仿生诱导骨促进兔横突间脊柱融合的研究

目的 探讨仿生诱导骨促进兔脊柱融合的可行性.方法 成骨诱导化培养兔脂肪基质细胞、制备释放重组人骨形成发生蛋白(rhBMP)-2的壳聚糖/胶原蛋白/B磷酸三钙基质,联合生物凝胶+动态种植,构建仿生诱导骨;兔腰4～5横突间融合模型移植4组,A1仿生骨诱导骨;A2自体髂骨;B1 rhBMP-2的复合基质;B2空白支架.行组织学、免疫组织化学、新骨及诱导因子定量分析、手动触检,生物力学检查.结果 A1组骨性融合能力最强,融合率、新骨面积、BMP含量及力学指标均高于其他组,差异有统计学意义(P<0.05);A2组次之,B1组新骨生长不明显,融合相对延缓.B2组未融合.结论 仿生诱导骨能强化骨诱导、传导效应,超越自体骨修复效果,促进脊柱融合.     

仿生活性骨膜及工程化骨修复兔桡骨缺损的实验研究

目的 探讨仿生诱导骨膜与工程化骨联用修复兔桡骨缺损的可行性,以了解其骨再生特点.方法 兔脂肪基质细胞种植于释放rhBMP-2的生物衍生型复合载体,三维构建工程化骨;壳聚糖,胶原蛋白/β磷酸三钙支架,复合rhBMP-2及兔脂肪基质细胞构建仿生活性骨膜.建立兔桡骨10 mm骨缺损模型,随机移植4组,A组仿生骨膜及工程化骨;B组工程化骨;C组仿生骨膜;D组空白支架,即生物衍生型复合骨.行X线、组织学、免疫组化、双能X线吸收法及透射电镜检查.结果 在新生组织再生、成熟骨替代程度及骨修复完善方面,A组占明显优势、12周时已完成缺损修复,B组初步完成缺损修复,C组仍处于塑形改建期,D组修复能力较差,骨再生延缓;A组骨矿物含量(BMC)及骨密度(BMD)值均高于其它组,具有统计学意义(P＜0.05).结论 仿生骨膜与工程化骨可强化骨再生能力,兼具诱导、引导、传导作用,通过两者协同效应,促进缺损修复.     

PRP及RGD联合修饰表面改性后仿生基质对ADSCs生物学行为的调控

目的 评估富血小板血浆(platelet rich plasma,PRP)及精氨酸-甘氨酸-天冬氨酸(Arg-Gly-Asp,RGD)联合修饰表面改性后支架的细胞生物学特征,验证优化界面整合的方法.方法 兔脂肪基质干细胞成骨诱导化,nβ-TCP/Cs/PCL仿生基质Nd:YAG激光处理,表面改性及细胞接种:A组(PRP凝胶加RGD修饰表面改性基质加ADSCs)、B组(RGD修饰表面改性基质加ADSCs)、C组(表面改性基质加ADSCs)、D组(未表面改性基质加ADSCs);第1、4、8、12、16、20、24、28天,显微镜和电镜、共聚焦技术观察和检测细胞的存活率、增殖活力、Westen blot测定碱性磷酸酶及Ⅰ型胶原蛋白表达活性,分析Runx2与OPG表达.结果 A组细胞生长旺盛、细胞外基质丰富,存活率高于其它组(88.16±1.29,P＜0.05),增殖活力、碱性磷酸酶及Ⅰ型胶原水平均高于其它组(0.92±0.13,87.27±3.08,93.27±3.91,P＜0.05),Runx2和OPG表达显著.结论 PRP及RGD修饰联合支架表面改性能促进细胞增殖,为理想的骨整合方法.     

