hBMP-2基因修饰自体BMSCs移植促进兔下颌骨牵张成骨新骨形成的X线分析

目的:探讨hBMP-2基因修饰自体BMSCs移植对兔下颌骨牵张成骨新骨形成的促进作用。方法:取新西兰白兔36只随机分为3组,每组12只。建立牵张成骨动物模型,在固定期第2天,实验组于牵张间隙注射200μl的BMP-2基因修饰的自体BMSCs(2×105个细胞)悬液;对照组注射200μl的自体BMSCs(2×105个细胞)悬液;空白组注射200μl生理盐水。分别于固定2、6周摄X线片观察骨质愈合、改建情况。结果:通过X线观察并经过灰度值统计软件分析,在固定期2周及6周实验组牵张区骨密度明显高于对照组和空白组(P0.01)。结论:BMP-2基因修饰的自体BMSCs移植能有效促进兔下颌骨牵张成骨新骨形成。     

引导组织再生膜促进兔下颌骨牵张成骨的实验研究

目的 探讨引导组织再生膜（GTRM）对兔下颌骨牵张成骨新骨形成的促进作用。方法 将20只新西兰大白兔随机分为2组,每组10只,建立兔下颌骨牵张成骨模型,A组单纯单侧下颌骨牵张成骨;B组将GTRM固定于牵张器内侧行单侧下颌骨牵张成骨。分别于固定期第2、6周时随机处死半数动物获取标本,通过X线、骨组织形态计量学比较2组牵张间隙内成骨效果。采用SPSS11.0软件包对数据进行两样本均数t检验。结果 通过X线及骨组织形态计量学检查并经过统计学分析发现,在固定期第2周和6周时,B组牵张区域内成骨质量显著好于A组（P<0.05）。结论 引导组织再生膜能有效促进兔下颌骨牵张成骨区域新骨的形成。     

Runt相关基因2修饰的骨髓间充质干细胞促进兔下颌骨牵张成骨的研究

目的探讨Runt相关基因2（Runx2）修饰的自体骨髓间充质干细胞（MSCs）促进兔下颌牵张成骨新骨形成的可行性。方法 将48只成年雄性新西兰大白兔随机分为3组：A组为Runx2重组质粒组,B组为不含Runx2重组质粒组,C组为生理盐水对照组。建立兔下颌牵张成骨模型;于牵张的第5天,A组兔牵张间隙内注射转染重组质粒adv-hRunx2-gfp的自体骨髓MSCs;B组注射转染adv-gfp质粒的自体骨髓MSCs;C组注射同体积生理盐水。在牵张结束后第8周处死所有动物,进行大体、影像学、组织学观察和三点弯曲测试。结果 CT平扫及组织学结果表明,A组牵张间隙内新骨形成和骨痂密度均明显高于B、C组;双能X线及三点弯曲测试表明,A组牵张间隙内新生骨组织密度、新生骨矿物质含量及最大载荷均高于B、C组（P<0.01）。结论 基于MSCs的Runx2体外基因治疗可有效地促进牵张成骨新骨形成,从而缩短固定期,为临床颅颌面骨缺损的重建提供一个极具价值的修复策略。     

局部应用β1转化生长因子对兔下颌牵张成骨的影响

目的：研究外源性β1转化生长因子对牵张成骨的作用。方法：将16口大耳白兔随机分为A、B两组，每组8只。在兔双侧下颌骨前部行骨切开术后用自行研制的牵张器延长双侧下颌骨6mm。从牵张开始第1天在A组动物牵张区每日注射TGF－β140ng，连续12d。不注射TGF－β1的B组动物用作对照。在牵张结束后第2和第4周分别处死A、B两组各4只动物，取下颌骨标本及牵张区新生骨痂进行X线和组织学检查。结果：注射TGF－β1的A组动物牵引间隙内新骨形成并不优于B组动物。相反，在第2周时还发现有较多的纤维组织和软骨形成。结论：导入外源性TGF－β1可能没有促进兔下颌牵张成骨的作用。     

丹参酮Ⅱa磺酸钠对兔下颌骨牵张成骨促进作用的研究

目的：探讨丹参酮Ⅱa磺酸钠注射液对兔下颌骨牵张成骨的作用。方法：将12只大耳白兔随机分为对照组和实验组,建立单侧兔下颌骨牵张成骨模型,实验组自牵张期开始每日注射丹参酮Ⅱa磺酸钠注射液3mL,并分别于固定期1周、4周、7周各处死2只对照兔和2只实验兔,对标本进行大体观察、X线观察、组织学观察和骨组织计量学观察。结果：实验组动物牵张间隙内新骨形成速度及质量均优于对照组,实验组成骨细胞活跃,骨小梁较成熟,单位面积内成骨细胞个数及骨小梁面积百分比均高于对照组。结论：丹参酮Ⅱa磺酸钠对兔下颌骨牵张成骨过程具有促进作用。     

