重组人骨形态发生蛋白4基因腺相关病毒载体对兔骨髓基质干细胞生物学行为的影响

目的应用重组人骨形态发生蛋白4基因腺相关病毒载体(AAV-hBMP4)转染兔骨髓基质干细胞(BMSCs),观察其对BMSCs生物学行为的影响,从而为骨组织工程寻找理想的病毒载体及种子细胞。方法全骨髓法培养兔BMSCs,按感染复数(MOI)值不同设定为四组,分别转染兔BMSCs,观察病毒量对细胞形态的影响。选取影响最小的MOI值,进行后续实验。转染兔BMSCs,MTT法描记细胞生长曲线,观察AAV对细胞增殖活性的影响。以重组增强型绿色荧光蛋白基因的腺相关病毒载体(AAV-EGFP)为参照,行流式细胞仪检测,计算转染效率。AAV-hBMP4与对照病毒AAV-EGFP分别转染细胞,观察细胞形态,行碱性磷酸酶(ALP)染色、Von Kossa染色及ALP含量测定,观察成骨活性。兔肌袋实验观察异位成骨情况。结果MOI值为5×10~4 vg/cell时,AAV对细胞形态影响最小,以此值进行后续实验。AAV转染后,细胞增殖活性良好,转染效率为55％～65％。AAV-hBMP4转染后,细胞形态呈现典型的成骨改变,ALP染色及Von Kossa染色均出现成骨的特征性改变,而AAV-EGFP组无上述改变。细胞上清ALP含量测定显示,实验组ALP含量显著增高,与对照组比较差异有统计学意义(t=218．65,P＜0．01)。兔肌袋实验术后4周组织学检测可见大量钙盐沉积,矿化结节形成。结论AAV-hBMP4转染效率高,对BMSCs的增殖活性影响小,AAV-hBMP4转染的BMSCs可望成为组织工程化骨的理想种子细胞。     

Unterkieferrekonstruktion mit autologem Knochen und einem induktiven Implantat beim G?ttinger Minischwein

ZusammenfassungFragestellung In der vorliegenden Studie am Göttinger Minischwein wurde die direkte Rekonstruktion des Unterkiefers nach ausgedehnter Kontinuitätsresektion mit autologem Knochen und einem osteoinduktiven Implantat untersucht.Methode An neun ausgewachsenen Göttinger Minischweinen wurde ein einseitiger, 5 cm langer Unterkieferkontinuitätsdefekt gesetzt. Die Rekonstruktion erfolgte bei vier Tieren mit einem 50×25×15 mm³ großen, kollagenen Träger, dotiert mit rhBMP-2 (400 µg/cm³). Bei zwei Tieren wurde nur der Träger alleine implantiert und bei drei Tieren das lokal resezierte Knochensegment replantiert. Die Knochenregeneration und Konsolidierung der Defekte wurde radiologisch und histologisch analysiert.Ergebnisse Nach Rekonstruktion mit dem osteoinduktiven Implantat zeigte sich bei allen Versuchstieren eine komplette knöcherne Konsolidierung des gesetzten Unterkieferkontinuitätsdefekts. Der gesamte Defekt wurde von einem biomechanisch hochwertigen, sich funktionell anpassenden Knochen überbrückt. Nach Replantation des ortsständigen autologen Knochens wird dieser nicht schnell genug knöchern integriert. In der Peripherie bilden sich nur unvollständige Knochenbrücken aus; dies führt zum vollständigen Versagen der Rekonstruktion. Ebenso findet bei der Implantation des Trägers alleine keine Konsolidierung statt.Schlussfolgerung Die direkte Rekonstruktion eines ausgedehnten, biomechanisch belasteten Defekts mit einem osteoinduktiven Implantat erwies sich als die überlegene Methode. Das hierbei entstehende knöcherne Regenerat erfährt eine unmittelbare funktionelle Strukturierung. Die Notwendigkeit zu extensiven adaptiven Umbauvorgängen wird hierdurch minimiert.     

