rhBMP-2临床应用研究新进展

1965年Urist发现脱钙骨中含有一种骨形态发生蛋白(bone morphogenetic protein,BMP),并发现这种蛋白具有成骨诱导作用。随着人们通过基因工程技术对这种蛋白的研究不断深入,到目前为止,已经发现的骨形态发生蛋白家庭成员已有43种,估计数量还在不断增加,其中BMP-2成骨活性最强。     

Improved healing of transected rabbit Achilles tendon after a single injection of cartilage-derived morphogenetic protein-2

BACKGROUND: Achilles tendon ruptures in humans might be treated more efficiently with the help of a growth factor. Cartilage-derived morphogenetic protein-2 has been shown to induce formation of tendon-like tissue. HYPOTHESIS: Cartilage-derived morphogenetic protein-2 has a positive effect on mechanical parameters for tendon healing in a rabbit model with Achilles tendon transection. STUDY DESIGN: Controlled laboratory study. METHODS: The right Achilles tendon of 40 rabbits was transected without tendon suture. Cartilage-derived morphogenetic protein-2 (10 micro g) or vehicle control (acetate buffer) was injected locally 2 hours postoperatively. All tendons were tested biomechanically at 8 and 14 days, and treated tendons were histologically and radiographically evaluated at 56 days. RESULTS: At 14 days, both failure load and stiffness of treated tendons were increased by 35%. The treated tendons had significantly larger callus size at 8 and 14 days. Histologic and radiographic examination showed no signs of ossification in the treated tendons after 56 days. CONCLUSIONS: A single injection of cartilage-derived morphogenetic protein-2 led to a stronger and stiffer tendon callus than that in the controls without inducing bone formation. Clinical Relevance: Similar results from a larger animal model would suggest a possible future use of cartilage-derived morphogenetic protein-2 in the treatment of human Achilles tendon ruptures.     

Augmentation of tendon healing in an intraarticular bone tunnel with use of a bone growth factor.

We hypothesized that an exogenous bone growth factor could augment healing of a tendon graft in a bone tunnel in a rabbit anterior cruciate ligament-reconstruction model. Seventy rabbits underwent bilateral anterior cruciate ligament reconstructions with a semitendinosus tendon graft. One limb received a collagen sponge carrier vehicle containing a mixture of bone-derived proteins while the contralateral limb was treated with either no sponge or a sponge without bone-derived proteins. The reconstruction was evaluated at 2, 4, or 8 weeks with histologic, biomechanical, and magnetic resonance imaging analysis. Histologic analysis demonstrated that specimens treated with bone-derived proteins had a more consistent, dense interface tissue and closer apposition of new bone to the graft, with occasional formation of a fibrocartilaginous interface, when compared with control specimens. The treated specimens had significantly higher load-to-failure rates than did control specimens. Treatment with bone-derived proteins resulted in an average increase in tensile strength of 65%. The treated specimens were stronger than control specimens at each time point, but the difference was greatest at 8 weeks. On the basis of signal characteristics and new bone formation, magnetic resonance imaging was useful for predicting which limb was treated, the site of failure, and the limbs with higher load-to-failure values. This study demonstrates the potential for augmenting tendon healing in an intraarticular bone tunnel using an osteoinductive growth factor.     

骨形态发生蛋白-2基因转染骨髓间充质干细胞移植对糖尿病大鼠骨折愈合的影响

目的 观察骨形态发生蛋白-2(BMP-2)基因转染大鼠骨髓间充质干细胞(BMSCs)移植对糖尿病骨折愈合的影响,探讨糖尿病骨折治疗的新方法.方法 Wistar雄性成年6周龄大鼠50只,按随机数字表法分为对照组和实验组,均制成糖尿病骨折模型,体外高糖环境下腺病毒介导的BMP-2转染BMSCs,分别于骨折局部移植相应的BMSCs.术后1,2,3,4,6周行X线检查;取骨痂行HE染色,免疫组化检测骨痂中BMP-2的灰度值;ELISA法检测血清中BMP-2的浓度.结果 第4周实验组骨痂BMP-2灰度值为83±3,对照组为118±4(P＜0.01).第4周实验组血清BMP-2浓度为(203.80±8.96)ng/L,对照组为(139.15±4.19)ng/L(P＜0.01).结论 BMP-2基因转染的BMSCs移植能促进糖尿病大鼠骨折的愈合.     

