Improved intervertebral bone union in ALIF rat model with porous hydroxyapatite/collagen combined with platelet-rich plasma

BACKGROUND CONTEXTPlatelet-rich plasma (PRP) can accelerate bone union in spinal fusion surgery with an autogenous bone graft. However, it is unclear whether bone union can be obtained by using artificial bone and PRP together in spinal interbody fusion surgery.PURPOSEThis study aimed to determine whether interbody fusion can be achieved by transplanting porous hydroxyapatite/collagen(HAp/Col) which is an artificial bone material frequently used in spinal fusion surgery, together with PRP in the intervertebral disc space in rats.STUDY DESIGN AND SETTINGA controlled laboratory study.METHODSA total of fourty 10-week old Sprague-Dawley rats were used in this study and assigned to three groups as follow: disc curettage only (control group, n=10), disc curettage + HAp/Col transplant (H group, n=10), and disc curettage + HAp/Col + PRP transplant (H+P group, n=10). The other 10 rats were sacrificed as blood donors for acquisition of PRP. Microcomputed tomography (μCT) examinations were performed to evaluate bone union, bone volume (BV), and bone mineral density (BMD) at 4, 8, and 12 weeks following surgery. Twelve weeks postoperatively, each group of three of L4–L5 spines was harvested to perform histological examination (hematoxylin & eosin stain) and the others were subjected to biomechanical testing (compression properties).RESULTSThe platelet count in PRP was approximately 4.1 times greater than that in whole blood (260.6±26.2 × 10⁴ mg/dL and 64.3±2.9 × 10⁴ mg/dL in PRP and whole blood, respectively). All the L4–L5 lumbar discs were fused in the H+P group, whereas only one case was fused in the H group and none in the control group at 12 weeks after surgery. BV was significantly higher in the H+P group than in the H group or control groups (both p<.01), although BMD was not significantly different among the three groups. Upon histological analysis, mature bone formation was observed at the transplanted space in all cases in the H+P group, whereas fibrous tissue was observed at the location in the H and control groups. Regarding biomechanical properties, the ultimate load to failure was significantly higher in the H+P group than in the H group or control group (p=.021 and .013, respectively), although stiffness was not significantly different between the three groups.CONCLUSIONThe combination of porous HAp/Col and PRP at an appropriate concentration can promote bone union in the intervertebral disc space without using an autologous bone graft in the rat model. Bone tissue formation was histologically confirmed, and it was mechanically strong.CLINICAL SIGNIFICANCEThis preclinical study showed that porous HAp/Col, when combined with PRP at an appropriate concentration, can induce bone union without autologous bone grafts. The results may eliminate the need for autologous bone collection for spinal fusion surgery in the future.     

Ectopic bone induction on and in porous hydroxyapatite combined with collagen and bone morphogenetic protein

Porous hydroxyapatite (HA-P) discs (5 mm in diameter; 1.5 mm thick; porosity, 80%; mean pore size, 200 micron) were impregnated with purified bovine skin collagen (1 mg/disc) and a small amount of semipurified bone morphogenetic protein (BMP) of sarcoma origin (100 micrograms/disc) and implanted into dorsal muscles of ddY mice. Within one week new ectopic cartilagenous tissue was consistently formed on the surface of the discs adjacent to the host tissue. The cartilage was resorbed and replaced by normal bone containing hematopoietic bone marrow four weeks after implantation and the discs became encased in the newly formed bone. HA-P discs impregnated with only collagen (HA-P/collagen) or only BMP (100 micrograms/disc; HA-P/BMP) did not evoke formation of new cartilage or bone. These results indicate that collagen is effective as a carrier of BMP for expression of the biologic activity of the latter in vivo and that it may be of practical use as a carrier of BMP with synthetic biomaterials.     

A resorbable porous ceramic composite bone graft substitute in a rabbit metaphyseal defect model.