凝胶包埋兔脂肪基质细胞接种三维支架动态构建组织工程化骨

目的 探讨高效的细胞种植方法,提高工程化骨构建质量.方法 成骨诱导化脂肪基质细胞,制备含rhBMP-2的纳米化壳聚糖/胶原蛋白/β磷酸三钙(Cs-Col-β-TCP)支架,构建工程化骨.静态种植静态培养(A组),振荡种植静态培养(B组),凝胶+振荡种植静态培养(c组),凝胶+静态种植静态培养(D组).第1、4、 8、 12、16、20、24、28天,扫描电镜、共聚焦显微镜观察,检测细胞存活率、细胞增殖、碱性磷酸酶、DNA、骨钙素水平.结果 C组细胞分布均匀、呈三维内生长、细胞外基质丰富,细胞存活率、增殖活力、碱性磷酸酶、DNA、骨钙素水平均高于其他组(79.53±2.67、0.59±0.20、65.16±6.85、207.62±19.36、55.22±8.51,P<0.05).结论 凝胶联合振荡种植可提高细胞接种效率及增强诱导成骨活性.     

骨髓基质干细胞联合血管束植入构筑血管化组织工程骨修复兔大段骨缺损

目的 研究兔骨髓基质干细胞(BMSCs)联合动静脉血管束植入异种脱蛋白松质骨(XDCB)构筑血管化组织工程骨修复兔桡骨中远段完全骨膜骨缺损的能力. 方法 从兔髂嵴捕骨髓培养制备兔BMSCs,将第5代BMSCs种植于多孔XDCB,并进行成骨诱导2周制备组织工程骨,手术中分离兔桡动、静脉血管束.动物模型为制备24只兔舣侧桡骨中远段完全骨膜骨缺损1.5 cm共48侧,分4组修复(n=12),A组为空白未治疗组,B组为单纯材料+血管束植入组(XDCB+VB),C组为组织工程骨组(XDCB+BMSCs),D组为组织工程骨+血管束植入组(XDCB+BMSCs+VB),符组交叉配对.分别于术后4、8、12周行X线片、大体解剖、组织切片、生物力学等检查,观察各组骨缺损修复效能及移植物血管化情况.结果 D组骨缺损修复效能(术后12周新骨面积比2.02%±0.16%)及血管化情况(术后12周血管面积比6.89%±0.32%)优于C组(1.50%±0.28%和3.17%±0.19%),而C组又优于B组(1.59%±0.19%和6.52%±0.23%),A组骨缺损未修复,各组结果差异有统计学意义(P<0.05).结论 BMSCs联合动静脉血管束植入构筑的血管化组织工程骨能促进成骨过程和新生骨的血管化,显著提高组织工程骨修复大段骨缺损的能力.     

组织工程化脂肪基质细胞诱导脊柱融合的实验研究

目的 探讨组织工程化脂肪基质细胞诱导脊柱融合的能力.方法 从成年大鼠腹股沟处无菌获取脂肪组织,消化分离培养出脂肪基质细胞.在大鼠脊柱融合模型中将32只大鼠随机分为4组,每组8只,分别植入骨形态发生蛋白-2(BMP-2)基因重组的腺病毒载体转染的脂肪细胞(A组)、LacZ基因重组的腺病毒载体转染的脂肪细胞(B组)、未转染脂肪细胞(C组)、胶原海绵对照(D组).测定A组和B组的BMP-2的定量表达.比较4组术后4、6、8周X线片评分.8周后处死大鼠,进行MicroCT扫描分析和组织学观察.结果 ELISA法结果显示A组BMP-2表达水平为3.46 ng/mL,而B组无BMP-2表达.A组4、6、8周X线片评分分别为(4.56±1.01)分、(4.72±0.24)分、(4.92±0.13)分,均明显高于其他组,差异有统计学意义(P＜0.05).Micro CT和组织学切片均显示A组全部获骨性融合,而其他组均未融合.结论 组织工程化的脂肪基质细胞能够很好地诱导脊柱融合,有望成为一种新颖、有效的治疗方法.

