仙灵骨葆胶囊联合阿仑膦酸钠对骨质疏松骨折大鼠骨痂血管形成的影响

目的探讨仙灵骨葆胶囊联合阿仑膦酸钠对骨质疏松性骨折大鼠骨痂血管形成及VEGF、BMP-2表达的影响。方法50只雌性SD大鼠随机分为假手术组(SHAM组)、模型组(MODEL组)、仙灵骨葆组(XLGB组)、阿仑膦酸钠组(ALLSN组)、联合药物组(LHYW组),10只/组,构建骨质疏松性骨折大鼠模型,放射性X线观察评估骨折愈合,双能X线检测骨密度,Mirco-CT检测骨结构形态学参数,番红O固绿染色观察骨痂组织形态学,免疫组化检测骨痂VEGF和BMP-2蛋白表达。结果所有实验大鼠均进入结果分析。与SHAM组比较,MODEL组大鼠骨折愈合评分、骨密度、骨组织形态学参数、骨痂VEGF和BMP-2表达均显著降低(P0.05),与MODEL组比较,XLGB组、ALLSN组和LHYY组骨折愈合评分、骨密度、骨组织形态学参数、骨痂VEGF和BMP-2表达均显著升高(P0.05),尤以LHYY组最高。SHAM组骨小梁结构正常,几乎均为骨性骨痂。MODEL组骨小梁明显稀疏、断裂,未见明显骨性骨痂。XLGB组和ALLSN组骨小梁增多,排列稍紊乱,大部分为骨性骨痂。LHYW组骨小梁明显增多,排列密集整齐,大量骨性骨痂。结论仙灵骨葆胶囊联合阿仑膦酸钠可能通过介导提高骨质疏松性骨折大鼠骨生长因子VEGF和BMP-2表达,促进骨痂血管形成,加速骨痂形成,增加骨密度,改善骨结构形态,促进骨折愈合。     

甲状旁腺激素联合降钙素对大鼠骨质疏松性骨折骨生长因子的影响

目的探讨甲状旁腺素联合降钙素对大鼠骨质疏松性骨折骨相关生长因子及骨折愈合的影响。方法75只雌性SD大鼠随机分为:假手术组、去势组、甲状旁腺素组、降钙素组、联合用药组,15只/组,首先构建去卵巢大鼠骨质疏松性骨折动物模型。观察并评估骨折愈合,测定骨痂BMD和BMC,检测血清BMP-2、VEGF、TGF-β、IGF-1水平和骨痂蛋白表达,观察骨痂形态结构变化。结果去势组较假手术组骨折愈合评分、骨痂BMD和BMC、血清BMP-2、VEGF、TGF-β、IGF-1水平和骨痂蛋白表达均显著降低(P均<0.05),而联合用药组较去势组上述指标均显著升高(P均<0.05)。去势组骨痂骨小梁明显减少,生长稀疏,排列紊乱,大量间充质干细胞及纤维软骨细胞,成骨细胞及微血管少见;联合用药组骨痂大量骨小梁,生长旺盛,互相交错网状,排列致密有序,可见较多成熟的骨细胞及成骨细胞。结论甲状旁腺素联合降钙素通过介导提高相关骨生长因子表达,提高骨质疏松性骨折骨密度和矿物含量,改善骨组织形态学,加快骨折愈合。     

