合并脑外伤的骨折愈合过程中PDGF的作用

目的 通过研究大鼠股骨干骨折合并脑外伤时骨痂组织内PDGF表达水平的变化,探讨脑外伤对骨折愈合的影响及作用机制. 方法 取12周雄性SD大鼠64只,体重(356 4±25)g,随机分成8组,每组8只.A1、B1、Cl、D1组分别为骨折合并脑外伤1、2、3、4周组,制作脑外伤和股骨干骨折模型;A2、B2、C2、D2组分别为单纯骨折1、2、3、4周组,制作单纯股骨干骨折模型作对照.各组摄CR片后截取骨痂,行HE染色观察骨痂生长情况及其组织形态,免疫组织化学染色测定PDGF表达,原位杂交检测PDGF mRNA表达. 结果 CR片示在同一时间点A1、B1、C1、D1组较A2、B2、C2、D2组骨折端骨痂形成早,骨痂多,骨折愈合快.HE染色示A1组可见较多早期软骨细胞、成纤维细胞,A2组骨折间隙可见成纤维细胞,仅夹杂有少量的早期软骨细胞;B1组骨折端有新生骨小梁长入,B2组未见骨小梁形成;C1组骨折端有大量编织骨形成且骨小粱间有少量成熟软骨细胞,C2组少量骨小梁向骨折端长入;D1组编织骨向板层骨转化,D2组骨折端为不成熟骨小梁.免疫组织化学染色和原位杂交实验均可见成纤维细胞、间充质细胞、血管内皮细胞、早期软骨细胞、成骨细胞、破骨细胞胞浆出现广泛的强阳性反应,A1、B1、C1、D1组PDGF和PDGF mRNA阳性细胞百分数均高于同一时间点A2、B2、C2、D2组,差异有统计学意义(P<0.05). 结论 脑外伤对骨折愈合有促进作用,可能与脑外伤后PDGF表达水平升高有关.     

颅脑损伤对股骨干骨折愈合的影响

目的 探讨颅脑损伤对股骨干骨折愈合的影响.方法 18例脑外伤合并股骨干骨折患者(A组)和18例单纯股骨干骨折患者(B组)均行髓内钉内固定治疗,记录骨痂量和骨折愈合时间,并进行统计学分析.结果 骨痂体积: A组(233.63 ±8.72)cm3 ,B 组(48.40±2.09)cm3,两组比较差异有统计学意义(P<0.01);骨折愈合时间:A组(11.83±1.58)周,B组(14.00±2.29)周,两组比较差异有统计学意义(P<0.01).结论 脑外伤和骨折愈合存在一定的联系,可以加快骨折愈合,增多骨痂量.     

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.     

NGF对骨折愈合影响的研究

目的 探讨NGF对骨折愈合的影响,以及对BMP-2诱导成骨的作用.方法 清洁级雄性6～8周昆明小鼠60只,体重23～25 g,随机分为4组,每组15只.建立股骨中段不稳定软骨内成骨骨折模型,A、B、C、D组分别于骨折断端局部使用NGF/生理盐水、BMP-2、BMP-2/NGF/生理盐水、生理盐水.各组分别于术后14、21和28 d各处死5只小鼠,并于骨折部位取材行大体、X线片观察、生化检测及组织学观察.处死小鼠前取小鼠眼球球后动静脉血液行血清ALP活性测定.结果 术后各时间点,大体观察A、B、C组局部新生硬组织大小、硬度,骨折断端骨痂生长依次增加,均高于D组.X线片观察A、B、C组骨折线逐渐模糊,钙化面积依次增大,均快于D组.组织学观察A、B、C组骨小梁成熟度依次增加,均优于D组.术后各时间点,A、B、c组成骨面积和成骨区灰度值、血清ALP含量、骨痂组织中ALP含量和钙含量、骨痂湿重均高于D组.术后各时间点各组成骨面积和成骨区灰度值比较、术后14 d各组血清中ALP含量比较、术后14 d和21 d各组骨痂组织中ALP含量比较、术后21 d和28 d各组骨痂组织中钙含量及骨痂湿重比较差异均有统计学意义(P<0.05).术后14d,B、C组与A、D组比较骨痂湿重差异有统计学意义(P<0.05).术后21、28d,骨组织形态计量分析显示各组新生骨骨小梁表面成骨细胞指数、平均骨小梁面积百分比和平均骨小梁宽度随时间增大依次减小,A、B、C组依次增大,均高于D组,各组间比较差异均有统计学意义(P<0.05).结论 NGF有促进骨折愈合作用,且具有协同BMP-2诱导成骨的作用.     