Abstract：

Objective To assess the capability of inducing spinal fusion by adipose derived stromal cells in a rat model. Methods Adipose derived stromal cells were isolated from inguinal fat pads in rats.In the rat model of spinal fusion,32 rats were randomized into 4 groups.In group A,the cells infected with AdBMP-2 were implanted.In group B,cells infected with AdLacZ were implanted.In group C,uninfected cells were implanted.In group D,only collagen sponges were implanted.Expressions of BMP-2 were quantified in groups A and B.The radiographic scores were compared at weeks 4,6 and 8 among the 4 groups.After sacrifice at week 8,the spine samples were assessed by radiographs,MicroCT,and histological analysis. Results ELISA revealed a 3.46 ng/mL expression of BMP-2 in group A but no expression in group B.The mean radiographic scores for group A at weeks 4,6 and 8 were 4.56 ± 1.01,4.72 ± 0.24,4.92 ±0.13,significantly higher than those for the other groups ( P ＜ 0.05) .MicroCT and histological analysis revealed spinal fusion in all the rats in group A.None of the rats in the control groups developed fusion.Conclusion Because tissue engineered adipose derived stromal cells can induce spinal fusion,they may be a promising strategy for spinal fusion in the future.     

血管化纳米诱导骨的构建及促进兔横突间融合的研究

我们研制释放血管内皮生长因子(VEGF)的人工膜材,作为毛细血管网的基质材料,同步构筑血管化纳米诱导骨,并应用于兔横突间融合部位,观察其能否促进脊柱融合,分析其作用机制,探讨应用潜力.     

重组人骨形态发生蛋合7、转化生长因子β1对兔脂肪基质细胞体外成骨潜能的初步实验研究

目的分离兔脂肪基质细胞并诱导培养其成骨表型,为扩增种子细胞提供实验依据。方法获取兔脂肪组织,胶原酶消化得到脂肪基质细胞,进行原代培养,消化传代后诱导培养,设置含重组人骨形态发生蛋白7(rhBMP-7)、转化生长因子β1(TGF-β1)的改良成骨诱导化培养组、常规成骨诱导培养组及对照非成骨诱导培养组,绘制生长曲线并对其成骨表型进行鉴定。结果与常规成骨诱导化方式相比,rhBMP-7、TGF-β1能明显促进的脂肪基质细胞增殖,并促使其向成骨细胞演变,碱性磷酸酶及VonKossa染色强阳性,群体倍增时间为32h;对照组未显示成骨细胞方向分化。结论采用多因子的成骨诱导培养利于脂肪基质细胞的增殖及诱导分化,是扩增组织工程种子细胞的有效方法。     

Ⅰ型胶原凝胶包埋脂肪干细胞复合PLGA-β-TCP支架修复兔自体桡骨缺损

目的 采用Ⅰ型胶原凝胶悬浮包埋兔脂肪干细胞并复合PLGA-β-TCP支架修复自体桡骨缺损。方法 使用Ⅰ型胶原凝胶悬浮兔脂肪干细胞并与PLGA-β-TCP复合构建脂肪干细胞-Ⅰ型胶原凝胶/PLGA-β-TCP复合体(ASCs-COL/PLGA-β-TCP)(A组),同时设立单纯细胞/PLGA-β-TCP材料复合体(ASCs/PLGA-β-TCP)(B组)、单纯Ⅰ型胶原凝胶/PLGA-β-TCP复合体(COL/PLGA-β-TCP)(C组)、单纯PLGA-β-TCP支架材料(D组)以及空白缺损(E组)作为对照,体外成骨诱导培养2周后植入桡骨1.5cm缺损部位。30只兔(60侧)采用两因素随机区组设计每只兔两侧骨缺损植入组别。分别于术后8、16、24周处死兔,取材,进行相关分析。结果 术后8周,大体观察、放射学、组织学检测示A组桡骨断端连续性基本恢复。术后16周,A组骨断端连续性完全恢复,髓腔未通。术后24周,A组髓腔再通,改建塑形趋于完成,材料基本降解。自术后16～24周,A组残存材料比例低于其他3组(P＜0.05,n=4);与A组比较,在整个观察周期内,B、C、D组均无明显成骨现象,E组保持骨缺损状态(P ＜0.05,n=4)。结论 通过应用Ⅰ型胶原凝胶悬浮包埋脂肪干细胞进而与PLGA-3-TCP多孔支架材料复合构建新型仿生骨组织工程复合体,可修复兔桡骨1.5 cm缺损。     