甲状旁腺激素联合阿仑膦酸钠对大鼠骨质疏松性骨折骨痂血管形成的影响

目的探讨甲状旁腺激素联合阿仑膦酸钠对大鼠骨质疏松性骨折骨痂血管形成及骨折愈合的影响。方法 75只雌性SD大鼠随机分为5组:假手术组、去势组、甲状旁腺素组、阿仑膦酸钠组、联合用药组,每组15只,首先行双侧卵巢切除术,术后4周行右侧股骨干骨折髓内固定术,以构建大鼠骨质疏松性骨折动物模型。观察并评估骨折愈合,检测骨痂生物力学和骨密度(bone mineral density,BMD),检测血清血管内皮生长因子(vascular endothelial growth factor,VEGF)和骨形成发生蛋白-2(bone morphogenetic protein-2,BMP-2)浓度,观察骨痂形态结构,检测骨痂VEGF表达。结果去势组较假手术组骨折愈合评分、骨痂生物力学强度、骨痂BMD、血清BMP-2和VEGF浓度、骨痂VEGF蛋白表达、骨痂微血管数均显著降低(P0.05),甲状旁腺素组、阿仑膦酸钠组、联合用药组较去势组上述指标均升高,其中以联合用药组升高最显著(P0.05)。结论甲状旁腺激素联合阿仑膦酸钠通过介导VEGF,上调BMP-2表达,促进骨质疏松性骨折大鼠骨痂血管形成,增加骨密度,改善生物力学强度及骨组织形态学,加快骨折愈合。     

葛根素对去卵巢大鼠骨质疏松性骨折骨痂血管形成的影响

目的探讨葛根素对骨质疏松性骨折骨痂血管形成的影响及对骨折愈合的作用。方法60只雌性SD大鼠分为3组:假手术组、去势组、葛根素组,20只/组,去卵巢大鼠骨质疏松性骨折动物。观察评估骨折愈合情况,检测血清BMP-2和VEGF浓度,观察骨痂形态结构变化,检测骨痂BMP-2和VEGF表达。结果去势组较假手术组骨折愈合评分、血清BMP-2和VEGF浓度、骨痂BMP-2和VEGF表达、微血管数均显著降低(P<0.05),葛根素组较去势组上述指标均显著增高(P<0.05)。去势组骨痂组织可见少量骨小梁,生长稀疏,排列紊乱,大量纤维软骨细胞等纤维组织,成骨细胞及新生小血管少见;葛根素组骨痂可见较多骨小梁,生长较旺盛,排列有序,可见较多成熟的骨细胞,新生微小血管较多。结论葛根素通过介导去卵巢大鼠骨质疏松性骨折骨痂BMP-2和VEGF表达,促进骨痂血管形成,改善骨组织形态学,加快骨折愈合。     

雌二醇对去卵巢骨质疏松大鼠骨组织Wnt/β-catenin信号通路的影响

目的观察雌二醇对去卵巢骨质疏松大鼠骨组织Wnt/β-catenin信号通路的影响,探讨其防治绝经后骨质疏松症的作用机制。方法将80只雌性SD大鼠随机分为对照组、假手术组、去势组、药物组,构建绝经后骨质疏松大鼠模型,检测骨组织骨密度(bone mineral density,BMD)和生物力学指标,运用qRT-PCR和免疫组化分别检测骨组织Wnt、β-catenin、BMP-2的mRNA和蛋白表达,通过HE染色观察骨组织形态学。结果去势组股骨BMD、生物力学指标较对照组和假手术组均显著降低(P0.05),药物组股骨BMD、生物力学指标较去势组均显著升高(P0.05)。去势组骨组织Wnt、β-catenin、BMP-2的mRNA和蛋白表达较对照组和假手术组均显著降低(P0.05),药物组骨组织Wnt、β-catenin、BMP-2的mRNA和蛋白表达较去势组均显著升高(P0.05)。去势组骨小梁明显减少,排列稀疏不规则,连接不完整,骨髓腔变大,有大量纤维组织。药物组骨小梁数量减少不明显,粗细均匀稍致密,排列尚规则,连续性完整性较好。结论雌二醇通过Wnt/β-catenin信号通路作用于BMP-2,增加了去卵巢骨质疏松大鼠骨密度,提高了骨生物力学性能,能够改善骨组织结构,发挥了抗骨质疏松作用。     

