rhBMP-2/Co/HA复合材料的制备及异位诱导成骨作用

目的：测定rhBMP-2/Co/HA复合制剂的生物活性,为其作为盖髓剂和根尖诱导剂提供理论依据。方法：将rhBMP-2、Ⅰ型胶原和羟基磷灰石复合,采用异位骨诱导实验检测复合材料的生物活性,并通过X线及组织学观察其效果。结果：rhBMP-2/Co/HA复合材料能够异位诱导成骨。结论：rhBMP-2/Co/HA复合材料可作为一种新型的、具有良好生物活性的材料进一步研究,为将来的临床开发和应用打下基础。     

rhBMP-2复合制剂用于犬牙根尖诱导成形术的实验研究

目的观察使用基因重组人骨形成蛋白-2、Ⅰ型胶原和羟基磷灰石（rhBMP-2/Co/HA）的复合制剂作为根尖诱导剂,用于犬牙牙根未发育完成的年轻恒牙的慢性根尖周炎的根管充填,观察其根尖诱导效果。方法制备杂种家犬牙根未发育完成的年轻恒牙慢性根尖周炎的动物实验模型。用rhBMP-2/Co/HA复合制剂作为根尖诱导剂充填到犬牙的根管内,对照组用氢氧化钙糊剂。12周后用X线片及组织学方法观察2种不同的根尖诱导剂根管充填后牙根延长、根尖封闭的效果。结果实验组全部牙根延长,根尖形成并闭合,闭合率100%;对照组45个牙根延长,根尖形成并闭合,闭合率93.75%;实验组和对照组的根尖闭合率无显著差异（P〉0.05）,且根尖均有不同程度的硬组织形成。结论 rhBMP-2/Co/HA复合制剂具有良好的生物活性和根尖诱导作用。     

rhBMP-2/hTGF-β1/胶原复合材料的活性研究

目的:检测不同浓度的rhBMP-2/hTGF-β1/胶原复合材料的生物学活性.方法:通过细胞计数(MTT法),碱性磷酸酶(ALP)活性检测,对不同浓度的rhBMP-2/bTGF-β1/胶原复合材料的活性进行检测.结果:不同浓度的rhBMP-2/hTGF-β1/胶原复合材料对体外培养的Beagle犬骨髓基质细胞(MSC)的增殖和碱性磷酸酶的含量的影响不同.结论:rhBMP-2/hTGF-β1/胶原复合材料的生物学活性有剂量依赖性.     

rhBMP-2/hTGF-β1/胶原复合材料的细胞活性研究

目的：检测不同浓度的rhBMP-2/hTGF-β1/胶原复合材料的生物学活性。方法：通过细胞计数（MTT法）,碱性磷酸酶（ALP）活性检测,对不同浓度的rhBMP-2/hTGF-β1/胶原复合材料的活性进行检测。结果：不同浓度的rhBMP-2/hTGF-β1/胶原复合材料对体外培养的Beagle犬骨髓基质细胞（MSC）的增殖和碱性磷酸酶的含量的影响不同。结论：rhBMP-2/hTGF-β1/胶原复合材料的生物学活性有剂量依赖性,和细胞浓度也有关。     

复合膜异位诱导成骨研究

目的将基因重组人骨形态发生蛋白-2（rhBMP-2）与胶原、聚乳酸/聚羟基乙酸共聚体（PLGA）复合,制成rhBMP-2/Co/PLGA复合膜,观察其异位诱导成骨情况,评价其生物特性。方法取雄性昆明小鼠48只,随机分成Co/PLGA复合膜组与rhBMP-2/Co/PLGA复合膜组。在小鼠右后肢大腿肌间隙内分别植入2种复合膜材料。术后7、14、28 d,各组分别处死8只动物,标本行肉眼、软X线照相和组织学观察。结果2组动物术后情况良好。Co/PLGA复合膜组术后7 d复合膜开始溶解,14 d膜结构不连续,28 d大部分溶解;软X线和组织学观察未见异位骨生成。rhBMP-2/Co/PLGA复合膜组软X线和组织学观察均有骨样结构形成,与Co/PLGA复合膜组相比复合膜降解速度缓慢。结论Co/PLGA是rhBMP-2的良好载体,参与rhBMP-2诱导成骨活性的表达,使其在局部发挥更持久的生物活性。     

