undifferentiated state and lineage commitment are being analyzed. Results point to the correlation between developmental and regenerative tissue morphogenesis, and suggest that these molecules and their down-stream signal mediators are potential targets for gene-based modulation of adult tissue genesis using mesenchymal stem cells. Novel methods have been developed for efficient gene transduction, using electrical field based and nucleofection protocols, to modulate the expression of these key factors and related signaling pathways in MSCs and to examine the effects on cellular differentiation. We are also currently developing clonal MSC cell lines harboring differentiation-specific marker gene constructs as read-out cell systems, e.g. for functional gene cloning applications, as well as gene microarray approaches to profile gene expression profiles during differentiation to identify key regulatory signals. Particularly noteworthy is our recent finding that MSCs possess transdifferentiation potential, underscoring the possibility of identifying ?stemness? regulatory genes.? ? (3) Cell-Based Cartilage Tissue Engineering. We are developing new methods to construct three-dimensional biodegradable scaffolds to seed MSCs under chondrogenic conditions ex vivo for cartilage tissue engineering applications. Specifically, our efforts are targeted towards articular cartilage of the joint, and fibrocartilage of the meniscus and rotator cuff. Several approaches are being investigated: a) development of novel three-dimensional nanofibrous scaffolds consisting of biodegradable polymers produced by electrospinning, and subsequent alignment of the fibrous network; b) application of hydrogels, such as agarose and collagen to encase MSCs and chondrocytes, for subsequent mechano-stimulation of tissue formation and cell-cell interaction. We are also custom-fabricating biomaterial scaffolds to assemble composite tissues of complex architecture. We believe that the knowledge gained from the study of developmental chondrogenesis will be important in designing approaches to modulate the cellular and molecular activities of chondrogenic MSCs in three-dimensional cartilage tissue engineering. Elucidating the biological characteristics of MSCs and how they respond to environmental signals is fundamental to the advancement of tissue engineering. ? ? (4) Cellular Mechanism of Wear Debris Mediated Osteolysis. Our recent studies have also addressed the cellular mechanisms responsible for implant wear debris mediated osteolysis, which is primarily responsible for aseptic implant loosening. Specifically, our results indicate that the presence of titanium particulate debris suppresses osteogenic differentiation and enhances apoptosis in cultures of MSCs in vitro, both of these responses likely contributing to compromised periprosthetic osteogenic tissue response and implant loosening. The identity and involvement of specific cytokines and signaling pathways in the affected cellular events are being investigated, using ex vivo approaches, including testing a tissue analogue to mimic bone-implant interaction in vitro. ? ? (5) Analysis of Physical Influences on Skeletal Biology. Skeletal tissues are uniquely adapted to responding to mechanical influences. We are currently designing mechanoactive bioreactor systems to apply both dynamic and static mechanical stimulation and using MSC-based tissue constructs to analyze the cellular and molecular basis of the biological responses. By varying the nature of the biomaterial scaffold, the mixture of cell types, and the growth factor treatment, we aim to decipher the crosstalk among various signal transduction pathways. We are also applying a mechanoactive bioreactor system for the tendon/ligament tissue engineering.? ? (6) Animal Models of Skeletal Injury Repair and Regeneration. Three models are being investigated: a) the role of GDF5 in mouse tibial fracture repair, b) cellular and molecular analysis of distraction osteogenesis in a mouse model, and c) the pathogenesis of osteoarthritis in a supraphysiological impact-induced cartilage degeneration rabbit model.? ? Information gathered from this project should provide a rational basis to functional skeletal tissue engineering.
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