BMP-2及Smad1在兔下颌骨垂直牵张中的表达和意义

目的：观察BMP-2及其信号传导分子Smad1在兔下颌骨垂直牵张后新骨组织中的分布和表达,探讨其在新骨形成中的作用。方法：36只兔子随机分成6组,用自制的种植型牵张器对兔下颌骨垂直牵张,以1mm/天的速度牵张4天,分别于牵后第1天、1周、2周、4周、6周取材,6只作为正常对照组。用ABC免疫组织化学法,检测兔下颌骨垂直牵张过程中不同时期BMP-2及Smad1的表达与分布。结果：免疫组化观察,牵张结束后BMP-2及Smad1阳性信号主要在间充质细胞和新生骨小梁边缘的成骨细胞表达,表达高峰在牵张后1周,以后逐渐下降,牵张后4周在骨细胞微弱表达,6周时无表达。正常对照组无表达。结论：BMP-2及Smad1/5参与兔下颌骨垂直牵张过程,并在牵张成骨的早期起作用;Smad1/5可能介导了BMP-2在牵张成骨中的信号传导。     

bFGF基因修饰的BMSCs促进大鼠下颌牵引成骨的研究

目的：探讨碱性成纤维生长因子（bFGF）基因修饰的自体骨髓间充质干细胞（BMSCs）促进大鼠下颌牵引成骨的作用。方法：36只雄性SD大鼠,随机分为实验组和对照组,同时对两组大鼠右下颌骨进行牵引成骨,牵引期最后1 d,实验组大鼠牵引间隙内注射转染重组质粒pcDNA-bFGF的BMSCs,对照组注射转染pcDNA空质粒的BMSCs。分别于固定期第2,4,8周分3批处死,并进行放射学检查、组织学检查及骨密度检测。结果：两组牵引间隙内均有新骨形成。各时间点实验组牵引间隙内新骨的形成和骨质密度均较对照组高（P〈0.05）。结论：bFGF基因修饰的BMSCs可有效促进DO新骨形成,为颌面部骨缺损的重建提供了一个新的修复方法。     

腺病毒介导的人骨形成蛋白2基因转染促进下颌牵张成骨的实验研究

目的探讨局部应用腺病毒介导的入骨形成蛋白2（adenovirus vectors containing human bone morphogenetic proteins 2, Ad-hBMP-2）对兔下颌骨牵张成骨的影响。方法24只新西兰大白兔随机分为实验组（12只）和对照组（12只），并建立下颌骨双侧牵张成骨模型。经过5d潜伏期后，以0．5mm／12h的速度牵张7d。固定期第1天，在实验组骨牵张区注射0．2ml滴度为10^12pfu／L的Ad-hBMP2，对照组骨牵张区注射0．2ml滴度为10^12pfu／L的腺病毒介导的增强型绿色荧光蛋白。在固定期第7、14、28天对下颌骨牵张区进行骨密度及新生骨量比较。结果Ad-hBMP-2治疗组牵张区骨密度及新生骨量明显高于对照组。结论腺病毒介导的人骨形成蛋白2具有促进兔下颌牵张成骨的作用。     

成纤维细胞生长因子对兔下颌牵张成骨的影响

目的 研究局部应用碱性成纤维细胞生长因子（bFGF)对兔下颌牵引张成骨的影响。方法 成年大耳白兔8只，随机分为A、B两组，每组4只。用自行研制的牵张器延长双侧下颌骨6mm，用bFGF（20ng/ml）注入A组动物的牵张区，不注射bFGF的B组动物作为对照。在牵张结束后4周处死所有动物，取双侧下颌骨标本进行X线和组织学检查。结果 组织学分析结果显示，局部给予bFGF的A组动物下颌牵张后新骨生成速度和数量优于B组动物。结论 外源性导入碱性成纤维细胞生长因了可能有促进下颌牵引张成骨的作用。     

