Hepatocyte growth factor/scatter factor modulates cell motility, proliferation, and proteoglycan synthesis of chondrocytes.

Hepatocyte growth factor/scatter factor (HGF/SF) is a multifunctional growth factor that promotes proliferation, motility, and morphogenesis in epithelial cells. Recently the HGF receptor, c-met protooncogene product, has been shown to be expressed in developing limb buds (Sonnenberg, E., D. Meyer, M. Weidner, and C. Birchmeiyer, 1993. J. Cell Biol. 123: 223-235), suggesting that some populations of mesenchymal cells in limb buds respond to HGF/SF. To test the possibility that HGF/SF is involved in regulation of cartilage development, we isolated chondrocytes from knee joints and costal cartilages of 23-d embryonic and 4-wk-old rabbits, and analyzed the effects of HGF/SF on migration and proliferation of these cells. We found that HGF/SF stimulated migration of cultured articular chondrocytes but did not scatter limb mesenchymal fibroblasts or synovial fibroblasts in culture. HGF/SF also stimulated proliferation of chondrocytes; a maximum three-fold stimulation in DNA synthesis was observed at the concentration of 3 ng/ml of HGF/SF. Moreover, HGF/SF had the ability to enhance proteoglycan synthesis in chondrocytes. The responsiveness of chondrocytes to HGF/SF was also supported by the observation that they expressed the HGF/SF receptor. Addition of the neutralizing antibody to rat HGF/SF affected neither DNA synthesis nor proteoglycan synthesis in rat chondrocytes, suggesting a paracine mechanism of action of HGF/SF on these cells. In situ hybridization analysis showed that HGF/SF mRNA was restrictively expressed in the areas of future joint regions in developing limb buds and in the intercostal spaces of developing costal cartilages. These findings suggest that HGF/SF plays important roles in cartilage development through its multiple activities.

Collagen integrin receptors regulate early osteoblast differentiation induced by BMP-2.

Studies in several cell types indicate that the actions of integrin receptors for extracellular matrix and receptors for growth factors are synergistic in regulating cellular differentiation and function. We studied the roles of the alpha1beta1 and alpha2beta1 integrin collagen receptors in regulating the differentiation of 2T3 osteoblastic cells in response to bone morphogenetic protein (BMP)-2. The immortalized 2T3 cell line was established from the calvaria of mice transgenic for a BMP-2 promoter driving SV40 T-antigen. These cells require exogenous BMP-2, as well as ascorbic acid and beta-glycerolphosphate, for expression of a mature osteoblast phenotype and formation of a mineralized matrix. To determine how integrin receptors for collagen-I affect BMP-2 signaling, function-perturbing anti-rat alpha1 and/or alpha2 integrin subunit, or anti-type I collagen (Col-I), antibodies were added to human recombinant (hr)BMP-2-treated 2T3 cultures at confluence (C0) or at 4 or 8 days postconfluence (C4, C8). After 4 days of exposure to the antibodies, cultures were assayed for alkaline phosphatase (ALP) mRNA levels and enzyme activity and for cAMP production in response to parathyroid hormone. Addition of anti-collagen-I or both anti-integrin-alpha1 and -alpha2 antibodies to C0 cultures blocked expression of these early osteoblast markers by more than 90%, and also blocked mineralization (0.5-1.8% control) of these cells. In all cases, adding anti-alpha1 or anti-alpha2 antibodies separately produced partial effects, while their combined effect approached that of anti-collagen-I. When antibodies were added to more differentiated 2T3 cells, the inhibitory effects decreased. 2T3 cells carrying constitutively active BMP receptor (caBMPR-IB) showed elevated ALP activity without hrBMP-2; this constitutive activity was also suppressed by alpha1 and alpha2 integrin antibodies and by anti-Col-I antibody. Together, our data suggest that a signal(s) from collagen integrin receptors regulates the response to BMP downstream of BMPR-IB and upstream of the regulation of ALP mRNA and other early markers of osteoblast differentiation.

Effects of parathyroid hormone and calcitonin on alkaline phosphatase activity and matrix calcification in rabbit growth-plate chondrocyte cultures.

