Tadashi Hayami

Expression of metalloproteinase-13 (Collagenase-3) is induced during fracture healing in mice.

In fracture healing, a large amount of cartilage is formed, then rapidly replaced by osseous tissue. This process requires the transition of extracellular matrix component from type II to type I collagen. We investigated the expression of matrix metalloproteinase-13 (MMP-13), which has a high potential to cleave type II as well as type I collagen, during fracture repair in mouse ribs. In situ hybridization demonstrated that MMP-13 mRNA was present throughout the healing process. It was detected in the cells of the periosteum at day 1. As fracture callus grew, strong MMP-13 mRNA signals were detected in cells of the cartilaginous callus. In the reparative and remodeling phases, both hypertrophic chondrocytes and immature osteoblastic cells in the fracture callus expressed MMP-13 mRNA strongly. These cells were located adjacent to tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts at the sites of cartilage/bone transition. In osteoclasts, MMP-13 expression was not detected. The level of MMP-13 mRNA peaked at day 14 postfracture by northern blotting. Immunohistochemical staining showed that MMP-13 was detected primarily in hypertrophic chondrocytes. These results indicate that MMP-13 is induced during fracture healing. The site- and cell-specific expression of MMP-13 and its enzymatic property suggest that MMP-13 initiates the degradation of cartilage matrix, resulting in resorption and remodeling of the callus. In conclusion, MMP-13 plays an important role in the healing process of fractured bone in mice.

Specific loss of chondromodulin-I gene expression in chondrosarcoma and the suppression of tumor angiogenesis and growth by its recombinant protein in vivo

Chondromodulin‐I (ChM‐I) was previously identified as an angiogenesis inhibitor in cartilage. Here, we demonstrated that the level of ChM‐I transcripts was substantially reduced to 100 or even less in the lower‐grade chondrosarcomas, in articular cartilage or other benign cartilage tumors. We implanted human chondrosarcoma OUMS‐27 cells into nude mice that reproducibly produced tumors with cartilaginous matrix. Tumor‐induced angiogenesis was evident when the tumors were excised 30 days after implantation. However, the local administration of recombinant human ChM‐I almost completely blocked vascular invasion and tumor growth in vivo. Moreover, ChM‐I also inhibited the growth of HT‐29 colon adenocarcinoma in vivo, implying its therapeutic potential for solid tumors.

In vivo bone-forming capacity of human bone marrow-derived stromal cells is stimulated by recombinant human bone morphogenetic protein-2

Abstract In the present study, we investigated whether the in vivo bone-forming capacity of human bone marrow-derived stromal cells (HMSCs) could be enhanced by recombinant human bone morphogenetic protein-2 (rhBMP-2). The HMSCs obtained from seven donors (5–54 years of age) were passaged three to six times. Passaged HMSCs exhibited the osteoblastic phenotype in vitro, including: (a) an increase in alkaline phosphatase (ALP) activity in response to dexamethasone, ascorbic acid, and β-glycerophosphate; and (b) mRNA expression for markers of osteoblastic lineage (ALP, osteopontin, osteocalcin, and parathyroid hormone-receptor) and BMP-2, -4, and -6 detected by reverse transcription–polymerase chain reaction. For the in vivo assay, transplants were subcutaneously implanted into nude mice as follows: group A (vehicle); group B (rhBMP-2); group C (HMSCs with vehicle); and group D (HMSCs with rhBMP-2). Transplants were obtained 2 and 4 weeks after implantation. Correlated radiographic findings, histological observations, and in situ hybridization using species-specific probes showed that the group B transplants contained bone tissue of mouse origin, which was observed at the periphery of the transplants. Four weeks after implantation, small amounts of HMSCs-derived bone tissue were detected at the periphery in two of seven transplants in group C. In contrast, five of seven group D transplants exhibited HMSCs-derived bone tissue, which was located at the center of the transplants and was surrounded by mouse bone tissue. Furthermore, HMSCs-derived chondrogenesis was detected in two of seven group D transplants. The results of the present study demonstrate that culture-expanded HMSCs preserve the osteoblastic phenotype, and the in vivo bone-forming capacity can be promoted by rhBMP-2.