放疗对兔下颌骨牵张成骨骨再生的影响

目的：探讨兔下颌骨放疗后牵张成骨（DO）的可行性及其骨再生的特点。方法：将12只成年新西兰大白兔随机分为放疗组和未放疗组，每组6只：放疗组用^60Co机照射大白兔下颌骨，5．4Gy／次，隔天1次，共5次，总剂量为27Gy。3个月后，在2组动物下颌骨的双侧截骨处安装牵张器，经5天延迟期开始牵张，速率为1mm／d，0．5mm／次，每天2次．连续7d，共延长下颌骨7mm．固定期的第4、6周拍摄下颌骨侧位X线片，取双侧新生骨痂行组织学和扫描电镜检查，观察其成骨特征。结果：X线片显示，2组动物同一固定时间牵张间隙内透射密度无明显差异；组织学观察显示，牵张区以膜内成骨为主，但放疗组有更多的软骨形成，放疗组较未放疗组新骨骨小梁细小、稀疏；扫描电镜示同定第6周时，放疗组新骨不如未放疗组致密、成熟。结论：在兔下颌骨放射损伤区行DO是可行的，但成骨质量较差，成骨方式以膜内成骨为主，放射线照射可促进软骨成骨。     

SDF-1α复合Pluronic F-127对兔上颌窦底提升术成骨影响的实验研究

目的 :通过制备基质细胞衍生因子(SDF1-α)的Pluronic F-127复合凝胶材料,观察这种复合材料在体外对兔骨髓间充质干细胞(BMSCs)的矿化及成血管作用和复合凝胶材料在兔上颌窦外提升术中的成骨及成血管效果。方法:将兔骨髓间充质干细胞、50ng/ml SDF1-α和质量百分比浓度0.1%的Pluronic F-127复合培养,MTT法检测其细胞毒性,碱性磷酸酶活性测定及茜素红染色。将18只新西兰大白兔双侧进行上颌窦外提升术,双侧植入不同的复合材料(共3种)并进行对照。材料分别为:β-磷酸三钙(β-TCP),生理盐水及SDF-1α复合Pluronic F-127凝胶。于术后4、8、12周处死,X线片观察双侧上颌窦骨质形成情况,行HE染色、CD31+、CD34+、BMP-2及VEGF免疫组织化学染色观察成骨及成血管作用,并进行统计学分析。结果:体外实验采用MTT法证实了0.1%w/w Pluronic F-127无细胞毒性(P>0.05)。BMSCs-SDF1α复合凝胶碱性磷酸酶染色下,细胞质可见大量碱性磷酸酶染色沉淀,其余3组未见沉淀。BMSCs-SDF1α复合凝胶、BMSCs-单纯凝胶、单纯BMSCs在成骨诱导培养条件下均产生矿化结节,非成骨诱导组未见矿化结节形成。体内实验,X线片结果显示BMSCs-SDF1-α复合凝胶及β-TCP充填侧均有阻射影,生理盐水组未见明显阻射影;HE染色结果显示BMSCs-SDF1-α复合凝胶成骨效果与β-TCP成骨效果明显,生理盐水组未见骨质形成,统计学分析表明BMSCs-SDF1-α复合凝胶成血管作用较β-TCP及生理盐水组明显增加。结论:SDF-1α复合Pluronic F-127凝胶能够成功诱导BMSCs分化为成骨细胞,无细胞毒性,在体外及兔上颌窦内成骨、成血管效果明显。     

Expression of bone morphogenetic proteins during mandibular distraction osteogenesis in rabbits.

PURPOSE: We examined the expression pattern of bone morphogenetic proteins (BMPs) during mandibular distraction osteogenesis in rabbits and also investigated the mechanism of membranous bone distraction. MATERIALS AND METHODS: Twenty-three rabbits underwent mandibular distraction (protocol; no latency period, a 1-week distraction at 0.5 mm/d, and a 2-week consolidation period). Samples were collected at 3, 5, and 7 days of distraction and at 1-week and 2-week consolidation. We prepared undecalcified fresh-frozen sections and immunohistochemically evaluated the expression of BMPs 2 through 8. RESULTS: Both endochondral ossification and intramembranous ossification were observed. The expression of BMPs 2, 4, 5, and 6 was observed continuously from the beginning of distraction. BMP-7 was expressed weakly. The expression of BMP-3 was not observed conspicuously during distraction but was strongly expressed at 1- and 2-week consolidation. CONCLUSION: The expression pattern of BMPs during membranous bone distraction was similar to that during long bone distraction, but it differed from the expression pattern of long bone distraction in that the expression of BMPs was maintained for 2 weeks after the completion of distraction.     

rhBMP载体系统复合骨髓基质干细胞修复犬颌骨缺损

目的 利用rhBMP载体系统与骨髓基质干细胞复合，体内构建组织工程骨，修复颌骨缺损的效能评价。方法将可吸收性聚乳酸（PLA）、重组人骨形成蛋白（rhBMP）以一定方式复合，种植体外培养扩增的犬骨髓基质干细胞（BMSc）后，植入颌骨缺损区。采用放射学、形态学、碱性磷酸酶（ALP）检测等方法对其成骨效应进行研究。结果A组实验侧具有高效的骨诱导活性，其成骨量显著高于对照组B、C、D及空白对照组。BC试验侧同样具有高效的骨诱导活性，其成骨量也显著高于空白对照组（P〈0．01）。结论rhBMP载体系统复合骨髓基质干细胞，具有高效的骨诱导活性，能修复颌骨缺损。     

