Chohei Shigeno

In vitro analysis of the stimulation of bone formation by highly bioactive apatite- and wollastonite-containing glass-ceramic: Released calcium ions promote osteogenic differentiation in osteoblastic ROS17/2.8 cells

We analyzed the mechanisms of the efficient bone formation on the osteoconductive surface of apatite- and wollastonite-containing glass-ceramic (AW) by using an in vitro system. AW releases Ca ions and bonds to bone via a submicron-thick hydroxycarbonate apatite (HCA) layer. AW disks were conditioned with simulated body fluid (SBF) to grow HCA layers, and the amount of released Ca ion was regulated by modulating the conditioning time from 24 to 240 h. Surface-transformed AW disks increased alkaline phosphatase (AP) activity in osteoblastic ROS17/2.8 cells by 1.5- to threefold over unconditioned disks. AW disks conditioned for 24 h [AW(24)], which had a homogeneous, submicron-thick apatite layer and increased extracellular ionized Ca concentration ([Ca(2+)](e)) in the culture medium to the greatest extent, enhanced the AP activity the most. High [Ca(2+)](e) promoted osteogenic differentiation in ROS17/2.8 cells: It increased AP activity in a dose-dependent manner by up to 1.6-fold, and up-regulated the expression of AP, osteocalcin (OC), and transforming growth factor-beta1 mRNAs in dose- and time-dependent manners. AW(24) enhanced AP activity in ROS17/2.8 cells as much as AW disks conditioned with SBF containing serum to exhibit in vivo surface-structure changes. AW(24) increased AP activity in ROS17/2.8 cells by 1.6-fold and enhanced the expression of AP and OC mRNAs significantly, compared with sintered hydroxyapatite (HA). After implantation of AW and HA in the distal metaphyses of rabbit femurs, thin, newly formed bone lined with cuboidal, osteoblast-like cells was characteristically observed adjacent to the AW surface within 8 days. These results provide evidence for the hypothesis that AW stimulates bone formation on its surface by increasing [Ca(2+)](e) to promote the HCA layer formation and the differentiation of osteogenic cells.

Human calcitonin gene-related peptide possesses weak inhibitory potency of bone resorption in vitro

SummarySynthetic human calcitonin gene-related peptide (CGRP) was examined for the action on the bonein vitro. Human CGRP inhibited bone resorption stimulated by both human parathyroid hormone and basal. The mode of inhibitory action of human CGRP seemed to be similar to that of calcitonin and the relative potency of human CGRP to inhibit bone resorption is one five-hundredth of that of human calcitonin. Thus, a novel pharmacological action of CGRP was demonstrated.

Molecular Cloning and Biological Activity of a Novel Ha-Ras Suppressor Gene Predominantly Expressed in Skeletal Muscle, Heart, Brain, and Bone Marrow by Differential Display Using Clonal Mouse EC Cells, ATDC5

We cloned a cDNA encoding a novel mouse protein, named A-C1, by differential display between two mouse cell lines: embryonic fibroblast C3H10T1/2 and chondrogenic ATDC5. The deduced amino acid sequence of A-C1 consists of 167 amino acids and shows 46% identity with that of a ras-responsive gene, rat Ha-rev107. Northern blot analysis showed a distinct hybridization band of 3.2 kilobases. Expression of A-C1 mRNA was detected in undifferentiated ATDC5 cells and myoblastic C2C12 cells, while none of C3H10T1/2 cells, NIH3T3 fibroblasts, Balb/c 3T3 fibroblasts, osteoblastic MC3T3-E1 cells, and ST2 bone marrow stromal cells expressed A-C1 mRNA in vitro. Moreover, A-C1 mRNA was expressed in skeletal muscle, heart, brain, and bone marrow in adult mice. By in situ hybridization, A-C1 gene expression was localized in hippocampus as well as bone marrow cells. By immunocytochemistry, A-C1 protein was detected in the cytoplasm as well as perinuclear region of the cells. Transfection of A-C1 cDNA into Ha-ras-transformed NIH3T3 cell line caused increase in the number of flat colonies and inhibition of cell growth. Our data indicate that A-C1 is expressed in some specific tissues in vivo and modulates Ha-ras-mediated signaling pathway.