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目的    检测rhBMP-2/hTGF-β1 /胶原复合材料与rhBMP-2/hTGF-β1生物学活性的差别。方法        本研究于2010年5—12月在福建医科大学附属口腔医院实验室进行。分离培养的Beagle犬骨髓基质细胞中加入质量浓度相同的rhBMP-2/hTGF-β1或rhBMP-2/hTGF-β1 /胶原复合材料后继续培养4 d,通过细胞计数（MTT法）、碱性磷酸酶（ALP）活性检测,分别对rhBMP-2/hTGF-β1 /胶原复合材料和rhBMP-2/hTGF-β1的生物学活性进行检测,并比较其活性差别。结果    rhBMP-2/hTGF-β1/胶原复合材料和rhBMP-2/hTGF-β1均具有生物学活性,并且其生物学活性差异无统计学意义（P > 0.05）。结论    这种合成rhBMP-2/hTGF-β1 /胶原复合材料的方法不会改变生长因子的生物学活性。     

rhBMP-2/Nha/Co复合膜异位成骨的实验研究

目的:观察rhBMP- 2/nHA/Co复合膜异位诱导成骨的能力.方法: 构建nHA/Co 复合膜和rhBMP- 2/nHA/Co复合生物膜,分别植入裸鼠后肢大腿肌囊,于10、20、30 d后观察成骨情况,并作比较.结果: rhBMP/nHA/Co组材料在裸鼠肌囊内出现骨样结构,异位成骨的能力较nHA/Co组强.结论: 动物实验中Co、nHA均为BMP的良好缓释载体,此复合膜能有效的诱导异位成骨.     

rhBMP-2/Co/PLGA复合生物膜对腭裂骨缺损修复的实验研究

目的:观察引导性骨再生(guidedboneregeneration,GBR)技术修复腭裂骨缺损的效果。方法:16只成年犬分为2组:实验组8只,对照组8只。实验组将rhBMP-2/Co/PLGA复合生物膜植入骨缺损区,对照组植入Co/PLGA复合生物膜。分别于术后第4、8、12、24周,对其上颌骨进行三维CT扫描观察,同时进行新骨组织学检查。结果:从影像学、组织学检查的结果看,实验组的新骨形成优于对照组。结论:植入rhBMP-2/Co/PLGA复合生物膜可用于修复腭裂骨缺损。     

消旋聚乳酸复合rhBMP-2/hTGF-β1诱导小鼠成骨及其机制的研究

目的:评价消旋聚乳酸(PDLLA)载体材料复合重组rhBMP-2和hTGF-β1诱导小鼠体内异位成骨的能力,初步探讨其作用机制。方法:建立诱导RCI小鼠股部肌内异位成骨的18只动物模型,实验组(各6只)分别植入PDLLA/rhBMP-2/hTGF-β1和PDLLA/rhBMP-2复合材料,对照组(6只)植入PDLLA材料。采用图像处理技术、组织病理学和RT-PCR等分子生物学方法,对消旋聚乳酸载体材料复合2种细胞因子诱导小鼠体内异位成骨能力进行评价;并对其诱导异位成骨过程中,II型胶原蛋白、骨桥蛋白和骨钙蛋白等骨基质蛋白基因表达进行检测。结果:PDLLA/rhBMP-2/hTGF-β1和PDLLA/rhBMP-2组复合材料均具有明显的诱导小鼠肌内异位成骨能力,而单纯PDLLA载体材料无诱导异位成骨能力,表现为小鼠股部复合材料植入区的相对密度指数存在显著性差异。II胶原、骨桥蛋白和骨钙蛋白及其基因在2种细胞因子复合材料植入组中均有较高表达,而在单纯PDLLA组则无表达。结论:PDLLA复合rhBMP-2/hTGF-β1具有诱导异位成骨作用,软骨内成骨是其诱导小鼠体内异位成骨的主要方式。     

rhBMP-2/hTGF-β1/胶原复合材料的初步研究

目的合成rhBMP-2／hTGF—β1／胶原复合材料，检测其生物相容性和活性。方法 用胶原复合rhBMP-2／hTGF—β1形成复合材料，通过兔眼结膜刺激试验和溶血试验对其生物相容性进行检测，观察复合材料对Beagle犬骨髓基质细胞（MSC）生长及增殖的影响，通过细胞计数（MTT法），碱性磷酸酶（ALP）活性检测，对复合材料的活性进行检测。结果 rhBMP-2／hTGF—β1／胶原复合材料呈白色棉絮状或团块状。对兔眼结膜无刺激作用，对兔血也无溶血作用。复合材料明显影响体外培养的Beagle犬骨髓基质细胞（MSC）的增殖和碱性磷酸酶水平。结论 按以上方法合成的rhBMP-2／hTGF—β1／胶原复合材料具有良好的生物相容性和生物学活性。     

Exogenous recombinant human BMP-2 has little initial effects on human osteoblastic cells cultured on collagen type I coated/noncoated hydroxyapatite ceramic granules.