The success of converted corals as a bone graft substitute relies on a complex sequence of events of vascular ingrowth, differentiation of osteoprogenitor cells, bone remodeling and graft resorption occurring together with host bone ingrowth into and onto the porous coralline microstructure or voids left behind during resorption. This study examined the resorption rates and bone infiltration into a family of resorbable porous ceramic placed bilaterally in critical sized defects in the tibial metaphyseal-diaphyseal of rabbits. The ceramics are made resorbable by partially converting the calcium carbonate of corals to form a hydroxyapatite (HA) layer on all surfaces. Attempts have been made to control the resorption rate of the implant by varying the HA thickness. New bone was observed at the periosteal and endosteal cortices, which flowed into the centre of the defect supporting the osteoconductive nature of partially converted corals. The combination of an HA layer and calcium carbonate core provides a composite bone graft substitute for new tissue integration. The HA-calcium carbonate composite demonstrated an initial resorption of the inner calcium carbonate phase but the overall implant resorption and bone ingrowth behaviour did not differ with HA thickness.     

17β-雌二醇对体外培养破骨细胞凋亡及其骨吸收调节作用

目的 观察17β-雌二醇对体外培养破骨细胞凋亡率的影响及其与骨吸收功能的关系。方法 在培养液中加入17β-雌二醇，透射电镜（transmission electron microscopy，TEM）观察破骨细胞内超微结构的改变，流式细胞仪（flow cytometry，FCM）观察不同浓度的17β-雌二醇在不同时间段对破骨细胞凋亡率的影响，同时用扫描电镜（scanning electron microscopy，SEM）观察破骨细胞在骨片上形成骨吸收陷窝数目及面积的变化。结果 17β-雌二醇可增加破骨细胞的凋亡率，这种作用具有剂量、时间依赖效应，同时，破骨细胞在骨片上形成的骨吸收陷窝的数目和面积减少。结论 17β-雌二醇可促进破骨细胞凋亡，从而抑制破骨细胞的骨吸收功能。     

Infection following the use of porous hydroxyapatite ceramic as a bone defect filler in articular fractures

Summary This article is a preliminary report focussed on infection after implantation of porous hydroxyapatite (Endobon®) as a bone defect filler. Eighteen adults received Endobon® implants for cancellous bone defects after trauma, tumoral excision or for arthrodesis. Four patients exhibited osteoarthritis. Infections required debridementand removal of osteosynthesis material and implants. Relevant infectious organisms were Staphylococcus and Enterobacteria. The incidence of postoperative infection, the nature of the organisms and indications are discussed. Further investigations are required in order to understand the causes of infection and undertake their prophylaxis.     

Compactin suppresses bone resorption by inhibiting the fusion of prefusion osteoclasts and disrupting the actin ring in osteoclasts.

Compactin (mevastatin), which inhibits 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, and thus biosynthesis of cholesterol and the prenylation of proteins, inhibits osteoclastic bone resorption. Although it has been suggested that compactin inhibits bone resorption by inducing apoptosis of osteoclasts, the pathway by which compactin inhibits resorption has not been established. We investigated the effect of compactin on the differentiation of osteoclasts and the relationship between the morphological changes elicited by compactin and its inhibitory effect on bone resorption. Compactin inhibited the differentiation of osteoclasts, interfering with the fusion process by which prefusion osteoclasts (pOCs) develop into multinucleated osteoclast-like cells (OCLs), and also disrupted the actin ring of OCLs. The potency of compactin to inhibit fusion of pOCs and to disrupt the actin ring of OCLs corresponded to that of compactin to inhibit bone resorption. The effects of compactin were prevented by the addition of MVA lactone or its downstream products farnesylpyrophosphate (FPP) and geranylgeranyl-pyrophosphate (GGPP) but not by squalene. Apoptosis of OCLs was not induced by the concentration of compactin that inhibited fusion of pOCs and disrupted the actin ring. The normal process of pOC fusion and the integrity of the actin ring were restored by the withdrawal of compactin from the cultures after they had been treated with compactin for 24 h, but they were not restored by the addition of zVAD-fmk, a caspase inhibitor. Compactin also reversibly inhibited interleukin-1beta (IL-1beta)-, 1alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3)-, and parathyroid hormone (PTH)-stimulated 45Ca release in bone organ cultures. Our results indicate that the inhibitory effects of compactin on bone resorption result from the inhibition of fusion of pOCs into OCLs and disruption of actin ring in OCLs and that apoptosis of OCLs is not necessary for these inhibitory effects of compactin. These effects of compactin are likely to be a consequence of the inhibition of prenylation of proteins that play an important role in the fusion of pOCs and in maintaining actin ring integrity in OCLs.     

