Comparison of mouse and human ankles and establishment of mouse ankle osteoarthritis models by surgically-induced instability.

OBJECTIVE: Prevalence of ankle osteoarthritis (OA) is lower than that of knee OA, however, the molecular mechanisms underlying the difference remain unrevealed. In the present study, we developed mouse ankle OA models for use as tools to investigate pathophysiology of ankle OA and molecular characteristics of ankle cartilage. DESIGN: We anatomically and histologically examined ankle and knee joints of C57BL/6 mice, and compared them with human samples. We examined joints of 8-week-old and 25-month-old mice. For experimental models, we developed three different ankle OA models: a medial model, a lateral model, and a bilateral model, by resection of respective structures. OA severity was evaluated 8 weeks after the surgery by safranin O staining, and cartilage degradation in the medial model was sequentially examined. RESULTS: Anatomical and histological features of human and mouse ankle joints were comparable. Additionally, the mouse ankle joint was more resistant to cartilage degeneration with aging than the mouse knee joint. In the medial model, the tibiotalar joint was markedly affected while the subtalar joint was less degenerated. In the lateral model, the subtalar joint was mainly affected while the tibiotalar joint was less altered. In the bilateral model, both joints were markedly degenerated. In the time course of the medial model, TdT-mediated dUTP nick end labeling (TUNEL) staining and Adamts5 expression were enhanced at early and middle stages, while Mmp13 expression was gradually increased during the OA development. CONCLUSION: Since human and mouse ankles are comparable, the present models will contribute to ankle OA pathophysiology and general cartilage research in future.

Lack of GPR40/FFAR1 does not induce diabetes even under insulin resistance condition.

AIMS: G protein-coupled receptor/free fatty acid receptor 1 (GPR40/FFAR1 ) regulates free fatty acid-induced insulin secretion. This study has been performed to clarify whether or not loss of GPR40/FFAR1 function exacerbates diabetes, that is, whether GPR40 has an essential physiological role in the development of diabetes or not. METHODS: We generated GPR40/FFAR1 knockout (KO) mice and analysed their phenotypes in vitro and in vivo under the condition of dietary or genetically induced insulin resistance. RESULTS: GPR40/FFAR1 KO mice kept on a high-fat diet became obese, developed glucose intolerance to a similar degree as GPR40/FFAR1 wild-type (WT) mice. In addition, the phenotype of KO mice harbouring diabetogenic KK background genes showed glucose intolerance at a level similar to level for control KK mice. In both mouse models with insulin resistance, insulin secretion after oral glucose load and homeostasis model assessment-insulin resistance (HOMA-IR) did not change between GPR40/FFAR1 KO and WT mice. Although glucose-induced insulin secretion under high palmitate concentration was significantly lower in KO than in WT islets, pancreatic insulin content and insulin secretion stimulated with glucose alone were not different between KO and WT mice. CONCLUSIONS: GPR40/FFAR1 has a major role in regulating fatty-acid-mediated insulin secretion, but the lack of GPR40/FFAR1 does not exacerbate glucose intolerance and insulin resistance induced by high-fat diet or diabetogenic KK gene. Our findings indicate that loss of GPR40/FFAR1 function does not play an important role in inducing or exacerbating diabetes.

Photorefractive multiple quantum wells at 1064 nm.

We have fabricated photorefractive InGaAs/GaAs multiple quantum wells that are sensitive at wavelengths near 1.06 mum for what is believed to be the first time. We have measured four-wave-mixing diffraction efficiency, using a Nd:YAG laser. A maximum diffraction efficiency of 7 x 10(-4) and a cutoff grating period of ~2 mum are obtained.

Enhancement of osteogenesis in vitro and in vivo by a novel osteoblast differentiation promoting compound, TAK-778.

