伊班膦酸钠对钛颗粒诱导的骨溶解作用

[目的]通过建立钛颗粒诱导的小鼠颅骨骨溶解动物模型,研究伊班膦酸钠对骨溶解的影响,并探讨其作用机制.[方法]24只昆明系小鼠随机分成4组,每组6只,建立钛颗粒诱导的小鼠颅骨骨溶解模型.空白对照组进行假手术;钛颗粒组在小鼠颅顶植入30 mg钛颗粒;1单位伊班膦酸钠组和10单位伊班膦酸钠组分别于植入钛颗粒第2 d腹腔注射伊班膦酸钠10 mg、100 mg.手术后第10 d,取出颅骨进行病理学分析.[结果]空白对照组骨溶解面积为(0.070±0.009)mm2,没有发生明显骨溶解.钛颗粒组骨溶解面积为(0.369±0.050)mm2,和空白对照组相比发生了更大面积的骨溶解(P<0.05).1单位伊班膦酸钠组和10单位伊班膦酸钠组骨溶解面积分别为(0.164±0.018)mm2、(0.018±0.075)mm2,均小于钛颗粒组骨溶解面积(P<0.05).而且10单位伊班膦酸钠组比1单位伊班膦酸钠组骨溶解面积更小(P<0.05).骨溶解区域破骨细胞数量各组比较结果和骨溶解面积结果类似.[结论]伊班膦酸钠能够有效抑制钛颗粒诱导的小鼠颅骨骨溶解.     

红霉素抑制磨损颗粒诱发体内骨溶解的研究

[目的]通过小鼠体内磨损颗粒颅骨溶解模型研究红霉素(erythromycin,EM)抑制磨损颗粒诱发骨溶解的效果。[方法]24只8周龄的C57BL/J6雄性小鼠随机分为4组:聚甲基丙烯酸甲酯(polymethyl-methacrylate,PM-MA)组接受PMMA30 mg颗粒植入颅顶部,PMMA 2EM组接受30 mg PMMA颗粒植入加每天EM2 mg/kg腹腔内注射,PMMA 10EM组接受PMMA30 mg颗粒植入加每天EM10 mg/kg腹腔内注射,对照组接受假手术。7 d后取出颅骨进行病理学分析。[结果]颅骨破坏面积对照组为0.079 mm2±0.011 mm2,PMMA组0.335 mm2±0.129 mm2,PM-MA 2EM组0.094 mm2±0.019 mm2,PMMA 10EM组0.091 mm2±0.028 mm2。对照组颅骨实验区域内破骨细胞计数为5.3±1.0个,PMMA组为19.2±5.3个,PMMA 2EM组为6.6±1.1个,PMMA 10EM组为6.1±1.9个。与对照组相比,PMMA颗粒可以诱发骨溶解(P<0.001),而EM治疗可以抑制PMMA颗粒诱发的颅骨溶解(P<0.001),及破骨细胞生成(P<0.001)。[结论]EM可以抑制PMMA磨损颗粒诱发的骨溶解。     