Effect of arachidonic acid metabolites on bone resorption by isolated rat osteoclasts

Arachidonic acid metabolites (eicosanoids) have major effects on bone but their role is unclear. Many are known to stimulate bone resorption in organ culture, but paradoxically, previous work has suggested that at least some of them act as direct inhibitors of osteoclastic function. In an attempt to clarify the role of eicosanoids in bone physiology, we have defined the duration of action and relative potencies of prostaglandin (PG) E1 and E2 and have extended the range of eicosanoids tested on isolated osteoclasts. We have found that PGE1 and PGE2 inhibited bone resorption by isolated osteoclasts for at least 6 h. Inhibition was followed by recovery to control, not supranormal levels. Bone resorption was inhibited in the range 10(-5)-10(-9) M for PGE1 and PGE2, and the rank order as resorption inhibitors was PGE1 greater than 6-keto PGE1 greater than PGE2 greater than PGA2 greater than PGB2. None of the products of lipoxygenase metabolism showed a significant direct effect. The effects of PGE1 and PGE2 were not antagonistic. Prostaglandin production does not seem to be implicated as a second messenger for the action of calcitonin. Although inhibition of osteoclasts by PGs was less prolonged than that observed in the presence of calcitonin, the sensitivity of osteoclasts to inhibition by PGs, and the duration of the effect without subsequent direct stimulation, suggests that inhibition of osteoclastic resorption is a major physiological role of PG production in bone.     

The effects of extracellular pH on bone resorption by avian osteoclasts in vitro

It has previously been reported that low extracellular pH stimulates the excavation of resorption lacunae by rodent osteoclasts in vitro. Using avian bone cells in a similar in vitro assay we have demonstrated that osteoclast activity is optimal at pH 7.20-7.40 and is inhibited at extremes of pH (less than 7.10 and greater than 7.60). Over the first 24 h of incubation at low pH there may be an increase in osteoclastic resorption but to a lesser extent than that reported for rodent cells. However, after 24-30 h in culture there is little or no further increase in bone resorption, presumably due to a cytotoxic effect of low pH acting either on the osteoclast directly or via nonosteoclastic bone cells. In contrast to a previous report, in which preincubation of wafers for 24 h had no effect on bone resorption, we found that preincubation of bone substrates at pH 6.50 for longer periods enhances subsequent resorption at pH 7.20.     

Effects of different doses of ferutinin on bone formation/resorption in ovariectomized rats

This study analyzes the effects of different doses of ferutinin on bone loss caused by estrogen deficiency in ovariectomized rats, in comparison with estradiol benzoate. Thirty female Sprague?CDawley rats were ovariectomized and treated for 30?days from the day after ovariectomy. Static/dynamic histomorphometric analyses were performed on trabecular and cortical bone of lumbar vertebrae and femurs. Very low weight increments were recorded only in all F-OVX groups, with respect to the others. Although the great differences in weight, that could imply a decrease of bone mass in F-OVX groups compared to the control ovariectomized group (C-OVX), trabecular bone in lumbar vertebrae did not show significant differences, suggesting that ferutinin, opposing estrogen deficiency, inhibits bone resorption. Newly formed cortical bone was always low in all F-OVX groups and high in C-OVX, suggesting that it is mainly devoted in answering mechanical demands. In contrast, in distal femoral metaphyses, trabecular bone was reduced and the number of osteoclasts was increased in C-OVX with respect to all other groups, suggesting that it is mainly devoted in answering metabolic demands; moreover, ferutinin dose of 2?mg/kg seemed to be more effective than the lower doses used and estrogens, particularly in those skeletal regions with higher metabolic activity. Our results suggest that the role of ferutinin in preventing osteoporosis caused by estrogen deficiency is expressed in decreasing bone erosion; moreover, in all F-OVX groups bone turnover is very low and seems correlated to the trivial body weight increase, which, in turn, depends on ferutinin treatment.     

