Fursultiamine, a vitamin B1 derivative, enhances chondroprotective effects of glucosamine hydrochloride and chondroitin sulfate in rabbit experimental osteoarthritis.

OBJECT AND DESIGN: The therapeutic effect of glucosamine hydrochloride (GH) and chondroitin sulfate (CS) in combination with fursultiamine, a vitamin B1 derivative, on the development of cartilage lesions was investigated in an animal model of osteoarthritis (OA). METHODS: The OA model was created by partial medial meniscectomy of the right knee joint (day 0). The rabbits were placed into three experimental groups: operated (OA) rabbits that received placebo treatment, OA rabbits that received GH (1000 mg/kg) + CS (800 mg/kg), and OA rabbits that received GH + CS + fursultiamine (100 mg/kg). Each treatment was initiated on day 3 and continued for 8 weeks. Macroscopic and histologic analyses were performed on the cartilage. The level of MMP-1 in OA cartilage chondrocytes was evaluated by immunohistochemistry. RESULTS: Only the group receiving combined treatment with GH + CS + fursultiamine showed a significant reduction in the severity of macroscopic and histologic lesions on tibial plateau, which is the weight bearing cartilage surface of the tibia, compared with placebo-treated OA rabbits. This treatment group also revealed a small, but significant, decrease in the body weight gain of the rabbits. In cartilage from placebo-treated OA rabbits, a significantly higher percentage of chondrocytes in superficial layer stained positive for MMP-1 compared with unoperated control. Rabbits treated with the GH + CS + fursultiamine revealed a significant reduction in the level of MMP-1. CONCLUSION: These results suggest that the chondroprotective effect of GH + CS is enhanced by the addition of fursultiamine in experimental OA. This effect was associated with a reduction in the level of MMP-1, which are known to play an important role in the pathophysiology of OA lesions.

The induction of cell death in human osteoarthritis chondrocytes by nitric oxide is related to the production of prostaglandin E2 via the induction of cyclooxygenase-2.

There is increasing evidence suggesting that chondrocyte death may contribute to the progression of osteoarthritis (OA). This study focused on the characterization of signaling cascade during NO-induced cell death in human OA chondrocytes. The NO generator, sodium nitroprusside (SNP), promoted chondrocyte death in association with DNA fragmentation, caspase-3 activation, and down-regulation of Bcl-2. Both caspase-3 inhibitor Z-Asp(OCH3)-Glu(OCH3)-Val-Asp(OCH3)-CH2F and caspase-9 inhibitor Z-Leu-Glu(OCH3)-His-Asp(OCH3)-CH2F prevented the chondrocyte death. Blocking the mitogen-activated protein kinase pathway by the mitogen-activated protein kinase kinase 1/2 inhibitor PD98059 or p38 kinase inhibitor SB202190 also inhibited the SNP-mediated cell death, suggesting possible requirements of both extracellular signal-related protein kinase 1/2 and p38 kinase for the NO-induced cell death. Furthermore, the selective inhibition of cyclooxygenase (COX)-2 by NS-398 or the inhibition of COX-1/COX-2 by indomethacin blocked the SNP-induced cell death. The chondrocyte death induced by SNP was associated with an overexpression of COX-2 protein (as determined by Western blotting) and an increase in PGE2 release. PD98059 and SB202190, but neither Z-DEVD FMK nor Z-LEHD FMK completely inhibited the SNP-mediated PGE2 production. Analysis of interactions between PGE2 and the cell death showed that PGE2 enhanced the SNP-mediated cell death, whereas PGE2 alone did not induce the chondrocyte death. These data indicate that NO-induced chondrocyte death signaling includes PGE2 production via COX-2 induction and suggest that both extracellular signal-related protein kinase 1/2 and p38 kinase pathways are upstream signaling of the PGE2 production. The results also demonstrate that exogenous PGE2 may sensitize human OA chondrocytes to the cell death induced by NO.

Enhancement of fracture repair in rats with streptozotocin-induced diabetes by a single injection of biodegradable microcapsules containing a bone formation stimulant, TAK-778.

The feasibility of using microcapsules containing a bone formation stimulant, (2R,4S)-(-)-N-(4-diethoxyphosphorylmethylphenyl)-1,2,4, 5-tetrahydro-4-methyl-7, 8-methylenedioxy-5-oxo-3-benzothiepin-2-carboxamide (TAK-778) to enhance fracture repair was assessed in rats with streptozotocin-induced diabetes. The release profile of the microcapsules was designed to mimic a dosing regimen of multiple injections of TAK-778 solution. The solution was injected locally every third day from day 0 (the day of operation) to day 27 according to several dosing regimens, and fracture repair was assessed at day 28. The production of callus was most prominent when TAK-778 solution was injected so that 50-75% of the total dose (5 mg TAK-778/site) was administered during the first half of the treatment period. Thus, injectable microcapsules of 30 micrometer in mean diameter were prepared in order to release TAK-778 over 4 weeks using a biodegradable polymer, poly(d,l-lactic/glycolic) acid, with a copolymer ratio of 85:15 (mol/mol) and an average molecular weight of 14,000. A single local injection of the microcapsules markedly enhanced fracture repair, which resulted in recovery of destructive bending strength of the bone at day 28. Histologically, the injection of TAK-778 microcapsules stimulated both fibrous and cartilaginous proliferation and periosteal ossification in the callus at day 7; bony bridge formation was observed at day 28. At day 56, the callus was remodeled and cortical bony union was evidenced in the microcapsule-treated fractures compared with the controls, which showed only fibrous union.