绿色荧光蛋白和Nel1型蛋白基因共表达腺病毒载体的构建及体外转染大鼠BMSCs的初步实验研究

目的构建同时表达绿色荧光蛋白(green fluorescent protein,GFP)报告基因和人类Nel1型蛋白[homo sapiens NEL-like1,NELL1)]基因的重组腺病毒载体pAdxsi-GFP-NELL1,并转染大鼠BMSCs,观察其表达情况,为进一步研究NELL1蛋白的成骨作用提供理论基础。方法设计特异性引物从NELL1质粒中扩增NELL1,将测序正确的片段用XhoI/BglII酶切处理,定向插入至pShuttle-GFP-CMV(-)TEMP重组穿梭载体,然后将验证正确的重组穿梭质粒中的插入片段转移至pAdxsi载体构建重组腺病毒载体质粒,用PacI限制性内切酶线性化后转染HEK293细胞,扩增纯化重组腺病毒,半数组织培养感染量法测定重组腺病毒滴度。培养大鼠BMSCs,采用流式细胞仪鉴定表面标志物,并行成骨、成脂诱导鉴定。用构建的pAdxsi-GFP-NELL1及空病毒pAdxsi-GFP(作为对照)转染鉴定正确的BMSCs,RT-PCR检测NELL1的表达,免疫荧光检测GFP基因及NELL1的表达情况,细胞计数试剂盒(cell counting kit-8,CCK-8)法检测对细胞增殖的影响。结果成功构建同时表达NELL1和GFP基因的重组腺病毒载体(pAdxsi-GFP-NELL1),纯化后获得滴度达1×1011pfu/mL的重组腺病毒。成功分离获得大鼠BMSCs并传代、纯化,经流式细胞仪鉴定表面标志物及成骨、成脂诱导鉴定为BMSCs。pAdxsi-GFP-NELL1转染BMSCs后,RT-PCR检测示NELL1mRNA阳性表达,荧光显微镜观察示细胞爬片GFP阳性表达,NELL1抗体免疫荧光观察示NELL1蛋白阳性表达。CCK-8法鉴定显示转染后对BMSCs生长无明显影响。结论构建并纯化后的重组腺病毒载体(pAdxsi-GFP-NELL1)可高效转染大鼠BMSCs,并稳定表达NELL1和GFP两种基因,为进一步研究新的成骨基因NELL1的作用,追踪其在体内、体外表达情况提供了新工具。     

假体周围骨溶解性骨缺损的转骨形态发生蛋白-2基因治疗

目的 模拟假体周围骨溶解环境,观察骨形态发生蛋白-2(BMP-2)基因治疗假体周围骨溶解性骨缺损的效果.方法 成年雄性Beagle犬6条,于股骨外髁造成假体周围3mm骨缺损区.1条动物的左侧缺损区植入1ml平均直径1μm的钛合金颗粒混悬液,右侧植入1ml磷酸盐缓冲液(PBS),观察造模结果;其他5条动物双侧植入1ml钛合金颗粒混悬液,于术后2个月取出假体,植入转BMP-2基因冻干骨或单纯冻干骨,二次术后3个月取材,行组织学、组织形态计量学观察植骨愈合替代及界面骨整合情况.结果 颗粒造模术后2个月可见典型的骨溶解界膜组织形成.翻修术后3个月,冻干骨组见较多植骨残余,假体-骨界面基本为软组织界膜,假体骨接触率(BIC)为(1.38±1.22)%;基因治疗组见少量植骨残余,假体-骨界面有点状骨接触,BIC为(12.96±1.61)%,两组差异有统计学意义(P<0.01).结论 采用BMP-2基因治疗可提高假体周围骨溶解性骨缺损的界面骨整合.     

Anti-CDllb单克隆抗体对超高分子聚乙烯磨损颗粒诱导骨溶解作用的研究

目的 通过建立小鼠颅骨溶解模型,探究anti-CDllb抗体对颗粒诱导骨溶解的作用。方法 取10周龄雌性C57BL/6J小鼠32只,平均分为4组,A(空白对照组）,切开颅顶皮肤后即缝合,于术后第二天灌胃生理盐水,并尾静脉注射PBS,B[脾酪氨酸激酶（Spleen tyrosine kinase,Syk)抑制剂组]、C( anti-CDllb单克隆抗体组）和D(颗粒组）组于烦骨骨膜外移植20μg超高分子聚乙烯磨损颗粒后缝合,B组术后第2大起以15 mg/kg灌胃Syk抑制剂,尾静脉注射PBS,C组术后第2天起尾静脉注射 anti-CDllb单克隆抗体,2 g/kg,灌胃生理盐水,D组术后第二天起每日灌胃生理盐水,尾静脉注射PBS。两周后颈椎脱臼法处死小鼠,摘眼球取血测血清肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)值,取颅骨做显微CT重建并测骨体积分数,取颅骨骨膜进行western blot分析。结果 建模小鼠全部存活,空白对照组、anti-CDl lb单克隆抗体组及Syk抑制剂组血清TNF-α值均低于颗粒组（P <0. 05);空白对照组、anti-CDllb单克隆抗体组与Syk抑制剂组骨体积分数均高于颗粒组（P < 0. 05 ) ; Syk抑制剂组及anti-CDllb单克隆抗体组Erk活性、c-fos及NFATcl表达均低于颗粒组而高于空白对照组（P < 0. 05 )。结论 Anti- CDllb单克隆抗体及Syk抑制剂均对颗粒诱导骨溶解具有抑制作用,CDllb分子通过激活Syk通路而发挥促破骨细胞分化作用。     

