Kunihiko Tokunaga

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.

The incidence of cervical and trochanteric fractures of the proximal femur in 1999 in Niigata Prefecture, Japan

Abstract. The purpose of this study was to determine the incidence of cervical and trochanteric fractures of the proximal femur in 1999 in Niigata Prefecture, Japan, and to compare this incidence with those previously reported in Niigata in 1985, 1987, 1989, and 1994. The authors visited all hospitals within Niigata Prefecture having an orthopedic department and reviewed the medical records and radiographs of all patients who sustained such fractures in 1999. The population of Niigata Prefecture was determined in 1999 to be 2 486 999 (1 208 195 males and 1 278 804 females). The population over 65 years of age was 515 290 (210 564 males and 304 726 females), representing 20.7% of the total population. In 1999, there were 1697 cervical or trochanteric fractures, in 400 males and 1297 females, with a male-to-female ratio of 1 : 3.2. The incidence of these fractures in persons over 65 years of age was 308.7 fractures per 100 000 per year. This incidence increased from 1985 to 1989 and from 1989 to 1994, but after that, the rate of increase in incidence from 1994 to 1999 slowed down slightly. This suggests that the prevention of fractures in the elderly population in Niigata Prefecture influenced the lower ratio.

High-Level Expression of the Coxsackievirus and Adenovirus Receptor Messenger RNA in Osteosarcoma, Ewing’s Sarcoma, and Benign Neurogenic Tumors among Musculoskeletal Tumors

Purpose: The sensitivity of human tumor tissues to infection with recombinant adenoviruses correlates with the expression of the coxsackievirus and adenovirus receptor (CAR). CAR has been shown to function as the primary receptor for adenoviruses and to play a critical role in adenovirus entry into host cells. It is important for clinical gene therapy to determine the expression level of CAR in tumor tissues. Experimental Design: We analyzed the expression of CAR mRNA in 154 musculoskeletal tumor tissues from 154 patients and 10 normal mesenchymal tissues from 3 patients using reverse transcription-PCR and real-time quantitative PCR. An adenovirus infection assay was performed in two cell lines that were established from CAR-positive osteosarcoma tissue and CAR-negative malignant fibrous histiocytoma tissue. Results: Ninety-nine of 154 tumors were detected as CAR positive by reverse transcription-PCR. We found that the expression levels of CAR mRNA varied markedly between different tumors as determined by real-time quantitative PCR. CAR mRNA was expressed at high levels in osteosarcoma, Ewing’s sarcoma, neurofibroma, and schwannoma; at intermediate levels in exostosis, giant cell tumor, liposarcoma, synovial sarcoma, malignant peripheral nerve sheath tumor, and hemangioma; and at low levels in alveolar soft part sarcoma and desmoid. Whereas the osteosarcoma cell line that expressed a high level of CAR mRNA, like its parent tumor, had a high efficiency of adenovirus infection, the malignant fibrous histiocytoma cell line with almost undetectable expression of CAR mRNA, like its parent tumor, had a low efficiency of infection. Conclusions: Our data showed the great variations in CAR mRNA expression among human musculoskeletal tumors and mesenchymal tissues and implicated the potential usefulness of adenoviral vectors in gene therapy for osteosarcoma, Ewing’s sarcoma, neurofibroma, and schwannoma. Efficient transduction with adenovirus for gene therapy could be realized in appropriate, sensitive tumor types.

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.

Intermittent administration of human parathyroid hormone (1–34) increases new bone formation on the interface of hydroxyapatitecoated titanium rods implanted into ovariectomized rat femora

