Morimichi Mizuno

Type I collagen‐induced osteoblastic differentiation of bone‐marrow cells mediated by collagen‐α2β1 integrin interaction

Bone marrow cells are multipotent cells. When bone marrow cells were cultured with type I collagen matrix gels, they showed high alkaline phosphatase activity, collagen synthesis, and formed mineralized tissues. Furthermore, cells expressed osteocalcin and bone sialoprotein genes, which are osteoblast‐specific genes. These findings indicate that type I collagen matrix gels induce osteoblastic differentiation of bone marrow cells. Type I collagen interacts with the α 2 β 1 integrin receptor on the cell membrane and mediates extracellular signals into cells. DGEA peptide is a cell‐binding domain of type I collagen molecule. When collagen–integrin interaction was interrupted by the addition of Asp‐Gly‐Glu‐Ala (DGEA) peptide to the culture, the expression of osteoblastic phenotypes of bone marrow cells was inhibited. Furthermore, anti‐α 2 integrin antibody, which interacts with α subunit of integrin and blocks the binding of integrin with collagen, suppressed the expression of osteoblastic phenotypes. These findings imply that collagen‐α 2 β 1 integrin interaction is an important signal for the osteoblastic differentiation of bone marrow cells. J. Cell. Physiol. 184:207–213, 2000.

Isolation and multilineage differentiation of bovine bone marrow mesenchymal stem cells

The bone marrow harbors a population of mesenchymal stem cells (MSCs) that possess the potential to differentiate into bone, cartilage, and fat, and along other tissue pathways. To date, MSCs from various species have been studied. Despite the bovine experimental model being widely used in experiments in vivo and in vitro, only a limited amount of information regarding bovine MSCs is available. The aim of this study was to isolate and induce the multilineage mesenchymal differentiation of bovine MSCs, thereby initiating further research on these cells. Bovine MSCs were isolated from eight calves, and osteogenic, chondrogenic, and adipogenic differentiation was induced by using a combination of previously reported protocols for other species. The level of differentiation was evaluated by histological examination and by analyzing the expression of tissue-specific genes by a quantitative “real time” reverse transcription/polymerase chain reaction technique. Following osteoinduction, the isolated fibroblast-like cells transformed into cuboidal cells and formed alkaline-phosphatase-positive colonies; during differentiation, these colonies transformed into mineralized nodules. In addition, osteogenesis was followed by osteocalcin and collagen type I mRNA expression. Chondrogenesis was confirmed by the demonstration of collagen type II, aggrecan, and sox9 mRNA expression in the cells stimulated by transforming growth factor β1 in monolayer culture. After being cultured in an adipogenesis-inducing medium, the MSCs responded by the accumulation of lipid vacuoles and the expression of adipocyte-specific genes. We have therefore demonstrated that cells harvested from bovine bone marrow are capable of in vitro extensive multiplication and multilineage differentiation, making them a relevant and invaluable model in the field of stem cell research.

Properties and cytotoxicity of water soluble Na2O–CaO–P2O5 glasses

Various compositions of Na2OCaO-P2O5 glasses are prepared to estimate glass formation, dissolution properties and cytotoxicity. In the wide composition range of 40 mol% of P2O5 or more, clear glass samples were obtained. The estimated glass forming region was consistent with other ternary phosphate glass systems. The glass transition temperatures and crystallization temperatures decreased with increasing P2O5 content and increased with CaO content. Dissolution properties in distilled water and simulated body fluid (SBF) were measured. In distilled water, CaO free glasses showed extremely fast dissolution. The dissolution rate decreased with increasing CaO content and decreasing P2O5 content. This composition effect results from cross-link formation between the non-bridging oxygens of two different chains by Ca2+ ions which improves the phosphate network strength. In SBF, the dissolution rate followed a similar trend, but glass dissolution was suppressed. This suppression occurred due to the existence of soluble species of glass such as Na+, Ca2+ and HPO(2-)4. The cytotoxicity decreased with increasing CaO content and with decreasing PO2.5 content. This was the result of a change in pH and ion concentration in the medium.

Two distinctive BMP-carriers induce zonal chondrogenesis and membranous ossification, respectively; geometrical factors of matrices for cell-differentiation.

A partially purified BMP preparation was combined with a fibrous glass membrane (FGM) or porous particles of hydroxyapatite (PPHAP), and then implanted subcutaneously into the backs of rats. As a control of these new carriers, a conventional carrier of insoluble bone matrix (IBM) was also used. These new geometrically different solid-state carriers induced tissues in quite different manners. FGM/BMP implants induced cartilage formation within the entire inner area of the membrane accompanied by a small amount of bone formation on the surface of the membrane. In contrast, PPHAP/BMP implants induced only bone within the pores of PPHAP without any detectable cartilage formation. Enzyme-linked immunosorbent assay revealed that the type II collagen content in FGM/BMP was six times higher than that in IBM/BMP, while there was no detectable type II collagen in PPHAP/BMP. The results were explained by the geometric properties of the two distinctive carriers.

3steogenesis by bone marrow stromal cells maintained on type I collagen matrix gels in vivo

In this study, we demonstrated that bone marrow stromal cells maintained on type I collagen matrix induced bone in vivo. The formed bone contained bone marrow, and the process of bone formation occurred without cartilage formation. Bone marrow stromal cells differentiated into osteoblasts on type I collagen matrix in vitro, but types II, III, and V collagens did not possess this activity. These findings imply that type I collagen matrix offers a suitable environment for the induction of osteoblastic differentiation in vitro and osteogenesis in vivo.

