Effects of platelet-derived growth factor isoforms on calcium release from neonatal mouse calvariae.

Platelet-derived growth factor (PDGF) is the major growth factor in serum for cells of mesenchymal origin and induces many different activities, including bone resorption. Since the initial report that PDGF stimulated calcium release from bone organ cultures, it has been shown that PDGF is a dimeric protein consisting of two disulfide bonded polypeptides encoded by different genes. Three isoforms of the two gene products have been isolated. We compared the capacity of each isoform to stimulate calcium release from radiolabeled mouse calvariae. PDGF-AB from human platelets and recombinant PDGF-BB isoforms significantly stimulated calcium release at 5 ng/ml, but not in lower doses. Recombinant PDGF-AA did not induce calcium release. Indomethacin blocked the stimulated bone resorption, suggesting a prostaglandin-mediated mechanism of action. PDGF-induced calcium release was compared to TGF-beta 1 in the organ culture system. Approximately a 10-fold greater concentration of PDGF-AB and PDGF-BB was required to achieve a similar degree of calcium release as found in TGF-beta 1 treated calvariae. Thus, TGF-beta 1, PDGF-AB, and PDGF-BB significantly stimulated calcium release from mouse calvariae. This response is specific in that PDGF-AA did not stimulate calcium release.

Mitogenic effects of growth factors on human periodontal ligament cells in vitro.

Periodontal regeneration is thought to require the migration and proliferation of periodontal ligament cells. Evidence suggests that the polypeptide growth factors PDGF, IL-1, and TGF-beta are mediators of these cellular events in wound healing. The purpose of this study was to determine the effects of these growth factors on human periodontal ligament (PDL) cell mitogenesis, and to identify the regulatory influences of TGF-beta on the response to PDGF and IL-1. Confluent, quiescent human PDL cells were cultured in vitro and treated with the polypeptide growth factors PDGF-AA and -BB, IL-1 beta, and TGF-beta in both a dose and time-dependent manner. Mitogenic activity, as a measure of proliferative potential, was determined by the quantitation of 3H-thymidine incorporation during DNA synthesis. The results of this study demonstrated that both PDGF-AA and -BB enhance mitogenic activity in a dose-dependent manner over a concentration range of 1.0 to 50.0 ng/ml. IL-1 beta (0.01 to 1.0 pM) resulted in no mitogenic enhancement, and at high concentrations (10.0 to 100.0 pM) demonstrated an inhibitory effect. TGF-beta produced a significant increase (P < 0.01) in mitogenic activity (although relatively much less than PDGF) in a delayed, bimodal, dose-dependent manner over a concentration range of 0.01 to 20.0 ng/ml, with a maximal response at a concentration of 1.0 ng/ml. Additionally, incubation with TGF-beta at 1.0 ng/ml prior to the addition of PDGF significantly enhanced (P < 0.01) the mitogenic response to both PDGF-AA and PDGF-BB.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of conditioned medium from connective tissue fibroblasts and epithelium on calcium release from mouse calvarial organ culture.

Fibroblasts from periodontal ligament and gingival explants were cultured in vitro. The conditioned media from four different periodontal ligament and four gingival explant cultures were examined to determine their effect on calcium release in mouse calvarial organ culture. All the cultures stimulated calcium release, with a range of 20.6-43% over control. Conditioned media from two periodontal ligament cultures and a gingival culture significantly stimulated calcium release from the bone organ culture. The stimulatory activity in the oral fibroblasts cell cultures was compared to that in conditioned medium from two non-oral, established, fibroblasts cell lines and an epidermal keratinocyte cell line. Similar to the oral fibroblast cultures, conditioned medium from all three cell lines resulted in stimulation of calcium release in the bone culture assay. In order to characterize the bone-stimulating activity, a 0.4 micron membrane was used to separate the cell cultures from the bone organ culture. Both gingival and periodontal ligament fibroblast cultures gave values for calcium release significantly less than control when the separating membrane was used. Both non-oral cell lines and the epidermal keratinocyte cell line gave values for calcium release similar to those when no membrane was used. These results suggest that oral fibroblasts, but not non-oral fibroblasts and epidermal keratinocytes, release a unique bone-resorption stimulating activity.

The induction of specific metabolic alterations in mouse calvarial organ cultures by glycosaminoglycans.

Glycosaminoglycans specifically regulate the amount of calcium released from bone cultures; the mechanisms responsible for this regulation are not known. Media from glycosaminoglycan-stimulated bone organ cultures were analysed to determine (1) if specific calcium-releasing substances were selectively produced, and (2) if protein synthesis was differentially affected by glycosaminoglycans. Chondroitin sulphate B, hyaluronic acid and keratan sulphate at 100 micrograms/ml significantly increased prostaglandin release when compared with control cultures. In combination with suboptimal concentrations of PTH, chondroitin sulphate B, heparin and keratan sulphate significantly stimulated prostaglandin release. When indomethacin was included in the test assays, the stimulated prostaglandin release was abolished. Heparin-treated cultures released the greatest percentage of latent collagenase activity followed by hyaluronic acid-treated cultures. Organ cultures treated with heparin and PTH amount of active collagenase. Stimulation increased interleukin-1 above control levels but with no significant difference among the glycosaminoglycans except for keratan sulphate cultures with which had the greatest amount of interleukin-1. Collagen protein decreased between 48 and 72 h under both control and experimental conditions. Examination of the predominant [35S]-methionine labelled proteins revealed that prostaglandin E2 treatment resulted in a relative shift in labelling to higher molecular-weight proteins as time in culture increased (up to 144 h). After 48 h, when equal amounts of labelled protein were analysed, there was a predominance in labelling of a 200,000 Da protein in the prostaglandin-treated cultures. These findings demonstrate that modulation of calcium release by glycosaminoglycans results in the selective release of molecules capable of stimulating calcium release.(ABSTRACT TRUNCATED AT 250 WORDS)