骨质疏松性骨折骨痂软骨细胞增殖和IGF-I表达的实验研究

目的：通过去势大鼠骨质疏松骨折模型，研究骨质疏松骨痂软骨细胞的增殖及IGF-I的表达情况。方法：建立去势大鼠骨质疏松及骨折模型，在术后10～40d内取材，行IGF-I和PCNA免疫组化染色，观察骨质疏松骨折骨痂内软骨细胞的增殖及IGF-I的表达情况。结果：骨质疏松性骨痂组织中IGF-I早期上调表达，可以促进骨膜生发层细胞和骨折断端间充质细胞增殖、分化；IGF-I在骨折后期一部分成熟软骨细胞中表达，可能延缓了软骨细胞向终末分化，在维持软骨细胞表型过程中发挥一定的作用。结论：IGF-I在骨质疏松性骨折愈合过程中可能发挥着重要的调节作用。     

雷尼酸锶促进大鼠股骨骨质疏松性骨折愈合的研究

目的 探讨雷尼酸锶(strontium ranelate,SR)对大鼠股骨骨质疏松性骨折愈合的影响。方法 6个月龄Wistar雌性处鼠66只,切除双侧卵巢制作骨质疏松动物模型,模型建立成功后,在大鼠股骨中段横行截骨,制作大鼠股骨骨质疏松性骨折动物模型,术后死亡6只,然后将剩余60只大鼠随机分为实验组(SR干预治疗组）和空白对照组,每组30只。骨折术后第一天起,实验组给予SR 400 mg/(kg d)灌胃,对照组给予同等体积的生理盐水灌胃。通过影像学、组织学和免疫组织化学（骨形态发生蛋白-2,BMP-2)观察,骨组织形态计量学、骨密度（bone mineral density,BMD)和生物力学测量,观察骨折愈合情况。结果 实验组腰4、5椎体BMD、BMP-2阳性表达细胞数、骨小梁面积比、骨小梁平均宽度、最大负荷和最大桡度高于同时期对照组,差异有统计学意义(P <0. 01);骨小梁平均间隔小于同期对照组,差异有统计学意义(P <0. 01)。结论 SR具有抑制骨吸收和促进骨形成的双重作用,能增加BMD,加快骨痴形成,促进骨质疏松性骨折愈合,改善骨的显微结构,提高骨折愈合后骨的生物力学特性。     

雷洛昔芬联合仙灵骨葆对骨质疏松骨微结构和生物力学性能的影响

目的探讨雷洛昔芬联合仙灵骨葆对骨质疏松大鼠骨微结构及其生物力学性能的影响。探讨其调控骨吸收与骨形成及其防治骨质疏松症的作用机制。方法 6月龄40只SD大鼠在骨质疏松造模之后,随机分为正常组、假手术组、模型组、干预1组、干预2组,每组8只。造模手术1周后,干预1组采用经口灌胃雷洛昔芬6.2 mg/(kg·d),干预2组采用经口灌胃雷洛昔芬6.2 mg/(kg·d)+仙灵骨葆250 mg/(kg·d),而正常组、假手术组、模型组采用经口灌胃注射用水。12周后应用组织病理学分析及组织计量学测定;利用Van Gieson胶原纤维染色法观察胶原纤维沉积变化;ELISA测定血清蛋白聚糖活性。分离右侧股骨进行三点弯曲试验,检测股骨生物力学性能。结果对照组骨小梁粗厚、有完整的成骨细胞区。模型组骨小梁稀疏细薄、细胞核固缩;与对照组相比,模型组胶原纤维明显减少,蛋白聚糖含量明显增加,最大载荷(FM)下降,差异均有统计学意义(均P0.05)。干预组骨小梁排列较紧密。干预2组较干预1组,胶原纤维明显增加,蛋白聚糖活性降低,最大载荷(FM)差异具有统计学意义(P0.01)。结论雷洛昔芬联合仙灵骨葆较雷洛昔芬单独用药可以显著改善骨质疏松大鼠的骨基质代谢,促进成骨,显著加强骨生物力学性能。     