骨形态发生蛋白-7复合纤维蛋白对大鼠骨质疏松性椎体骨折愈合的影响

目的:观察以纤维蛋白为载体的骨形态发生蛋白-7(BMP-7)对骨质疏松性椎体骨折愈合的影响,探讨局部应用BMP-7纤维蛋白复合物治疗骨质疏松性椎体骨折的可行性。方法:雌性SPF级8月龄SD大鼠54只,随机选取36只,经去除卵巢建立骨质疏松模型并随机分为A、B组,每组18只;另18只行伪手术,在背部卵巢周围切除等体积脂肪,作为对照组(C组)。术后3个月所有动物采用L5椎体手术开窗刮除术区内松质骨方法建立骨折模型,A组骨折区内放入含BMP-724μg和纤维蛋白20mg的凝胶状混合物,B组和C组于同部位放入等体积小牛血清,盖好窗口骨瓣。建立骨折模型后4、6、8周,每组处死动物3只,取L5椎体切片行HE染色;6、8、12周时每组处死动物3只,行L5椎体力学性能测试(载荷能力、弹性模量和最大应变量)。结果:骨折后4周3组骨折区均有纤维骨痂形成,但B组纤维骨痂量少于A、C组;6周时A、C组有较多骨性骨痂形成,B组仅有微量骨性骨痂;8周时A、C组均为成熟小梁骨,骨折基本愈合,B组骨性骨痂量少,仍以纤维骨痂为主,尚有部分骨缺损存在;各时间点A组与C组相似。骨折后6、8、12周A、C组L5椎体的最大载荷、弹性模量及8、12周时的最大应变量均明显高于B组(P0.05或0.01),B组6周时的最大应变量与A、C组比较无显著性差异(P0.05),各时间点A组与C组比较无显著性差异(P0.05)。结论:局部应用以纤维蛋白为载体的BMP-7能明显促进大鼠骨质疏松性椎体骨折的愈合,纤维蛋白可作为BMP-7的载体。     

可控性应力与微动对骨折愈合影响的组织学研究

目的 探讨骨折端在不同轴向应力作用下,不同骨折愈合时期所需轴向应力的适宜力值. 方法 32只青山羊均行股骨干中段横行截骨制作骨折模型,按骨折端施加实验动物自身体质量的0倍(对照组)、1/6(A组)、1/3(B组)、1/2(C组)应力分为4组,每组8只.术后4、8周分批处死,每次每组处死4只,行大体观察和组织学观察,测量骨折端骨外膜骨痂面积. 结果 对照组有1只动物骨折端发生成角畸形,实验A、B、C组分别有1、2、4只动物骨折端发生成角畸形.术后4周,对照组、A、B、C组骨折端骨外膜骨痂面积平均值分别为(1.15±0.34)、(1.86±0.28)、(2.18±0.36)、(1.99±0.33)cm~2,A、B、C组分别与对照组比较,骨折端骨痂生成多,骨外膜骨痂面积差异有统计学意义(P<0.05).术后8周,沿轴向排列骨外膜骨痂中骨性骨痂多、致密,皮质骨松化明显;对照组、A、B、C组骨折端骨外膜骨痂面积平均值分别为(1.38±0.31)、(2.09±0.23)、(2.69±0.28)、(2.71±0.31)cm~2,A、B、C组分别与对照组比较,B、C组分别与A组比较,差异均有统计学意义(P<0.05). 结论 骨折端施加轴向应力时能促进骨折端骨痂生长,较大的应力强度能更好地促进骨折端骨痂生长,但同时会造成骨折愈合成角畸形发生率增高.骨折端施加自身体质量的1/3应力时骨折端成角畸形发生率较低,最适宜促进骨折端骨痂生长.     

