人骨髓间充质干细胞定向诱导分化为成骨细胞及其鉴定

目的探讨将成人骨髓间充质干细胞(MSCs)定向诱导分化为成骨细胞的方法,并对所诱导细胞的成骨特性进行鉴定. 方法分离人骨髓,梯度离心并结合全骨髓法进行培养,培养基中添加成骨诱导剂地塞米松、β-甘油磷酸钠及抗坏血酸.贴壁细胞传代,取第3代细胞鉴定其成骨特性;在倒置相差显微镜下观察细胞形态,钙-钴法染色检测碱性磷酸酶(ALP)表达,免疫组织化学检测Ⅰ型胶原表达,原位杂交检测骨连接素(ON)、骨桥素(OP)表达,Von Kossa 染色检测钙结节形成. 结果第3代人MSCs呈典型的成骨细胞形态,可连续传代10次;ALP染色阳性率达85%;Ⅰ型胶原、ON和OP表达阳性;Von Kossa 染色可见钙结节形成. 结论成功地将人MSCs诱导分化为成骨细胞,所诱导的细胞具有典型的成骨细胞特性.     

骨形成蛋白-2基因转染人骨髓间质干细胞诱导异位成骨的实验研究

目的 评价腺病毒介导的人骨形成蛋白—2基因(Adv—hBMP—2)转染人骨髓间质干细胞(MSCs)的诱导成骨能力。方法 从人骨髓中分离培养获取MSCs，分为三组：①Adv—hBMP—2转染细胞组；②Adv—βgal转染细胞组；③未转染细胞组。分别于体外行western immunoblot试验、碱性磷酸酶(ALP)和Von Kossa染色、ALP定量测定，以及裸鼠肌内诱导成骨试验。共9只裸鼠，双例股后肌群内注射，每组6例。结果 Adv—hBMP—2组MSCs可分泌BMP—2蛋白；转染后第9天ALP染色多数细胞为阳性，其它两组ALP染色阳性细胞少见；ALP活性第3天开始升高，12天达高峰为14．76单位，与其它两组比较有统计学意义(P＜0．05)；于第21天后出现钙结节，而其它两组未见。裸鼠肌内注射后4周Adv—hBMP—2组X线片均有异位成骨，Adv—βgal转染细胞组未见明显成骨，未转染细胞组仅有少量骨形成。组织学检查发现：Adv—hBMP—2组也可见典型的板层骨和骨髓腔形成，Adv—βgal组主要为纤维组织，未转染组也可见散在骨小粱形成。结论 Adv—hBMP—2基因转染可诱导人骨髓问质干细胞成骨。     

重组人骨形成蛋白2诱导的骨膜细胞构建组织工程骨的实验研究

目的通过组织工程技术方法，研究细胞-材料复合体体内骨再生的能力。方法采用材料学自组装技术的原理．以Ⅰ型胶原蛋白为分子模板．引导钙磷盐在液相中的矿化，制备具有天然骨基质层状结构的羟基磷灰石／胶原复合材料，并以热致分相法制备羟基磷灰石／胶原-聚乳酸[hydroxyapatite／collagen—poly-(L—lactic acid)，HAC—PLA]复合三维多孔框架，与重组人骨形成蛋白2(recombinant human bone morphogenetic protein2，rhBMP-2)诱导分化后的兔骨膜细胞构建组织工程骨，进行体外电镜及组织学观察。将3月龄裸鼠18只，分为3组，每组6只。A组皮下注射经rhBMP-2处理后的骨膜细胞悬液，B组植入未复合细胞的HAC—PLA，C组植入rhBMP-2处理后的细胞-材料复合体。术后8周取材行组织学观察，C组与B组及A组进行比较。结果三维多孔HAC—PLA框架材料孔隙率大于90％，孔径为50～300μm。骨膜细胞经500ng／ml的rhBMP-2处理后，与改善亲水性后的HAC—PLA三维多孔框架复合，构建组织工程骨。电镜显示接种的细胞能在此组织工程骨内部生长增殖，术后8周C组有新骨形成，A组注射部位表面平整，无新生物，B组为纤维组织，无骨及软骨形成。结论所构建的组织工程骨有望成为骨创伤修复治疗一种新的技术。     