The effects of PTH and calcitonin (CT) on the expression of mineralization-related phenotypes by chondrocytes were examined. In cultures of pelleted growth-plate chondrocytes. PTH caused 60-90% decreases in alkaline phosphatase activity, the incorporation of 45Ca into insoluble material, and the calcium content during the post-mitotic stage. These effects of PTH were dose-dependent and reversible. In contrast, CT increased alkaline phosphatase activity, 45Ca incorporation into insoluble material, and the calcium content by 1.4- to 1.8-fold. These observations suggest that PTH directly inhibits the expression of the mineralization-related phenotypes by growth-plate chondrocytes, and that CT has the opposite effects.

Effects of cyclic adenosine 3',5'-monophosphate on chondrocyte terminal differentiation and cartilage-matrix calcification.

We examined the effects of cyclic AMP on terminal differentiation and calcification in rabbit growth plate chondrocyte cultures. Dibutyryl cAMP (dbcAMP), as well as 8-bromo-cAMP abolished the increases in chondrocyte size, alkaline phosphatase activity, type X collagen synthesis, 1 alpha, 25-dihydroxyvitamin D3 receptor synthesis, the incorporation of 45Ca into insoluble material, and the calcium content. All of these occurred in parallel untreated cultures during the hypertrophic (terminal) stage. The inhibition of alkaline phosphatase by dbcAMP was detectable after 24 h, and this effect was reversible. dbcAMP and 8-bromo-cyclic AMP inhibited alkaline phosphatase induction and calcification at low concentrations (3-5 microM), whereas 10-30-fold higher concentrations were required to stimulate proteoglycan synthesis. These findings suggest that cAMP plays a crucial role in suppressing terminal differentiation of chondrocyte and cartilage-matrix calcification.

Effects of X-ray irradiation on terminal differentiation and cartilage matrix calcification of rabbit growth plate chondrocytes in culture.

The retardation of long bone growth caused by irradiation is thought to be closely related to the impairment of growth plate function, but its mechanism remains unclear. In this study, we examined the effects of irradiation on the terminal differentiation of growth plate chondrocytes and on calcification. Chondrocytes were isolated from the growth plate of the ribs of four-week-old rabbits and inoculated at a high density on a type-I collagen-coated dish. Following logarithmic proliferation, they reached confluence (premature chondrocytes), then matured (mature chondrocytes), and became hypertrophied (hypertrophic chondrocytes). 10 Gy or less irradiation of the premature chondrocytes potently inhibited the terminal differentiation and matrix mineralization. Irradiation inhibited chondrocyte hypertrophy and suppressed alkaline phosphatase induction and the expression of type-X collagen without changing the protein composition profile of any other cell layer. Premature cells had the highest radiosensitivity. The sensitivity was decreased as the cells differentiated; the effects of irradiation on hypertrophic chondrocytes with terminal differentiation-related phenotypes were reduced. This study showed that 10 Gy or less irradiation of growth plate chondrocytes impaired terminal differentiation and mineralization. Since chondroclasts and bone marrow cells invade only to the mineralized cartilage, the induction of calcification in cartilage matrices is one of the most important steps in endochondral ossification. Therefore, it is conceivable that the damage in the growth plate induced by irradiation could account for the subsequent abnormal bone and skeletal growth.

Analysis of genomic instability in squamous cell carcinoma of the head and neck using the random amplified polymorphic DNA method.

Using the random amplified polymorphic DNA (RAPD) method, we identified genomic instability in head and neck squamous cell carcinoma (HNSCC) tissues. We extracted DNA from tumor and corresponding normal tissues of 30 HNSCC patients and amplified with ten random 10-mer arbitrary primers by the RAPD method. Genomic instabilities, which appeared as banding pattern changes between normal and tumor DNA, were detected by at least one primer in all tumor tissues. Moreover, there was significant correlation between the frequency of genomic instability and the degree of tumor differentiation. These results indicate a possible association of genomic instability with malignant potential of head and neck cancer.

Effect of X irradiation on secondary palate development in mice.

