M. Thavarajah

Studies on type II collagen and aggrecan production in human articular chondrocytes in vitro and effects of transforming growth factor-β and interleukin-1β*

Type II collagen and aggrecan are major components of the extracellular matrix of articular cartilage. Their biosynthesis and catabolism are regulated by chondrocytes. They may be used as markers of chondrocyte phenotype for cells cultured in vitro. Type II collagen gene expression was detected by amplification of type II collagen-specific sequences, using cDNA produced by reverse transcription of mRNA extracted from freshly isolated and cultured human articular chondrocytes by the polymerase chain reaction (PCR). The synthesis of gene product was confirmed by immunohistochemical localization of type II collagen in cartilage sections and in cultured chondrocytes. Aggrecan core protein was also immunolocalized in cartilage sections and in chondrocytes in culture. Expression of type II collagen or aggrecan was not detected immunohistochemically in skin or bone. These results demonstrate that human articular chondrocytes can be characterized in culture, by the combined application of PCR and immunohistochemistry. Interleukin-1beta (IL-1beta) may play an important role in the destruction of cartilage matrix in arthritis, whereas transforming growth factor-beta (TGFbeta) may have an opposing effect and their combined actions may modulate chondrocyte phenotype. The effect of rhIL-1beta and rhTGFbeta on the production of type II collagen by chondrocytes in culture was investigated. It was shown that TGFbeta enhanced the production of type II collagen, localized immunocytochemically, in cultured chondrocytes. IL-1beta inhibited expression of mRNA for type II collagen. The implications of this study, in terms of a better understanding of degenerative cartilage disease, are discussed.

Involvement of prostaglandin E2 in the inhibition ofosteocalcin synthesis by human osteoblast-like cells in response to cytokines and systemic hormones

The stimulation of the production of osteocalcin by human osteoblast-like cells in response to 1,25(OH)2D3 is antagonized by several agents that induce the synthesis of prostaglandin E2 (PGE2) including interleukin 1 (IL-1), tumour necrosis factor (TNF) and parathyroid hormone (PTH). The mechanism whereby these agents inhibit the synthesis of osteocalcin is not known. In this report we show that exogenous PGE2 inhibits this stimulatory action of 1,25(OH)2D3 on human osteoblast-like cells in a dose-dependent manner, suggesting that PGE2 may contribute to the inhibition of osteocalcin synthesis in response to these agents. Assessment of the inhibitory role of endogenous PGE2 synthesis in the action of rhIL-1 alpha, rhIL-1 beta and rhTNF alpha on the production of osteocalcin demonstrated that the inhibition by these agents could be partially overcome by the addition of indomethacin, an inhibitor of PGE2 synthesis. In contrast, the inhibitory action observed with bPTH (1-84) was unaffected by indomethacin. These observations indicate that endogenous PGE2 synthesis mediates, in part, some of the inhibitory actions of the cytokines on the induction of osteocalcin synthesis in response to 1,25(OH)2D3, but not of PTH. Since the antagonism of the synthesis of osteocalcin by rhIL-1 alpha, rhIL-1 beta and rhTNF alpha was not completely abolished following the inhibition of PGE2 synthesis this would indicate that additional PGE2-independent mechanisms also account for the action of these cytokines on osteocalcin production. The nature of these mechanisms is currently not known.

1,25(OH)2D3 INDUCES DIFFERENTIATION OF OSTEOCLAST-LIKE CELLS FROM HUMAN BONE MARROW CULTURES

Multinucleated cells were generated from human bone marrow cultured in the presence of 10(-6)M 1,25(OH)2D3 and 10(-6)M all-trans-retinoic acid for 3-4 weeks. These multinucleated cells have the phenotypic and functional characteristics of osteoclasts as judged by (a) immunostaining with osteoclast specific monoclonal antibodies 13C2 and 23C6 (b) expression of tartrate resistant acid phosphatase, an enzyme marker of osteoclast differentiation; and (c) the ability to resorb bone in vitro. The multinucleated cells appeared to form by fusion of large mononuclear cells. The monoclonal antibodies 13C2 and 23C6 stained 60-90% of the multinucleated cells, and 40-60% of the large mononuclear cells. Tartrate resistant acid phosphatase activity was expressed by 80-95% of the multinucleated cells and 60-80% of the large mononuclear cells. Scanning electron microscopy of bone wafers co-incubated with the multinucleated cells, for 7 days, revealed resorption pits. These findings suggest that in the presence of 1,25(OH)2D3 the marrow cells differentiated into multinucleated and large mononucleated cells in which a proportion of them expressed osteoclast phenotype and resorbed bone.

Immunoreactivity and proliferative actions of β2 microglobulin on human bone-derived cells in vitro

Recent studies have demonstrated homology between bone-derived growth factor and beta 2 microglobulin. We have shown that beta 2 microglobulin has proliferative actions on human bone-derived cells in vitro and that these cells also show immunogenicity for beta 2 microglobulin. beta 2 microglobulin stimulated the incorporation of 3H-thymidine into DNA of human bone cells in a dose-dependent manner. In contrast to this stimulatory action, beta 2 microglobulin had no detectable activity with the same concentration on the production of osteocalcin, alkaline phosphatase activity or prostaglandin E2 synthesis. The possibility that the human bone-derived cells could also produce beta 2 microglobulin was examined. Under basal conditions these cells exhibit immunoreactivity for beta 2 microglobulin, the expression of which could be enhanced following treatment with interferon gamma in a dose-dependent manner. The co-localization of staining for beta 2 microglobulin and alkaline phosphatase, a marker of the osteoblast phenotype, indicate that human osteoblast-like cells represent a source of activity of this factor. The production of beta 2 microglobulin by human osteoblast-like cells and the subsequent action of this factor on cells within the bone microenvironment may indicate a role for beta 2 microglobulin as a local regulator of bone metabolism.