仙灵骨葆对卵巢切除大鼠股骨骨折愈合影响的实验研究

目的 研究仙灵骨葆对卵巢切除大鼠股骨骨折愈合的作用.方法 健康12周龄雌性SD大鼠40只,体重(258±14)g,随机分为4组,每组10只.A组仅作切口暴露腹腔;B组仅切除卵巢;C、D组切除卵巢同时制备右侧股骨中段横型骨折,髓内针固定,术后分别采用生理盐水及250 mg/(kg·d)仙灵骨葆灌胃.于术后即刻、1、2、3、4及5周测量A、B组大鼠体重.术后5周取各组右侧股骨标本,双能X线骨密度仪测量股骨全长骨密度(total femur bone mineral density,tBMD)、远1/3段骨密度(distal femur bone mineral density,dBMD)和中1/3段骨密度(middle femur bone mineral density,mBMD),并行C、D组CR摄片、HE染色和免疫组织化学染色.结果 B组大鼠体重从第3周开始显著高于A组(P＜0.05),绝经后骨质疏松模型制备成功.CR摄片示D组骨痂量相对较多,骨折线模糊;C组骨痂量较少,骨折线清晰.B组tBMD和dBMD明显低于A组,D组mBMD明显高于C组,差异均有统计学意义(P＜0.05);D组tBMD和dBMD较C组有增高趋势,但差异无统计学意义(P＞0.05).组织学观察D组与C组比较,骨折端大部分骨性愈合,骨髓腔可见较多毛细血管植入.免疫组织化学染色示C、D组BMP-2的积分吸光度(IA)值分别为2.2366±0.1818和3.7273±0.8742,VEGF的IA值分别为2.8355±0.5370和3.8396±0.2230,差异均有统计学意义(P＜0.05).结论 仙灵骨葆可促进卵巢切除大鼠股骨骨折愈合,加快编织骨向板层骨的转化,这可能与上调BMP-2和VEGF表达相关.     

去势对骨折早期愈合过程的影响

目的 通过对去势大鼠股骨骨折模型愈合早期骨痂的组织学、骨密度以及转化生长因子β1(Transforming growth factor beta 1.TGF-β1)、碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF）、骨形态发生蛋白-2(bone morphogenetic proteins-2，BMP-2)合成变化的观察．探讨骨质疏松骨折早期愈合过程的变化。方法 将60只雌性SD大鼠分为去势组和对照组，3个月后制成股骨干骨折模型．伤后不同阶段处死．分别进行组织学、骨密度以及TGF-β1,bFGF,BMP-2免疫组化染色方法观察。结果 大鼠去势后3个月全身骨密度检查证实去势组骨质疏松形成。骨折后第3d两组均开始形成原始骨痂；第4～6周，去势组骨痂比对照组少，且软骨痂比例较高．骨密度较低，免疫组化染色显示，在两组间bFGF、BMP-2的表达与分布、高峰出现与持续时间差异无显著性．去势组骨小梁附近表达TGF-β1的成骨细胞数目减少，结论 骨质疏松使大鼠骨折早期骨痂的数量与质量降低，自软骨性骨痂至骨性骨痂演变过程减缓．对骨折愈合过程的影响与BMP-2、bFGF的表达无明显关联。TGF-β1在成骨细胞中的表达减少可能是引起骨质疏松骨折愈合质量下降的因素之一．     

Bone morphogenetic protein-7 accelerates fracture healing in osteoporotic rats

Background:

Osteoporosis is characterized by low bone mass, bone fragility and increased susceptibility to fracture. Fracture healing in osteoporosis is delayed and rates of implant failure are high with few biological treatment options available. This study aimed to determine whether a single dose of bone morphogenetic protein-7 (BMP-7) in a collagen/carboxy-methyl cellulose (CMC) composite enhanced fracture healing in an osteoporotic rat model.

Materials and Methods:

An open femoral midshaft osteotomy was performed in female rats 3 months post-ovarectomy. Rats were randomized to receive either BMP-7 composite (n = 30) or composite alone (n = 30) at the fracture site during surgery. Thereafter calluses were collected on days 12, 20 and 31. Callus cross-sectional area, bone mineral density, biomechanical stiffness and maximum torque, radiographic bony union and histological callus maturity were evaluated at each time point.