Origin of bone-forming cells in human osteosarcomas transplanted into nude mice—which cells produce bone, human or mouse?

Osteosarcomas are malignant tumours producing osteoid and/or bone. It is difficult to distinguish tumour bone formation from reactive, based on their morphological features alone. The objective of this study was two‐fold: to clarify the origins of bone‐forming cells in human osteosarcoma transplanted into nude mice; and to examine the role of bone morphogenetic proteins (BMPs) in the tumour‐induced osteogenesis. DNA in situ hybridization was carried out with digoxigenin (DIG) polymerase chain reaction (PCR) labelled DNA probes for human‐specific ‘Alu’ and mouse‐specific ‘mouse L1 (m‐L1)’ genes. Human osteosarcoma cells, established cell lines of NOS‐1, NOS‐2, and HuO9, were transplanted separately into nude mice. Bone‐forming cells of the bone in the NOS‐1 or NOS‐2 tumours were positive for Alu, while they were negative for m‐L1. The cells lining the surface of trabeculae in the HuO9 tumour were positive for Alu, but a few of them were also positive for m‐L1. The m‐L1‐positive cells expressed mouse osteocalcin and type I collagen mRNAs. These facts suggest that the mouse cells were involved in osteoid synthesis of the HuO9 tumour. The NOS‐1 or NOS‐2 tumours expressed human BMP 2–7 mRNAs, whereas the HuO9 tumour expressed human BMPs 2, 4, 5, and 7. The osteogenetic potential of the tumours may depend on the expression patterns of BMPs. These results demonstrate two distinct types of bone formation, by tumour cells and by an admixture of tumour and non‐tumour cells. The present study showed that the HuO9 tumour produces chimeric bone formation. This is the first report to demonstrate the relationships between tumour cells and non‐tumour cells in bone formation, using genetic markers.

Chondromodulin-1 directly suppresses growth of human cancer cells

BackgroundChondromodulin-1 (ChM1), an endogenous anti-angiogenic factor expressed in cartilage, has been suggested to inhibit invasion of endothelial cells into cartilage. In addition, the ectopic administration of ChM1 has been reported to suppress tumorigenesis in vivo. However, it is unclear whether the anti-tumor effect is due to not only the anti-vascularization effect of ChM1, but also its direct action against oncocytes. In the present study, we sought to determine whether ChM1 has a direct action on tumor cells.MethodsBrdU incorporation assay was performed on human umbilical vein endothelial cells (HUVECs), normal human dermal fibroblasts (NHDFs), HepG2 cells and HeLa cells in the presence or absence of recombinant human ChM1 (rhChM1). An adenovirus that expresses ChM1, Ad-ChM1, was established and applied to the tumor xenografted in vivo, and to in vitro tumor cells cultured on plates or in soft agar. Cell cycle-related proteins and the phosphorylation of Erk, Akt, and GSK3β, the downstream molecules of the extracellular matrix-integrin signaling pathways, in HepG2 cells treated with or without Ad-ChM1 were detected by western blot analysis. Luciferase reporter assays of STAT, GAS, and ISRE, which participate in another cytokine signaling pathway, ware performed in HepG2, HeLa, and HUVEC cells.ResultsChM1 suppressed BrdU incorporation in HUVECs and in HepG2 cells dose-dependently, but did not suppress BrdU incorporation in NHDFs and HeLa cells cultured on plates. In soft agar, however, ChM1 suppressed the growth of HeLa cells, as well as HepG2 cells. Western blot analyses demonstrated that ChM1 decreased the levels of cyclin D1, cyclin D3, and cdk6 and increased those of p21cip1 without affecting the phosphorylation levels of Erk, Akt, and GSK3β in HepG2 cells. The luciferase reporter assay demonstrated that ChM1 suppressed the transcriptional activities of STAT and GAS but not of ISRE.ConclusionChM1 directly suppressed the proliferation of tumor cells in an anchorage-independent manner. However, ChM1 did not alter the phosphorylation of downstream molecules, at which the signaling pathways through growth factor and cytokine receptors converge with the anchorage-dependent pathway. Our results show that ChM1 has a direct anti-tumor effect; moreover, this effect occurs by inhibiting the STAT signaling pathway.