重组人BMP-2聚乳酸缓释纳米微球对体外培养兔成骨细胞增殖和矿化的影响

目的 观察重组人骨形态发生蛋白-2聚乳酸纳米微球缓释系统(rhBMP-2-PLA-Ns)对体外培养的兔成骨细胞的生物学效应. 方法 体外培养兔成骨细胞并鉴定,将rhBMP-2-PLA-Ns和第3代成骨细胞一起培养,免疫荧光法检测成骨细胞核中增殖细胞核抗原(PCNA)的表达,茜素红染色方法观察矿化结节的形成,Western blot检测rhBMP-2-PLA-Ns对成骨细胞自分泌血管内皮细胞生长因子(VEGF)的作用,并与单纯rhBMP-2组和空白组进行比较. 结果 培养5 d后,各组PCNA阳性细胞差异无统计学意义.培养10 d后,rhBMP-2-PLA-Ns组阳性细胞数明显高于单纯rhBMP-2组和空白组,表明rhBMP-PLA-Ns能够明显促进PCNA在成骨细胞核中的表达.与其他两组比较,rhBMP-2-PLA-Ns能显著促进矿化结节的形成(P<0.05);对Western blot检测结果进行半定量分析显示:三组成骨细胞均有VEGF的自分泌,加入rhBMP-2-PLA-Ns成骨细胞自分泌VEGF的量超过单纯rhBMP-2组以及空白对照组(P<0.05).结论 rhBMP-PLA-Ns有较好的生物学活性,能促进成骨细胞的增殖、矿化和VEGF自分泌的增加,具有一定的临床应用价值,为加快骨创伤的愈合提供了新的思路.     

腺病毒介导的人骨形态发生蛋白2基因修复兔桡骨缺损

目的 评价腺病毒介导的人骨形态发生蛋白2（Adv-hBMP-2)基因对兔骨髓间充质干细胞（BMSCs)的诱导成骨活性及修复长骨干骨缺损的效果。方法 （1）抽取兔骨髓行BMSCs培养，经Adv-hBMP-2转染后分别行免疫沉淀加Western印迹法和碱性磷酸酶（ALP）检测及von Kossa染色，并行裸鼠肌内诱导成骨试验。（2）修复兔桡骨缺损：15只兔30侧、1.5cm的骨缺损分为Adv-hBMP-2转染BMSCS加珊瑚及胶原载体组、β半乳糖苷酶（Adv-βgal)转染BMSCs加载体组、未转染BMSCs加载体组、载体组和未治疗组，术后行X线、组织学检查。结果 （1）Adv-hBMP-2组BMSCs表达hBMP-2，其ALP活性升高，并有钙结节形成,裸鼠肌内注射后有异位成骨。（2）在兔桡骨缺损处，Adv-hBMP-2转染组有明显骨痂形成，5个组的愈合率分别为4／5、2／5、2／5、0／5、0／5。结论 Adv-hBMP-2基因转染的BMSCs是修复骨缺损的好方法。     

优化配方的CPC/DBM/rhBMP-2复合材料在体内的超微结构和组织学特征

目的探讨磷酸钙骨水泥(CPC)/脱钙骨基质颗粒(DBM)/重组人骨形成蛋白-2(rhBMP-2)复合材料在骨内的超微结构和组织学特征,评价该材料的成骨性能。方法预制兔DBM,按0.2的DBM质量比制作CPC/DBM/rhBMP-2复合材料。将复合材料(A组,n=12)、CPC(B组,n=10)、医用骨水泥(C组,n=7)分别植入兔股骨髁部骨缺损,在植入后第6、12、24周取材进行组织学和扫描电镜观察。结果术后第6周,A组中复合材料-骨界面模糊,宿骨发出纤维连续通过界面,编织骨样结构开始向材料内部长入。第12周,A组中血管和成骨细胞在复合材料内部生长,产生新骨。第24周,A组中骨缺损已修复,形成骨性连接,材料大部分被新骨替代。B组术后第6周CPC-骨界限清晰,未见新骨向材料内部长入,第24周骨缺损部位仍为CPC填充,材料内部未见新骨出现。C组材料直至第24周尚未与宿骨形成任何连接。A组材料在成骨细胞活性、血管化程度和新骨生长速度方面均优于B组。结论将DBM质量比为0.2的复合材料植入骨缺损内可以促进细胞、血管、新骨的长入,且易降解及被自体骨替代。     