双相陶瓷生物活性骨的制备及其细胞相容性研究

[目的]制备一种双相陶瓷牛物活性骨,并了解其细胞相容性.[方法]分离培养兔骨髓基质干细胞(mesenchymalstemcells,MSCs),将浓度为1×106/ml目的第3代MSCs接种于复合有Ⅰ型胶原、骨形态发生蛋白(bone morphogenetk protein,BMP)和碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)目的双相陶瓷生物骨(bniphask ceramic bioiogic bone,BCBB)上,联合培养,用倒置差显微镜、扫描电子显微镜观察,测定光密度(OD)值,了解双褶陶瓷牛物活性骨BCBB/BMP/bFGF目的细胞相容性.[结果]BCBB孔内充满Ⅰ型胶原、BMP及bFGF.MSCs在双相陶瓷生物活性骨BCBB/BMP/bFGF上黏附生长,第6 d时细胞在材料表面形成致密细胞层,增殖达稳定状态.[结论]该研究制备目的双相陶瓷生物活性骨BCBB/BMP/bFGF具有良好目的细胞相容性.     

转染人骨形成蛋白-7基因的骨髓基质细胞异位成骨实验研究

目的 观察转染人骨形成蛋白-7（Bone morphogenetic protein-7,BMP-7）基因的骨髓基质细胞（Bone marrow stromal cells,BMSCs）与胶原膜（BME-10X）复合培养后的异位成骨能力。方法将hBMP-7基因转染Beagle犬BMSC,与胶原膜复合培养后,植入裸鼠皮下,8周后取材,HE、Mallory染色及Ⅰ型胶原免疫组化染色,观察其异位成骨能力。结果各组均有不同程度Ⅰ型胶原的表达,转染组和未转染组显著高于单纯膜组,转染组又显著高于未转染组（P〈0．05）。结论转染了hBMP-7基因的BMSCs具有更强的异位成骨能力,有望成为牙周组织工程理想的种子细胞,     

纤维蛋白胶复合骨形成蛋白的注射型骨修复材料对兔骨髓基质细胞增殖和分化的影响

目的观察以纤维蛋白胶（fibrin sealant，FS）为载体复合骨形成蛋白（bone morphogenetic protein，BMP）的注射型骨修复材料对体外培养的兔骨髓基质细胞（marrow stromal cells，MSCs）增殖和分化的影响，为其临床应用提供实验依据。方法取3日龄新西兰兔骨髓进行培养传代，采用第3代细胞进行研究。实验分为3组，实验组：以含1μg／ml rhBMP-2注射型FS培养细胞；FS对照组：以单纯FS培养细胞；空白对照组：不作任何处理。采用细胞培养、组织化学及电镜等方法对各组兔MSCs的增殖、贴壁率、碱性磷酸酶（alkaline phosphatase，ALP）活性及染色、Ⅰ型胶原表达、超微结构及细胞在材料中的生长情况进行观察。结果各组细胞促增殖作用由强到弱依次是：FS对照组→实验组→空白对照组，各组间差异均有统计学意义（P〈0.05）；对细胞贴壁率的影响总体由强到弱依次是：FS对照组→实验组→空白对照组，各组间差异均有统计学意义（P〈0.05）。各组ALP活性由强到弱依次是：实验组→FS对照组→空白对照组，各组间差异均有统计学意义（P〈0.05）；各组细胞的Ⅰ型胶原表达水平由高到低依次是：实验组→FS对照组→空白对照组，差异均有统计学意义（P〈0.05）。扫描电镜观察，材料表面粗糙，有微孔存在，FS对照组、实验组细胞与材料融合生长。透射电镜观察：实验组细胞向成骨细胞分化程度高，但细胞增殖活性较FS对照组稍差；FS对照组细胞向成骨细胞分化的程度较实验组低，细胞增殖活性较好；空白对照组的细胞增殖活性较差，胞外基质少。结论以FS为载体复合BMP的注射型骨修复材料可显著提高MSCs向成骨细胞方向的分化水平，但对MSCs促增殖作用不明显。     