肝癌中骨形成蛋白2对PTEN蛋白水平影响的研究

目的用骨形成蛋白2(BMP2)干预肝癌细胞系HepG2细胞,从而观察肝癌细胞中PTEN蛋白水平的变化,观察BMP2对PTEN蛋白表达的影响,探讨BMP2与PTEN蛋白水平之间的关系,探索肝癌治疗的新途径。方法将受试对象分为3个组:对照组、BMP2100ng/ml组、BMP2300ng/ml组,作用时间为3个水平:6、12、24h。用免疫组织化学方法测定细胞中PTEN蛋白水平。结果免疫组织化学结果显示在不同浓度BMP2组中HepG2细胞中PTEN蛋白表达差异有统计学意义。不同时间组中PTEN蛋白表达差异有统计学意义。PTEN蛋白阳性表达率在300ng/ml组与100ng/ml组、对照组之间差异有统计学意义(P<0.05),在6h组与12h组、24h组之间差异有统计学意义(P<0.05)。在300ng/ml、24h组中PTEN蛋白阳性表达最高。浓度与时间之间无交互作用。结论在肝癌中BMP2可以增加PTEN蛋白水平,且呈浓度时间依赖。     

Global gene profiling reveals a downregulation of BMP gene expression in experimental atrophic nonunions compared to standard healing fractures.

Nonunion is a challenging problem that may occur following certain bone fractures. However, there has been little investigation of the molecular basis of nonunions. Bone morphogenetic proteins (BMPs) play a significant role in osteogenesis. However, little is known about the expression patterns of BMPs in abnormal bone healing that results in nonunion formation. These facts prompted us to investigate and compare the gene expression patterns of BMPs and their antagonists in standard healing fractures and nonunions using rat experimental models. Standard closed healing fractures and experimental atrophic nonunions produced by periosteal cauterization at the fracture site were created in rat femurs. At postfracture days 3, 7, 10, 14, 21, and 28, total RNA was extracted from the callus of standard healing fracture and fibrous tissue of nonunion (n=4 per each time point and each group). Gene expression of BMPs, BMP antagonists, and other regulatory molecules were studied by methods including Genechip microarray and real-time quantitative RT-PCR. Gene expression of BMP-2, 3, 3B, 4, 6, 7, GDF-5, 7, and BMP antagonists noggin, drm, screlostin, and BAMBI were significantly lower in nonunions compared to standard healing fractures at several time points. Downregulation in expression of osteogenic BMPs may account for the nonunions of fracture. The balance between BMPs and their endogenous antagonists is critical for optimal fracture healing.     

Regional gene therapy for full-thickness articular cartilage lesions using naked DNA with a collagen matrix.

A novel gene therapy approach for treating damaged cartilage is proposed that involves placing endotoxin-free cDNA containing the gene for bone morphogenetic protein-2 (BMP-2) in type I collagen sponges and then transferring the naked plasmid DNA construct to the injury site. A full-thickness cartilaginous defect in rabbits implanted with plasmid containing a marker gene (beta-galactosidase) showed expressed protein as detected by immunostaining. At 1 week postimplantation, mesenchymal cells subjacent to the defect had incorporated the implanted naked plasmid DNA and, once transfected, served as local bioreactors, transiently producing the gene product. Plasmids containing the gene for BMP-2 implanted in collagen sponges in cartilage lesions stimulated hyalinelike articular cartilage repair at 12 weeks postimplantation, nearly equivalent in quality to that induced by collagen sponges with recombinant BMP-2 protein. Our approach circumvents the risks of inflammation and immunogenic response associated with the use of viral vectors. Naked plasmid DNA as a vehicle for transferring therapeutic genes has been shown to be effective in a therapeutic model within rabbit articular cartilage and appears to be safe and cost effective.     

Osteoclastogenesis in the nonadherent cell population of human bone marrow is inhibited by rhBMP-2 alone or together with rhVEGF.