Bone Demineralization Following Urinary Intestinal Diversion Assessed by Urinary Pyridinium Cross-Links and Dual Energy X-Ray Absorptiometry

PURPOSE We investigated the acid-base balance and bone mineral status in patients with 3 types of urinary intestinal diversion. MATERIALS AND METHODS Of 46 men with urinary intestinal diversions 20 had a Kock pouch, 15 had an Indiana pouch and 11 had an ileal conduit. Acid-base balance was assessed by arterial blood gas analysis. Bone mineral status was measured by urinary pyridinium cross-links and dual energy x-ray absorptiometry. In addition, urinary deoxypyridinoline was measured in 79 patients. RESULTS Of the 46 patients 7 (15%) with the Kock pouch (1), Indiana pouch (5) and ileal conduit (1) had metabolic acidosis associated with significantly lower bone mineral densities (p < 0.05) and higher urinary pyridinium cross-links (p < 0.005) than did those with normal acid-base status. No difference was found in metabolic acidosis and bone demineralization among the 3 groups. Additionally, in 79 patients urinary deoxypyridinoline reached the highest level immediately postoperatively and then gradually decreased to the stable level within 1 or 2 years. CONCLUSIONS Metabolic acidosis following urinary intestinal diversion results in bone demineralization. The types of diversion did not cause differences in metabolic acidosis and bone resorption. Bone has a major role in buffering acid overload in the early postoperative period.

Synovial Fluids from Patients with Osteoarthritis and Rheumatoid Arthritis Contain High Levels of Parathyroid Hormone–Related Peptide†

High levels of immunoreactive and biologically active parathyroid hormone–related peptide (PTHrP) were detected in synovial fluids from patients with osteoarthritis (OA) and rheumatoid arthritis (RA). The levels of PTHrP immunoreactivity in synovial fluids, measured by a two‐site immunoradiometric assay (IRMA) which detects hPTHrP(1–72) or longer peptides and a radioimmunoassay (RIA) specific to the carboxy‐terminal portion of hPTHrP, were 3.2 ± 0.3 pmol of hPTHrP(1–86)/l and 61 ± 7.0 pmol of hPTHrP(109–141)/l in OA patients (mean ± SE, n = 23), and 4.8 ± 0.8 pmol of hPTHrP(1–86)/l and 164 ± 30 pmol of hPTHrP(109–141)/l in RA patients (n = 26). Synovial fluid PTHrP levels distributed above the normal plasma reference ranges in each assay (0.7–2.6 pmol of hPTHrP(1–86)/l; 16–60.6 pmol of hPTHrP(109–141)/l). After concentration using sequential cation‐exchange and reverse‐phase chromatography, synovial fluid exhibited the activity that stimulated cyclic adenosine monophosphate (cAMP) accumulation in osteoblastic ROS 17/2.8 cells expressing PTH/PTHrP receptors. The cAMP accumulation activity in synovial fluid was sensitive to coincubation with excess hPTHrP(3–40), a PTH/PTHrP receptor antagonist, and was completely neutralized by preincubation with a monoclonal antibody specific to hPTHrP but not PTH. Immunohistochemical analysis of RA synovium revealed that PTHrP was localized in fibroblast‐like cells in the synovial pannus invading articular cartilage. Our data show that PTHrP is produced locally by the diseased synovial tissue and released into synovial fluid at high concentrations, allowing us to hypothesize that PTHrP plays a novel role as a paracrine/autocrine factor in the pathology of OA and RA.

Bone morphogenetic proteins (BMP‐2 and BMP‐3) induce the late phase expression of the proto‐oncogene c‐fos in murine osteoblastic MC3T3‐E1 cells

Here we report that bone morphogenetic proteins 2 and 3 (BMP‐2 and BMP‐3) induced marked expression of c‐fos mRNA in a biphasic manner, i.e. the late phase (48 to 60 h) as well as the immediate‐early phase (0.5 h), in murine osteoblastic MC3T3‐El cells in vitro. The BMP‐induced late phase c‐fos gene expression was temporally associated with the onset of marked expression of the genes for osteocalcin and alkaline phosphatase, differentiation markers of mature osteoblasts. In contrast, none of TGF‐β1, 10% FBS, IGF‐I and IGF‐II, which induced only the immediate‐early c‐fos mRNA expression, stimulated the expression of osteocalcin and alkaline phosphatase genes. These data suggest that in osteoblasts BMP‐2 and BMP‐3 induce the late phase expression of c‐fos, which may play a role in transcriptional activation of the genes involved in differentiation of osteoblasts.

Fracture healing induces expression of the proto-oncogene c-fos in vivo. Possible involvement of the Fos protein in osteoblastic differentiation.