Effect of nano-hydroxyapatite／collagen composite and bone morphogenetic protein-2 on lumbar intertransverse fusion in rabbits

Objective: To investigate the effect of nanohydroxyapatite/collagen (nHA/collagen) composite as a graft extender and enhancer when combined with recombinant human bone morphogenetic protein-2 (rhBMP-2) on lumbar intertransverse fusion in rabbits. Methods: Sixty-four adult female New Zealand white rabbits, aged 1 year and weighing 3.5-4.5kg, underwent similar posterolateral intertransverse process arthrodesis and were randomly divided into 4 groups based on different grafts: autogenous cancellous bone alone (ACB group), nHA/collagen alone ( HAC group ), half autogenous cancellous bone and half nHA/collagen (ACB HAC group) and nHA/collagen combined with rhBMP-2 (HAC BMP group ). The fusion masses were analyzed by manual palpation, radiography, biomechanical testing and histological examination. Results: Fusion was observed in 4 cases in the 6th week and in 5 cases in the 10tb week after surgery in ACB group. No case showed fusion in HAC group. In ACB HAC group, there was fusion in 3 cases in the 6th week and in 4 cases in the 10th week after surgery. In HAC BMP group, fusion in 1 case was found in the 4th week, in 5 cases in the 6th week and in 6 cases in the 10th week after surgery. It suggested that ACB, ACB HAC and HAC BMP groups showed similar fusion ratio and mechanical strength in the 6th and 10th week after surgery. According to the microstructure analysis of the samples, nHA/collagen had no negative effect when implanted together with ilium autograft. In HAC BMP group, new bone-like tissue was observed in the 2nd week postoperatively, and nearly all of the implanted composites were replaced by mature bone matrix and new bones in 10th week postoperatively. Conclusions：The nHA/collagen, especially combined with rhBMP-2, is a promising bone substitute, for it has quick biodegradation, fine bone-bending ability, and high osteoconductivity on posterolateral spinal fusion in rabbits.     

谷康泰灵对骨质疏松模型大鼠骨形成和骨吸收功能的影响

目的　观察药物谷康泰灵对骨质疏松 (OP)模型大鼠骨形成和骨吸收功能的影响 ,为其临床治疗骨质疏松提供实验依据。方法　SD大鼠 4 4只 ,随机分为正常组 (13只 )和骨质疏松模型组 (31只 )。以维甲酸 80mg·kg- 1 ·d- 1 灌胃 15d,诱导OP模型。模型复制成功后 ,各组处死 5只 ,正常组 (8只 )继续观察 ,模型组又随机分为无措施对照组 (8只 )、谷康泰灵治疗组 (10只 ,0 0 8mg·kg- 1 ·d- 1 谷康泰灵腹腔注射 )和雌二醇治疗组 (8只 ,每只 0 0 5mg,每周 3次苯甲酸雌二醇腹腔注射 )。治疗期 30d。观察股骨松质骨区成骨细胞及破骨细胞数量和血清中AKP、TRAP活性变化。结果　与正常组相比 ,维甲酸诱导的OP模型大鼠股骨松质骨区成骨细胞功能活跃 ,数量变化不大 ;破骨细胞数量、活跃程度显著增加 ;血清中AKP和TRAP明显增高。谷康泰灵治疗后 ,OP大鼠活跃成骨细胞数量明显增加 ,破骨细胞数量显著减少 ,血清AKP活性明显增高 ,TRAP活性下降。结论　谷康泰灵对OP大鼠骨形成和骨吸收功能有显著影响 ,可刺激成骨细胞的产生增加成骨细胞的活性 ,减少破骨细胞的数量抑制破骨细胞的活性。     

Increased bone formation in a rabbit long-bone defect model after single local and single systemic application of erythropoietin

Background and purpose — Delayed bone healing with non-union is a common problem. Further options to increase bone healing together with surgery are needed. We therefore evaluated a 1-dose single application of erythropoietin (EPO), applied either locally to the defect or systemically during surgery, in a critical-size rabbit long-bone defect.