TAK-778 [(2R,4S)-(-)-N-(4-diethoxyphosphorylmethylphenyl)-1,2,4, 5-tetrahydro-4-methyl-7, 8-methylenedioxy-5-oxo-3-benzothiepin-2-carboxyamide; mw 505.53], a novel osteoblast differentiation promoting compound, was characterized in vitro and in vivo models. TAK-778 at doses of 10(-6) M and higher promoted potently bone-like nodule formation in the presence of dexamethasone in rat bone marrow stromal cell culture. This was accompanied by increases in cellular alkaline phosphatase activity, soluble collagen release, and osteocalcin secretion. Under the culture conditions, TAK-778 also stimulated the secretion of transforming growth factor-beta and insulin-like growth factor-I, indicating that TAK-778 may exert regulatory effects on osteoblast differentiation via autocrine/paracrine mechanisms. Furthermore, the in vivo osteogenic potential of TAK-778 was studied in bony defect and osteotomy animal models, using sustained release microcapsules consisted of a biodegradable polymer, poly (dl-lactic/glycolic) acid (PLGA). Single local injection of TAK-778/PLGA-microcapsules (PLGA-MC) (0.2-5 mg/site) to rat skull defects resulted in a dose-dependent increase in new bone area within the defects after 4 weeks. When the pellet containing TAK-778/PLGA-MC (4 mg/pellet) was packed into place to fill the tibial segmental defect in rabbit, this pellet induced osseous union within 2 months, whereas the placebo pellet did not. In addition, single local application of TAK-778/PLGA-MC (10 mg/site) to rabbit tibial osteotomy site enhanced callus formation accompanied by an increase in breaking force after 30 days. These results reveal for the first time that a nonendogenous chemical compound promotes potently osteogenesis in vitro and enhances new bone formation during skeletal regeneration and bone repair in vivo and should be useful for the stimulation of fracture healing.

Studies on disease-modifying antirheumatic drugs. III. Bone resorption inhibitory effects of ethyl 4-(3,4-dimethoxyphenyl)-6,7-dimethoxy-2-(1,2,4-triazol-1-ylmethyl) quinoline-3-carboxylate (TAK-603) and related compounds.

In the course of our studies aimed at obtaining new drugs for treatment of bone and joint diseases, chemical modification of the potent bone resorption inhibitors justicidins, was performed and various naphthalene lactones, quinoline lactones and quinoline derivatives bearing an azole moiety at the side chain were prepared. Their inhibitory effects on bone resorption were evaluated by Raisz's method, and several compounds, including ethyl 4-(3,4-dimethoxyphenyl)-6,7-dimethoxy-2-(1,2,4-triazol-1-ylmeth yl)quinoline -3-carboxylate (6c, TAK-603), were found to have activities comparable with or superior to the justicidins. The 4-(3-isopropoxy-4-methoxy)-phenyl derivative (6d), in particular, displayed a marked increase in potency. TAK-603 and compound 6d were very effective in preventing osteoclast formation and bone resorption by mature osteoclasts. Further, TAK-603 was shown to be effective in preventing bone loss in ovariectomized mice.

Synthesis of novel 2-benzothiopyran and 3-benzothiepin derivatives and their stimulatory effect on bone formation.

In a search for therapeutic agents for the treatment of osteoporosis and bone fracture, we found that 2-benzothiopyran-1-carboxamide derivatives 1, derived from ipriflavone as a lead compound, increase cellular alkaline phosphatase activity in cultures of rat bone marrow stromal cells. Further modification of 1 has led to the discovery of more potent 3-benzothiepin-2-carboxamide derivatives 2. Of these, 3-benzothiepin derivatives bearing a 4-(dialkoxyphosphorylmethyl)phenyl group on the 2-carboxamide moiety such as 2h and 2q exhibited significant improvement of activity compared to ipriflavone. Asymmetric synthesis of 2h and 2q revealed that the (-)-isomers possessed activities superior to those of the (+)-isomers. Further evaluation of these compounds using the mouse osteoblastic cell line MC3T3-E1 revealed that (-)-2q enhanced the effect of bone morphogenetic protein. In addition, application of a sustained-release agent containing 2q increased the area of newly formed bone in a rat skull defect model. Based on these findings, (-)-2q was selected for further investigation as a new drug stimulating bone formation. Synthesis and structure-activity relationships for this novel series of 2-benzothiopyran and 3-benzothiepin derivatives are detailed.