人骨形成蛋白7基因修饰的兔骨髓基质干细胞异位成骨实验

[目的]通过人骨形成蛋白7(human bone morphogenetic protein 7,BMP7)基因修饰的兔骨髓基质干细胞(m esenchymal stem cells,MSCs)与聚乳酸-羟基乙酸(polycleclide-co-plycoclide,PLGA)支架复合,进行自体皮下异位成骨实验,探索BMP-7基因治疗与组织工程技术相结合的可行性。[方法]PLGA与腺病毒Ad-BMP-7感染后的兔MSCs共培养,电镜观察了解细胞在PLGA上黏附、生长和合成分泌骨基质成分的情况;将转染基因的细胞和未转染基因的细胞分别与PLGA支架复合,构建组织工程化骨并植入体内,通过组织观察新骨形成情况。[结果]制备得到的PLGA为疏松多孔的网格样结构,具有一定的韧性和强度。将腺病毒感染的骨髓基质干细胞接种于修剪成一定形状的PLGA上,经电镜观察可见细胞贴附于材料表面和孔隙内并增殖。异位成骨实验证实空白对照PLGA孔隙内无新骨形成,对照细胞组内有部分新生骨形成,而Ad-BMP-7实验组形成的新生骨组织面积更大(P<0.05)。[结论]应用hBMP-7基因修饰的MSCs作为骨组织工程的种子细胞,可望取得更强的成骨能力。     

复合骨形态发生蛋白(BMP)的微小颗粒骨修复兔桡骨缺损的实验研究

目的探讨自体或深低温冷冻同种异体微小颗粒骨复合胶原、骨形态发生蛋白(BMP)修复节段性兔桡骨缺损的效果。方法将兔自体或同种异体骨研磨成微小颗粒，分别与BMP及Ⅰ型胶原复合，并采用兔桡骨干1．5cm缺损的动物模型，通过X线、组织学、骨密度、生物力学等检测手段，与自体微小颗粒骨复合胶原修复节段性骨缺损的疗效比较。结果自体或深低温冷冻同种异体微小颗粒骨复合BMP胶原比自体微小颗粒骨复合胶原成骨效果优良，其中复合BMP组在8周即可使骨缺损修复，髓腔通畅，在骨缺损修复各时期，其成骨速度及成骨量均好于未复合BMP组。结论自体或深低温冷冻同种异体微小颗粒骨复合胶原BMP均可有效地修复节段性骨缺损，两种方法促进新骨形成无明显差异，异体微小颗粒骨复合胶原BMP是良好的骨缺损修复材料。     

脂肪干细胞/Ⅰ型胶原凝胶/聚乳酸聚乙醇酸-β-磷酸三钙骨组织工程复合体的构建及其异位成骨研究

目的 构建基于脂肪干细胞、Ⅰ型胶原凝胶以及聚乳酸聚乙醇酸-β-磷酸三钙支架(PLGA-β-TCP)的骨组织工程复合体并对其异位成骨进行研究.方法 设计构建脂肪干细胞-Ⅰ型胶原凝胶/PLGA-β-TCP复合体(A组),同时设立单纯细胞/PLGA-β-TCP材料复合体(B组)、单纯Ⅰ型胶原凝胶/PLGA-β-TCP复合体(C组)以及单纯PLGA-β-TCP支架材料(D组)作为对照.扫描电镜、相差显微镜观察细胞材料复合情况并对脂肪干细胞增殖以及成骨分化进行分析.体外成骨诱导培养2周后移植于自体股部肌袋,8周后取出,依次行放射学、组织学定性及半定量分析.结果 (1)体外成骨诱导2周,A组细胞增殖率慢于B组(P<0.05,n=4),但其细胞总数远高于后者(P<0.01,n=4).A组碱性磷酸酶(ALPase)活性、细胞外基质矿化程度均显著高于B组(P<0.01,n=4).A组细胞悬浮于Ⅰ型胶原凝胶并均匀分布于材料孔隙中而B组细胞仅见黏附于材料表面.(2)移植8周,X线片示A组高密度钙化影形成,B、C、D组未见有阳性结果.A组材料孔隙中均匀充满新生骨组织,可观察到骨小梁样结构.B组仅在少数孔隙中有类骨组织形成,同时伴有结缔组织长入.A组新生骨面积百分比显著高于B组(P<0.01,n=4).结论 通过应用Ⅰ型胶原凝胶来实现脂肪干细胞与PLGA-β-TCP多孔支架材料的均匀复合,能够有效促进脂肪干细胞在材料孔隙中的成骨分化及均质骨组织形成.     