Here we report marked in vivo expression of the c‐fos gene in the external soft callus (ESC) and periosteal hard callus (PHC) at the fracture site of adult rat tibia. Northern‐blot analysis showed that the ESC expressed a high level of c‐fos mRNA from post‐fracture day 10 to day 28, the time when endochondral ossification progressed, and that the ossifying PHC also expressed c‐fos mRNA. This c‐fos expression was followed by sequential expression of the genes for alkaline phosphatase, osteopontin and osteocalcin, which are osteoblastic markers. Immunohistochemical analysis showed that the c‐Fos protein was predominantly located in osteoblasts in the ossifying calluses.

Osteoclast-mediated osteolysis in bone metastasis from renal cell carcinoma

Osteolytic characteristics of bone metastasis from renal cell carcinoma were morphologically and biochemically investigated. First, undecalcified ground sections of bone metastases were made from four patients with renal cell carcinoma. Second, renal cell carcinoma cell line (RCC‐K1) was established from one of the four patients, and its effect on bone resorption in vitro was examined. Marked proliferation and activation of osteoclasts around the tumor cells was histologically demonstrated. Conditioned medium from the RCC‐K1 cells contained potent bone‐resorbing activity in vitro. The activity was reduced to basal level by calcitonin, but was not blocked by indomethacin. The activity was lost after dialysis (MW cutoff 3500), while it was retained after 2 weeks of storage. Levels of prostaglandin E2 and 1,25‐dihydroxyvitamin D of the RCC‐K1‐conditioned medium were insufficient to cause bone resorption in vitro. The conditioned medium did not stimulate cAMP accumulation in rat osteoblastic cells. These results suggest that renal cell carcinoma causes bone destruction through the stimulation of osteoclasts by locally secreting an unknown humoral factor or factors.

Bone morphogenetic protein-6 and parathyroid hormone-related protein coordinately regulate the hypertrophic conversion in mouse clonal chondrogenic EC cells, ATDC5

We evaluated the roles of bone morphogenetic protein (BMP)-6, BMP-4 and parathyroid hormone-related protein (PTHrP) in the hypertrophic conversion using mouse chondrogenic EC cells, ATDC5. In ATDC5 cells, the expression of BMP-6 and PTHrP receptor mRNAs increased in parallel with the progression of chondrogenic differentiation of these cells, exhibiting a time course similar to that of type II collagen, a phenotypic marker of proliferating chondrocytes, while BMP-4 mRNA was continuously expressed throughout the differentiation processes. The expression of type X collagen mRNA, a phenotypic marker of hypertrophic chondrocytes, was upregulated by BMP-6 and BMP-4, and downregulated by PTHrP(1-141). The expression of BMP-6 mRNA was upregulated while that of BMP-4 mRNA was downregulated by both BMP-6 and BMP-4. Moreover, the expression of BMP-6 mRNA was downregulated by PTHrP(1-141). Furthermore, even in the presence of PTHrP(1-141), BMP-6 increased the transcript level of type X collagen in a dose-dependent manner. These results indicate that transiently expressed BMP-6 promotes the hypertrophic conversion in association with the augmentation of BMP-6 gene expression by BMP signals and that both BMP-6 and PTHrP coordinately regulate the rate of the hypertrophic conversion of ATDC5 cells.

Effects of transforming growth factor-β signaling on chondrogenesis in mouse chondrogenic EC cells, ATDC5

Cellular condensation of chondroprogenitors is a distinct cellular event in chondrogenesis. During this process, N-cadherin mediates cell-cell interactions responsible for the initial stage of cellular condensation and subsequently fibronectin contributes to cell-matrix interactions mediating a progression of chondrogenesis. We previously showed that chondrogenesis in mouse chondrogenic EC cells, ATDC5, was induced, at a high incidence in the presence of insulin, through formation of cellular condensation. In this study, we took advantage of the sequential progression of chondrogenesis in ATDC5 cells and evaluated, in vitro in these cells, the role of endogenous transforming growth factor (TGF)-beta in chondrogenesis. ATDC5 cells expressed TGF-beta2 mRNA at a cellular condensation stage. The treatment of undifferentiated ATDC5 cells with anti-TGF-beta32 neutralizing antibody inhibited the accumulation of Alcian blue stainable proteoglycan in a dose-dependent manner. Transfection of a dominant-negative mutant of mouse TGF-beta type II receptor to undifferentiated ATDC5 cells completely inhibited cellular condensation. Moreover, exogenously administered TGF-beta2 upregulated the expression of fibronectin and type II collagen (a phenotypic marker gene of chondrogenesis) mRNAs and downregulated that of N-cadherin mRNA in time- and dose-dependent manners. These results indicate that TGF-beta stimulates chondrogenesis via initiation of cellular condensation by transition from an initial N-cadherin-contributing stage to a fibronectin-contributing stage during processes of chondrogenesis in ATDC5 cells.