Advanced glycation end products enhance osteoclast-induced bone resorption in cultured mouse unfractionated bone cells and in rats implanted subcutaneously with devitalized bone particles.

Advanced glycation end products (AGE) are formed in long-lived matrix proteins by a nonenzymatic reaction with sugar. The presence of AGE in beta 2-microglobulin-amyloid fibrils of dialysis-related amyloidosis, one of the characteristic features of which is an accelerated bone resorption around amyloid deposits, was recently demonstrated. This suggested a potential link of AGE in bone resorption and initiated this investigation of whether AGE enhance bone resorption. When mouse unfractionated bone cells containing osteoclasts were cultured on dentin slices, both AGE-modified beta 2-microglobulin and BSA increased the number of resorption pits formed by osteoclasts, whereas their normal counterparts of those modified with the early glycation products did not. AGE proteins, however, did not increase the number of newly formed osteoclasts, even in the coculture of mouse bone marrow cells with osteoblastic cells isolated from mouse calvaria. Enhanced bone resorption was also observed when unfractionated bone cells were cultured on AGE-modified dentin slices. AGE-enhanced bone resorption was effectively inhibited by calcitonin and ipriflavone, both of which are inhibitors of bone resorption. AGE-enhanced bone resorption was further supported by in vivo evidence that rat bone particles-upon incubation with glucose for 60 days (AGE-bone particles)-when implanted subcutaneously in rats, were resorbed to a much greater extent than control bone particles upon parallel incubation without glucose. These findings suggest that AGE enhance osteoclast-induced bone resorption. Although the mechanism remains unknown, AGE are unlikely to promote differentiation of osteoclast progenitors into osteoclasts, suggesting that AGE activate osteoclasts or alter microenvironments favorable for bone resorption by osteoclasts. The modification of bone matrices with AGE might play a role in the remodeling of senescent bone matrix tissues, further implicating a pathological significance of AGE in dialysis-related amyloidosis or osteoporosis associated with diabetes and aging.

Studies on disease-modifying antirheumatic drugs: synthesis of novel quinoline and quinazoline derivatives and their anti-inflammatory effect.

In the course of our study aimed at developing new types of DMARDs (disease-modifying antirheumatic drugs), we found that quinoline derivative 1a had a potent anti-inflammatory effect in an adjuvant arthritis (AA) rat model, starting from the potent bone resorption inhibitors justicidins as the lead compounds. Further modification of 1a was performed, and various quinoline and quinazoline derivatives having a heteroaryl moiety on the alkyl side chain at the 2-position of the skeleton were prepared. These compounds were evaluated for anti-inflammatory effects using the AA rat model. Most of these compounds, especially those having an imidazole or a triazole moiety on the 2-alkyl chain, exhibited a potent effect. Among the compounds synthesized, ethyl 4-(3,4-dimethoxyphenyl)-6,7-dimethoxy-2-(1,2, 4-triazol-1-yl-methyl)quinoline-3-carboxylate (12d), having an ED50 value of 2.6 mg/kg/day (anti-inflammatory effect in an AA rat model, po), was selected as a candidate for further investigation. In vitro, 12d inhibited mitogen-induced proliferation at 10(-7)-10(-5) M but not prostaglandin E2 production at 10(-5) M. Moreover, 12d preferentially inhibited the IFN-gamma production by Th1-type clones over the IL-4 production by Th2-type clones. This preferential suppression of Th1 cytokine production is considered the essential immunomodulating action of 12d for the present. Synthesis and structure-activity relationships for this novel series of quinoline and quinazoline derivatives are detailed.