多孔聚乳酸-聚乙醇酸共聚物作为缓释重组人骨形态发生蛋白-2载体的实验研究

目的 探讨多孔聚乳酸-聚乙醇酸共聚物(PLGA)作为重组人骨形态发生蛋白-2(rhBMP-2)的载体，通过体外释放试验和体内活性试验来评价rhBMP-2释放的动力学过程及活性。方法 采用乳液冷冻干燥法制作含rhBMP-2缓释系统的PLGA支架，支架进行扫描电镜观察；采用高效液相色谱分析仪检测不同时间点释放液中rhBMP-2的含量，进行累积释放量的动态观察；将缓释rhBMP-2支架植入SD大鼠大腿股部肌袋内，分别在不同时间点进行组织学观察。结果 支架材料的形态学观察显示，材料表面呈多孔状；rhBMP-2从支架中释放的动力学过程为第1天表现为爆发性释放(30.0％)，以后缓慢持续释放，至1个月左右释放量达80.6％；活性评价结果显示，rhBMP-2缓释支架组可见新生骨组织形成伴较多的骨母细胞排列，无rhBMP-2支架组则无成骨现象。结论 多孔PLGA可作为rhBMP-2的缓释载体，并具有良好的生物活性，可作为骨组织工程研究中的新型支架，同时具有临床应用的可行性。     

MRI of transforaminal lumbar interbody fusion: imaging appearance with and without the use of human recombinant bone morphogenetic protein-2 (rhBMP-2)

Purpose

To describe the vertebral endplate and intervertebral disc space MRI appearance following TLIF, with and without the use of rhBMP-2, and to determine if the appearance is concerning for discitis/osteomyelitis.

Materials and methods

After institutional review board approval, 116 TLIF assessments performed on 75 patients with rhBMP-2 were retrospectively and independently reviewed by five radiologists and compared to 73 TLIF assessments performed on 45 patients without rhBMP-2. MRIs were evaluated for endplate signal, disc space enhancement, disc space fluid, and abnormal paraspinal soft tissue. Endplate edema-like signal was reported when T1-weighted hypointensity, T2-weighted hyperintensity, and endplate enhancement were present. Subjective concern for discitis/osteomyelitis on MRI was graded on a five-point scale. Generalized estimating equation binomial regression model analysis was performed with findings correlated with rhBMP-2 use, TLIF level, graft type, and days between TLIF and MRI.

Results

The rhBMP-2 group demonstrated endplate edema-like signal (OR 5.66; 95 % CI [1.58, 20.24], p?=?0.008) and disc space enhancement (OR 2.40; 95 % CI [1.20, 4.80], p?=?0.013) more often after adjusting for the TLIF level, graft type, and the number of days following TLIF. Both groups had a similar temporal distribution for endplate edema-like signal but disc space enhancement peaked earlier in the rhBMP-2 group. Disc space fluid was only present in the rhBMP-2 group. Neither group demonstrated abnormal paraspinal soft tissue and discitis/osteomyelitis was not considered likely in any patient.

Conclusions

Endplate edema-like signal and disc space enhancement were significantly more frequent and disc space enhancement developed more rapidly following TLIF when rhBMP-2 was utilized. The concern for discitis/osteomyelitis was similar and minimal in both groups.     

The fate of host and graft cells in early healing of bone tunnel after tendon graft

BACKGROUND: The behavior of host and graft cells during the healing process after autologous tendon graft has not been elucidated. HYPOTHESIS: Host cells will integrate into the bone-tendon interface and contribute to cellular repopulation of the graft. STUDY DESIGN: Controlled laboratory study. METHODS: Twelve-week-old, genetically identical, female green fluorescent protein transgenic rats (n = 20) and wild-type rats (n = 20) were used. The rats were divided into 2 experimental groups. In group A, the Achilles tendons of wild-type rats were harvested and transplanted into the transcondylar femoral bone tunnels of green fluorescent protein rats. In group B, the Achilles tendons of green fluorescent protein rats were transplanted into a transcondylar femoral bone tunnel of wild-type rats. Immediately after transplantation (time zero) and at 1, 2, and 4 weeks after the transplantation, distal femoral epiphyses were harvested and cut into 14-mum serial sagittal frozen sections. The sections were examined with a confocal laser-scanning microscope to quantify green fluorescent protein-positive cell survival. RESULTS: At time zero, only host cells in group A and only graft cells in group B demonstrated green fluorescent protein signals. At 1 week in group A, many green fluorescent protein-positive cells were found in the graft. In group B, a few green fluorescent protein-positive cells were found in the graft. At 2 and 4 weeks in group A, many green fluorescent protein-positive cells were detected in the graft, but green fluorescent protein-positive cells had disappeared completely in group B. CONCLUSION: Host cells, rather than graft cells, contribute to repair of the bone-tendon interface and the remodeling of grafts after simulated autologous tendon graft.     