The mechanisms whereby X irradiation induces palatal clefting were investigated in vivo and in an in vitro organ culture system. When pregnant mice at day 12.5 of gestation were exposed to a 4-Gy dose of whole-body X radiation, the incidence of palatal clefting in their offspring was 91%. The volume of the irradiated palatal shelves was too low for them to make contact with each other. On gestational day 13.5 after labeling, bromodeoxyuridine-positive cells were sparse and apoptotic cells were abundant in the irradiated shelves. To prevent secondary effects of irradiation from the injured maternal body, fetal palatal explants were immediately transferred to an organ culture system after X irradiation in utero. The incidence of palatal clefting was 24%, much lower than the incidence in vivo. The addition of 10(-4) M of dexamethasone to the culture medium increased the incidence of palatal clefting to 56%. These findings indicated that X irradiation inhibited cell proliferation and induced apoptosis, resulting in small-volume palatal shelves that could not fuse with each other. The organ culture data also indicated that 4 Gy of irradiation appears to produce its effects both by a direct action on the fetus and indirectly by affecting the metabolism of the pregnant dam.

Changes in phenotypic expression of osteoblasts after X irradiation.

Changes in the phenotypic expression of osteoblasts after X irradiation were investigated. Osteoblast-like MC3T3-E1 cells at the actively proliferating, confluent and postproliferation stages were subjected to 10 Gy X irradiation. Irradiation at the confluent stage enhanced accumulation of type I collagen normalized to the DNA content. Irradiation at all stages down-regulated the expression of osteocalcin, but the levels of osteopontin and osteonectin mRNAs were unchanged from the control level. After irradiation at the later stages, the time-dependent increase in alkaline phosphatase activity per cell exceeded that in the control cells. The localization of alkaline phosphatase-positive cells was concordant with that of calcification. In addition, the quality of the calcium deposits was found to be similar to that in control cells as determined by energy dispersive spectrometry and the ratio of calcium to phosphorus, even if the cells were not exactly the same morphologically. The changes in phenotypic expression observed here are closely related to the enhancement of calcification observed in a previous study.

Effects of X irradiation on metabolism of proteoglycans.

The effects of X irradiation on matrix formation by growth-plate and articular chondrocytes, as reflected by metabolism of proteoglycans and type II collagen, were examined in a rabbit chondrocyte culture system. Irradiation with 1 to 10 Gy selectively inhibited synthesis of proteoglycans (incorporation of [35S]sulfate) depending on the stage of differentiation of the irradiated cells; however, synthesis of type II collagen was not affected. Irradiation of an immature culture, in which chondrocytes had just reached confluence, suppressed incorporation of [35S]sulfate into the glycosaminoglycan, 10 Gy inducing approximately 45-50% inhibition. In contrast, the irradiation of mature cultures, in which chondrocytes had already secreted extensive cartilage matrix, did not affect the rate of synthesis of proteoglycans (incorporation of [35S]sulfate). We also found that here irradiation stimulated the degradation of proteoglycans, but with the effect differing in growth-plate chondrocytes and articular chondrocytes. In growth-plate chondrocytes, cleavage from a site close to the G1 globular domain induced by 10 Gy enhanced the release of 35S-labeled proteoglycans into the medium, whereas in articular chondrocytes, irradiation had only marginal effects on the release of 35S-labeled proteoglycans. Our results show that irradiation with 1-10 Gy impaired proteoglycan metabolism in cartilage, with differing effects according to the stage of cell differentiation and the type of chondrocyte.

Expression of vascular endothelial growth factor in human oral squamous cell carcinoma: its association with tumour progression and p53 gene status.

AIMS: To correlate vascular endothelial growth factor (VEGF) expression in oral squamous cell carcinoma with the clinicopathological characteristics and prognosis; and to assess whether p53 gene status is associated with VEGF expression in human cancers. METHODS: Tumour specimens from 45 patients with oral squamous cell carcinomas were examined. Expression of VEGF was determined using an immunohistochemical method, and a tumour was considered positive when more than 5% of the neoplastic cells showed VEGF immunoreactivity. The p53 gene status was screened using a polymerase chain reaction--single strand conformation polymorphism analysis. RESULTS: VEGF positive staining was detected in 19 (42.2%) of the 45 cases. VEGF immunoreactivity did not correlate with the histological degree of tumour differentiation, clinical stages, or lymph node metastasis. The patients with VEGF positive tumours had a significantly worse prognosis than those with VEGF negative tumours. The five year overall survival rate of the VEGF negative patients was 76.5%, as compared with 48.8% for the VEGF positive patients. No significant association between VEGF expression and the p53 gene status of the tumours was found. CONCLUSIONS: VEGF is a good prognostic indicator of the survival of patients with oral squamous cell carcinoma. The p53 gene status does not seem to be associated with VEGF expression in these cancers.