Results:

There were statistically significant increases in bone mineral density and callus cross-section area at all time points in the BMP-7 group as compared to controls and biomechanical readings showed stronger bones at day 31 in the BMP-7 group. Histological and radiographic evaluation indicated significant acceleration of bony union in the BMP-7 group as compared to controls.

Conclusion:

This study demonstrated that BMP-7 accelerates fracture healing in an oestrogen-deficient environment in a rat femoral fracture healing model to scientific relevance level I. The use of BMP-7 composite could offer orthopedic surgeons an advantage over oestrogen therapy, enhancing osteoporotic fracture healing with a single, locally applied dose at the time of surgery, potentially overcoming delays in healing caused by the osteoporotic state.     

骨质疏松性骨折愈合与骨量、骨结构及力学性能相关性的实验研究

目的探明骨质疏松性骨折愈合程度与骨量、骨结构与力学性能的相互关系,以期能为临床治疗提供科学指导与理论依据.方法选择8月龄雌性SPF级SD大鼠130只,随机分为2组：骨质疏松性骨折组（OPFM）与一般性骨折组（CFM）,每组各65只.手术方法建立骨质疏松性骨折与一般性骨折的实验模型,于术后1、2、4、6、8、12、16周作骨痂组织形态计量学、骨密度与力学性能测试等观察.结果①骨痂组织骨组织形态计量学结果发现：OPFM组成熟小梁骨占骨痂面积比CFM组小,且小梁骨厚度变薄、小梁骨间距较宽;同时骨小梁动力学参数显示：OPFM组骨小梁表面荧光标记百分比（LS）较CFM组低,而骨矿化沉积率（MAR）却较高.②骨密度（BMD）测定显示：两组BMD值8周时均达到高峰（P＞0.5）,随后均降低,OPFM组下降尤为明显;12周时两组间差异有显著性（P＜0.05）.③骨痂组织扭转力学强度测试结果表明：8、12周OPFM组扭转强度均较CFM组低.结论在骨质疏松性骨折修复过程中,骨痂组织的有机成份组成、显微结构、骨矿代谢与骨量的异常改变导致了其力学强度乃至骨折愈合质量的降低,是再次骨折发生的主要原因.     

Relationship among bone mineral density, collagen composition, and biomechanical properties of callus in the healing of osteoporotic fracture

Objective： To study the change and relationship among bone mineral density （BMD）, collagen composition and biomechanical properties of the callus in the healing process of osteoporotic fracture. Methods： The osteoporotic rat model and fracture model were established through bilateral ovariectomy （OV＇X） and osteotomy of the middle shaft of the right hind tibiae, respectively. Ninety female SD rats were randomly divided into OVX group and sham group. With the samples of blood and callus, roentgenoraphic and histological observation were performed for the assessment of the healing progress of the fracture, and the serum concentration of TRAP-5b, proportion of type I collagen, BMD and biomechanical properties of the callus were measured. Results： The OVX group experienced a significant delay of fracture healing. The mean serum concentration of TRAP-5b of rats in the OVX group was much higher than that in the sham group after the operation （P 〈 0.05）, but the difference at the same time point after fracture was smaller than that before fracture （P 〈 0.05）. The BMD of the callus in both groups reached the peak value at the 6 th week after fracture while the proportion of the type I collagen and the biomechanical strength reached the peak at the 8th week. Conclusions. The deficiency of estrogen after the ovariectomy could induce the up-regulation of the osteoclasts activities, whereas the potency of further activation after fracture was depressed. Although the synthesis of collagen together with its mineralization determines the biomechanical properties of new bone, the accumulation of collagen could be assessed as an index in the prediction of biomechanical strength of bones independent of the bone mineral deposition.     