RANKL/RANK/OPG信号通路参与调控磷酸三钙磨损颗粒诱导假体周围骨溶解的研究

目的探讨RANKL/RANK/OPG信号通路在磷酸三钙磨损颗粒诱导假体周围骨溶解中的作用。方法将30只小鼠随机分为假手术组、模型组和OPG组,制备小鼠颅骨溶解模型,OPG组于术后第2天在颅顶局部注射骨保护素(osteoprotegerin,OPG)(3 mg/kg),每隔2 d 1次;假手术组和模型组给予等量生理盐水,共2周。测定破骨细胞数和骨溶解面积,检测白细胞介素-1β(Interleukin-1β,IL-1β)、白细胞介素-6(Interleukin-6,IL-6)和肿瘤坏死因子-α(tumor necrosis factorα,TNF-α)、OPG、NF-kappa B的受体激活因子(receptor activator of nuclear factor-κB,RANK)和RANK配体(receptor activator of nuclear factor-κB ligand,RANKL)的水平。结果与假手术组比较,模型组小鼠颅骨破骨细胞数、骨溶解面积均增加,IL-1β、IL-6和TNF-α水平均增加,RANKL和RANK mRNA的相对表达量均增加,OPG mRNA的相对表达量降低(P0.05);与模型组比较,OPG组小鼠颅骨破骨细胞数和骨溶解面积减少,IL-1β、IL-6和TNF-α水平减少,RANKL和RANK mRNA的相对表达量均降低,OPG mRNA的相对表达量增加(P0.05)。结论 RANKL/RANK/OPG信号通路参与调控磷酸三钙磨损颗粒诱导假体周围骨溶解,通过给予外源性OPG能显著抑制磷酸三钙磨损颗粒诱导的骨溶解。     

Anti-CD11b单克隆抗体对超高分子聚乙烯磨损颗粒诱导骨溶解作用的研究

目的通过建立小鼠颅骨溶解模型,探究anti-CD11b抗体对颗粒诱导骨溶解的作用。方法取10周龄雌性C57BL/6J小鼠32只,平均分为4组,A(空白对照组),切开颅顶皮肤后即缝合,于术后第二天灌胃生理盐水,并尾静脉注射PBS,B[脾酪氨酸激酶(Spleen tyrosine kinase,Syk)抑制剂组]、C(anti-CD11b单克隆抗体组)和D(颗粒组)组于颅骨骨膜外移植20μg超高分子聚乙烯磨损颗粒后缝合,B组术后第2天起以15 mg/kg灌胃Syk抑制剂,尾静脉注射PBS,C组术后第2天起尾静脉注射anti-CD11b单克隆抗体,2 g/kg,灌胃生理盐水,D组术后第二天起每日灌胃生理盐水,尾静脉注射PBS。两周后颈椎脱臼法处死小鼠,摘眼球取血测血清肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)值,取颅骨做显微CT重建并测骨体积分数,取颅骨骨膜进行western blot分析。结果建模小鼠全部存活,空白对照组、anti-CD11b单克隆抗体组及Syk抑制剂组血清TNF-α值均低于颗粒组(P0.05);空白对照组、anti-CD11b单克隆抗体组与Syk抑制剂组骨体积分数均高于颗粒组(P0.05);Syk抑制剂组及anti-CD11b单克隆抗体组Erk活性、c-fos及NFATc1表达均低于颗粒组而高于空白对照组(P0.05)。结论 AntiCD11b单克隆抗体及Syk抑制剂均对颗粒诱导骨溶解具有抑制作用,CD11b分子通过激活Syk通路而发挥促破骨细胞分化作用。     