An analysis of the prognostic significance of p53 status for squamous cell carcinoma of the oral cavity treated by radiotherapy.

The status of the p53 gene in biopsy specimens was analyzed to determine whether it is predictive of the outcome of radiotherapy of squamous cell carcinomas of the oral cavity. Biopsy materials were obtained from 45 patients, and the p53 status of each patient was determined using a single-strand conformation polymorphism analysis. Fourteen of the patients were treated with radiation therapy alone; the other 31 patients underwent radiotherapy in combination with surgery or chemotherapy. Twenty-seven patients had tumors with wild-type p53 and 18 patients had a tumor with mutant p53. The initial tumor response was not significantly different between these two groups. Kaplan-Meier survival plots (log-rank test) showed that the probability of survival was not significantly different between two groups although the patients with mutant p53 had a tendency for longer survival (P = 0.2941). However, among the patients with stage III/IV tumors (n = 24), those with a wild-type p53 status tended to have longer survivals.

Differences in the biological effects of in vitro irradiation by 65 MeV protons and 137cesium gamma-rays.

Using the irradiation system constructed for research in radiation biology, we have investigated the differences in the biological effects of in vitro irradiation by 65 MeV protons and by 137Cs gamma-rays. Survival curves were generated using V79 cells. The effects on biological parameters (SF2, RBE) of protons were greater than gamma-rays. Furthermore, 3H-thymidine incorporation in KOSC-3 cells, which display the p53 gene mutation, was inhibited by protons much more than by gamma-rays. On the other hand, in bleomycin-sensitive SCCKN cells, 3H-thymidine incorporation decreased more than in bleomycin-resistant SCCTF cells, however, both were inhibited by protons much more than by gamma-rays. In this study, the biological parameters and 3H-thymidine incorporation caused by 65 MeV protons were more severe than those caused by 137Cs gamma-rays.

Relationship of glucose intolerance and indocyanine green clearance to respiratory enzyme levels in human cirrhotic liver.

The relationship of glucose intolerance and indocyanine green clearance to respiratory enzyme levels in liver mitochondria was studied along with standard liver function tests in 40 patients (8 cirrhosis, 19 cirrhosis with hepatoma, 13 non-cirrhotic with hepatoma). There was a negative correlation between cytochrome a(+a3) concentrations and phosphorylative activity per unit of cytochrome a(+a3) (r = -0.75, p less than 0.01), but no correlation between ICG-K and cytochrome a(+a3) concentrations. Cytochrome a(+a3) concentrations in cirrhotic patients with linear oral glucose tolerance pattern, characterized with no return toward normal glucose levels within 120 minutes after an oral glucose load, increased to 1.45 +/- 0.11 (10(-10) mol/mg of protein) compared with 0.90 +/- 0.07 in cirrhotic patients with parabolic OGTT pattern, characterized with a return toward normal glucose levels within 120 minutes (p less than 0.01) (0.82 +/- 0.02 in control patients without liver diseases). The former had high operative mortality regardless of ICG-K value and the latter had virtually uneventful clinical courses. It was suggested that increased cytochrome a(+a3) concentrations and impaired glucose tolerance might be responsible for decreased hepatic functional reserve and poor prognosis in cirrhotics.

Short-term changes in blood ketone body ratios in the phase immediately after hepatic artery embolization: their clinical significance.

Changes in arterial and hepatic venous blood ketone bodies were investigated following transcatheter hepatic artery embolization (THAE) in patients with hepatocellular carcinoma. Acetoacetate/ beta-hydroxybutyrate ratio (ketone body ratio) in arterial blood was positively correlated with those of hepatic venous blood (r = 0.960, p less than 0.001), which reflects the mitochondrial redox potential in the embolized lobe. Nine cirrhotic patients were classified into three groups according to the changes in arterial blood ketone body ratio following THAE: Type A without decrease to below 0.7; Type B with a transient decrease to below 0.7, followed by its restoration within 5 hours; and Type C with decrease to below 0.7 without recovery within 5 hours. There were no serious complications in Type A and B patients. By contrast, severe sepsis and hepatic failure developed in Type C patients, possibly due to the extended embolization of both lobes. It is suggested that THAE can be successfully performed even in severely cirrhotic patients, as long as the embolized area is restricted to one lobe. In addition, changes in arterial blood ketone body ratios can give early information about the likely consequences of the THAE procedure just performed.