PURPOSE: Tissue engineering of bone entails the successful interplay between osteoinductive factors, osteogenic cells, their extracellular environment, and an osteoconductive biomaterial scaffold. Naturally produced ceramics, like hydroxyapatite (HA) calcified from red algae, are the most promising materials for use as scaffolds in this field. We hypothesized that extracellular matrix compartments and osteoinductive factors could further ameliorate the bioactivity of the scaffold. MATERIALS AND METHODS: Osteosarcoma cells with proven osteogenic phenotype (SaOS-2) were cultured onto type I collagen coated (Coll I/HA) and noncollagen coated HA granules (NC/HA) gained from red algae (C GRAFT/Algipore). Cells grown on tissue culture polystyrene dishes (TCPS) were used as controls. Second, SaOS-2 cells cultured on Coll I/HA, NC/HA, and TCPS were treated with recombinant human bone morphogenetic protein-2 (rhBMP-2) in different concentrations (10, 100, and 500 ng/mL). Non rhBMP-2-treated cultures were used as controls. Cultures of both experiments were grown under osteogenic differentiation conditions and after 24, 48, and 72 hours assays for cell viability, apoptosis, alkaline phosphatase activity (ALP), and osteocalcin (OC) secretion were done. RESULTS: Coating of HA granules with type I collagen showed higher cell viability in rhBMP-2-treated and nontreated cells. Supplementation of cultured cells with exogenous rhBMP-2 showed a dose-dependent effect only in the TCPS group. No alterations of the apoptotic rate within 1 investigation group were found. Addition of rhBMP-2 did not significantly alter the specific OC secretion of cells grown on Coll I/HA and TCPS. CONCLUSION: These in vitro findings show that in the initial period of cultivation and up to 72 hours, the coating of HA granules with collagen type I had positive effects on cell viability and osteoblastic characteristics of osteoblastic cells. In contrast, the supplementation with exogenous rhBMP-2 shows no dose-dependent effects. The combination of collagen type I and exogenous rhBMP-2 did not ameliorate the bioactivity of hydroxyapatite calcified from red algae in the initial period of cultivation.     

Bone morphogenetic protein-2 enhances bone formation when delivered by a synthetic matrix containing hydroxyapatite/tricalciumphosphate

PURPOSE: The aim of the present study was to test whether or not a synthetic matrix consisting of a polyethylene glycol (PEG) hydrogel containing recombinant human bone morphogenetic protein-2 (rhBMP-2) combined with grafting materials enhances bone regeneration compared with grafting alone or empty control sites. MATERIAL AND METHODS: In each of 10 rabbits, four titanium cylinders were screwed in perforated slits made in the external cortical bones of the calvaria. The following four treatment modalities were randomly allocated: (1) empty control, (2) a combination of a PEG matrix and hydroxyapatite/tricalciumphosphate (HA/TCP) granules and a combination of a PEG matrix containing either 10 microg/ml (3) or 30 microg/ml (4) of BMP-2 and HA/TCP granules. After 8 weeks, the animals were sacrificed and ground sections were obtained for histological analysis. For statistical analysis repeated measures ANOVA and subsequent pairwise Student's t-test were applied (P<0.01). RESULTS: Histomorphometric analysis showed an average area fraction of newly formed bone of 13.96+/-5.98% for the empty control, 15.16+/-7.95% for the PEG and HA/TCP group, 26.32+/-8.56% for the group containing 10 mug rhBMP-2/ml, and 30.15+/-7.63% for the group containing 30 microg rhBMP-2/ml. Statistical analysis revealed significantly more newly formed bone in the two rhBMP-2 groups compared with the PEG and HA/TCP group and with the empty control. Regarding the surface fraction of the HA/TCP graft particles covered with newly formed bone the addition of rhBMP-2 revealed a more than two-fold increase compared with cylinders containing HA/TCP granules without rhBMP-2. This difference reached statistical significance. CONCLUSIONS: It is concluded that rhBMP-2 significantly enhances bone regeneration in rabbits when delivered by a synthetic matrix containing HA/TCP. This synthetic PEG matrix containing HA/TCP granules apparently fulfills a number of criteria required for an ideal carrier system for rhBMP-2.     

