Understanding of the regulation of growth, differentiation and mineralization in skeletal tissues is key to developing effective therapies for calcific diseases. A key system critical to bone growth and fracture repair is growth plate (GP) cartilage. Expression of local growth factors, and modulation by endocrine agents, plays a critical role in normal development and calcification of GP and fracture-callous cartilage. The ability now to grow cultures of GP chondrocytes in which normal matrix synthesis and mineralization occur, provides and opportunity to explore the effects of these factors in isolation from unknown systemic factors. The long-term objective of this proposal is to utilize this culture system to elucidate the mechanism of biomineralization in GP cartilage and to clarify the role that growth factors and hormones play in regulating GP development. Humoral factors have been selected for study based on their known effects on skeletal cells and GP chondrocytes, and on our recent findings. These include the lipophilic hormones, the calcific hormones, local growth factors, mineralization, when added alone or in combination to primary cultures of avian GP chondrocytes at both pre-and post-confluent stages of culture, will be analyzed using several biochemical and morphological parameters. The three major goals in this proposal are: 1) to study the effect of these factors on GP chondrocytes evaluating their influence on: a) cell growth (total cellular protein, RNA and DNA levels), b) extracellular matrix production (rate of synthesis of collagen and proteoglycans), c) differentiation (expression of alkaline phosphatase activity, type X collagen, the acidic phospholipid-dependent Ca2+ binding proteins (annexins), and their co-localization in the cells and extracellular matrix), and d) mineralization of the cultures (Ca2+ and Pi content and matrix vesicle production). 2) to develop a rapidly mineralizing GP cultures system using a programmed sequence using a combination fracture repair, but also be a paradigm for development of """"""""tissue engineered"""""""" cartilage for replacement in diseased joints. 3) to establish spatial and temporal relationships between key cellular proteins, enzymes, lipids, and mineral ions calcification of this complex tissue. State-of-the-art fluorescent probes will be used to monitor the location of these key entities using powerful new laser confocal microscopes to study their localization in fixed tissues and in living cells with a degree of precision heretofore impossible. These studies should enable major strides toward the goal of understanding the processes occurring during growth plate development, and should uncover critical parameters required for the rational design of treatments to promote cartilage repair and rapid bone fracture healing.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR042359-04
Application #
2006329
Study Section
Orthopedics and Musculoskeletal Study Section (ORTH)
Project Start
1993-12-01
Project End
1998-11-30
Budget Start
1996-12-01
Budget End
1998-11-30
Support Year
4
Fiscal Year
1997
Total Cost
Indirect Cost
Name
University of South Carolina at Columbia
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
111310249
City
Columbia
State
SC
Country
United States
Zip Code
29208
Wu, Licia N Y; Genge, Brian R; Wuthier, Roy E (2008) Analysis and molecular modeling of the formation, structure, and activity of the phosphatidylserine-calcium-phosphate complex associated with biomineralization. J Biol Chem 283:3827-38
Genge, Brian R; Wu, Licia N Y; Wuthier, Roy E (2008) Mineralization of annexin-5-containing lipid-calcium-phosphate complexes: modulation by varying lipid composition and incubation with cartilage collagens. J Biol Chem 283:9737-48
Genge, Brian R; Wu, Licia N Y; Wuthier, Roy E (2007) In vitro modeling of matrix vesicle nucleation: synergistic stimulation of mineral formation by annexin A5 and phosphatidylserine. J Biol Chem 282:26035-45
Wu, L N Y; Genge, B R; Ishikawa, Y et al. (2006) Effects of 24R,25- and 1alpha,25-dihydroxyvitamin D3 on mineralizing growth plate chondrocytes. J Cell Biochem 98:309-34
Wu, Licia N Y; Ishikawa, Yoshinori; Genge, Brian R et al. (2005) Chondrocytes isolated from tibial dyschondroplasia lesions and articular cartilage revert to a growth plate-like phenotype when cultured in vitro. J Cell Physiol 202:167-77
Ishikawa, Y; Genge, B R; Wuthier, R E et al. (1998) Thyroid hormone inhibits growth and stimulates terminal differentiation of epiphyseal growth plate chondrocytes. J Bone Miner Res 13:1398-411
Sauer, G R; Nie, D; Wu, L N et al. (1998) Induction and characterization of metallothionein in chicken epiphyseal growth plate cartilage chondrocytes. J Cell Biochem 68:110-20
Nie, D; Ishikawa, Y; Guo, Y et al. (1998) Inhibition of terminal differentiation and matrix calcification in cultured avian growth plate chondrocytes by Rous sarcoma virus transformation. J Cell Biochem 69:453-62
Nie, D; Ishikawa, Y; Yoshimori, T et al. (1998) Retinoic acid treatment elevates matrix metalloproteinase-2 protein and mRNA levels in avian growth plate chondrocyte cultures. J Cell Biochem 68:90-9
Wu, L N; Wuthier, M G; Genge, B R et al. (1997) In situ levels of intracellular Ca2+ and pH in avian growth plate cartilage. Clin Orthop Relat Res :310-24

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