外源性重组人骨形态发生蛋白-2应用于兔腰椎植骨融合中细胞增殖的实验研究

目的 探讨外源性重组人骨形态发生蛋白-2(rhBMP-2)应用于兔腰椎后路横突植骨融合中的促成骨效应细胞增殖作用及其成骨机制. 方法 45只新西兰大白兔随机分为三组(n=15),建立腰椎后路横突间植骨融合模型,分别植入rhBMP-2/异体骨复合骨条(复合骨组)、自体髂骨条(自体骨组)、单纯异体髂骨条(异体骨组).用流式细胞仪检测2、7、14、28、35 d具有成骨效应的骨髓基质细胞(MSCs)、成骨细胞、血管内皮细胞的增殖量. 结果复合骨组MSCs增殖量在术后2、7、35 d均比自体骨组和异体骨组高,差异均有统计学意义(P<0.05).复合骨组成骨细胞增殖量除在术后2 d高于自体骨组,差异有统计学意义(P=0.028)外,在其他时间点差异均无统计学意义(P>0.05),但复合骨组成骨细胞增殖量在术后2、7、14、28、35 d时均高于异体骨组,差异有统计学意义(P<0.05).复合骨组血管内皮细胞增殖量在术后2、7、28 d均高于自体骨组和异体骨组,差异有统计学意义(P<0.05).结论 在脊柱融合的不同时间段,外源性rhBMP-2能有效地促进MSCs、成骨细胞、血管内皮细胞增殖.     

构建组织工程骨修复兔颅骨极限缺损的实验研究

目的观察以胶原缓释重组人骨形成蛋白2(recom b inan t hum an bone m orphogenetic prote in 2,rhBM P-2)复合骨髓间充质干细胞(m arrow m esenchym a l stem ce lls,M SC s)及珊瑚构建的组织工程骨修复兔颅骨极限缺损的能力。方法新西兰大白兔40只,制备颅骨极限缺损,按植入的修复物不同随机分为5组,每组8只。Ⅰ组:自体髂骨,为阳性对照组;Ⅱ组:珊瑚,为阴性对照组;Ⅲ组:rhBM P-2+珊瑚;Ⅳ组:胶原+rhBM P-2+珊瑚;Ⅴ组:M SC s+胶原+rhBM P-2+珊瑚。将其分别植入兔颅骨极限缺损处,术后8、16周行大体观察、X线片、HE染色及M asson三色染色法观察比较骨缺损修复的情况。结果术后Ⅴ组材料与Ⅰ组修复颅骨极限缺损的效果相近,缺损区大体标本可见骨样组织充填,硬度与周边骨质相近,并与周边骨质形成明显骨融合;X线阻射程度高,16周时达80.45%±2.52%;组织学观察为板层状结构的新骨组织,空白孔隙区较少。Ⅳ组修复效果次之,Ⅲ组材料成骨能力较弱,Ⅱ组大部为半透明的纤维薄膜,缺损区界限清晰。结论胶原是rhBM P-2适宜的缓释载体,胶原及M SC s对促进复合支架材料修复骨缺损有重要意义。以M SC s+胶原+rhBM P-2+珊瑚构建的组织工程骨可成为一种良好的骨缺损修复材料。     