磨损颗粒诱导骨溶解的两种动物模型形态学比较研究

目的建立两种磨损颗粒诱导骨溶解模型,比较炎症水平、骨溶解情况以及破骨细胞计数等指标,为骨溶解相关实验动物模型的选择提供依据。方法分别采用小鼠颅骨模型、小鼠气囊植骨模型制备两组磨损颗粒诱导骨溶解模型。通过HE染色法进行形态学观察;利用扫描电子显微镜观察骨溶解情况;采用TRAP染色法进行破骨细胞计数。结果小鼠气囊植骨模型组骨吸收面积为(385.30±5.11)nm2,明显高于小鼠颅骨模型组的(288.60±4.89)nm2(P0.05);小鼠气囊植骨模型组破骨细胞数目为(16.75±0.53)个,明显高于小鼠颅骨模型组的(9.55±0.48)个(P0.05)。结论两种动物模型均构建成功;小鼠气囊植骨模型在炎症水平、骨溶解情况及破骨细胞计数等方面均优于小鼠颅骨模型,因此在模拟磨损颗粒诱导的骨溶解病理过程中小鼠气囊植骨模型模拟度更高。     

NF-κB受体激活因子配体抗体防治人工关节无菌性松动的实验研究

目的人工关节无菌性松动与破骨细胞激活后假体周围骨溶解有关,而NF-κB受体激活因子配体(receptor activator of NF-κB ligand,RANKL)/NF-κB受体激活因子(receptor activator of NF-κB,RANK)信号通路是激活破骨细胞的主要途径,通过建立小鼠气囊植骨模型模拟人工关节无菌性松动环境,观察RANKL抗体抑制炎性反应、减轻溶骨反应的作用。方法取8～10周龄雌性BALB/c小鼠60只(体重18～20 g),取其中20只小鼠颅骨制备骨片,作为气囊植骨供体;剩余40只小鼠随机分为阴性对照组(A组)、阳性对照组(B组)、RANKL抗体低剂量组(C组)和RANKL抗体高剂量组(D组),每组10只。于各组小鼠背部制备2 cm×2 cm大小的气囊后植入1片骨片;于植骨后1 d,A组气囊内注射0.5 mL PBS液;B、C、D组注射0.5 mL钛颗粒悬液。钛颗粒悬液注射前2 d,C、D组气囊内分别注射0.1 mL浓度为50μg/mL和500μg/mL的RANKL抗体,每天1次,连续2 d;A、B组注射0.1 mL PBS液。于植骨后14 d处死全部小鼠,取出植入颅骨骨片和周围囊壁组织,进行大体及组织学观察,并行颅骨骨溶解、骨胶原含量及破骨细胞含量分析。结果各组小鼠均存活至实验完成,切口愈合良好。大体观察见A、C、D组小鼠囊壁炎性反应较轻,偶见渗出和新生血管增生;B组炎性反应严重,见渗出以及新生血管增生。组织学观察见A、C、D组小鼠囊壁炎性细胞浸润及增厚不明显,骨胶原丢失量和破骨细胞含量少;B组大量炎性细胞浸润,囊壁增厚,骨胶原丢失量和破骨细胞含量多。B组炎性细胞数、囊壁厚度、骨胶原丢失量以及破骨细胞含量与A、C、D组比较,差异均有统计学意义(P<0.05)。结论 RANKL抗体可阻断小鼠气囊植骨模型的炎症信号传导通路,有效抑制磨损颗粒所致骨溶解。     

微小颗粒骨异位成骨过程中TGF-β1的表达

目的 通过观察转化生长因子β1(TGF-β1)在自体微小颗粒骨(300-500μm)异位成骨过程中的表达，探讨微小颗粒骨移植的成骨机制。方法 采用日本大耳白兔造股二头肌肌袋模型，分别植入自体微小颗粒骨及自体块骨。术后1、3、5、7、11、14、21、28d取材，进行组织学、免疫组化染色及原位杂交。检测TGF-β1及TGF-β1 mRNA表达并作图像分析。结果 ①术后5d，颗粒骨组开始有软骨生成，7d时达到高峰．21d后有骨吸收；块骨组则以骨吸收为主。②术后1d见基质及血肿中TGF-β1阳性染色，亦见骨细胞TGF-β1阳性染色。5～11d颗粒骨组见成骨细胞、软骨细胞、间充质细胞及骨细胞表达大量TGF-β1。14d以后TGF-β1渐减少，21d以后渐平稳。颗粒组TGF-β1表达高峰出现早、强度高、持续时间长。③颗粒骨组TGF-β1 mRNA阳性表达细胞出现早，高峰在术后5-11d，主要为软骨细胞、成骨细胞、骨细胞及问充质细胞。结论 颗粒骨异位成骨能力强于块骨，TGF-β1表达高峰出现时间早、量大、持续时间长。TGF-β1 mRNA定位细胞广泛，信号明显强于各期块骨组。     