Spatiotemporal change of rat collagenase (MMP-13) mRNA expression in the development of the rat femoral neck.

Abstract: The interepiphyseal region between the greater trochanter and the capital femoral epiphysis and the medioproximal portion of the femoral neck exhibit extensive morphological changes during the first 4 weeks after birth in rats. Previous reports show that matrix metalloproteinase-13 (MMP-13, rat collagenase) mRNA is expressed in bone and cartilage during embryonal development and fracture healing. We examined MMP-13 mRNA expression and compared it with the distribution of osteopontin and osteocalcine mRNA in the femoral neck. Moreover, we examined histomorphometric analysis in the femoral neck where the morphology changes rapidly. Histomorphometric analysis of the 4-week-old rat femoral neck showed a high rate of bone formation and resorption in the region where shape changed rapidly. Osteopontin mRNA was expressed diffusely along the endosteum. In contrast, MMP-13 mRNA expression was restricted to the medial endosteal portion near the cartilage–bone interface of the femoral neck in 15- and 28-day-old rats and in the deepest endosteal interepiphyseal region of 15-day-old rats. MMP-13 mRNA-expressing osteoblastic cells were also expressing osteopontin but not osteocalcin mRNA. MMP-13 mRNA-expressing cells differ from tartrate-resistant acid phosphatase (TRAP) -positive cells, and MMP-13 mRNA-positive cells are located adjacent to TRAP-positive cells. The results of the site- and cell-specific expression of MMP-13, taken together with its enzymatic property, suggest that MMP-13 plays an important role in morphological changes in the rat femur, at least during the third and fourth week after birth, and that MMP-13 itself is involved in the interaction between osteoblastic and TRAP-positive cells.

Parathyroid hormone increases the expression level of matrix metalloproteinase-13 in vivo.

Abstract Parathyroid hormone (PTH) increases serum calcium (Ca) by enhancing bone resorption and renal Ca reabsorption. However, detailed mechanisms of enhanced bone resorption by PTH remain to be elucidated. Although PTH has been shown to increase the expression level of osteoblastic matrix metalloproteinase (MMP)-13 in vitro, only limited results are available regarding the in vivo regulation of MMP expression. In the present study, we have examined expression levels of MMPs in PTH-infused rats. Infusion of 1.5 or 2.0 nmol/kg/day rat PTH(1–34) for 3 days resulted in a dose-dependent increase in serum Ca. PTH infusion also decreased serum phosphate levels and increased urinary excretion of Ca and phosphate. Infusion of PTH for 7 days resulted in less severe hypercalcemia and hypophosphatemia. Urinary Ca and phosphate excretion in rats infused for 7 days was less than that in rats infused for 3 days. Northern blot analysis showed that PTH infusion increased the expression level of MMP-13 in calvaria, although it did not affect MMP-2 expression. Furthermore, the time-course and severity of hypercalcemia and hypercalciuria correlated with the expression level of MMP-13. In situ hybridization also showed that PTH infusion increased the expression level of MMP-13 in femora. These results indicate that PTH enhances MMP-13 expression in vivo and suggest that PTH stimulates bone resorption at least partly by enhancing MMP-13 expression.