煅烧骨与诱导的MSCs复合的实验研究

目的：观察大鼠骨髓基质干细胞（MSCs）与经胶原涂层处理的煅烧骨（CCB）复合行体外培养时贴附、增殖能力的变化,探讨构建组织工程骨体外培养的最适时间。方法：制备鼠尾胶原（I型胶原）,无菌条件下,将煅烧骨置于胶原液中,2h后取出骨块,置滤网中将孔内外的胶原溶液沥干。体外培养的大鼠MSCs经矿化液诱导向成骨细胞分化。将分化后的成骨细胞滴加到涂胶原的煅烧骨块上,同时以未涂胶原的煅烧骨块为对照组。3天,7天时取材,扫描电镜下观察实验组与对照组成骨细胞在煅烧骨表面的贴附数量及形态,用细胞计数法测定细胞在两组材料的生长曲线,组织化学方法检测成骨细胞的ALP活性。结果：扫描电镜发现实验组成骨细胞的贴附数量显著高于对照组（P〈0．01）。细胞计数认为实验组细胞在材料上粘附数量多,增殖速度快。实验组ALP活性明显高于对照组（P〈0．01）。结论：胶原修饰的煅烧骨具有良好的组织相容性,能明显提高成骨细胞的贴附,并且促进成骨细胞活性维持,体外培养7～8天时,材料上的细胞数达到最大,应尽快植入体内。     

Effect of regional gene therapy with bone morphogenetic protein-2-producing bone marrow cells on spinal fusion in rats

BACKGROUND: Bone morphogenetic proteins (BMPs) are now being used as bone-graft substitutes to enhance spinal fusion. However, the large doses of BMP required to induce a spinal fusion in humans suggests that the delivery of these proteins should be improved. We used ex vivo adenoviral gene transfer to create BMP-2-producing bone marrow cells, and these autologous cells were found to induce a posterolateral fusion of the spine in syngeneic rats. METHODS: Intertransverse spinal arthrodesis (L4 and L5) was attempted in ten groups of Lewis rats with 5 x 10 (6) BMP-2-producing rat bone marrow cells (Ad-BMP-2 cells), created through adenoviral gene transfer with guanidine hydrochloride-extracted demineralized bone matrix as a carrier (Group I); 5 x 10 (6) Ad-BMP-2 cells on a collagen sponge carrier (Group II); 10 micro g of recombinant BMP-2 (rhBMP-2) in a guanidine hydrochloride-extracted demineralized bone matrix carrier (Group III); 10 micro g of rhBMP-2 in a collagen sponge carrier (Group IV); autogenous iliac crest bone-grafting (Group V); 5 x 10 (6) beta-galactosidase-producing rat bone marrow cells, created through adenoviral gene transfer with guanidine hydrochloride-extracted demineralized bone matrix as a carrier (Group VI); decortication of the transverse processes alone (Group VII); 5 x 10 (6) uninfected rat bone marrow cells with a guanidine hydrochloride-extracted demineralized bone matrix carrier (Group VIII); guanidine hydrochloride-extracted demineralized bone matrix only (Group IX); or a collagen sponge alone (Group X). Each specimen underwent plain radiography, manual palpation, and histological analysis. RESULTS: All spines in Groups I and II (BMP-2-producing bone marrow cells) and all spines in Groups III and IV were fused at four weeks postoperatively. In contrast, none of the spines in the other groups had fused at a minimum of eight weeks after implantation. Histological analysis of the specimens revealed that the spines that had received BMP-2-producing bone marrow cells (Groups I and II) were filled with coarse trabecular bone postoperatively, whereas those that had received rhBMP-2 (Groups III and IV) were filled with thin, lace-like trabecular bone. All of the other spines, including those that had been treated with autogenous iliac crest bone-grafting (Group V), produced little or no new bone. CONCLUSION: BMP-2-producing bone marrow cells, created by adenoviral gene transfer, produce sufficient BMP to induce an intertransverse fusion in the rat spine model.     