BMP-2联合BMP-7在截骨延长时促进成骨的实验研究

目的探究在截骨延长成骨过程中联合应用BMP-2和BMP-7对成骨的促进作用。方法采用健康成年日本大耳白兔30只,完全随机分为三组。A组作为对照组正常牵拉不做其他处理;B组在截骨后于截骨处加入rhBMP-2药片;C组在截骨后于截骨处加入rhBMP-2药片,7 d后在延长区经皮注射rhBMP-7溶解液。在术后第7天、第38天分别拍摄三组模型的右侧胫骨全长X线片;术后38 d取三组标本分别测量三组延长区及远近截骨端的骨密度;将取下的三组标本进行HE染色,光镜下观察。结果术后第38天X线观察:A组见截骨两端形成一定量的骨组织,延长区的骨痂影密度低,有透亮区;B组延长区新生骨组织较为均匀,可见骨折线;C组骨延长区形成的骨组织影密度高于其他两组,未见明显骨折线。使用SPSS 17.0软件对各组的骨密度进行统计学分析:B、C组的骨密度测定值明显高于A组(P<0.05),C组骨密度测定值优于B组(P<0.05);术后第38天HE染色:A组骨小梁内骨细胞稀少,B、C组骨小梁密度体积增大明显,骨痂向骨性骨痂改建明显,髓腔再通。结论BMP-2能促进兔胫骨牵张成骨的形成和愈合;BMP-7可加快启动软骨内成骨,使软骨内成骨时间提前,联合应用BMP-2和BMP-7比单纯应用BMP-2可更快速地形成新的骨组织,二者在成骨上具有协同作用。     

黄骨髓对兔骨折愈合影响的实验研究

[目的]研究自体黄骨髓一期移植对骨折愈合作用的实验疗效,观察黄骨髓是否具有成骨能力,为进一步临床应用提供理论依据。[方法]36只新西兰大白兔随机分为3组,建立左侧胫骨骨折延迟愈合模型,A组仅以钢板内固定;B组钢板内固定后,植入空白明胶海绵,C组钢板内固定后,植入黄骨髓与明胶海绵复合物。术后每组分别于2、4、8、12周各处死3只动物,进行影像学检测、大体观察及HE染色检查,观察骨折愈合情况及骨痂形成情况。[结果]A组及B组在术后4周只可见少量骨痂形成,而C组在术后2周即可见骨痂形成,术后4周可见大量骨痂形成,至术后8周C组骨折线消失,而A、B组骨折线仍可见;术后12周,A、B组仍可见骨折线存在。[结论]黄骨髓在骨折后能明显促进骨折愈合,在预防骨不连或骨折延迟愈合中具有重要作用。     