转染骨形态发生蛋白-2基因的人骨髓间质干细胞复合异种骨支架异位成骨的效果

目的 观察骨形态发生蛋白-2(BMP-2)罐因转染的人骨髓间质干细胞复合异种骨支架异位成骨效果。方法 分组：(1)BMP-2基因转染细胞 异种骨支架[去抗原牛松质骨(BCB)]；(2)对照基因转染细胞 BCB；(3)未转染细胞 重组BMP-2 BCB；(4)未转染细胞 BCB；(5)BCB。分别将各组人工骨植入裸鼠皮下，于术后4，8周行组织学观察．结果 体内植入后3d，细胞持续表达外源基因BMP-2基因转染组8周时完全由新形成的编织骨构成，血管丰富。结论 BMP-2基因转染后细胞复合BCB支架，可以在异位形成骨组织并诱导毛细血管长入。     

血管内皮生长因子在骨形态发生蛋白-2诱导成骨细胞过程中的作用

目的探讨血管内皮生长因子（vascular endothelial growth factor，VEGF）在骨形态发生蛋白-2（BMP-2）诱导成骨过程中的作用。方法（1）取小鼠胚胎成骨细胞进行体外培养，RT—PCR法检测rhBMP-2（300ng／m1）诱导成骨细胞分化过程中VEGF受体（VEGF receptors，VEGF—R）的表达。（2）采用VEGF反义寡核苷酸（antisense oligodeoxynucleotides，ASODNs）和苏拉明分别阻断VEGF的合成及功能，观察对成骨细胞碱性磷酸酶（ALP）活性和钙结节形成的影响。（3）在rhBMP-2诱导成骨的同时，添加不同浓度的外源性VEGF，观察对成骨细胞体外矿化能力的影响。结果（1）VEGF—R（Flk-1和Flt-1）mRNA在成骨细胞呈稳定弱表达，rhBMP-2干预24h后VEGF—R的表达量无明显变化。（2）应用VEGF ASODNs和苏拉明可抑制rhBMP-2所诱导的ALP活性和钙结节形成，并存在剂量依赖效应。（3）单独应用VEGF并不能使成骨细胞分化为钙结节，外源性VEGF的加入亦不影响rhBMP-2刺激钙结节形成的能力。结论VEGF在成骨细胞可以自分泌的形式发挥作用，VEGF至少部分参与了rhBMP-2的诱导成骨活性。     

重组人骨形态发生蛋白-2缓释凝胶微球的研制及其溶胀、降解、载药、释药特征

目的制备载人骨形态发生蛋白-2(rhBMP-2)的甲基丙烯酸缩水甘油酯右旋糖酐(dex-GMA)凝胶微球并初步考察其体外溶胀、降解、载药与释药特征。方法以液体石蜡为油相,Span-80为乳化剂,采用乳化化学交联技术制备载rhBMP-2的凝胶微球(BMP-HMs)并通过正交设计法优化其制备工艺;观察BMP-HMs形态和粒径,测定其包封率与载药量;用微球的吸水能力表示微球的溶胀率(Rs),扫描电镜观察微球的体外降解,动态观察体外释药特征及其与微球溶胀、降解的关系。结果所制备的BMP-HMs形态规整,粒径40～50μm,分布均匀;rhBMP-2载药量(10.6±4.8)%,包封率(88.9±1.0)%,BMP-HMs冻干剂4℃以下存放6个月性能稳定,但在磷酸盐缓冲液(PBS)中20～40d内可以完全降解。微球Rs随反应促进剂四甲基乙二胺(TEMED)用量的增大而减小,0.3mlTEMED制备的BMP-HMs体外释药实验表明80%的rhBMP-2在前20d左右释放。结论BMP-HMs对rhBMP-2具有确定的缓释作用,并可以通过制备工艺的改变控制其释药。     

Enhancement of bone formation with a synthetic matrix containing bone morphogenetic protein-2 by the addition of calcium citrate

Purpose

The aim of the study was to test whether calcium citrate combined with rhBMP-2 was able to enhance bone regeneration compared with a matrix containing only rhBMP-2.