骨髓基质细胞与关节软骨细胞生物学特性的比较研究

目的观察兔骨髓基质细胞(MSCs)诱导和基因修饰后的主要生物学特性,并与关节软骨细胞进行比较. 方法抽取成年雄性新西兰大白兔髂骨骨髓,密度梯度离心获得骨髓基质细胞,培养传至第5代,按处理方法分为常规培养液组(A组)、条件培养液组(B组)及重组缺陷型腺病毒携带肝细胞生长因子cDNA转染组(C组).条件培养液为常规培养液中含转化生长因子-β1(10 ng/ml)、碱性成纤维细胞生长因子(25 ng/ml)和地塞米松(10-7 mol/L).切取兔膝关节软骨,3 mg/ml Ⅱ型胶原酶消化传代培养至第3代(D组).观察原代MSCs及第5代MSCs(体外培养8～10周后)细胞形态,对第5代MSCs及第3代软骨细胞进行Ⅰ、Ⅱ型胶原免疫组织化学染色,MTT法检测细胞增殖情况.阿利新蓝法检测细胞培养上清液中糖胺多糖(GAG)含量.提取各组培养细胞总RNA,RT-PCR检测Ⅰ、Ⅱ型胶原表达. 结果原代MSCs为短梭形、簇状生长,传代细胞呈长梭形、旋涡样生长.A组细胞爬片Ⅰ型胶原免疫组织化学染色阳性,Ⅱ型胶原免疫组织化学染色阴性,GAG含量低,与D组比较,差异有统计学意义(P＜0.05).B组细胞爬片Ⅰ、Ⅱ型胶原免疫组织化学染色阳性,GAG含量升高,与D组比较差异无统计学意义(P＞0.05);C组转染后第4天增殖率降低,与A组比较差异有统计学意义(P＜0.05),其余时间点各组间无统计学意义(P＞0.05).RT-PCR表明A、B、C组均表达Ⅰ型胶原,B、D组可表达Ⅱ型胶原,C组有较弱的Ⅱ型胶原表达. 结论 MSCs体外培养过程中自然转归趋向于成骨.传代后经向成软骨方向诱导,具有向软骨分化的能力;体外传代培养的MSCs具有干细胞自我增殖和定向分化的特性,可作为靶细胞接受外源目的基因转染并能有效表达.     

The use of rhBMP-2 in interbody fusion cages. Definitive evidence of osteoinduction in humans: a preliminary report

STUDY DESIGN: A prospective randomized controlled human clinical pilot trial. OBJECTIVES: To determine the feasibility of using rhBMP-2/collagen as a substitute for autogenous bone graft inside interbody fusion cages to achieve arthrodesis in humans. SUMMARY OF BACKGROUND DATA: Preclinical studies have shown rhBMP-2 to be an effective substitute for autogenous bone graft, but there are no studies to date documenting such efficacy for human spine fusion. METHODS: Fourteen patients with single-level lumbar degenerative disc disease refractory to nonoperative management were randomized to receive lumbar interbody arthrodesis with a tapered cylindrical threaded fusion cage filled with rhBMP-2/collagen sponge or autogenous iliac crest bone. Patients were evaluated with radiographs, sagittally reformatted computed tomography scans, and Short Form-36 and Oswestry outcome questionnaires. RESULTS: All 11 patients who received rhBMP-2 were judged by three independent radiologists to have solid fusions (at 6, 12, and 24 months postimplantation), whereas only 2 of the 3 control patients, who received the standard treatment of autogenous iliac crest bone, were deemed to be fused. The Oswestry Disability Questionnaire scores of the rhBMP-2 group improved sooner (after 3 months) than those of the autograft group, with both groups demonstrating similar improvement at 6 months. Short Form 36 scores continued to improve up to 24 months. CONCLUSION: The arthrodesis was found to occur more reliably in patients treated with rhBMP-2-filled fusion cages than in controls treated with autogenous bone graft, although the sample size was limited. There were no adverse events related to the rhBMP-2 treatment. This study is one of the first to show consistent and unequivocal osteoinduction by a recombinant growth factor in-humans.     