重组人BMP-2缓释作用于基质血管成分细胞促进大鼠脊柱融合的实验研究

目的将重组人BMP-2(recombinant human BMP-2,rh BMP-2)缓释作用于新鲜分离的大鼠基质血管成分细胞(stromal vascular fraction cells,SVFs),移植到大鼠腰椎横突后外侧,观察其促进脊柱融合的效果。方法取健康4月龄SD大鼠双侧腹股沟区皮下脂肪,采用Ⅰ型胶原酶消化法提取SVFs。通过仿生磷灰石涂层方法,将rh BMP-2与β-磷酸三钙(β-tricalcium phosphate,β-TCP)通过共价键结合制成缓释载体,通过BCA法测定其缓释效应。将32只大鼠随机分成4组,每组8只。采用L_4、L_5横突去皮质化方法制备大鼠腰椎后外侧融合模型,分别于A、B、C、D组植入PBS+脱钙骨基质(decalcified bone matrix,DBX)、rh BMP-2/β-TCP缓释载体+DBX、SVFs+DBX和SVFs+rh BMP-2/β-TCP缓释载体+DBX。术后8周获取腰椎融合标本,进行手法判断和X线片观察评价脊柱融合情况,以及micro-CT评价脊柱融合情况,并分析骨密度(bone mineral density,BMD)及骨体积分数;行HE染色、Masson三色组织学染色,以及免疫组织化学染色检测骨钙蛋白(osteocalcin,OCN)。结果 2周时rh BMP-2/β-TCP缓释载体的rh BMP-2体外累计释放量为40%左右,起到缓慢释放效果;而未经仿生磷灰石涂层的对照组2周时几乎停止释放rh BMP-2。手法判断、X线片和micro-CT评估一致,显示D组成骨能力最强,达100%融合(8/8),之后依次为B组(75%、6/8)、C组(37.5%、3/8)、A组(12.5%、1/8)。Micro-CT分析示,D组BMD和骨体积分数均显著高于其余3组,B组显著高于A、C组,差异均有统计学意义(P0.05)。HE、Masson三色染色和OCN免疫组织化学染色示,D组有大量软骨细胞连接,伴有骨基质沉积、有活跃的成骨过程,类似长骨的矿化过程;B组的骨形成相对D组较弱;A、C组缺乏有效的新骨形成。结论新鲜分离的SVFs在rh BMP-2缓释作用下,显示出良好的骨生成能力,可有效促进脊柱融合,为临床应用提供理论基础。     

HA复合rhBMP-2转染的BMSCs对羊胫骨延长骨愈合的影响

目的 评价HA复合rhBMP-2的腺病毒(adenovirus mediated rhBMP-2,Adv-rhBMP-2)转染的羊BMSCs对骨痂延长骨愈合的影响. 方法 成年山羊19只,雌雄不限,体重15～20 kg.于每只羊髂嵴上穿刺抽取骨髓10 mL,常规传代培养BMSCs.取第3代BMSCs,以感染复数200转染腺病毒.取0.25%胰蛋白酶消化转染后3 d的细胞1×108个,与HA充分混匀制备Adv-rhBMP-2/BMSCs/HA.建立山羊右侧胫骨延长模型,术后立即于截骨部位注射自体细胞复合物.按术后注入物质不同随机分成4组A组为Adv-rhBMP-2/BMSCs/HA组(n=6),B组为Adv-rhBMP-2/BMSCs组(n=5),C组为Adv-β-gal/BMSCs/HA组(n=4),D组为空白组(n=4).术后第7天开始行胫骨延长,速度1 mm/d,共延长4周.术后5、8、12周摄X线片观察骨痂生长情况,12周处死动物,取标本分别行骨密度检测、生物力学测定、组织学观察和骨形态计量学分析. 结果 X线片检查示,术后5、8周A、B组骨痂生成量明显多于C、D组,X线片定量评分A、B组明显高于C、D组,差异均有统计学意义(P＜0.05);12周各组均在骨延长部位形成连续骨痂.骨密度测定术后12周A、B、C、D组延长部位骨矿物质含量分别为(4.175 ±1.921)、(2.600 ±0.638)、(2.425±0.826)和(1.175 ±0.574)g,骨矿物质密度分别为(0.612 ±0.196)、(0.630 ±0.159)、(0.450 ±0.166)和(0.266 ±0.113)g/cm2,A、B组显著高于C、D组(P＜0.05).生物力学测定A、B、C、D组最大载荷分别为(490.20 ±155.08)、(350.59±80.48)、(221.95±68.79)和(150.65±92.29)N,弹性模量为(178.24±105.80)、(105.88 ±27.09)、(81.18±48.67)和(50.35±47.64)Mpa,各指标A组显著高于C、D组(P＜0.05).组织学观察见A组骨延长处大量新生骨组织,骨小梁多为纵行网状排列.骨形态计量分析示A、B、C、D组新骨体积分别为72.35%±5.68%、67.58%±7.42%、49.63%±4.87%和38.87%±2.35%,A组新生骨生成量明显比D组多(P＜0.05). 结论 HA复合rhBMP-2修饰的BMSCs制备的组织工程骨可促进羊胫骨延长骨愈合.     