Novel ipriflavone receptors coupled to calcium influx regulate osteoclast differentiation and function.

Ipriflavone (7-isopropoxyisoflavone) is an effective antiresorptive agent used to treat osteoporosis. However, the mechanism of its action on osteoclasts and their precursor cells is not well understood. To determine whether the mechanism involves direct effects on osteoclasts or their precursors, we examined the effects of ipriflavone on cytosolic free calcium ([Ca2+]i) in osteoclasts and their precursors and measured specific binding of 3H-labeled ipriflavone. Highly purified chicken osteoclast precursors, which spontaneously differentiate into multinucleated osteoclasts in 3-6 days, were loaded with fura-2, and the subcellular [Ca2+]i distribution was monitored by videoimaging. Ipriflavone induced a rapid increase in [Ca2+]i followed by a sustained elevation [EC50 = 5 x 10(-7) M, 263 +/- 74 nM (SE) (n = 8) above basal levels, by 10(-6) M ipriflavone, sustained phase]. The responses were the same in differentiated chicken osteoclasts and isolated rabbit osteoclasts. An influx of extracellular Ca2+ is likely to be responsible for the ipriflavone-induced change in [Ca2+]i because the response was abolished by 0.5 mM LaCl3, or by Ca-free medium containing EGTA. Moreover, high [Ca2+]i levels were detected adjacent to the cell membrane after ipriflavone addition. Ipriflavone induced Ca influx mainly through dihydropyridine-insensitive Ca2+ channels, because nicardipine (10(-7)M) and verapamil (10(-7)M) had no effects on ipriflavone-induced [Ca2+]i responses. [3H]Ipriflavone binding studies indicated the presence of specific ipriflavone binding sites (two classes), both in precursor cells [dissociation constant (Kd), 7.60 x 10(-8)M, 2.67 x 10(-6)M] and in mature osteoclasts (Kd, 4.98 x 10(-8)M, 3.70 x 10(-6)M). Specific ipriflavone binding was not displaced by various modulators of avian osteoclast function, such as estradiol (10(-8)M) or retinoic acid (10(-6)M), indicating that ipriflavone receptors differ from the receptors for these Ca-regulating hormones. The fusion of osteoclast precursor cells was significantly inhibited by ipriflavone, which led to dose-dependent inhibition of bone resorption and tartrate-resistant acid phosphatase activity. Novel specific ipriflavone receptors that are coupled to Ca2+ influx were demonstrated in osteoclasts and their precursor cells. These ipriflavone receptors may provide a mechanism to regulate osteoclast differentiation and function.

Increase in femoral bone mass by ipriflavone alone and in combination with 1 alpha-hydroxyvitamin D3 in growing rats with skeletal unloading.

We assessed the possibility that ipriflavone treatment might result in bone restoration in immobilized rats. We also investigated the effect of combined treatment with ipriflavone and vitamin D3 on the bone. Male Sprague-Dawley rats, 6 weeks of age, were subjected to unilateral sciatic neurectomy. Three weeks after the operation, ipriflavone (100 mg/kg), 1 alpha-hydroxyvitamin D3 [1 alpha (OH)D3, 25 ng/kg], or both ipriflavone and 1 alpha (OH)D3 were orally administered every day for 12 or 24 weeks. After 12 weeks of treatment, only the group receiving combined treatment with ipriflavone and 1 alpha (OH)D3 showed increases in total femur calcium content (+16.4%, compared with the control). After 24 weeks, both animals treated with ipriflavone alone and those that had received the combination of ipriflavone and 1 alpha (OH)D3 showed significant increases in femur calcium content (+18.0% and +23.8%, respectively). In these treatment groups, X-ray analysis revealed an increase in bone mineral density over the entire length of the femur, and an increase in cortical diameter at the midshaft without affecting medullary width. Administration of 1 alpha (OH)D3 (25 ng/kg) alone had no effect. Body weight, femur length, and serum markers of calcium and bone metabolism were not affected in any group. We evaluated the relationship between ipriflavone and vitamin D3 in bone cells in a culture system using rat bone marrow stromal cells in which the cells subsequently form mineralized bone-like tissue. Continuous treatment with ipriflavone (10(-5) M) for 21 days resulted in an increase in osteocalcin secretion, and enhanced its response to 1 alpha, 25-dihydroxyvitamin D3 (10(-11) M-10(-8 M)). These findings indicate that ipriflavone treatment increases the femoral bone mass in immobilized rats. In addition, a low dose of 1 alpha (OH)D3, which did not induce hypercalcemia, in combination with ipriflavone, augmented the stimulatory effect of ipriflavone alone on the bone mass, possibly due to a direct effect of each agent on osteoblastic cells.