Attachment of osteoblastic cells to hydroxyapatite crystals by a synthetic peptide (Glu7-Pro-Arg-Gly-Asp-Thr) containing two functional sequences of bone sialoprotein

We investigated activity of bone sialoprotein (BSP) to mediate attachment of cells to hydroxyapatite using a model peptide, Glu7-Pro-Arg-Gly-Asp-Thr, which contains a putative hydroxyapatite-binding site (poly-Glu) and a cell-attachment site. The peptide has affinity to hydroxyapatite with a dissociation constant of 13.5 microM. The peptide affected in vitro mineralization in a gel system, indicating interaction between this peptide and calcium phosphate. The osteoblastic cell line MC3T3-E1 was incubated with hydroxyapatite powder coated with the peptide or proteins. Attachment of the cells was observed on the powder coated with BSP, but not on the powder coated with serum albumin. The cells were attached to the powder coated with the peptide. The cells were flattened on the powder, and pseudopods developed. The attachment of the cells was inhibited by an excessive amount of Gly-Arg-Gly-Asp-Ser peptide. In conclusion, BSP mediated attachment of osteoblastic cells to hydroxyapatite, and this activity could be accomplished only by the poly-Glu sequence and the Arg-Gly-Asp sequence.

Bone Sialoprotein (BSP) is a Crucial Factor for the Expression of Osteoblastic Phenotypes of Bone Marrow Cells Cultured on Type I Collagen Matrix

Abstract In this study, we demonstrated that type I collagen matrix induced the expression of osteoblastic phenotypes of bone marrow cells, and that antibone sialoprotein (BSP) monoclonal antibody suppressed the expression of these phenotypes. On the other hand, BSP accelerated the expression of osteoblastic phenotypes of bone marrow cells. The adherent bone marrow cells were harvested from rat femur and cultured on type I collagen matrix gels in medium containing 15% fetal calf serum, neither β-glycerophosphate nor glucocorticoid. Cells showed osteoblastic phenotypes (high alkaline phosphatase activity, osteocalcin synthesis, and responsiveness against parathyroid hormone) on collagen matrix gels at week 3 after the inoculation, and simultaneously, BSP was detected in the conditioned medium by Western blotting using an anti-BSP monoclonal antibody. However, cells in the conventional culture dishes did not show osteoblastic phenotypes during the experimental period. To investigate the physiological function of BSP in osteoblastic differentiation, bone marrow cells were cultured on collagen matrix with an anti-BSP monoclonal antibody for 3 weeks. This treatment suppressed the expression of the osteoblastic phenotypes, and the effect of the antibody was abolished by the addition of bovine bone BSP. Furthermore, bovine bone BSP stimulated the expression of osteoblastic phenotypes of bone marrow cells. Our results indicate that BSP plays a crucial role in the expression of osteoblastic phenotypes of bone marrow cells.

Stimulation by bone sialoprotein of calcification in osteoblast-like MC3T3-E1 cells

Bone sialoprotein (BSP) containing an Arg-Gly-Asp cell-binding sequence was purified from bovine bone 4 M guanidine-HCl extract after HCl demineralization by a series of chromatographic procedures. When this protein was coated on culture dishes in the presence of type I collagen, it increased both DNA content and alkaline phosphatase (ALP) activity in osteoblast-like MC3T3-E1 cells, and stimulated calcification in the cells, whereas fibronectin, another cell-binding protein, showed a marked increase in the DNA content but had little effect on the ALP activity. These findings suggest that BSP is mitogenic for preosteoblasts and differentiating the cells into osteoblasts, thereby stimulating bone calcification

Time-dependent changes of collagen cross-links and their precursors in the culture of osteogenic cells

SummaryThe early stage of cross-link formation in bone collagen was studied in a cell culture system. An osteogenic cell line that produces and accumulates a remarkably high amount of collagen, and that eventually forms bone-like structures, was used in this study for its time-dependent development of reducible cross-links. It was found that precursors of the cross-link, dehydro-dihydroxynorleucine and dehydro-hydroxynorleucine became detectable as soon as the cells attained a confluent state. They showed maximal amounts at day 3–5 after confluence, but substantially disappeared at day 10 after confluence. In contrast, two characteristic cross-links of bone collagen, dehydrodihydroxylysinonorleucine dehydro-DHLNL and dehydrohydroxylysinonorleucine (dehydro-HLNL), which were present in trace amounts at the stage of cell confluence, gradually increased in amount and reached a plateau at day 10, just when their precursors disappeared. Thus, it was found that there was a time lag of about a week between the maximal formations of precursors and cross-links of bone collagen in this system. The significance of this time lag was interpreted in terms of the minimum essential accumulation of collagen for the precursor-product transition. The ratio of dehydro-DHLNL to dehydro-HLNL was as low as 0.7 at day 3 after confluency, increased to 4.2 at day 20, the period just before mineralization began, and decreased thereafter, suggesting a qualitative change in bone collagen associated with mineralization.

Effects of a bone lysine-rich 18 kDa protein on osteoblast-like MC3T3-E1 cells

A lysine-rich 18 kDa protein was isolated from bovine bone and examined for its effects on osteoblast-like MC3T3-E1 cells. This protein is homologous to a heparin-binding protein in brain and uterus. This protein enhanced cell attachment independent of the Arg-Gly-Asp cell-binding sequence and stimulated proliferation during the growth phase. Addition of this protein to cell cultures on days 11, 12, and 13 after confluency resulted in a 1.6-2.0-fold increase in the alkaline phosphatase activity and little increase in the DNA content. These findings suggest that the 18 kDa protein may be functional in promoting the proliferation and differentiation of osteoblasts.