Material and methods — 19 New Zealand White rabbits received a 15-mm defect in the radius diaphysis. An absorbable gelatin sponge was soaked with saline (control group and systemic treatment group) or EPO (local treatment group) and implanted into the gap. The systemic treatment group received EPO subcutaneously. In vivo micro-CT analysis was performed 4, 8, and 12 weeks postoperatively. Vascularization was evaluated histologically.

Results — Semiquantitative histomorphometric and radiological evaluation showed increased bone formation (2.3- to 2.5-fold) in both treatment groups after 12 weeks compared to the controls. Quantitative determination of bone volume and tissue volume showed superior bone healing after EPO treatment at all follow-up time points, with the highest values after 12 weeks in locally treated animals (3.0- to 3.4-fold). More vascularization was found in both EPO treatment groups.

Interpretation — Initial single dosing with EPO was sufficient to increase bone healing substantially after 12 weeks of follow-up. Local application inside the defect was most effective, and it can be administered directly during surgery. Apart from effects on ossification, systemic and local EPO treatment leads to increased callus vascularization.     

Inhibition of bone resorption by inorganic phosphate is mediated by both reduced osteoclast formation and decreased activity of mature osteoclasts

High concentrations of inorganic phosphate (Pi) are known to inhibit bone resorption, although the mechanism(s) underlying this effect is unclear. To investigate whether Pi can inhibit the formation of osteoclasts we studied the effects of changes in Pi concentration between 1 and 4 mM on osteoclast-like cell formation in 1 week cultures of mouse bone marrow. Osteoclast-like cells were identified by multinuclearity, positive staining for tartrate-resistant acid phosphatase (TRAP), and contraction in response to calcitonin. Increasing concentrations of Pi inhibited formation of these cells in a dose-dependent manner. To study effects of Pi on the bone-resorbing activity of mature osteoclasts we isolated osteoclasts from calcium-deficient egg-laying hens or rat pups and incubated them on sperm whale dentine slices. High Pi concentrations markedly reduced both the number of resorption pits formed per dentine slice and the mean area of each pit in both avian and mammalian systems. These data indicate that high concentrations of Pi act on bone directly, both to inhibit generation of new osteoclasts from their precursor cells and to inhibit bone resorption by mature osteoclasts. These effects of extracellular Pi concentration may play an important modulatory role on bone turnover in vivo and have potential importance in several disease states in which Pi metabolism is perturbed.     

Effects of acetylsalicylic acid on heterotopic bone resorption and formation in rats

The effects of acetylsalicylic acid (ASA) on bone metabolism have been studied in a bone transplantation model using radioisotopic and biochemical parameters. Isografts (femora) from infant inbred rats, extensively prelabeled with collagen and mineral-tracing radioisotopes, were transplanted to muscle pouches in young male rats. Bones from the opposite side of the donor rats served as nonimplanted reference bones. The recipients were given 150 mg/kg/12 h of ASA by gavage for 18 days. The serum concentrations obtained were comparable with the recommended anti-inflammatory levels in humans. Twenty-four hours before being killed the animals were labeled again with other collagen and mineral radioisotopes. After 18 days of medication the resorption of the transplanted bone was inhibited by about 15% in the ASA treated rats compared with controls, as measured by the losses of collagen (14C-hydroxyproline) and mineral (strontium-85). Also, the net gains of mineral and collagen in the ASA-treated transplants were reduced by about 15% and 11% respectively compared with controls during the medication period. During the last 24 h of the study the rates of mineral incorporation (calcium-47 uptake) and collagen synthesis (3H-hydroxyproline) were reduced to an even greater degree in the ASA-treated transplants. These results indicate an inhibitory effect of ASA on bone metabolism.     