Possible involvement of advanced glycation end-products in bone resorption.

Advanced glycation end-products (AGEs) are formed in long-lived matrix proteins by a non-enzymatic reaction with sugar. We recently demonstrated the presence of AGEs in amyloid fibrils of dialysis-related amyloidosis, one of the characteristic features of which is an accelerated bone resorption around amyloid deposits. This suggested a potential link of AGEs in bone resorption and led us to investigate whether AGEs enhance bone resorption. An immunohistochemical study using anti-AGE antibody revealed positive immunostaining for AGEs in bone tissues from elderly subjects. AGE-modified proteins were shown to stimulate monocyte/macrophage to secrete bone-resorbing cytokines such as interleukin-1 beta, interleukin-6 and tumour necrosis factor- alpha. AGE-modified proteins enhanced net calcium efflux in cultured neonatal mouse calvariae to a much greater extent than unmodified proteins. Furthermore, when mouse unfractionated bone cells containing osteoclasts were cultured on dentin slices, AGE-modified proteins increased the number of resorption pits formed by osteoclasts, whereas their normal counterparts or those modified with the early glycation products did not. These findings suggest that AGEs enhance bone resorption by osteoclasts. The modification of bone matrices with AGEs might, therefore, play a pathophysiological role not only in the remodelling of senescent bone matrix tissues, but also in dialysis-related amyloidosis or osteoporosis associated with diabetes and ageing.

Potent ectopic bone-inducing activity of bone morphogenetic protein-4/7 heterodimer.

We have purified and characterized recombinant Xenopus bone morphogenetic proteins (xBMPs): homodimers of xBMP-4, 7 and heterodimers (xBMP-4/7) produced by a baculovirus expression system. Highly purified xBMPs had homogeneous NH2-termini predicted from a consensus motif, Arg-X-X-Arg, while they possessed diverse sugar chains. Implantation of xBMPs together with pure collagen carrier in rats induced new bone formation in a dose-dependent manner. The xBMP-4/7 heterodimer showed the strongest activity, with an effective dose of 1-30 micrograms, while more than 10 micrograms of xBMP-4 or 7 homodimer was required for a significant effect. Histological examination revealed that xBMP-4/7 implants showed intramembranous ossification without chondrogenesis. In primary cultures of rat bone marrow stromal cells, xBMP-4/7 induced alkaline phosphatase 3-fold more strongly than xBMP-7 and 20-fold more than xBMP-4. These results suggest that the heterodimeric form of BMP would generate the strongest signal triggering osteogenic differentiation of osteoprogenitor cells in adult tissues.

Similarities and differences between the effects of ipriflavone and vitamin K on bone resorption and formation in vitro.