BackgroundAs hydroxyapatite (HA) has good osteoconductive properties, HA is used as coating material for the implants in cementless arthroplasty. However, its effect is not sufficient for osteoporotic bone. Parathyroid hormone (PTH) is known to have anabolic effects on bone formation. Intermittent administration of PTH increases both cancellous and cortical bone mass. The aim of this study was to confirm the effect of the fixation strength of HA-coated implants in the osteoporotic condition with a mechanical test and a bone histomorphometric method.MethodsFemale Sprague-Dawley rats were used for this study. Four weeks after ovariectomy (OVX) or sham surgery, HA-coated titanium rods were inserted into the distal femoral canal (Sham+HA group and OVX+HA group). PTH was administered immediately after the implantation of the HAcoated rods (OVX+HA+P group). We measured the shear strength at the bone-implant interface by a push-out test and the newly formed bone volume on the implant (BV.Im) by bone histomorphometry at 2 and 4 weeks after implantation.ResultsThe bone-implant shear strength in the OVX+HA group was significantly lower than that in the Sham+HA group at 2 weeks after implantation of the rods. In the OVX+HA+P group, the strength was significantly higher than that in the other groups. Similarly, at 4 weeks, statistically significant differences were confirmed in the bone-implant shear strength among the Sham+HA group, the OVX+HA group, and the OVX+HA+P group. BV.Im in the OVX+HA group was significantly lower than that in the Sham+HA group at 2 weeks after implantation. BV.Im was significantly higher in the OVX+HA+P group than that in the OVX+HA group. However, there was no difference in BV.Im between the Sham+HA group and the OVX+HA+P group. At 4 weeks after implantation, BV.Im was significantly lower in the OVX+HA group than that in the other groups, but no difference was found between the Sham+HA group and the OVX+HA+P group.ConclusionsIntermittent administration of PTH has an effect to increase new bone formation on the surface of HA-coated implants in the osteoporotic condition. This finding suggests that PTH administration is useful to improve the initial fixation of HA-coated implants even in osteoporotic patients.

Human osteosarcoma (OST) induces mouse reactive bone formation in xenograft system

To distinguish the origin of bone-forming cells in the osteosarcoma (OST) tumor inoculated into nude mice, we have developed a novel in situ hybridization technique. The system used digoxygenin (DIG) labeled DNA probes that encoded human specific repetitive gene, Alu, and mouse specific repetitive gene, mouse L1 (m-L1). The chondrogenic and osteogenic cells in the tumor had strongly positive signals for m-L1 probe without any signals for Alu probe. The expression of bone matrix proteins was also examined by in situ hybridization. The bone-forming cells were positive for mRNAs of mouse osteonectin, osteopontin, and osteocalcin relating to calcification during bone formation, while these were negative for human mRNAs of these bone matrix proteins. The OST cells in the tumor expressed the human bone morphogenetic proteins (BMPs) mRNAs by RT-PCR. These data indicated that the mouse cells, not the human sarcoma cells, are responsible for cartilage and bone formation in the OST tumor inoculated into nude mice, and we speculated that BMPs, at least in part, could play an important role in this ossification.

Activating Gs α mutation at the arg201 codon in liposclerosing myxofibrous tumor

Abstract Liposclerosing myxofibrous tumor (LSMFT) is a benign fibro-osseous lesion that is characterized by mixture of histologic elements including lipoma, fibroxanthoma, myxoma, ischemic ossification, and fibrous dysplasia (FD)-like features. These tissue components are seen in the original reports of FD; however, the relationship between LSMFT and FD is not clear. Point mutation of the α subunit of G protein (Gs α), which increases cyclic adenosine monophosphate formation, has been recognized as the cause of McCune-Albright syndrome as well as polyostotic and monostotic FD of bone. Gs α mutation at the Arg 201 codon in 2 patients of LSMFT was demonstrated in the present study. Although direct sequencing analysis using the fresh-frozen materials could not detect the mutation, the polymerase chain reaction fragmentation length polymorphism (PCR-RFLP) disclosed the missense point mutation Gs α at the Arg 201 codon in 2 cases involving LSMFT. This result strongly suggests that a subset of LSMFT is a variant form of FD.

Coxsackievirus and adenovirus receptor (CAR)-positive immature osteoblasts as targets of adenovirus-mediated gene transfer for fracture healing

Adenovirus vectors are expected to be a powerful tool for gene therapy to treat severe fractures. Adenovirus invades cells through binding to the coxsackievirus and adenovirus receptor (CAR) on the cell membrane. CAR expression is low in normal adult animals, but it is induced on regenerating cells in some experimental models. We made a rib fracture model in mice and evaluated the histological changes and CAR mRNA expression by RT-PCR 1, 5, 10, 14, and 21 days after the fracture. CAR mRNA was expressed exclusively in the fractured ribs at each time point, but not in the normal ribs. We detected the CAR protein immunohistochemically in fibroblast-like cells in the fracture callus on days 10 and 14 after fracture. In situ hybridization showed that these fibroblast-like cells expressed mRNA of type I collagen and osteopontin, but not osteocalcin, defining the cells as immature osteoblasts. We then transferred small doses (104–108 PFU) of lacZ-expressing adenovirus vector into immature osteoblasts on day 14. β-galactosidase was detected only on the immature osteoblasts at every dose. Immature osteoblasts play an important role in the matrix replacement step in fracture healing. CAR-mediated gene transfer into immature osteoblasts can be reasonable for adenovirus-mediated treatment of fracture healing.