Parathyroid hormone-activated volume-sensitive calcium influx pathways in mechanically loaded osteocytes.

This paper documents for the first time a volume-sensitive Ca(2+) influx pathway in osteocytes, which transmits loading-induced signals into bone formation. Stretch loading by swelling rat and chicken osteocytes in hypo-osmotic solution induced a rapid and progressive increase of cytosolic calcium concentration, [Ca(2+)](i). The influx of extracellular Ca(2+) explains the increased [Ca(2+)](i) that paralleled the increase in the mean cell volume. Gadolinium chloride (Gd(3+)), an inhibitor of stretch- activated cation channels, blocked the [Ca(2+)](i) increase caused by hypotonic solutions. Also, the expression of alpha1C subunit of voltage-operated L-type Ca(2+) channels (alpha1C) is required for the hypotonicity-induced [Ca(2+)](i) increase judging from the effect of alpha1C antisense oligodeoxynucleotides. Parathyroid hormone (PTH) specifically potentiated the hypotonicity-induced [Ca(2+)](i) increase in a dose-dependent manner through the activation of adenyl cyclase. The increases induced by both PTH and hypotonicity were observed primarily in the processes of the osteocytes. In cyclically stretched osteocytes on flexible-bottomed plates, PTH also synergistically elevated the insulin-like growth factor-1 mRNA level. Furthermore, Gd(3+) and alpha1C antisense significantly inhibited the stretch-induced insulin-like growth factor-1 mRNA elevation. The volume-sensitive calcium influx pathways of osteocytes represent a mechanism by which PTH potentiates mechanical responsiveness, an important aspect of bone formation.

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.

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.

Activation by alpha 1-adrenergic agonists of the progression phase in the proliferation of primary cultures of smooth muscle cells in mouse and rat aorta.

The effects of catecholamines on the competence and progression phases in the proliferation of vascular smooth muscle cells (SMCs) in mouse and rat were investigated in primary cultures: alpha,beta-Adrenergic agonists such as epinephrine and norepinephrine, and the alpha-adrenergic agonist, phenylephrine, stimulated proliferation of primary cultured SMCs, whereas the alpha 2-adrenergic agonist, clonidine, and beta-adrenergic agonist, isoproterenol, did not. The stimulating effect of epinephrine was maximal at 0.54 microM and was then decreased at higher concentrations. The alpha-adrenergic antagonist, phentolamine, and alpha 1-adrenergic antagonist, prazosin, inhibited epinephrine-induced SMC proliferation, while the alpha 2-adrenergic antagonist, yohimbine, and beta-adrenergic antagonist, propranolol, did not. In primary cultured and synchronized SMCs at the G0 phase, norepinephrine accelerated the rate of SMC proliferation, but did not change the starting time of DNA synthesis and proliferation. These results show that catecholamines activate the progression phase in primary cultured aortic SMCs alpha 1-adrenergic receptors.

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.

Effect of sterilization on bone morphogenetic protein.

Demineralized bone matrix and bone morphogenetic protein have been used clinically to accelerate bone regeneration. However, the best method of sterilization has been the subject of controversy. Some investigators have used ethylene oxide, but others have reported that doses adequate for sterilization destroyed the osteoinductivity of demineralized bone matrix and that gamma irradiation was less harmful in this respect. We used partially purified bone morphogenetic protein and type-I collagen to investigate the effects of sterilization by ethylene oxide and gamma irradiation on the activity of bone morphogenetic protein. Osteoinductivity was reduced considerably after sterilization by gamma irradiation at 2.5 Mrad and by ethylene oxide at 37 degrees C for 4 hours and at 55 degrees C for 1 hour; however, the reduction induced by ethylene oxide at 29 degrees C for 5 hours was about half of the control values. This study showed that ethylene oxide at 29 degrees C for 5 hours can be used clinically for sterilization of bone morphogenetic protein. We also investigated the effect of gamma irradiation on bone morphogenetic protein and the collagen carrier separately and found that collagen was far more labile than bone morphogenetic protein.

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.

Antiproliferative effects of the traditional Chinese medicine shimotsu-to, its component cnidium rhizome and derived compounds on primary cultures of mouse aorta smooth muscle cells.

Antiproliferative effects of the Japanese-Sino medicine Shimotsu-to (a combined prescription of cnidium rhizome, angelica root, peony root and rehmannia root) were investigated in the primary culture of smooth muscle cells (SMC) of mouse aorta. Fetal bovine serum (10%)-induced proliferation of primary cultured SMC was inhibited by Shimotsu-to at 4, 20, 100 or 500 micrograms/ml. The inhibitory effect was selective on SMC and due to cnidium rhizome or angelica root. The IC50 values of senkyunolide H, senkyunolide A, ligustilide and butylidenephthalide derived from cnidium were below 0.1, 1.52, 1.68 and 3.25 micrograms/ml, respectively. These results indicate that the antiproliferative effect of Shimotsu-to may depend on these cnidium-derived phthalides.

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.