The effects of ipriflavone and vitamin K on bone metabolism were examined using a culture system. Vitamin K1 and vitamin K2 (10(-7)M-10(-5)M) inhibited both the activation of mature osteoclasts and the formation of new osteoclasts without affecting the growth of progenitor cells in cultures of mouse unfractionated bone cells. The inhibitory effects of vitamin K on bone resorption were similar to those of ipriflavone and were not affected by the vitamin K antagonist warfarin. When ipriflavone was added to the culture medium in combination with vitamin K2, an additive inhibitory effect on bone resorption was observed. An additive effect was also observed in organ cultures of mouse calvaria. On the other hand, ipriflavone, but neither vitamin K1 nor vitamin K2, stimulated cellular alkaline phosphatase (ALP) activity on rat bone marrow stromal cells under culture conditions in which cells subsequently form mineralized bone-like tissue. Vitamin K1 and vitamin K2 also did not modulate the stimulatory effect of ipriflavone on the ALP activity of the cells. These results suggest that the inhibitory effects of vitamin K on bone resorption are similar to those of ipriflavone through mechanisms that may be independent of the gamma-carboxylation system, while the effects of vitamin K on osteoblast phenotype expression are different from those of ipriflavone.

Production of human PTH(1-34) via a recombinant DNA technique.

The production of hPTH(1-34) by site specific chemical cleavage of [Cys35]hPTH(1-84) obtained by a biotechnology technique is described. The amino-peptide bond of S-cyanylated cysteine residues in peptides or proteins is specifically cleaved by exposure to an alkaline solution (Stark, G. R. (1977) Methods Enzymol, 47, 129-132). However, when applying this method to [Cys35]hPTH(1-84), we observed many by-products. Formation of these by-products was suppressed using a short reaction in 0.03N NaOH containing 6M urea at low temperature, and hPTH(1-34) was specifically produced. The product was indistinguishable from standard hPTH(1-34) with respect to chemical and biological characterization.

Effect of ipriflavone on expression of markers characteristic of the osteoblast phenotype in rat bone marrow stromal cell culture.

The effects of ipriflavone on cellular proliferation and differentiation of osteoblasts were investigated using stromal cells isolated from the femoral bone marrow of young rats. To induce the formation of mineralized bone-like tissue in vitro, the cells were cultured in the presence of beta-glycerophosphate and dexamethasone. Ipriflavone was added when subculturing was started. After 14 days of culturing with ipriflavone (10(-7)-10(-5) M), increases in both the alkaline phosphatase activity and the hydroxyproline content per culture dish and a slight decrease in the saturated cell density were observed. Furthermore, continuous treatment with ipriflavone for 14-33 days resulted in an increase in the area of bone-like mineralized tissue accompanied by an increase in the secretion of osteocalcin. When culture medium lacking dexamethasone was used, rat bone marrow stromal cells neither differentiated into osteoblasts nor formed bone-like tissue, and under these conditions, ipriflavone had no effect on the proliferation or the phenotypic expression of the cells. These results suggest that ipriflavone directly stimulates markers of the osteoblast phenotype at a certain stage in bone formation without affecting undifferentiated cells that have not been committed to the osteogenic lineage.

Inhibitory effect of ipriflavone on osteoclast-mediated bone resorption and new osteoclast formation in long-term cultures of mouse unfractionated bone cells.

To study the effect of ipriflavone on osteoclast-mediated bone resorption and new osteoclast formation, we used an unfractionated bone cell culture system containing mature osteoclasts from femur and tibia of newborn mice. Ipriflavone (10(-5) M) inhibited pit formation on dentin slices and caused a decrease in the number of tartrate-resistant acid phosphatase (TRAP)-positive (+) multinucleate cells (MNCs) in a 4-day culture period in which no increase in the number of TRAP(+)-MNCs was observed in the presence of 5% fetal bovine serum (FBS) and 10(-8) M 1 alpha,25-dihydroxy-vitamin D3 (1 alpha,25(OH)2D3). During the following 12 days, both the total area of the pits and the number of TRAP(+)-MNCs increased in the control. Continuous treatment with ipriflavone also inhibited the increase in pit area during this period. These effects of ipriflavone were reversible. Furthermore, the differentiation of osteoclasts was examined when preexisting TRAP(+)-MNCs were removed by incubation in the absence of 1 alpha,25(OH)2D3 for the initial 4 days in culture dishes without dentin slices. When 1 alpha,25(OH)2D3 and ipriflavone were added to the medium on the 4th day, ipriflavone inhibited new TRAP(+)-MNC formation stimulated by 1 alpha,25(OH)2D3 in a dose-dependent manner. However, pretreatment of the cells with ipriflavone before the addition of 1 alpha,25(OH)2D3 did not inhibit TRAP(+)-MNC formation. These results indicate that ipriflavone inhibits both the activation of mature osteoclasts and the formation of new osteoclasts without affecting growth of TRAP-negative progenitor cells.