Qtracker体外标记兔成骨诱导分化后细胞的初步研究

目的 探讨Qtracker体外标记兔成骨诱导分化后细胞的特点及可行性.方法 抽取3个月龄健康新西兰大白兔骨髓,贴壁培养骨髓基质干细胞(BMSCs),传至第3代后向成骨细胞诱导,并做鉴定.Qtracker分别以1、2、4、8、16和32 nmol/10~6细胞的浓度标记成骨诱导分化后细胞,分别记为A、B、C、D、E、F组;未标记的细胞作为空白对照组(G组).分别利用荧光显微镜计数和流式细胞术两种方法枪测标记阳性率,台盼蓝拒染法检测标记后细胞存活率,甲基噻唑基四唑(MTT)法观察Qtracker染料对细胞增殖的影响.结果 兔BMSCs经诱导后能向成骨细胞诱导分化.经Qtracker标记后,荧光显微镜下胞浆呈绿色荧光.随着标记浓度的增加,A、B、C、D、E、F组细胞标记阳性率逐渐增高,于8 nmol/10~6细胞的浓度标记时,在荧光显微镜下计数,标记率可达到(93.58±2.08)%;通过流式细胞仪检测,其标记率为(95.24±1.31)%,经两种方法测定,D、E、F组间标记率差异均无统计学意义(P>0.05),且与其他各组比较差异均有统计学意义(P<0.05);G组各时间点标记阳性率均为0.以不同浓度标记后各组细胞存活率均在96%以上,且各组之间差异无统计学意义(P>0.05).标记Qtracker后对细胞的增殖无影响(P>0.05).结论 Qtracker可用于兔成骨诱导分化后细胞的体外标记,在浓度为8 nmol/10~6细胞下可得到最佳的标记率,且其对成骨诱导分化后细胞的增殖无明显影响.     

Successful Spinal Fusion by E. coli-derived BMP-2-adsorbed Porous β-TCP Granules: A Pilot Study

Bone morphogenetic proteins (BMPs) were originally identified as osteoinductive proteins. With cloning of BMP genes, studies of BMPs and their clinical application have advanced. However, with increasing clinical applications, drug delivery systems and production costs have become more important issues. To address these issues, we asked whether E. coli-derived rhBMP-2 (E-BMP-2)-adsorbed porous β-TCP granules could achieve posterolateral lumbar fusion in a rabbit model similar to autogenous bone grafts. Lumbar spinal fusion masses were evaluated by 3-D computed tomography, mechanical testing, and histological analyses 8 weeks after surgery. By these measures E-BMP-2-adsorbed β-TCP granules achieved lumbar spinal fusion in dose-dependent fashion in a rabbit model as well as autogenous bone graft. Our preliminary findings suggest E-BMP-2-adsorbed porous β-TCP could be a novel, effective alternative to autogenous bone grafting for generating new bone and promoting regenerative repair of bone, and potentially utilizable in the clinical setting for treating spinal disorders.     

