Matrix mineralization in MC3T3-E1 cell cultures initiated by beta-glycerophosphate pulse.
ABSTRACT MC3T3-E1 cells, grown in the presence of serum and ascorbate, express alkaline phosphatase and produce an extensive collagenous extracellular matrix that can be mineralized by the addition of beta-glycerophosphate (beta-GP). In the present work, we study the influence of concentration and duration of beta-GP treatment on the mineralization pattern in 4-week-old cell cultures. Amount and structure of mineral deposition were monitored by von Kossa staining, light, and electron microscopy, as well as small-angle X-ray scattering (SAXS) of unstained specimens. SAXS measures the total surface of the mineral phase and is therefore preferentially sensitive to very small crystals (typically <50 nm). It was used to determine the ratio (M) of small crystals to collagen matrix. A variety of mineralization patterns was observed to occur simultaneously, some associated with collagen within nodules or in deeper layers of the cultures and some independent of it. At a beta-GP concentration of 10 mmol, mineralization was initiated after about 24 h and continued to increase, irrespective of whether the high level of beta-GP was maintained or reduced to 2 mmol. With shorter pulses (<24 h), no significant mineralization was observed in the week following beta-GP pulse. With continuous treatment at 5 mmol beta-GP, the first signs of mineralization were detected 14 days after the beginning of treatment in the 4-week-old cultures, but no mineralization at all occurred at lower beta-GP concentrations. When cells were grown without ascorbic acid for 4 weeks, only two cell layers without collagen matrix were found. In these cultures, no mineralization detectable by SAXS could be induced with beta-GP. These data indicate that, in viable cells, high doses of beta-GP are essential for the nucleation of mineral crystals, but not for the progression of mineralization once crystals had been nucleated. In contrast, when 4-week-old cell cultures were devitalized, M was found to increase immediately, even at 2 mmol beta-GP. These results suggest that, in MC3T3-E1 cell cultures, cell viability is essential for prevention of spontaneous mineralization of the extracellular matrix.
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ABSTRACT: During bone formation, osteoblasts deposit an extracellular matrix (ECM) that is mineralized via a process involving production and secretion of highly specialized matrix vesicles (MVs). Activin A, a transforming growth factor-β (TGF-β) superfamily member, was previously shown to have inhibitory effects in human bone formation models through unclear mechanisms. We investigated these mechanisms elicited by activin A during in vitro osteogenic differentiation of human mesenchymal stem cells (hMSC). Activin A inhibition of ECM mineralization coincided with a strong decline in alkaline phosphatase (ALP) activity in extracellular compartments, ECM and MVs. SILAC-based quantitative proteomics disclosed intricate protein composition alterations in the activin A ECM, including changed expression of collagen XII, osteonectin and several cytoskeleton-binding proteins. Moreover, in activin A osteoblasts MV production was deficient containing very low expression of annexin proteins. ECM enhanced hMSC osteogenic development and mineralization. This osteogenic enhancement was significantly decreased when hMSC were cultured on ECM produced under activin A treatment. These findings demonstrate that activin A targets the ECM maturation phase of osteoblast differentiation resulting ultimately in the inhibition of mineralization. ECM proteins modulated by activin A are not only determinant for bone mineralization but also possess osteoinductive properties that are relevant for bone tissue regeneration.Molecular & Cellular Proteomics 06/2013; · 7.25 Impact Factor
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ABSTRACT: The hydroxyapatite (HAP) with variable chemical substitutions has been considered as the major component in the mineralized part of bones. Various metastable crystalline phases have been suggested as transitory precursors of HAP in bone, but there are no consensuses as to the nature of these phases and their temporal evolution. In the present study, we cultured rat calvarial osteoblasts with ascorbate and β-glycerophosphate to explore which calcium phosphate precursor phases comprise the initial mineral in the process of osteoblast mineralization in vitro. At the indicated time points, the deposited calcium phosphate was analyzed after removing organic substances from the extracellular matrix with hydrazine. The features comparable to dicalcium phosphate dihydrate (DCPD) and octacalcium phosphate (OCP), in addition to HAP, were detected in the mineral phases by high resolution transmission electron microscopy. And there was a trend of conversion from DCPD- and OCP-like phases to HAP in the course of mineralization, as indicated by Fourier-transform infrared microspectroscopy, energy-dispersive X-ray spectroscopy and synchrotron X-ray powder diffraction analyses. Besides, biochemical assay showed a progressive decrease in the ratio of mineral-associated proteins to calcium with time. These findings suggest that DCPD- and OCP-like phases are likely to occur on the course of osteoblast mineralization, and the mineral-associated proteins might be involved in modulating the mineral phase transformation.Journal of inorganic biochemistry 11/2013; 131C:109-114. · 3.25 Impact Factor
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ABSTRACT: RELAXIN (RLN) is a polypeptide hormone of the insulin-like hormone family; it facilitates birth by softening and widening the pubic symphysis and cervix in many mammals, including humans. The role of RLN in bone metabolism was recently suggested by its ability to induce osteoclastogenesis and activate osteoclast function. RLN binds to RELAXIN/INSULIN-LIKE FAMILY PEPTIDE 1 (RXFP1) and 2 (RXFP2), with varying species-specific affinities. Young men with mutated RXFP2 are at high risk for osteoporosis, as RXFP2 influences osteoblast metabolism by binding to INSULIN-LIKE PEPTIDE 3 (INSL3). However, there have been no reports on RLN function in osteoblast differentiation and mineralization or on the functionally dominant receptors for RLN in osteoblasts. We previously described Rxfp1 and 2 expression patterns in developing mouse oral components, including the maxillary and mandibular bones, Meckel's cartilage, tongue, and tooth primordia. We hypothesized that Rln/Rxfp signaling is a key mediator of skeletal development and metabolism. Here, we present the gene expression patterns of Rxfp1 and 2 in developing mouse calvarial frontal bones as determined by in situ hybridization. In addition, RLN enhanced osteoblastic differentiation and caused abnormal mineralization and extracellular matrix metabolism through Rxfp2, which was predominant over Rxfp1 in MC3T3-E1 mouse calvarial osteoblasts. Our data suggest a novel role for Rln in craniofacial skeletal development and metabolism through Rxfp2.Bone 05/2014; · 4.46 Impact Factor