Methods

In each of experimental mice, one cylinder of calcium citrate–rhBMP-2 or rhBMP-2 alone was implanted into the thigh muscle pouches of the mouse. The following two treatment modalities were randomly allocated: (1) empty control with rhBMP-2 alone in a gelatin matrix and (2) a gelatin matrix including both calcium citrate and BMP-2. After several weeks, bone granules were obtained by histological analysis.

Results

Histomorphometric analysis showed the greatest amount of newly formed bone was observed in the group that contained 10.0 mg calcium citrate with 2.0 mg rhBMP-2 (p < 0.05). Quantitative histomorphometry revealed in the calcium citrate–rhBMP-2 group an obvious increase in the fractional area and the average new bone mineral density of newly formed bone at 2, 4 and 6 weeks than in the rhBMP-2 group (p < 0.05). At 2 weeks time-point, the mature cancellous bone had formed in the calcium citrate–rhBMP-2 group.

Conclusions

From this study, it can be concluded that calcium citrate combined with rhBMP-2 significantly enhances bone regeneration in muscle. This synthetic gelatin matrix containing calcium citrate/gelatin granules fulfils a number of criteria required for an ideal carrier system for rhBMP-2. The calcium ions that calcium citrate releases into the surrounding environment can activate bone formation when used as part of a combination of calcium citrate and BMP-2.     

Dynamic magnetic resonance tomography (MRI): a follow-up study after femur core decompression and instillation of recombinant human bone morphogenetic protein-2 (rhBMP-2) in avascular femur head necrosis

OBJECT: The aim of the study was to test the use of dynamic magnetic resonance imaging study with Gd-DTPA-application and the dynamic changes of signal intensity at patients with avascular femoral head necrosis after having installed rhBMP-2 and/or decompressed the core. MATERIAL AND METHODS: Six patients with avascular necrosis of the femoral head ARCO-stage I- or II-lesions were treated surgically by femoral head core decompression. Three of these patients were additionally treated with rhBMP-2-instillation. The progression or regression could be confirmed by T1- and T2-weighted spinecho-sequences (zero, four, ten, sixteen weeks and 24 months follow up). RESULTS: Corresponding ARCO-classification with partly more sensitive measurement of vitality signs in comparison to the optical x-ray classification. The objective, quantitative measurement of signal intensity post contrast medium reduces the influence of experience and level of education. The dynamic sequences results are reproducible. CONCLUSION: The dynamic magnetic resonance imaging study after Gd-DTPA-application and the dynamic changes of signal intensity after Gd-DTPA enhancement in the necrotic areas of the femoral head were the important subject of our study and it seems, that these sequencies and the ascertainment of signal intensity changes will be an efficient method for judgement of vitality, vascularisation and perfusion after therapeutical intervention. Combination of femoral head core decompression and rhBMP-2-instillation for the purpose of osseous regeneration seems to stabilize the affection.     

Use of bone marrow stromal cells for tendon graft-to-bone healing: histological and immunohistochemical studies in a rabbit model

BACKGROUND: Despite increasing attention on the issue of tendon-to-bone integration, there has been no animal study on the use of cell therapy for promoting the insertion healing of tendon to bone. PURPOSE: To determine the efficacy of using a large number of bone marrow stromal cells (bMSCs) to enhance tendon-to-bone healing. STUDY DESIGN: Controlled laboratory study. METHODS: The hallucis longus tendons were translated into 2.5-mm diameter calcaneal bone tunnels in a New Zealand white rabbit model. The bone tunnels were treated with or without bMSCs. Three specimens from each group were harvested at 2, 4, and 6 weeks postoperatively and evaluated by conventional histological and immunohistochemical methods. RESULTS: At 4 weeks, the specimens with bMSCs exhibited more perpendicular collagen fiber formation and increased proliferation of cartilage-like cells, which was indicated by positive collagen type-II immuno-staining of the tendon-bone interface. In contrast, the specimens without bMSCs demonstrated progressive maturation and reorganization of fibrous tissue aligned along the load axis. CONCLUSION: Introduction of a large number of bone marrow stromal cells to the bone tunnel have shown to improve the insertion healing of tendon to bone in a rabbit model through formation of fibrocartilagenous attachment at early time points.