Similarities and differences between porcine mandibular and limb bone marrow mesenchymal stem cells

ObjectiveResearch has shown promise of using bone marrow mesenchymal stem cells (BMSCs) for craniofacial bone regeneration; yet little is known about the differences of BMSCs from limb and craniofacial bones. This study compared pig mandibular and tibia BMSCs for their in vitro proliferation, osteogenic differentiation properties and gene expression.DesignBone marrow was aspirated from the tibia and mandible of 3–4 month-old pigs (n = 4), followed by BMSC isolation, culture-expansion and characterization by flow cytometry. Proliferation rates were assessed using population doubling times. Osteogenic differentiation was evaluated by alkaline phosphatase activity. Affymetrix porcine microarray was used to compare gene expressions of tibial and mandibular BMSCs, followed by real-time RT-PCR evaluation of certain genes.ResultsOur results showed that BMSCs from both locations expressed MSC markers but not hematopoietic markers. The proliferation and osteogenic differentiation potential of mandibular BMSCs were significantly stronger than those of tibial BMSCs. Microarray analysis identified 404 highly abundant genes, out of which 334 genes were matched between the two locations and annotated into the same functional groups including osteogenesis and angiogenesis, while 70 genes were mismatched and annotated into different functional groups. In addition, 48 genes were differentially expressed by at least 1.5-fold difference between the two locations, including higher expression of cranial neural crest-related gene BMP-4 in mandibular BMSCs, which was confirmed by real-time RT-PCR.ConclusionsAltogether, these data indicate that despite strong similarities in gene expression between mandibular and tibial BMSCs, mandibular BMSCs express some genes differently than tibial BMSCs and have a phenotypic profile that may make them advantageous for craniofacial bone regeneration.     

骨形态发生蛋白-2与碱性成纤维细胞生长因子在异位和原位成骨中的作用

目的通过骨髓基质细胞（BMSCs）的体外增殖和分化、异位成骨和原位成骨实验来观察骨形态发生蛋白-2（BMP-2）和碱性成纤维细胞生长因子（bFGF）在成骨过程中的作用。方法分别用含BMP-2、bFGF和BMP-2+bFGF的培养液体外培养Beagle犬的BMSCs,通过甲基噻唑基四唑（MTT）比色法测定细胞增殖水平,通过测定碱性磷酸酶（ALP）活性观察细胞的分化情况。将BMSCs与多孔磷酸钙（CPC）分别在含BMP-2、bFGF和BMP-2+bFGF的培养液中复合培养,制成复合材料,一部分植入裸鼠皮下,观察异位成骨情况,另一部分植入Beagle犬的种植体周围骨缺损区,经过荧光标记观察原位成骨情况。结果含有BMP-2+bFGF的培养液促进BMSCs增殖和分化的能力最强。异位成骨情况：BMP-2+bFGF组的成骨量较其他组明显增加,其新骨形成百分比为48.79%±11.31%,高于单一BMP-2组（30.71%±10.85%）和bFGF组（27.33%±9.67%）以及对照组（10.65%±6.05%）。原位成骨术后12周,BMP-2+bFGF组的矿化沉积率高于其他组,其差异有统计学意义（P<0.01）。结论在促进成骨方面,BMP-2和bFGF共同作用优于单一因子。     

Effect of distraction rates on expression of bone morphogenetic proteins in rabbit mandibular distraction osteogenesis.

BACKGROUND: The expression of bone morphogenetic proteins can be regarded as an indicator for biological interaction in distraction osteogenesis. It was hypothesized that different distraction rates may produce different patterns of BMP expression. MATERIAL AND METHODS: This study compared the expression of BMP-2, -4 and -7 at routine (0.9mm once daily) and rapid (2.7mm once daily) distraction rates using the rabbit mandibular lengthening model. Mandibular samples were harvested at days 1, 7 and 14 of consolidation. RESULTS: The expression of BMP-2 and BMP-4 was found throughout the experiment, their expression intensity and location being essentially similar, whereas BMP-7 had not been expressed. Expression of BMP-2 and -4 was more intense at the routine distraction rate than at the rapid distraction rate. CONCLUSIONS: BMP-7 had no expression during the consolidation period of mandibular distraction. The change in the mechanical environment created by different distraction rates led to different expression of BMP-2 and -4. In the early consolidation stage the biological environment for bone formation created by distraction at a routine rate was superior to that created by the rapid distraction rate.