中枢神经损伤对大鼠股骨骨折愈合影响观察

摘要：[目的] 观察中枢神经损伤后大鼠股骨骨折愈合速度和骨痂量的变化，分析中枢神经损伤对大鼠股骨骨折愈合速度影响的机制。[方法] 54只雄性Wistar大鼠随机分成3组，（1）脑外伤合并股骨骨折组18只；（2）脊髓损伤合并股骨骨折18只；（3）单纯股骨骨折18只。术后7、14、28d分批处死动物，大体体积测量骨痂，X线片评分，并做HE染色比较。[结果] 脑外伤组，脊髓损伤组形成骨痂体积、X线片评分高于单纯股骨骨折组，差异有显著性（P〈0．05）。HE染色结果显示脑外伤组、脊髓损伤组骨折愈合速度较单纯骨折组加快。[结论] 中枢神经损伤后大鼠骨折愈合加速，骨痂量增多，中枢神经损伤后引起骨痂量和骨折愈合速度的改变，表明中枢神经损伤对骨折愈合有促进作用。     

仙灵骨葆胶囊对骨质疏松性骨折大鼠骨生长因子及骨折愈合的影响

目的 探讨仙灵骨葆胶囊对骨质疏松性骨折大鼠骨生长因子BMP-2、IGF-1表达及骨折愈合的影响。方法 48只雌性SD大鼠随机分为：假手术组、模型组、雌二醇组、仙灵骨葆组，12只/组，采用“双侧卵巢切除术+右侧股骨干骨折髓内固定术”构建骨质疏松性骨折大鼠模型，评估骨折愈合情况，检测股骨骨痂BMD、股骨骨生物力学指标和血清骨代谢相关指标，检测骨痂BMP-2、IGF-1蛋白表达。结果 模型组较假手术组骨折愈合评分、股骨痂BMD、股骨骨生物力学指标（最大载荷、最大应力、最大位移）、骨痂BMP-2和IGF-I阳性表达均显著降低（P<0.05），雌二醇组、仙灵骨葆组较模型组骨折愈合评分、股骨痂BMD、股骨骨生物力学指标、骨痂BMP-2和IGF-I阳性表达均显著升高（P<0.05），均以仙灵骨葆组最高。模型组较假手术组血清骨代谢指标（BGP、PICP、TRACP-5b）均显著升高（P<0.05），雌二醇组、仙灵骨葆组较模型组血清骨代谢指标均显著降低（P<0.05），以仙灵骨葆组最低。结论 仙灵骨葆胶囊可能通过介导提高骨质疏松性骨折大鼠骨生长因子BMP-2和IGF-1表达，改善骨代谢，加速骨痂形成，增加骨密度，提高骨生物力学，促进骨折愈合。     

中枢神经损伤大鼠骨痂中神经肽的表达

[目的]研究中枢神经损伤后大鼠股骨骨折骨痂中神经肽的表达。[方法]45只雄性Wistar大鼠随机分成3组，脑外伤合并股骨骨折组15只，脊髓损伤合并股骨骨折15只，单纯股骨骨折15只。术后4、7、14、21、28d分批处死动物，行神经肽免疫组织化学染色。[结果]脑外伤组，脊髓损伤组免疫组化染色结果显示骨折愈合过程中骨痂骨膜内、外层骨祖细胞、软骨细胞、成骨细胞内CGRP、SP阳性表达，与单纯骨折组相比，差异具有显著性。[结论]中枢神经损伤大鼠骨痂中神经肽有显著性改变，推测神经因素参与调节骨折愈合过程。     