In vivo stimulation of endosteal bone formation by basic fibroblast growth factor in rats.

Intravenous administration of human basic fibroblast growth factor (bFGF) for 2 weeks stimulated osteoblast proliferation and new bone formation in various skeletal bones in young and aged rats at dosage levels of 0.1 mg/kg/day and greater. Morphometry of the soft X-ray radiograms of cross sections of the tibia indicated about a 20% increase in the calcified bone area of the diaphysis at 0.1 mg/kg/day. The Ca and hydroxyproline contents showed statistically significant increases at this dosage. The new bone formation was found only on the endosteal side, and no periosteal bone formation was found. Similar systemic osteogenic potential was seen after intravenous administration of other growth factors of the FGF family, human acidic FGF and human heparin-binding secretory transforming protein-1. The above results suggest a potential therapeutic role for these growth factors in bone-loss diseases such as osteoporosis.

Stimulatory effect of ipriflavone on formation of bone-like tissue in rat bone marrow stromal cell culture.

The effects of ipriflavone (IP) (10(-5) M) on bone formation were studied in stromal cells from the femoral bone marrow of young adult rats cultured for 21 days in the presence of beta-glycerophosphate and dexamethasone. Stereoscopic microscopy showed nodule formation after 14 days of culturing, and both the number and the size of the nodules increased with time. The alizarin-red-stained calcified area in the nodules in the IP group was nearly 4 times as large as that in the control after 21 days. Light and electron microscopy revealed the presence of many osteoblast-like cells with developed rough endoplasmic reticulum and Golgi apparatus in the nodules in the control group after 14 days, and a collagenous fibril network was seen among the cells. After 21 days, calcification of the dense collagenous fibril network and bone matrix-like tissue were observed in many nodules, resulting in the formation of bone-like tissue containing osteocyte-like cells. In the IP group, the collagenous fibril network area in the nodules was greater than that in the control after 14 days, and a further increase in both the dense collagenous fibril network area and calcified bone-like tissue area was observed after 21 days. These findings indicate that IP stimulates bone-like tissue formation in the rat bone marrow stromal cell culture, suggesting that the promotion of collagen production by osteoblasts is involved in the stimulation of bone-like tissue formation by IP.

Inhibitory effect of ipriflavone on pit formation in mouse unfractionated bone cells.

Effects of ipriflavone (7-isopropoxyisoflavone) on osteoclast-induced bone resorption were evaluated using an unfractionated bone cell culture system containing mature osteoclasts from the femur and tibia of newborn mice. When cells were cultured for 4 days on dentin slices in the presence of 5% fetal bovine serum and 10(-8) M 1 alpha, 25(OH)2D3, ipriflavone (3 x 10(-7) -3 x 10(-5) M) inhibited pit formation and caused a decrease in the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs). The lowest significant effect was observed at a concentration of 10(-6) M. Unlike ipriflavone, calcitonin inhibited pit formation 4 days after the culture was started without affecting the number of TRAP-positive MNCs. Ipriflavone still inhibited pit formation when the culture period was 13 days, when new osteoclasts were expected to be formed. These findings suggest that ipriflavone inhibits new osteoclast formation and bone resorption at the cellular level.