NELL1 promotes high-quality bone regeneration in rat femoral distraction osteogenesis model

NELL1 (NEL-like molecule-1; NEL [a protein strongly expressed in neural tissue encoding epidermal growth factor like domain]) is a cranisynostosis-associated molecule directly regulated by Runx2, the master molecule in controlling osteoblastic differentiation. NELL1 has exhibited potent osteoinductive activity for bone regeneration in several animal models. However, its capacity for promoting repair of long-bone defects remains unknown. In this study, we investigated the osteogenic effects of NELL1 on femoral distraction osteogenesis using adenoviral gene delivery and multiple approaches of in vivo analysis. Thirty Sprague-Dawley (SD) rats were randomly assigned to 3 groups for treatment (n=10 each): adenovirus-green fluorescent protein (Ad-GFP)-NELL1 or Ad-GFP at 1×10? plaque-forming units/ml diluted in saline, or saline alone. The femoral distraction was at a speed of 0.25 mm every 12h for 14 days, and a single injection of Ad-GFP-NELL1 or Ad-GFP was given at the mid-distraction period. The effective NELL1 delivery in vivo after Ad-GFP-NELL1 injection was evaluated by optical imaging. The bone regeneration was assessed quantitatively at days 21, 28, 42, and 56 by live 3-D micro-computed tomography (micro-CT), and animals were sacrificed at day 56 for biomechanical testing and histological analysis. Exogenous NELL1 was expressed in the distracted gap for at least 14 days after Ad-GFP-NELL1 transfection. The bone union rate in the distracted gap was significantly higher with Ad-GFP-NELL1 than with Ad-GFP (9/9 vs. 4/9 rats) or saline alone (5/9 rats) at day 56. The serial 3-D micro-CT images and quantitation obtained with the development and application of radiolucent external fixators showed less callus but more mature cortical bones formed with Ad-GFP-NELL1 than with Ad-GFP transfection and saline administration during distraction osteogenesis. The biomechanical properties of femur samples with Ad-GFP-NELL1 transfection were better than samples with Ad-GFP transfection or saline treatment, and were similar with unoperated femurs. Histology revealed cartilaginous tissues in the middle of distraction gaps with Ad-GFP transfection and saline treatment but only bony bridges with Ad-GFP-NELL1 transfection at the final time point (day 56). Coincidently, the expression of Runx2, BMP2, and BMP7 did not differ among groups at day 56, whereas the expression of osteocalcin and osteopontin was slightly higher with Ad-GFP-NELL1 transfection. Thus, sustained Ad-NELL1 protein delivery into a local area of a rat femoral distraction osteogenesis model remarkably improved regeneration of good-quality bones and accelerated bone union at a high rate. Acquiring serial micro-CT data during rat femoral distraction osteogenesis and regional adenovirus delivery of NELL1 may facilitate future in vivo studies.     

Direct subcutaneous injection of polyethylene particles over the murine calvaria results in dramatic osteolysis

Purpose

The murine calvarial model has been widely employed for the in vivo study of particle-induced osteolysis, the most frequent cause of aseptic loosening of total joint replacements. Classically, this model uses an open surgical technique in which polyethylene (PE) particles are directly spread over the calvarium for the induction of osteolysis. We evaluated a minimally invasive modification of the calvarial model by using a direct subcutaneous injection of PE particles.

Methods

Polyethylene (PE) particles were injected subcutaneously over the calvaria of C57BL6J ten-week-old mice (“injection” group) or were implanted after surgical exposure of the calvaria (“open” group) (n = 5/group). For each group, five additional mice received no particles and served as controls. Particle-induced osteolysis was evaluated two weeks after the procedure using high-definition microCT imaging.

Results

Polyethylene particle injection over the calvaria resulted in a 40 % ± 1.8 % decrease in the bone volume fraction (BVF), compared to controls. Using the “open surgical technique”, the BVF decreased by 16 % ± 3.8 % as compared to controls (p < 0.0001).