Phagocytosis of bone collagen by osteoclasts in two cases of pycnodysostosis

Summary Electron microscopic examination of bone biopsies obtained from two patients suffering from pycnodysostosis revealed that osteoclasts contained (sometimes large) cytoplasmic vacuoles filled with bone collagen fibrils. These vacuoles stained positive for acid phosphatase activity, thereby suggesting that bone matrix had been phagocytosed and subsequently exposed to hydrolytic enzymes of the lysosomal apparatus. Collagen-containing vacuoles were not observed in osteoclasts of individuals not suffering from this disease.     

Effects of acetylsalicylic acid on heterotopic bone resorption and formation in rats

Summary The effects of acetylsalicylic acid (ASA) on bone metabolism have been studied in a bone transplantation model using radioisotopic and biochemical parameters. Isografts (femora) from infant inbred rats, extensively prelabeled with collagen and mineral-tracing radioisotopes, were transplanted to muscle pouches in young male rats. Bones from the opposite side of the donor rats served as nonimplanted reference bones. The recipients were given 150mg/kg/12h of ASA by gavage for 18 days. The serum concentrations obtained were comparable with the recommended anti-inflammatory levels in humans. Twenty-four hours before being killed the animals were labeled again with other collagen and mineral radioisotopes. After 18 days of medication the resorption of the transplanted bone was inhibited by about 15% in the ASA treated rats compared with controls, as measured by the losses of collagen (¹⁴C-hydroxyproline) and mineral (strontium-85). Also, the net gains of mineral and collagen in the ASA-treated transplants were reduced by about 15% and 11% respectively compared with controls during the medication period. During the last 24h of the study the rates of mineral incorporation (calcium-47 uptake) and collagen synthesis (³H-hydroxyproline) were reduced to an even greater degree in the ASA-treated transplants. These results indicate an inhibitory effect of ASA on bone metabolism.

---

Zusammenfassung Es wurde die Wirkung analysiert, die eine 18tägige Behandlung mit Azetylsalizylsdure (ASS) in einer Dosis von 150 mg/kg/12 Std auf den Knochenstoffwechsel isogener Knochentrans plantate in der Muskulatur junger Ratten hat. Die Knochen waren vor der Transplantation mit Isotopen des Knochenkollagens and -minerals markiert worden und wurden am Ende der Behandlung erneut markiert. Gemessen an der Abnahme der Kollagenund Mineralisotope, zeigten die mit ASS behandelten Transplantate eine Herabsetzung der Knochenresorption. Beurteilt nach der Nettozunahme des Kollagen- und Calziumgehaltes als auch nach der Kollagensynthese and des Mineraleinbaus wurde nach diesem Zeitraum eine gleichzeitige Verminderung des Knochenneubaus gefunden. Diese Ergebnisse zeigen, da13 die Knochenumbauvorgdnge durch ASS herabgesetzt werden.

     

Inhibition of bone resorption by bisphosphonates: Interactions between bisphosphonates,osteoclasts, and bone