During bone development and repair, angiogenesis, osteogenesis, and bone remodeling are closely associated processes that share some common mediators. In the present study nonadherent human bone marrow mononuclear cells under the induction of sRANKL and M-CSF, differentiated into osteoclasts with TRAP-positive staining, VNR expression, and Ca-P resorptive activity. The effects of various combinations of rhBMP-2 (0, 3, 30, and 300 ng/mL) and rhVEGF (0 and 25 ng/mL) on osteoclastogenesis potentials were examined in this experimental system. The percentages of TRAP-positive multiple nucleated cells represent osteoclast differentiation potential, and the percentages of resorptive areas in the Ca-P coated plates resemble osteoclast resorption capability. The presence of rhBMP-2 at 30 and 300 ng/mL showed inhibitory effects on osteoclast differentiation and their resorptive capability in the human osteoclast culture system. rhVEGF (25 ng/mL) enhanced the resorptive function of osteoclast whenever it was used alone or combined with 3 ng/mL rhBMP-2. However, rhVEGF-induced resorptive function was inhibited by 30 ng/mL and 300 ng/mL rhBMP-2 in a dose-dependent manner. Statistical analysis demonstrated that an interactive effect exists between rhBMP-2 and rhVEGF on human osteoclastogenesis. These findings suggested that an interactive regulation may exist between BMPs and VEGF signaling pathways during osteoclastogenesis; exact mechanisms are yet to be elucidated.     

骨髓间充质干细胞在丝素蛋白/骨形态发生蛋白中的生物学行为观察

目的 观察丝素蛋白与牛骨形态发生蛋白的载体系统与骨髓间充质干细胞的体外相容性.方法 将多孔丝素蛋白修剪成20×10×5 mm长方体状物,置入6孔板中;将粉状bBMP用PBS液溶解制成2mg/ml混悬液,0.5ml无菌移液管滴定于多孔丝素蛋白上(0.5毫升/个).骨髓间充质干细胞以107/ml接种到载体系统上,复合4小时后加入生长液继续体外培养,每1、4、8天时进行碱性磷酸酶、矿化结节染色观察以及扫描电镜和激光共聚焦显微镜观察;另外同时设立单纯丝素蛋白与细胞复合培养对照组进行观察.结果 倒置显微镜、扫描电镜和共聚焦显微镜下观察到细胞在两种材料上生长形态、活性正常,体外培养24小时时细胞已贴附材料上,呈匍匐状伸展,以后逐渐融合生长,8天时大量细胞成片状攀附在材料表面,并分泌有大量的网状细胞外基质,实验组可见结节状基质生成;碱性磷酸酶、矿化结节染色观察发现实验组细胞碱性磷酸酶和矿化结节染色阳性反应,对照组阴性.结论 丝素蛋白是一种良好细胞外基质材料,也是一种良好生长因子的缓释载体.     

牦牛骨形态发生蛋白及其与陶瓷化异种骨复合修复兔桡骨缺损的实验研究

本实验采用yBMP和yBMP/CBB材料,修复1.5cm长的兔桡骨缺损。分yBMP和yBMP/CBB二个实验组。材料植入20天,缺损区均分别生成多量软骨及交织骨。40天,大量交织骨生成,其间出现骨髓组织。60天,板层骨增多。80天,板层骨进一步成熟,骨缺损修复。与之对照,空白组各期仅见两端形成交织骨。这表明:yBMP及yBMP/CBB对骨缺损的修复作用非常显著。     

Regeneration of periodontal ligament and cementum by BMP-applied tissue engineering

Previously, we demonstrated that the inductive properties of bone morphogenetic protein (BMP) highly depend on the nature of the carrier material used for implantation. In this paper, we show that administration of BMP incorporated in a fibrous collagen membrane can help to regenerate periodontal ligament and cementum both in cat canines and in monkey molars. The partially purified bovine BMP was combined with one or two layers of a fibrous collagen membrane. Although the single layer approach showed partial regeneration of periodontal defects, it also quite often led to ankylosis. The double layer technique in artificially prepared class III furcation defects in monkey molars gave favorable results. After 12 wk, not only the alveolar process but also the periodontal ligament and cementum had regenerated along the entire treated dentin surface. Collagen fibers were arranged more or less perpendicular to the surface of the new cementum. Ankylosis was not seen. It is concluded that the double-layer approach is superior to the single-layer technique in regenerating cementum.