纤维蛋白凝胶复合rhBMP-2及骨髓基质干细胞应用于兔脊柱融合的实验研究

目的观察纤维蛋白凝胶（fibrin glue，FG）复合rhBMP-2及骨髓基质干细胞（mesenchymal stem cells，MSCs）用于兔横突间脊柱融合的效果。方法取1月龄日本大耳白兔股骨MSCs体外原代培养，传代培养时加入成骨诱导物向成骨方向诱导分化。40只1岁龄日本大耳白兔随机分为4组，根据植入的材料分别为：MSCs／rhBMP-2／FG组，MSCs／FG组，rhBMP-2／FG组，FG组。复合物置于皮质已打磨的左侧k-6横突上。脊柱标本均于术后6周取材，应用手法检测、DR拍片、CT扫描三维重建、灰度分析、生物力学检测、组织学染色等方法观察各组脊柱融合效果。结果手法检测和DR片示MSCs/rhBMP-2/FG组融合率分别为70％和80％，rhBMP-2/FG组经两种方法检测融合率均为40％。MSCs/FG组和FG组无脊柱融合标本。MSCs／rhBMP-2／FG组与rhBMP-2／FG组在融合率上虽无统计学差异，但在成骨密度和面积上存在显著差异，生物力学检测结果示MSCs，rhBMP-2/FG组在各方向上的融合强度均显著高于其他三组。CT扫描三维重建显示MSCs／rhBMP-2／FG组k-6横突间有连续骨组织生成。组织学染色示MSCs／rhBMP-2／FG组和rhBMP-2/FG组融合标本横突间为成熟骨组织。结论MSCs／rhBMP-2／FG复合物应用于兔横突间脊柱融合可以取得良好成骨质量。     

复合骨在兔腰椎融合过程中相关基因表达调控的影响

目的 观察复合骨即重组人骨形态发生蛋白-2(rhBMP-2)/异体骨不同时间点融合骨组织中BMP-2、血管内皮生长因子(VEGF)的表达.方法 将新西兰大白兔60只随机分为3组,在L5、L6横突间行后路植骨融合术,分别植入复合骨条、自体骨条及异体骨条,于术后第1、2、3、4、5周取融合标本,用实时荧光定量逆转录聚合酶链反应(real time RT-PCR)分析内源性BMP-2和VEGF基因水平的变化.结果 术后第3周,复合骨组BMP-2为(5.3519±1.0384),VEGF为(0.9257±0.2534),均达到峰值且高于异体骨组和自体骨组(P<0.05),之后则缓慢下降.第4周后,内源性BMP-2表达仍保持较高水平,但VEGF的水平与自体骨组和异体骨组差异无统计学意义(P>0.05).结论 复合骨能有效地诱导内源性BMP-2和VEGF的表达,促进了成骨效应.     

重组人骨形态发生蛋白-2/异体骨复合骨用于兔腰椎融合的显微CT研究

目的 观察重组人骨形态发生蛋白-2/异体骨复合骨、自体骨与异体骨分别用于兔腰椎融合后,不同时间点融合骨组织微结构的变化.方法 成年雄性新西兰大白兔45只,随机分为3组,每组15只.在每只兔的L5、L6横突间行腰椎后路植骨融合术,各组分别植入复合骨条,自体髂骨条以及单纯异体髂骨条,每组于术后第3、4、5周各处死5只大白兔,分离保存融合节段标本.用显微cT扫描后行骨组织定量分析.结果 术后3个时间点中,复合骨组和自体骨组新生骨小梁的强度和形态均要优于异体骨组且差异有统计学意义(P＜0.05).第3周,复合骨组的组织骨密度(TMD)为(433.98±2.64)mg/cm3,高于自体骨组(424.81±4.69)mg/cm3(P＜0.05);第4周,复合骨组的骨小梁厚度(Tb.Th)为(0.097±0.004)mm,高于自体骨组(0.082±0.003)mm(P＜0.01);第5周,复合组的组织矿含量(TMC)为(7.70±0.30)mg,高于自体骨组(7.00±0.24)mg(P＜0.01).结论 在兔腰椎后路横突间植骨融合中,重组人骨形态发生蛋-2/异体骨复合骨的成骨效应不低于自体骨,优于异体骨.     

Efficacy of interspinous process lumbar fusion with recombinant human bone morphogenetic protein-2 delivered with a synthetic polymer and β-tricalcium phosphate in a rabbit model

Introduction

As a powerful bone-inducing cytokine, rhBMP-2 has been used as a bone graft substitute in combination with animal-derived collagen to achieve interbody or posterolateral spinal fusion. Successful interspinous process fusion using rhBMP-2 in combination with synthetic carrier materials would offer a safe, minimally invasive spinal fusion option for the treatment of spinal disorders. The aims of the present study were to achieve interspinous process fusion by implanting rhBMP-2-retaining degradable material instead of bone grafting and to evaluate efficacy for vertebral stabilization.