1,25(OH)2D3 and calcipotriol (MC903) have similar effects on the induction of osteoclast-like cell formation in human bone marrow cultures

MC903 is a novel analogue of 1,25(OH)2D3 which exhibits similar inhibitory effects on cell proliferation and like, 1,25(OH)2D3, stimulates synthesis of osteoblast specific proteins by osteoblast-like cells in vitro. It is less active than 1,25(OH)2D3 in causing hypercalcemia in vivo. Since 1,25(OH)2D3 is known to stimulate bone resorption and increase the number of osteoclasts in several systems (in vivo and in vitro) we examined the effects of MC903 on the formation of osteoclast-like cells in vitro. As reported previously 1,25(OH)2D3 promoted the formation of multinucleated cells with phenotypic and functional characteristics of osteoclasts from adult human bone-marrow cultures at concentrations between 10(-8)M to 10(-12)M. Higher doses consistently suppressed multinucleated cell formation to values seen in the absence of 1,25(OH)2D3. Cells cultured in the presence of MC903 or for three weeks consistently induced the formation of multinucleated cells at concentrations 10(-8)M to 10(-12)M. As seen with 1,25(OH)2D3, MC903 also inhibited multinucleated cell formation at very high concentrations (10(-6)M). In two separate experiments MC903 appeared to be more potent than 1,25(OH)2D3 at lower concentrations (10(-10)M - 10(-12)M). From this study we conclude that MC903 is at least as potent as 1,25(OH)2D3 in inducing the formation human osteoclast-like cells in vitro. The decreased ability of MC903 to induce hypercalcemia in vivo is not therefore a result of a less marked effect than 1,25(OH)2D3 on the regulation of osteoclast formation.

Differentiation of heterogeneous phenotypes in human osteoblastt cultures in response to 1,25-dihydroxyvitamin D3

1,25(OH)2D3 (1,25-dihydroxyvitamin D3) inhibits the cell proliferation of human osteoblast-like cell cultures, but stimulates the synthesis of two of the phenotypic markers of the osteoblast, alkaline phosphatase and osteocalcin. It is not known whether all cells which synthesize alkaline phosphatase also synthesize osteocalcin in response to 1,25(OH)2D3. In this study we addressed this question by examining the response of human osteoblast-like cell cultures to 1,25(OH)2D3, using concurrent histochemical and immunochemical staining for alkaline phosphatase and osteocalcin, respectively. The cells were grown in the presence or absence of 1,25(OH)2D3 (10(-9) M) for 48 h. Co-localisation of osteocalcin and alkaline phosphatase in osteoblast-like cell cultures showed that not all cells which synthesize osteocalcin (about 9%) in response to 1,25(OH)2D3 synthesize alkaline phosphatase (about 24%) and vice versa. There was also a proportion of osteoblast-like cells which produce both osteocalcin and alkaline phosphatase simultaneously (about 12%). These findings suggest that during differentiation of bone-derived cells in cultures, in response to 1,25(OH)2D3, heterogeneous phenotypes with respect to expression of alkaline phosphatase and osteocalcin appear.

Bone loss and age-related fractures

The aging of the skeleton represents an important cause of both morbidity and mortality in the Western World. A great deal is known about the epidemiology, pathogenesis, and structural alterations which occur during the osteoporotic process to increase the risk of fracture with age. Whereas osteoporosis is associated with a decrease in bone mass, factors other than bone mass are clearly important. These include the turnover of bone and the destruction of trabecular elements, the repair of fatigue damage, as well as extraskeletal factors. An improved understanding of the aging of bones will form a basis for improved strategies for its prevention and treatment.

Treatment of Paget’s Disease with the New Bisphosphonates

The increased availability of the bisphosphonates over the past 15 years has revolutionized the medical treatment of Paget’s disease and also stimulated renewed interest in its surgical management. Etidronate is the most widely available bisphosphonate, and it is against a wide experience of this agent that new bisphosphonates are being evaluated.

Increased potency of 1,25-dihydroxyvitamin D3 on human osteoblast-like cells following structural side-chain modification

Structural modifications of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) appear to alter its biological activity. We have investigated whether the position of the C = C bond in the side chain of fluorinated analogues can alter the spectrum of activity of 1,25(OH)2D3. For this purpose we compared the actions of 26,27-hexafluoro-1,25-dihydroxy-delta 22-vitamin D3 (1,25(OH)(2)26,27F6 delta 2D3), 26,27-hexafluoro-1,25-dihydroxy-delta 23-vitamin D3 (1,25(OH)(2)26,27F6 delta 23D3) and 1,25(OH)2D3 on human osteoblast-like cells. Both analogues and 1,25(OH)2D3 stimulated the production of osteocalcin and alkaline phosphatase activity in a dose-dependent manner. Both analogues were markedly more potent than 1,25(OH)2D3 in these respects. At high concentrations the vitamin D3 analogues and metabolite inhibited DNA synthesis in a dose-dependent manner. A correlation between the inhibition of cell growth and expression of the two osteoblast markers was observed, and apart from a difference in potency, did not differ from 1,25(OH)2D3. These studies indicate that hexafluorination and the C = C bond increase the potency of 1,25(OH)2D3 on human bone-derived osteoblast-like cells in vitro, but without changing their relative activity on these various aspects of osteoblastic function tested.