Conclusions

Direct subcutaneous injection of PE particles over the murine calvaria produced more profound resorption of bone. Polyethylene particle implantation by injection is less invasive and reliably induces osteolysis to a greater degree than the open technique. This subcutaneous injection method will prove useful for repetitive injections of particles, and the assessment of potential local or systemic therapies.     

Particle-Induced Osteolysis in Three-Dimensional Micro-Computed Tomography

Small-animal models are useful for the in vivo study of particle-induced osteolysis, the most frequent cause of aseptic loosening after total joint replacement. Microstructural changes associated with particle-induced osteolysis have been extensively explored using two-dimensional (2D) techniques. However, relatively little is known regarding the 3D dynamic microstructure of particle-induced osteolysis. Therefore, we tested micro-computed tomography (micro-CT) as a novel tool for 3D analysis of wear debris-mediated osteolysis in a small-animal model of particle-induced osteolysis. The murine calvarial model based on polyethylene particles was utilized in 14 C57BL/J6 mice randomly divided into two groups. Group 1 received sham surgery, and group 2 was treated with polyethylene particles. We performed 3D micro-CT analysis and histological assessment. Various bone morphometric parameters were assessed. Regression was used to examine the relation between the results achieved by the two methods. Micro-CT analysis provides a fully automated means to quantify bone destruction in a mouse model of particle-induced osteolysis. This method revealed that the osteolytic lesions in calvaria in the experimental group were affected irregularly compared to the rather even distribution of osteolysis in the control group. This is an observation which would have been missed if histomorphometric analysis only had been performed, leading to false assessment of the actual situation. These irregularities seen by micro-CT analysis provide new insight into individual bone changes which might otherwise be overlooked by histological analysis and can be used as baseline information on which future studies can be designed.     

Local delivery of mutant CCL2 protein‐reduced orthopaedic implant wear particle‐induced osteolysis and inflammation in vivo

Total joint replacement (TJR) has been widely used as a standard treatment for late‐stage arthritis. One challenge for long‐term efficacy of TJR is the generation of ultra‐high molecular weight polyethylene wear particles from the implant surface that activates an inflammatory cascade which may lead to bone loss, prosthetic loosening and eventual failure of the procedure. Here, we investigate the efficacy of local administration of mutant CCL2 proteins, such as 7ND, on reducing wear particle‐induced inflammation and osteolysis in vivo using a mouse calvarial model. Mice were treated with local injection of 7ND or phosphate buffered saline (PBS) every other day for up to 14 days. Wear particle‐induced osteolysis and the effects of 7ND treatment were evaluated using micro‐CT, histology, and immunofluorescence staining. Compared with the PBS control, 7ND treatment significantly decreased wear particle‐induced osteolysis, which led to a higher bone volume fraction and bone mineral density. Furthermore, immunofluorescence staining showed 7ND treatment decreased the number of recruited inflammatory cells and osteoclasts. Together, our results support the feasibility of local delivery of 7ND for mitigating wear particle‐induced inflammation and osteolysis, which may offer a promising strategy for extending the life time of TJRs. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:58–64, 2016.     

Polyethylene Particle-Induced Bone Resorption in Substance P-Deficient Mice

Aseptic loosening is the major cause of total joint replacement failure. Substance P (SP) is a neurotransmitter richly distributed in sensory nerve fibers, bone, and bone-related tissue. The purpose of this study was to investigate the potential impact of SP on bone metabolism in polyethylene particle-induced osteolysis. We utilized the murine calvarial osteolysis model based on ultrahigh molecular weight polyethylene (UHMWPE) particles in 14 wild-type mice (C57BL/J6) and 14 SP-deficient mice. Group 1 (C57BL/J 6) and group 3 (SP-knockout) received sham surgery, and group 2 (C57BL/J6) and group 4 (SP-knockout) were treated with polyethylene particles. Analytical methods included three-dimensional micro-computed tomographic (micro-CT) analysis and histomorphometry. Bone resorption was measured within the midline suture. The number of osteoclasts was determined by counting the tartrate-resistant acid phosphatase-positive cells. UHMWPE-particle treated SP-deficient mice showed significantly reduced osteolysis compared to wild-type mice, as confirmed by histomorphometry (P < 0.001) and micro-CT (P = 0.035). Osteoclast numbers were significantly reduced in groups 3 and 4 compared to groups 1 and 2 (P < 0.001). Unexpectedly, SP-deficient mice (group 3) showed a significantly increased absolute bone mass compared to wild-type mice (group 1) (P = 0.02). The findings of our murine calvaria model lead to the assumption that SP is a promoter in particle-induced osteolysis. The pathophysiology of aseptic loosening is complex, and neuropeptides are not solely responsible for the progress of implant loosening; however, we conclude that there could be coherence between neurotransmitters and particle-induced osteolysis in patients with aseptic loosening.     

