The repair of cartilage disorder with parathyroid hormone related peptide.

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Parathyroid hormone-related peptide (PTHrP) was originally identified as a causative agent of hypercalcemia associated with malignancy. PTHrP binds to and activates the same receptor as PTH does. Mice missing both copies of the PTHrP or the PTH/PTHrP receptor gene die immediately after birth, and exhibit a multitude of skeletal abnormalities. The chondrodysplastic phenotype in both PTHrP-less, and PTH/PTHrP receptor-less mice clearly demonstrates important physiological roles of PTHrP and its receptor in regulating endochondral bone development. Consistent with the data obtained with these mutant mice, PTH has been shown to exert mitogenic effects on chondrocytes isolated from fetal cartilage. Contrary to the stimulatory effects on the proliferation of fetal chondrocytes, it has been shown that PTH does not have mitogenic effects on chondrocytes isolated from post-natal animals. The goal of this study is to test the hypotheses that PTHrP and its receptor play a role (s) in regulating g … More rowth of growth-plate and articular cartilage in postnatal animals, and that PTHrP is involved in disease processes that affter the cartilage. We examined the effects of various analogues of PTH/PTHrP including C-terminal peptides on the growth of postnatal growth-plate and articular chondrocytes in serum free culture, and then we examined PTHrP expression in cartilage from patients with osteoarthritis and rheumatoid arthritis.Materials and Methods : Rabbits chondrocytes were isolated from growth plates of ribs and articular cartilage of knee joints of 4-wk-old rabbits by trypsin and collagenase digestion. Chondrocytes were seeded at 10,000 per type I collagen coated 96-well plates, and maintained in alpha MEM supplemented with egg lipoprotein (20mug/ml), transferrin (10mug/ml), hydrocortisone (10-8M), bFGF (ing/ml) and insulin (10mug/ml) under 5% CO2 in air at 37*C.When cultures became preconfluent, the cells were preincubated for 24 h in same medium with above factors except insulin. We then measured [3H] thymidine incorporation into DNA after the additions of PTH [1-34] or other fragments. We also measured DNA contents, [35S] sulfate incorporation into proteoglycans (PG) in same condition. Furthermore, cartilages obtained at total knee arthroplasties were examined for localization of PTHrP by immunostaining (n=8).Results : PTH [ 1-34], PTHrP [1-34] and PTH [1-84] dramatically increased DNA synthesis by postnatal articular chondrocytes dose-dependently with an ED 100 of 10-7M.Furthermore, PTH/PTHrP increased the DNA content of articular chondrocytes and [35S] sulfate incorporation into PG by growth plate and articular chondrocytes. Immuno-histochemically PTHrP was detected in chondrocytes in affected cartilage with high ratio (6/8).Discussion : In several reports, PTH/PTHrP showed little effect on DNA synthesis by postnatal chondrocytes in cell cultures, probably because the effect of PTH/PTHrP may be masked by 10% fetal calf serum included in conventional culture systems. In this study we could demonstrate that PTH/PTHrP peptide stimulates the growth of postnatal chondrocytes in chemically defined medium. This mitogenic effect of PTH/PTHrP is more prominent in articular chondrocytes. PTHrP is detected immunohistochemically in cartilage from patients with both osteoarthritis and rheumatoid arthritis. Although several explanations are plausible for these results, more investigations on receptor expression or post receptor signaling pathway will be necessary. Less