Summary Bisphosphonates are nonbiodegradable pyrophosphate analogues that are being used increasingly to inhibit bone resorption in disorders characterized by excessive bone loss. We have previously found that dichloromethylene bisphosphonate (Cl₂MBP) inhibits bone resorption through injury to the cells that resorb Cl₂MBP-contaminated surfaces. 3-amino-1-hydroxypropylidene-1,1-bisphosphonate (AHPrBP) is a more potent inhibitor of bone resorptionin vivo, and we have attempted to identify a step in the resorptive pathway that accounts for this increased potency. We found that when osteoclasts, isolated from neonatal rat long bones, were incubated on bone slices in the presence of bisphosphonates, AHPrBP was less, rather than more potent as a resorption-inhibitor than Cl₂MBP. The greater sensitivity of resorption to AHPrBPin vivo could neither be attributed to an effect of AHPrBP on the ability of osteoblastic cells to stimulate resorption in response to calcium-regulating hormonesin vitro nor to an effect on osteoclast generation: osteoclast formation was unaffected by concentrations of AHPrBP 10-fold higher than those of Cl₂MBP which inhibit bone resorption in the bone slice assay. We also found no evidence for impaired osteoclast generationin vivo in AHPrBP-treated rats. These results suggest that the comparisons of potencyin vitro do not include all the factors responsible for determining bisphosphonate potencyin vivo. Because bisphosphonates owe the specificity of their actions to their ability to bind to bone surfaces, we performed experiments using bone slices that had been immersed in bisphosphonates before use. Bone resorption was virtually abolished on bone slices preincubated in 10⁻³ M AHPrBP. Inhibition was associated with degenerative changes in osteoclasts and a more rapid decrease in the number remaining on the bone surface than occurred with Cl₂MBP. The effect was specific for osteoclasts, could be prevented if bone resorption was suppressed by calcitonin, and was not seen in osteoclasts incubated in AHPrBP on plastic coverslips. These observations suggest that AHPrBP inhibits bone resorption through injury to osteoclasts when they solubilize bisphosphonate-contaminated bone. We found that the concentration of AHPrBP used in the preincubation phase could be reduced by an order of magnitude if the volume of the AHPrBP solution was correspondingly increased. This implies that the concentration of bisphosphonate is less relevant to potency comparisons than the density of bisphosphonate on the bone surface. The latter will be strongly influencedin vivo not only by affinity for bone but by the pharmacokinetic and other properties of the compound.     

Inhibition of bone resorption by alendronate and risedronate does not require osteoclast apoptosis

Bisphosphonate inhibition of bone resorption was proposed to be due to osteoclast apoptosis. We tested this hypothesis for both the N-containing bisphosphonates alendronate and risedronate, which inhibit farnesyldiphosphate synthase and thus protein isoprenylation, and for clodronate and etidronate, which are metabolized to adenosine triphosphate (ATP) analogs. We found, in dose-response studies, that alendronate and risedronate inhibit bone resorption (in pit assays) at doses tenfold lower than those reducing osteoclast number. At an N-bisphosphonate dose that inhibited resorption and induced apoptosis, the antiapoptotic caspase inhibitor, Z-VAD-FMK, maintained osteoclast (Oc) number but did not prevent inhibition of resorption. Furthermore, when cells were treated with either alendronate alone or in combination with Z-VAD-FMK for 24 or 48 h, subsequent addition of geranylgeraniol, which restores geranylgeranylation, returned bone resorption to control levels. On the other hand, Z-VAD-FMK did block etidronate and clodronate inhibition of resorption. Moreover, in cells treated with etidronate, but not alendronate or risedronate, Z-VAD-FMK also prevented actin disruption, an early sign of osteoclast inhibition by bisphosphonates. These observations indicate that, whereas induction of apoptosis plays a major role in etidronate and clodronate inhibition of resorption, alendronate and risedronate suppression of bone resorption is independent of their effects on apoptosis.     

淫羊藿苷对小鼠骨髓源性破骨细胞诱导生成及骨吸收功能的影响

目的探讨淫羊藿苷对破骨细胞诱导产生及骨吸收功能的影响。方法用终浓度分别为25ng·mL^-1、30ng·mL^-1、10^-8mol·L^-1的M—CSF、RANKL、1，25（OH）2VitD3体外诱导培养小鼠骨髓源性破骨细胞，在此过程中加入终浓度分别为0、10^-7mol·L^-1、10^-6mol·L^-1、10^-5mol·L^-1的淫羊藿苷。倒置相差显微镜下观察活体细胞、HE染色、TRAP染色及降钙素受体染色鉴定破骨细胞，计数骨片上骨吸收陷窝数及面积，玻片上TRAP阳性多核细胞数。结果加药组随淫羊藿苷浓度的增加，骨片上形成的骨吸收陷窝数及面积，玻片上的TRAP阳性多核细胞数呈量的依赖性的减少，与非加药组比较，10^-5mol·L^-1、10^-5mol·L^-1浓度的淫羊藿苷组，差异有显著性（P〈0．05）。结论淫羊藿苷具有抑制破骨细胞诱导产生及骨吸收功能的作用，并随浓度增加抑制作用增强。