Age-dependent effects of hedgehog protein on chondrocytes.

Bone morphogenetic protein (BMP) has a crucial role in osteochondrogenesis of bone formation as well as in the repair of fractures. The interaction between hedgehog protein and BMPs is inferred from recent molecular studies. Hedgehog genes encode secreted proteins which mediate patterning and growth during skeletal development. We have shown that Indian hedgehog gene (Ihh) is expressed in cartilage anlage and later in mature and hypertrophic chondrocytes. This finding suggests that Ihh may regulate the development of chondrocytes. Our results in this study have shown that Ihh transcripts were expressed in hypertrophic chondrocytes in mice at three days but not at three weeks, although a similar expression pattern of alpha1 (X) collagen could be observed in both types of cartilage. To investigate the possibility that there are direct and age-dependent functions of Ihh in chondrocytes, cultured chondrocytes were treated with the amino-terminal fragment of Sonic hedgehog protein (Shh-N) which can functionally substitute for Ihh protein. Shh-N did not affect the proliferation and differentiation of chondrocytes from three-week-old mice but had a significant effect on three-day-old mice. It enhanced proliferation up to 128% of the control culture in a dose-dependent manner. Although there was no effect in Shh-N-treated cultures, Shh-N enhanced the stimulatory effect of parathyroid hormone (PTH) on the synthesis of proteoglycans. Because the effects of Shh-N on chondrocyte differentiation in this culture system differed from those of bone morphogenetic protein-2 (BMP2) and PTH, in terms of proteoglycan synthesis and ALPase activity, it is unlikely that BMP2 or PTH/PTH-related protein mediates the direct effects of Ihh in chondrocytes. Our study shows that Ihh can function in chondrocytes in a direct and age-dependent fashion.

Cemento-osseous dysplasia of the jaws in 54 Japanese patients: a radiographic study.

OBJECTIVE: The aim of this study was to describe the radiographic patterns of cemento-osseous dysplasia. STUDY DESIGN: Fifty-four patients affected with benign fibro-osseous jaw lesions that showed periapical radiopacities and/or radiolucencies in a focal or a multiplex form were studied. The clinical, radiographic, and histopathologic features of the patients with cemento-osseous dysplasia were retrospectively studied. Radiographic features of the cemento-osseous dysplasia lesions were classified according to the appearance of calcified bodies. Radiographic visibility of periodontal ligament spaces of related teeth was assessed. RESULTS: Forty-nine (91 %) of the 54 patients were women. The mean age of the total group was 50.8 years, and that of the male group was 64.6 years. The cemento-osseous dysplasia lesions could be classified into 6 types radiographically. Eighteen patients had at least 2 or more types of cemento-osseous dysplasia lesions. Of 147 related teeth, 142 had periodontal ligament spaces clearly visible. Six of 9 patients who had a total of 25 teeth with active hypercementosis showed concomitant occurrence of other types of cemento-osseous dysplasia lesions. Biopsy specimens showed various amounts of bonelike and cementumlike tissues. CONCLUSIONS: It is likely that cemento-osseous dysplasia consists of 3 variations of a single entity, all with the same unknown cause. In one variation, the entity originates from the periodontium; in another, it is of medullary bone origin; and in the third it results from the simultaneous involvement of both tissues.

Differential expression of the splicing regulatory factor genes during two-step chemical transformation in a BALB/3T3-derived cell line, MT-5.

Although the alternative splicing of various genes is a common event in human tumors, the mechanisms behind it have not been characterized. We hypothesized that the expression of splicing regulatory factors would be changed during cellular transformation. Gene expression of three splicing regulatory factors, alternative splicing factor/splicing factor 2 (ASF/SF2), heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2) and the 65 kDa subunit of U2 small nuclear ribonucleoprotein particles auxiliary factor (U2AF(65)), were examined by northern blotting in a two-step chemical transformation model. This in vitro model is composed of BALB/3T3 cells and a BALB/3T3-derived N-methyl-N-nitro-N-nitrosoguanidine (MNNG)-initiated cell line (MT-5). MT-5 cells can be transformed on exposure to 12-O-tetradecanoylphorbol-13-acetate (TPA). ASF/SF2 mRNA levels were decreased 2-fold in both MNNG-initiated cells and TPA-induced transformed cells compared with the normal parental cells, whereas hnRNP A2 mRNA expression did not significantly change between these three types of cells. U2AF(65) mRNA levels were markedly increased ( approximately 4.7-fold) associated with progression of cellular transformation. Moreover, RT-PCR analysis showed that distinct forms of ASF/SF2 mRNA were present in the MNNG-initiated cells and TPA-induced transformed cells but not in the parental cells. These findings indicate that ASF/SF2 or U2AF(65) gene expression is altered during in vitro two-step chemical transformation. The data suggest that the differential expression of splicing regulatory factors is one cause of aberrant expression of alternatively spliced mRNAs encoded by various genes in tumor cells.