诱导骨髓间充质干细胞向软骨细胞分化的体外研究

目的 探讨转化生长因子β1（transforming growth factor β1，TGF—β1）、胰岛素样生长因子1（insulinlike growth factor1，IGF-1）在诱导骨髓间充质干细胞（marrow mesenchymal stem ceils，MSCs）向软骨细胞分化过程中的相互作用，并研究细胞密度对MSCs向软骨细胞分化的影响。方法 取健康昆明种小白鼠骨髓，用全骨髓贴壁法筛选获得MSCs，体外培养传代。采用特定的诱导培养使MSCs向软骨细胞分化，按培养基内添加生长因子的不同分成3个实验组和对照组。实验组分别为：TGF—β1＋IGF-1联合应用组（TGF—β1 10ng／ml、IGF-1 50ng／m1）；TGF—β1单独应用组（TGF—β1 10ng／m1）；IGF-1单独应用组（IGF-1 50ng／m1）；对照组不添加任何生长因子。TGF—β1＋IGF-1联合应用组于诱导14d和21d，分别进行甲苯胺蓝染色及免疫荧光双染法鉴定；于诱导7、14和21d各组分别提取诱导细胞总RNA，进行RT—PCR扩增，检测TGF—β1、IGF-1对诱导细胞Ⅱ型胶原表达量的影响；比较MSCs在平板培养及细胞团培养时，Ⅱ型胶原表达量的差异。结果TGF—β1＋IGF-1联合应用组诱导培养14d，诱导软骨细胞甲苯胺蓝染色呈阳性，免疫荧光染色可见诱导软骨细胞的细胞外基质含有Ⅱ型胶原。各组基因扩增产物的凝胶电泳可见，TGF—β1＋IGF-1联合应用组和TGF—β1单独应用组Ⅱ型胶原扩增片段呈阳性；IGF-1单独应用组和对照组，未见Ⅱ型胶原扩增条带；凝胶成像系统灰度扫描示Ⅱ型胶原表达量TGF—β1＋IGF-1联合应用组各时间点均比TGF—β1单独应用组明显增加（P〈0．05）。细胞团培养模式下，诱导细胞表达Ⅱ型胶原比平板培养模式更加显著。结论 MSCs向软骨细胞诱导分化时，IGF-1对TGF—β1有明显的促进作用；细胞培养密度提高有利于MSCs成软骨细胞表型。     

Clinical and radiographic outcomes of posterolateral lumbar spine fusion in humans using recombinant human bone morphogenetic protein-2: an average five-year follow-up study

The objectives of this study were to determine whether recombinant human bone morphogenetic protein-2 (rhBMP-2) can be used as the sole stimulator of osteogenesis with success equal to an autologous graft in posterolateral lumbar fusion (PLF) at the same level and to describe the progress until bone union. This study included 11 patients who underwent PLF of L4-5. On the right side, only rhBMP-2, for which polylactic/glycolic acid (PLGA) was used as a carrier, was used, whereas, on the left side, autogenous bone was used. The bone union rate was 73 and 82% at 12 and 24 months after surgery, respectively, on the right BMP side, while the rate on the autogenous bone side was 91%. There was no statistically significant difference in the bone union rate. rhBMP-2 can be used as the sole source of osteogenesis with success equivalent to an autologous graft of the PLF.     

Additive enhancement of implant fixation following combined treatment with rhTGF-beta2 and rhBMP-2 in a canine model