Migration of polyethylene particles around stable implants in an animal model

The aim of this study was to test the hypothesis that a tight seal between bone and implant will eliminate the avenue of particle migration around stable implants. Three types of implants were used in rabbits (polished press-fit Ti-6Al-4V or plasma-sprayed hydroxyapatite [HA]-coated Ti-6Al-4V) or doughy stage polymethyl methacrylate (PMMA). Implants were placed in the condylar notch. Each animal received an intra-articular injection of high density polyethylene (PE) particles (10(8) in 0.4 mL; mean size 4.7 microns) at 4 and 6 weeks postoperatively. Eight weeks postoperatively, peri-implant tissues were examined for PE particles and osteolysis. In all cases, intracellular PE particles were seen at the bone-implant interface and within marrow. No osteolysis was observed. Bone apposition was determined by computerized image analysis. There was no significant difference in the percentage of bone apposition (+/- SD) among the three groups of implants: Ti-6Al-4V (68% +/- 19%), HA-coated Ti-6Al-4V (70% +/- 10%), and PMMA (59% +/- 12%). These results indicate that a polished Ti-6Al-4V surface is as effective as PMMA or HA coating in limiting migration of PE particles around stable osseointegrated implants in rabbits.     

Effect of partially purified bone morphogenetic protein on DNA synthesis and cell replication in calvarial and fibroblast cultures

The effects of bone morphogenetic protein (BMP), a molecule extracted from demineralized bone, were observed in organ cultures of 21-day fetal rat calvariae. The effects of BMP on cell replication in cultures of normal rat kidney (NRK) fibroblasts were studied for comparison. At concentrations of 0.1-10 micrograms/ml for periods of 24-96 hours, BMP stimulated the incorporation of 3H-thymidine into acid-insoluble residues (DNA) in calvariae by 25%-159%, and at 1-10 micrograms/ml it increased bone DNA content by 20%-23%. BMP at 1 micrograms/ml also increased the number of calvarial mitoses after colcemid arrest by 1.5-1.8-fold. The effect of BMP on calvarial DNA synthesis was observed in the periosteal bone. In contrast to its effects on DNA synthesis, BMP did not stimulate the incorporation of 3H-proline into collagenase-digestible and noncollagen protein and did not alter calvarial alkaline phosphatase activity. BMP at 1-10 micrograms/ml caused a marked increase in 3H-thymidine incorporation into DNA in cultured NRK fibroblasts and increased DNA content and cell number by 1.5-2-fold. These studies indicate that BMP stimulates DNA synthesis and cell replication in calvarial and fibroblast cultures but does not stimulate postdifferentiated bone cells in incubated calvariae.     

Porous gelatin/tricalcium phosphate/genipin composites containing lumbrokinase for bone repair

Bone cell activities are very important in bone remodeling. This study investigates the effects of lumbrokinase on bone cell activities in cultures. Moreover, a biodegradable composite (GGT) containing genipin-crosslinked gelatin and β-tricalcium phosphate was prepared to carry lumbrokinase (GGTLK). Rat calvarial bone defects were filled with GGT and GGTLK composites. Bone healing was monitored in vivo by bioluminescence imaging and micro-CT. Lumbrokinase was found to have a dose-dependent effect on bone cell activities. Low concentrations (< 1 μg/ml) of lumbrokinase increased the viability, total alkaline phosphatase activity and mobility of osteoblasts, the number of total calcified nodules and the expression of osteopontin and osteocalcin; however, they considerably reduced the total tartrate-resistant acid phosphatase activity of osteoclasts. IVIS images revealed a stronger fluorescent signal in GGTLK-treated animals than in GGT-treated animals. Micro-CT analysis revealed that GGTLK induced more new bone formation than did GGT. These observations suggest that lumbrokinase released from GGTLK composite can enhance bone tissue regeneration.     