降脂、抗凝药物预防激素所致股骨头骨细胞坏死、凋亡及对BcL-2表达的影响

目的 通过建立动物实验模型，探讨同时应用降脂、抗凝药物对激素性股骨头坏死的防治作用及病理机制。方法 选用大白鼠60只，体重约200g，随机分为A、B、C、D组，A、B、C组给予不同的药物，D组不给药，空白对照。实验8周处死动物，取股骨头作组织学、超微结构观察，并用TUNEL及免疫组化技术分别检测骨细胞凋亡和BcL-2表达情况。结果 ①光镜下B、C两组较A组股骨头骨小梁变细、稀疏、空骨陷窝明显增多，脂肪细胞增大，A组接近D组。②电镜下B、C两组细胞体积缩小，核固缩，染色质边聚较多见于A组，A组接近D组。③C组凋亡骨细胞多见于其它组，A组接近D组，A组BcL-2表达多于其它组。结论 大剂量应用激素同时给予降脂、抗凝药预防股骨头坏死的作用优于单纯应用降脂药组；激素性股骨头坏死是骨细胞坏死和凋亡共同作用的结果。     

Systemic recruitment of osteoblastic cells in fracture healing.

We hypothesise that following a bone fracture there is systemic recruitment of bone forming cells to a fracture site. A rabbit ulnar osteotomy model was adapted to trace the movement of osteogenic cells. Bone marrow mesenchymal stem cells from 41 NZW rabbits were isolated, culture-expanded and fluorescently labelled. The labelled cells were either re-implanted into the fracture gap (Group A); into a vein (Group B); or into a remote tibial bone marrow cavity 48 h after the osteotomy (Group C) or 4 weeks before the osteotomy was established (Group D), and a control group (Group E) had no labelled cells given. To quantify passive leakage of cells to an injury site, inert beads were also co-delivered in Group B. Samples of the fracture callus tissue and various organs were harvested at discrete sacrifice time-points to trace and quantify the labelled cells. At 3 weeks following osteotomy, the number of labelled cells identified in the callus of Group C, was significantly greater than following IV delivery, Group B, and there was no difference in the number of labelled cells in the callus tissues, between Groups C and A, indicating the labelled bone marrow cells were capable of migrating to the fracture sites from the remote bone marrow cavity. Significantly fewer inert beads than labelled cells were identified in Group B callus, suggesting some of the bone-forming cells were actively recruited and selectively chosen to the fracture site, rather than passively leaked into the circulation and to bone injury site. This investigation supports the hypothesis that some osteoblasts involved in fracture healing were systemically mobilised and recruited to the fracture from remote bone marrow sites.     

Manipulation of the anabolic and catabolic responses with BMP-2 and zoledronic acid in a rat femoral fracture model

Bone repair involves a complex set of regulated signaling pathways that control the formation of new bone matrix and the resorption of damaged bone matrix at the fracture site. It has been reported that the optimal time point for single-dose zoledronic acid (ZA) administration systemically increased the strength of bone morphogenetic protein (BMP)-7-mediated callus. However, its repair mechanism during bone fracture healing remains unknown. We aimed to investigate the synergic effect of recombinant human (rh) BMP-2 and ZA in a rat femoral fracture model. Fifty-eight rats were divided into 4 groups. Group I (n = 14) animals were implanted with a carrier alone. Group II (n = 15) animals were implanted with a carrier containing 1-μg rhBMP-2. Group III (n = 14) animals were implanted with a carrier and a subcutaneous systemic ZA injection 2 weeks after surgery. Group IV (n = 15) animals were implanted with a carrier containing 1-μg rhBMP-2 and ZA subcutaneous injection 2 weeks after surgery. The rats were euthanized after 6 weeks and their fractured femurs were explanted and assessed by manual palpation, radiographs, and high-resolution micro-computerized tomography (micro-CT) and were subjected to biomechanical and histological analysis. The fusion rates in Group IV (93.3%) were considerably higher than those in Groups I (28.6%), II (53.3%), and III (57.1%). Additionally, the radiographic scores of Group IV were higher than those in Groups I, II, and III. In micro-CT analysis, the tissue volume (TV) of the callus was higher in Group IV than in Groups I and II (p < 0.05). New bone volume (BV) and trabecular spacing (Tb.Sp) also showed essentially the same trend as that of TV. The ratio of BV to TV (BV/TV), the trabecular number (Tb.N), and the trabecular thickness (Tb.Th) was higher in Groups III and IV than in Groups I and II (p < 0.05). In biomechanical analysis, the ultimate loads at failure and stiffness in Groups III and IV were on average higher than those in Groups I and II (p < 0.05), while the energy absorption of Group IV was higher than those of Groups I and II (p < 0.05). The synergic effect of rhBMP-2 and ZA given systemically as a single dose at the optimal time was efficacious for fracture repair and significantly enhanced bone fusion. Our results suggest that this combination facilitates bone healing and has potential clinical application.     