不同类型老年女性髋部骨折血清IGF-1及IGFBP-3,4,5表达相关性研究

目的研究不同类型老年女性髋部骨折中外周血清中血清胰岛素样生长因子1（IGF-1）及血清胰岛素样生长因子结合蛋白3,4,5（IGFBP-3,4,5）表达差异及同骨密度的相关性。方法选取年龄大于65岁老年女性髋部骨折患者,检测并比较外周血清中IGF-1及IGFBP-3,4,5表达差异及同骨密度的相关性。结果转子间骨折组转子区骨矿物质密度（BMD）较股骨颈骨折组低（P〈0.001）,股骨颈组血清中IGFBP-4含量较转子间骨折组低（P〈0.001）,而IGFBP-5含量高于转子间骨折组（P〈0.001）。在血清指标相关性表达中,血清IGF-1表达与股骨颈区BMD成正相关（R=0.46,P=0.002）,血清IGFBP-4同转子区BMD成负相关（R=-0.634,P〈0.001）,血清IGFBP-5同股骨颈区BMD成正相关（R=0.235,P〈0.001）。结论外周血清中IGF-1及其结合蛋白表达的差异可能是导致不同类型老年髋部骨折的病理机制之一,但由于IGF-1及其结合蛋白调节机制相当复杂,对于IGF-1及IGFBPs表达机制同骨折类型的关系尚需大量的实验研究进一步证实。     

Early period of fracture healing in ovariectomized rats

Objective. To evaluate the effect of osteoporosis on fracture healing through observing the hlstomorphological changes, bone mineral density of callus and expression and distribution of transforming growth factor beta 1 (TGF-β1 ), basic fibroblast growth factor (bFGF)and bone morphogenetic protein.2 (BMP-2) in ovariectomized rats. Methods. Sixty female Sprague-Dawley rats ( aged 12 weeks and weighing 235 g on average ) were randomly divided into an ovariectomized (OVX) group (n =30) anda sham-operated (SO) group ( n = 30). Ovariectomy was performed in the OVX rats and same incision was made in the SO rats. Three months later, fracture of femoral shaft was made on all the rats. Then they were killed at different time points. Callus formation was observed with histological and imethods. Results: A reduction in callus and bone mineral density in the healing femur and a decrease of osteoblasts expressing TGF-β1 near the bone trabecula were observed in the OVX rats 3-4 weeks after fracture.Histomorphological analysis revealed a higher content of soft callus in the OVX rats than that in the SO rats.Immunohistochemistry results showed that no remarkable difference in expression and distribution of BMP-2 and bFGF between the OVX and SO groups was found. Conclusions: Osteoporosis influences the quantity and quality of callus during the early period of fracture healing. The effect of osteoporosis on fracture healing has no relationship with the expression of BMP-2 or bFGF. The decreased expression of TGF-I31 in osteoblasts may cause a decrease in quality of facture healing after osteoporosis.     

ERK5在小鼠骨质疏松性骨折愈合过程中作用的实验研究

目的通过构建小鼠骨质疏松骨折模型,研究ERK5在骨质疏松性骨折愈合过程中的作用。方法将108只6周龄雌性昆明小鼠随机分成4组,通过手术切除小鼠双侧卵巢及小鼠股骨离断分别构建骨质疏松(OVX)和骨折模型(Fracture),然后给实验组小鼠每日腹腔注射ERK5特异性阻断剂XMD8-92,于1 w、2 w、4 w后分别处死一定数量的小鼠,取术侧股骨标本,行X线片检查、股骨骨痂Micro-CT、HE染色及免疫组织化学染色检查,观察骨折端骨小梁生长情况,骨痂内成骨相关蛋白及ERK5表达情况。结果给予实验小鼠注射XMD8-92后第2周及第4周,Fracture组小鼠骨痂生长较快,骨小梁数目较多,厚度较大,成骨相关蛋白ALP、Runx2的表达相对较多(P0.05),而Fracture+XMD8-92组小鼠骨痂生长则相对缓慢,骨小梁稀少且绯薄,结构相对较乱,碱性磷酸酶(alkaline phosphatase,ALP)、Runx2表达较少(P0.05);且OVX+Fracture+XMD8-92组小鼠与OVX+Fracture组小鼠相比,骨小梁生成更少且紊乱,骨痂生长明显延迟,ALP、Runx2表达量显著减少(P0.05)。结论 ERK5影响骨折端骨痂形成的速度和质量,在促进骨质疏松性骨折愈合过程中起着十分重要的生理作用。