TGF-beta promotes the establishment of renal cell carcinoma bone metastasis.

Bone metastases develop in approximately 30% of patients with RCC, and the mechanisms responsible for this phenomenon are unknown. We found that TGF-beta1 stimulation of RCC bone metastasis cells promotes tumor growth and bone destruction possibly by stimulating paracrine interactions between tumor cells and the bone. INTRODUCTION: Bone metastasis is a frequent complication and causes marked morbidity in patients with renal cell carcinoma (RCC). Surprisingly, the specific mechanisms of RCC interaction with bone have been scarcely studied despite the inability to prevent or effectively treat bone metastasis. Bone is a reservoir for various growth factors including the pleiotropic cytokine TGF-beta1. TGF-beta1 has been shown to have tumor-supportive effects on advanced cancers and evidence suggests its involvement in promoting the development of breast cancer bone metastasis. Here, we studied the potential role of TGF-beta1 in the growth of RCC bone metastasis (RBM). MATERIALS AND METHODS: To inhibit TGF-beta1 signaling, RBM cells stably expressing a dominant-negative (DN) TGF-betaRII cDNA were generated. The in vivo effect of TGF-beta1 on RBM tumor growth and osteolysis was determined by histological and radiographic analysis, respectively, of athymic nude mice after intratibial injection of parental, empty vector, or DN RBM cells. The in vitro effect of TGF-beta1 on RBM cell growth was determined after TGF-beta1 treatment by MTT assay. RESULTS: TGF-beta1 and the TGF-beta receptors I and II (TGF-betaRI/II) were consistently expressed in both RBM tissues and cell lines. Inhibition of TGF-beta1 signaling in RBM cells significantly reduced tumor establishment and osteolysis observed in vivo after injection into the murine tibia, although no effect on tumor establishment was observed after injection of RBM cells subcutaneously or into the renal subcapsule. Treatment of five RBM cell lines with TGF-beta1 in vitro either had no effect (2/5) or resulted in a significant inhibition (3/5) of cell growth, suggesting that TGF-beta1 may promote RBM tumor growth indirectly in vivo. CONCLUSIONS: TGF-beta1 stimulation of RBM cells plays a role in promoting tumor growth and subsequent osteolysis in vivo, likely through the initiation of tumor-promoting paracrine interactions between tumor cells and the bone microenvironment. These data suggest that inhibition of TGF-beta1 signaling may be useful in the treatment of RBM.     

Micro-CT imaging analysis for the effect of celecoxib,a cyclooxygenase-2 inhibitor,on inflammatory bone destruction in adjuvant arthritis rats

Cyclooxygenase (COX)-2 is known to play an important role in the differentiation and maturation of osteoclasts. However, the role of COX-1 in bone metabolism has not been well explored. In this study, the bone-conserving effects of COX-2-specific (celecoxib), COX-nonselective (loxoprofen), and COX-1-specific agents (SC-58560) were compared using an adjuvant-induced arthritis (AIA) rat model. Arthritis was induced by injecting 50 microl liquid paraffin containing 1 mg Mycobacterium butyricum into the left footpad of Lewis rats. Drugs were given orally twice daily for 10 days beginning 15 days after adjuvant injection. Celecoxib was administered at the rate of 3 mg/kg per day, loxoprofen at 3 mg/kg per day, and SC-58560 at 10 mg/kg per day. The therapeutic effects on 3-D architectural bone changes in the arthritic condition, e.g., the bone volume/total tissue volume ratio and the amount of trabecular bone pattern factor, were determined by analyzing the hindpaw calcaneus of AIA rats using microcomputed tomography (micro-CT). In addition, dual-energy X-ray absorptiometry 2-D bone analysis was performed to compare with micro-CT analysis. AIA rats are prone to substantial bone erosion, which allows for significant changes in the 3-D architectural index. This inflammatory bone destruction was suppressed potently by celecoxib, only moderately by loxoprofen, and not at all by SC-58560. These data suggest that COX-2 plays an important role in the inflammatory bone destruction that occurs with rheumatoid arthritis. The results also suggest that COX-2 is more effective than COX-1 at suppressing the destruction of bone associated with arthritis.