BACKGROUND: Gaps at the interface between implant and bone increase the risk of diminished implant fixation and eventual loosening. The purpose of the present study was to determine if combined use of recombinant human transforming growth factor-beta 2 (rhTGF-beta2) and bone morphogenetic protein 2 (rhBMP-2) led to greater implant fixation strength in the presence of interface gaps than the use of either growth factor alone. METHODS: Twenty-eight skeletally mature adult male dogs received one porous-coated titanium implant in the proximal part of each humerus, for a total of fifty-six implantation sites. Spacers were used to establish an initial 3-mm gap between the implant and the host bone at all fifty-six sites. Forty-two implants were coated with hydroxyapatite-tricalcium phosphate and were used in three growth-factor-treatment groups in which the implants placed in the left humerus were loaded with 12 microg of rhTGF-beta2 (Group 1, seven animals), 25 microg of rhBMP-2 (Group 2, seven animals), or 12 microg of rhTGF-beta2 combined with 25 microg of rhBMP-2 (Group 3, seven animals). In these animals, the twenty-one implants that were placed in the right humerus were loaded with buffer only to serve as contralateral controls. In Group 4 (seven animals), the implants were not coated with hydroxyapatite-tricalcium phosphate, the gap in the left humerus was lightly packed with autogenous bone graft, and the gap in the right humerus was left empty to serve as a contralateral control. All animals were killed at twenty-eight days. The primary end points included three mechanical variables: fixation strength, interface stiffness, and energy to failure. Secondary end points included bone ingrowth and bone volume and trabecular architecture in the gap and in a region located 2 mm medial to the implantation site. RESULTS: The hydroxyapatite-tricalcium phosphate coating had no effect on implant fixation, bone ingrowth, or bone formation in the 3-mm gap. Individual growth factor treatments led to 2.3 to 3.2-fold increases in fixation strength and stiffness as compared with the values for the contralateral controls (p < 0.05). The combined growth factor treatment led to 5.7-fold increases in fixation strength and stiffness compared with the values for the contralateral controls (p < 0.01). Autogenous bone graft treatment was associated with 4.5 to 6.4-fold increases in implant fixation strength and stiffness as compared with the values for the contralateral controls (p < 0.01). Compared with the relevant contralateral controls, energy to failure was increased 3.5-fold in association with TGF-beta2 alone (p < 0.05), 4.5-fold in association with TGF-beta2 combined with BMP-2 (p < 0.01), and 2.5-fold in association with autogenous bone-grafting. As much as 63% of the variance in the mechanical end points was associated with variance in bone volume and architecture in the 3-mm gap and in the region of interest located 2 mm medial to the implantation site (p < 0.01). CONCLUSIONS: In this animal model, the combined use of TGF-beta2 and BMP-2 led to more secure mechanical fixation of the implant than did the use of either growth factor alone and demonstrated results that were similar to those associated with the use of autogenous bone graft.     

大鼠骨髓间充质干细胞与多孔双相磷酸钙陶瓷支架体外黏附的实验研究

目的研究大鼠骨髓间充质干细胞(marrowmesenchymalstemcells,MSCs)诱导培养后在多孔双相磷酸钙(biphasiccalciumphosphate,BCP)陶瓷支架材料上的黏附和生长。方法SD大鼠MSCs经矿化诱导培养、扩增,具有成骨细胞表型后,与多孔BCP陶瓷支架及普通多孔羟基磷灰石(hydroxyapatite,HA)陶瓷支架体外复合培养,扫描电镜观察比较MSCs在两种材料支架表面的黏附数量和形态;同时以0.5、1.0、2.0、3.0和4.0×106/ml浓度细胞悬液接种于多孔BCP支架材料,检测适宜的接种浓度及单位体积支架材料可黏附MSCs数量。结果大鼠MSCs经诱导培养14d后,行矿化沉积茜素红染色、型胶原免疫细胞化学染色及碱性磷酸酶细胞化学染色,结果均为阳性。大鼠MSCs黏附于多孔BCP陶瓷上的细胞数(88.00±6.58)明显高于HA陶瓷组(39.00±3.65),两组比较差异有统计学意义(P<0.01)。当接种浓度为2.0×106/ml时,单位体积的陶瓷支架材料最多可黏附MSCs数量为1.28×107个/cm3,为细胞适宜接种浓度。结论大鼠MSCs在体外经矿化诱导培养可表达成骨细胞表型,细胞浓度为2.0×106/ml时与多孔BCP陶瓷支架材料具有良好的黏附能力。