Materials and methods

A polymer gel (200 mg), β-tricalcium phosphate powder (400 mg), and rhBMP-2 (0, 30, 60 or 120 μg) were mixed to generate a plastic implant, which was then placed during surgery to bridge the L5–6 interspinous processes of 58 rabbits. Control animals received implants either without rhBMP-2 or with autogenous bone chips from the iliac crest. L5–6 vertebrae were recovered 8 weeks postoperatively. Interspinous process fusion was evaluated by radiography, biomechanical bending test, intradiscal pressure (IDP) measurement, and histology.

Results

In bending tests, strength of fusion was significantly greater in BMP60 and BMP120 groups than in sham, BMP0, BMP30 or autogenous bone groups. IDP at L5–6 was significantly reduced in BMP60 and BMP120 groups compared to sham, BMP0, BMP30, and autograft groups. Histologically, coronal sections of the fusion mass showed a bone mass bridging both spinous processes.

Conclusion

Solid interspinous process fusion was achieved in rabbit models by 8 weeks after implanting the biodegradable bone-inducing material. These results suggest a potential new less-invasive option without bone grafting for the treatment of lumbar disorders.     

Accelerated repair of a bone defect with a synthetic biodegradable bone-inducing implant

Background Nothing has ever had osteoinductive capacity and degradability equivalent to that of autogenous bone, although many types of biomaterials have been developed. To address this issue, we constructed a new bone graft substitute with osteogenic potential and degradability by using porous beta-tricalcium phosphate (β-TCP) granules, bone morphogenetic protein (BMP), and a synthetic block copolymer composed of poly-d,l-lactic acid with randomly inserted p-dioxanone and polyethylene glycol (PLA-DX-PEG). In this experimental study, the bone-inducing capacity and degradation properties of the composite implant during the bone healing process were examined in vivo in a cortical and cancellous bone defect model in rabbits. Methods The advantages of this type of implant have been examined in a cortical bone defect model created in the distal femur of rabbits. The defects (6.5 × 5 mm) were filled with 30 mg of various implants: BMP-H [rhBMP-2, 0.0025% (w/w)], BMP-L [rhBMP-2, 0.000625% (w/w)], control A (β-TCP alone), and control B (no implant). The distal femurs were harvested at scheduled intervals after surgery and examined for the evaluation of the bony repair of the defects by three-dimensional computed tomography and histology. Results The repair of both cortical and cancellous bone occurred predominantly in the BMP-H group, and only minor cortical bone repair and cancellous bone formation were noted in the BMP-L and control A groups. Most of the β-TCP was resorbed in the BMP-H group at 6 weeks after surgery, whereas a significant amount of β-TCP remained in the BMP-L and control A groups. Conclusions β-TCP granules coated with a BMP-retaining synthetic polymer appear to be effective in enhancing the repair of both cancellous and cortical bone defects. The early disappearance of the implanted β-TCP and restoration of the normal anatomy of bone tissue are two notable features of this approach.     

Hydroxyapatite granule graft combined with recombinant human bone morphogenic protein-2 for solid lumbar fusion

The purpose of this study was to evaluate the availability of recombinant human bone morphogenetic protein-2 (rhBMP-2) combined with hydroxyapatite (HA) and autogenous bone. Posterolateral intertransverse fusion between the fifth and sixth lumbar vertebrae was performed in 27 adult Japanese white rabbits. These 27 rabbits were classified into three groups: the autogenous bone group, the HA group, and the bone morphogenic protein (BMP) group. In the HA group, HA (0.5 g) mixed with iliac bone was grafted. In the BMP group, HA (0.5 g) soaked with rhBMP-2 (100 mg) and iliac bone was grafted. At 6 weeks after the procedure, bone union was evaluated. In the BMP group, all cases showed solid bone union, and fusion masses were stiffer than the masses obtained in the other group. Biomechanically and histologically, grafts of HA soaked with rhBMP-2 and iliac bone was clearly effective in obtaining a solid intertransverse arthrodesis.