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.     

Distraction osteogenesis enhanced by osteoblastlike cells and collagen gel

Femoral distraction was done in rats to determine whether the injection of osteoblastlike cells with collagen gel into the distracted callus was useful for new bone formation. The cells were obtained from the femoral marrow of Sprague-Dawley rats and cultured for approximately 3 weeks. These rats were divided into four groups. The rats in Group A received injections of physiologic saline, those in Group B received injections of collagen gel, those in Group C received injections of cells, and those in Group D received injections containing a mixture of cells and collagen gel. The distracted areas were harvested and evaluated by histologic analysis, radiography, three-point bending testing, and the weight of femoral ash. Histologic evaluation did not show an immunoreaction between the donor and recipient. Radiographs showed that Group D had the most callus, and the fracture strength in this group as determined by the three-point bending test was higher than in Group A at 2, 4, and 6 weeks after elongation was completed. Group D showed a significant difference in the ash weight of the distracted femurs at 2 weeks. The current study showed that osteoblastlike cells with collagen gel promoted new bone formation in the distracted gap, and shortened the consolidation period.     

骨形成蛋白2活性多肽体外定向诱导骨髓间充质干细胞向成骨方向分化的剂量依赖性研究

目的探讨自行合成的骨形成蛋白2(bone morphogenetic protein2,BMP-2)活性多肽对大鼠骨髓间充质干细胞(marrow mesenchymal stem cells,MSCs)体外定向诱导成骨的能力,评价BMP-2活性多肽的成骨诱导性及诱导成骨的剂量依赖性。方法取4周龄Wistar大鼠分离培养MSCs,传至第3代时改用条件培养基,培养基中分别加入浓度为300、200、100、50及0μg/ml的BMP-2活性多肽,并依次记为A、B、C、D和E组。细胞培养至5、10、15及20d,检测碱性磷酸酶(alkaline phosphatase,ALP)活性,测定钙含量,采用实时荧光定量PCR(fluorescent quantitativePCR,FQ-PCR)检测型胶原、骨桥蛋白(osteopontin,OPN)及骨钙素(osteocalcin,OCN)mRNA的表达,检测不同浓度BMP-2活性多肽体外诱导成骨的能力。结果倒置相差显微镜下观察,用条件培养基培养3～4d后,培养细胞由长梭形转变为短梭形或多角形。随条件培养基中BMP-2活性多肽浓度的增加,细胞发生成骨样改变的时间提前。ALP活性和钙含量检测显示,A组和B组较其他组增加明显,且差异有统计学意义(P<0.05),但A、B组间比较差异无统计学意义(P>0.05)。FQ-PCR检测发现不同浓度条件培养基培养14d后,型胶原、OPN和OCN mRNA在各组均有表达。Ct值表明其表达量的大小顺序为A、B、C、D组,E组无表达,A组和B组的Ct值明显大于C组和D组,差异有统计学意义(P<0.05),但A组和B组之间差异无统计学意义(P>0.05)。结论BMP-2活性多肽能有效地促进MSCs向成骨方向分化,其诱导作用存在剂量依赖关系,最佳诱导剂量为200μg/ml。     

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。