Human PTH (1-34) induces longitudinal bone growth in rats

Abstract. The growth plate is a specialized structure that is responsible for longitudinal bone growth (LGR). Growth plate organization is altered with loading in rats. Parathyroid hormone (PTH) is known to induce mitogenic effect on chondrocytes in vitro. Type I PTH/PTH related peptide (rP) receptor is expressed in growth plate cartilage in rats. We therefore investigated the effect of PTH administration on the organization and longitudinal growth rate of the growth plate in rats. We also investigated the effect of PTH on the changes induced by unloading in the organization and growth of the growth plate. Thirty 6-week-old and 30 15-week-old male Sprague-Dawley rats were randomly assigned to five groups (n = 6 per group), i.e., basal controls, control (i.e., normally loaded), PTH-treated control (i.e., PTH-treated under normal loading), unloaded, and PTH-treated under unloading. PTH-treated animals received human PTH (1–34) at a dose of 80 μg/kg per day five times per week for 3 weeks, for the duration of unloading. In young loaded rats treated with the systemic administration of PTH, growth plate thickness, chondrocyte number, and LGR were increased in the proximal tibiae compared with findings in young loaded rats without PTH administration. Hindlimb unloading induced a reduction in growth plate thickness, chondrocyte number, and LGR. In young rats, systemic administration of PTH partly prevented these changes induced by unloading. These preventive effects of PTH were observed only in young rats; not in adult rats. These results show that the systemic administration of PTH stimulates longitudinal bone growth, and diminishes the reduction in growth plate growth induced by unloading in young rats.

Origin of histiocyte-like cells and multinucleated giant cells in malignant fibrous histiocytoma: Neoplastic or reactive?

The origin of histiocyte‐like cells in malignant fibrous histiocytoma (MFH) remains controversial. To determine whether histiocyte‐like cells and multinucleated giant cells show reactive or neoplastic proliferation, we transplanted human storiform‐pleomorphic MFH to nude mice and investigated the origin of histiocyte‐like cells using the DNA in situ hybridization (ISH) system. In addition, we analyzed the mRNA expression of mouse c‐fms and human colony stimulating factor‐1 (CSF‐1); immunohistochemical expression of markers detectable in cells of monocyte/macrophage lineage. The DNA ISH revealed neoplastic proliferation of fibroblastic cells and bizarre multinucleated giant cells of human origin. Monocyte/macrophage lineage cells were seen in parental tumors, whereas they did not participate in neoplastic proliferation in transplanted tumors. The parental tumors expressed human CSF‐1 mRNA and the histiocyte‐like cells in transplanted tumors expressed ‘mouse’c‐fms mRNA. These results suggest that MFH induce infiltration of monocyte/macrophage and CSF‐1 is one of the mediators involved in this phenomenon, because the human CSF‐1 can act as a ligand to the mouse c‐fms. Histiocyte‐like cells in MFH should be considered as a reactive monocyte/macrophage lineage rather than as an element of neoplasm.

The varus alignment and morphologic alterations of proximal tibia affect the onset of medial knee osteoarthritis in rural Japanese women Case control study from the longitudinal evaluation of Matsudai Knee Osteoarthritis Survey

BACKGROUND Relationship malalignment of the knee and the onset of knee OA are unclear. Moreover, it has not been clarified whether malalignment of the knee affects the morphological change of the proximal tibia. The purpose of current study was to investigate whether varus knee alignment and morphological change of the proximal medial tibia influence the onset of medial knee OA and to clarify the relationship between them. METHODS The subjects comprised 736 knees from 390 women that went under both the first survey in 1979 and the fourth survey in 2000 in the Matsudai district in Niigata Prefecture in Japan. None of the subjects demonstrated radiographic knee OA at the first survey. OA was graded according to the Kellgren-Lawrence classification and the subjects were divided according to the OA grades in the forth survey into the 3 groups (non-OA, early OA, and advanced OA). Age, BMI, Femorotibial angle (FTA), the tibial plateau angle and medial tibial cortical bone thickness (MTCBT) were measured. We performed multivariate logistic regression analysis using the stepwise method to identified the risk factors regarding onset of knee OA and calculated the odds ratio (OR) and 95% confidence intervals (95% CI). RESULTS Advanced OA had a significantly higher OR compared to non-OA in 1979 with the tibial plateau angle (OR: 1.15) and MTCBT (OR: 2.11). And also advanced OA had a significantly higher OR compared to early-OA in 1979 with the tibial plateau angle (OR: 1.17) and MTCBT (OR: 1.62). CONCLUSIONS In the advanced OA, varus alignment of the proximal tibia had already existed before the onset of knee OA. In addition, we thought that varus alignment due to the proximal tibia had influenced the morphologic alterations of proximal medial tibia.