Inhibition of chondrocyte terminal differentiation and matrix calcification by soluble factors released by articular chondrocytes.

Chondrocytes do not undergo terminal differentiation in normal articular cartilage, whereas growth plate chondrocytes synthesize ALPase and induce matrix calcification terminally. Articular chondrocytes in osteoarthritic joints have been reported to express the terminal differentiation phenotypes, suggesting that terminal differentiation of articular chondrocytes is inhibited in normal joints. In the present study, we investigated the underlying inhibitory mechanism of the terminal differentiation in articular cartilage using a culture on type II collagen-coated dishes or a novel culture model on Millipore filters. ALPase activity increased from day 7 to day 8 in growth plate chondrocyte cultures on the collagen-coated dishes, but not in articular chondrocyte cultures. The ALPase expression of growth plate chondrocytes on the collagen-coated dish was completely inhibited when the same number of articular chondrocytes was mixed in the growth plate chondrocyte cultures. When articular chondrocytes or growth plate chondrocytes were maintained on Millipore filters held in 16-mm dishes, they started to synthesize ALPase. The ALPase expression of the chondrocytes on Millipore filters was inhibited by the presence of articular chondrocytes maintained on the bottom collagen-coated substratum in the same dishes. These results indicate that factors that diffused into the medium through the Millipore filters are involved in the inhibition of terminal differentiation. Since the conditioned medium from articular chondrocyte cultures did not affect the ALPase expression, it is considered that the soluble factors, which are continuously released from articular chondrocytes, are responsible for the inhibition of terminal differentiation.

Characterization of the mineralization process in cultures of rabbit growth plate chondrocytes.

The present study was undertaken to investigate the mineralization process in chondrocyte cultures. Chondrocytes were isolated from the growth plate of ribs of 4-week-old rabbits. The nature and properties of mineral crystals precipitated in chondrocyte cultures were compared with those of crystals formed in the hypertrophic zone and bone of rabbit rib growth plates in vivo. The chondrocytes were maintained at high density on type II collagen-coated dishes in Eagle's medium, alpha-modification, with 10% fetal bovine serum and 50 micrograms/ml of ascorbic acid. These cells differentiated into hypertrophic cells 10 days after seeding and produced alkaline phosphatase and 1,25-dihydroxyvitamin D3 receptors on Days 30-70 at levels as high as those in the lower hypertrophic zone in vivo. Mineralization was initiated between Days 20 and 30 and advanced progressively throughout the culture period. However, mineralization was suppressed by the addition of parathyroid hormone (2 x 10(-8) M) or by the presence of fibroblasts. Examination by electron microscopy and Fourier transform infrared (FTIR) spectroscopy verified that mineralized nodules formed in vitro were composed of small apatite crystals. Importantly, FTIR spectral features of the apatite crystals (e.g., the prominent PO4 bands at 1125 and 1032 cm-1) were similar to those of cartilage apatites formed in vivo and differed markedly from those of carbonated bone apatites. These results suggest that growth plate chondrocytes cultured on collagen-coated dishes are an appropriate model for studies on cartilage mineralization.

Effect of X-ray irradiation on proliferation and differentiation of osteoblast.

We exposed the osteoblast-like cell line, MC3T3-E1, to 1-to 10-Gy X-ray. Irradiation at doses of 5-Gy dose or stage, confluence, and post-proliferation stages. The alkaline phosphatase activity, conversely, was increased by irradiation, and the calcium content of irradiated cells was greater than that of nonirradiated. These findings suggest that irradiation induces terminal differentiation and calcification of osteoblasts.