rhBMP-2及其纳米微球缓释系统对成骨细胞钙结节影响的实验研究

目的 :观察rhBMP 2聚氰基丙烯酸正丁酯纳米微球缓释系统对成骨细胞钙结节的影响。方法 :培养诱导成骨细胞形成钙结节 ,并对其行特异性染色 ;在rhBMP 2纳米微球对钙结节作用后 ,采用扫描电镜观察其表面形态的改变 ,采用能谱仪分析比较其组成成分的改变。结果 :rhBMP 2纳米微球能明显促进钙结节形成 ,且rhBMP 2纳米微球作用后钙结节中钙磷含量显著高于单纯rhBMP 2组。结论 :rhBMP 2纳米微球缓释系统促进成骨的作用优于单纯rhBMP 2。     

Experience with freeze-dried PGLA/HA/rhBMP-2 as a bone graft substitute.

We investigated bone induction by recombinant human bone morphogenetic protein-2 in rodents. The purpose of this study was to evaluate the osteoinductive potential of a resorbable bone substitute fabricated from freeze-dried poly(glycolic acid-co-lactic acid) (PGLA) mixed with hydroxyapatite particles incorporated with bone morphogenetic protein-2 in skull defects of rats (FD-PGLA/HA/rhBMP-2). The FD-PGLA/HA/rhBMP-2 composite or as a control, the FD-PGLA/HA by itself were implanted in skull defects (psi 8 mm) of rats. The samples were harvested at 2 or 4 weeks postoperatively and were studied radiographically and histologically. Four weeks after implantation, the FD-PGLA/HA/rhBMP-2 discs were completely replaced by newly-formed bone possessing bone marrow. In contrast, the defects implanted with FD-PGLA/HA were filled only with fibrous connective tissue. The results suggest that the FD-PGLA/HA/rhBMP-2 composite could be an optimum bone substitute with osteoinductive potential and could function as an alternative bone graft material for autogenous bone in humans.     

Comparative study of intramuscular and intraskeletal osteogenesis by recombinant human bone morphogenetic protein-2

Objective. The purpose of this study was to compare the osteoinducing activity of recombinant human bone morphogenetic protein-2 (rhBMP-2) at intramuscular and intraskeletal sites in rats. Study Design. Five μg of rhBMP-2 was implanted into the right calf muscle of each of 20 rats and into a hole (4 mm in diameter, 1.5 mm in depth) that was made in the mandibular body of each of 20 other rats, with atelopeptide type I collagen as a carrier. The alkaline phosphatase activity and calcium content were quantitatively analyzed 1, 3, 7, and 21 days after the implantation of rhBMP-2 into either mandibular bone (in the intraskeletal group) or calf muscle (in the intramuscular group). The new bone formation was evaluated histologically 21 days after implantation. Results. On days 1 and 3, the alkaline phosphatase activity and calcium content in the intraskeletal group showed no significant differences from those in the intramuscular group. On the 7th and 21st days after implantation, however, the alkaline phosphatase activity and calcium content in the intraskeletal group were significantly higher than those in the intramuscular group. Histometry of the microscopic views showed that the mean trabecular area was 0.87 mm² in the intramuscular group and 2.66 mm² in the intraskeletal group. Conclusions. These results suggest that the new bone formation stimulated by rhBMP-2 in the intraskeletal group was greater than in the intramuscular group.(Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1999;87:34-8)     

Evaluation of recombinant human bone morphogenetic protein-2 on the repair of alveolar ridge defects in baboons

BACKGROUND: The objective of this study was to evaluate alveolar ridge augmentation following surgical implantation of recombinant human bone morphogenetic protein-2 (rhBMP-2) using two novel space-providing carrier technologies in the baboon (Papio anubis) model. METHODS: Standardized alveolar ridge defects ( approximately 15 x 8 x 5 mm) were surgically produced in maxillary and mandibular edentulous areas in four baboons. The defect sites were implanted with rhBMP-2 (0.4 mg/mL) in a tricalcium phosphate/hydroxyapatite/ absorbable collagen sponge composite (TCP/HA/ACS) or calcium phosphate cement (alpha-BSM). Control treatments were TCP/HA/ACS and ?-BSM without rhBMP-2 and sham surgery. Stainless steel pins were placed at the mid-apical and coronal level of the defect sites to provide landmarks for clinical measurements pre- and post-implantation. Impressions were obtained pre- and postimplantation to determine changes in alveolar ridge volume. Radiographic registrations were obtained pre- and post-implantation. Block sections of the defect sites were harvested at week 16 postimplantation and processed for histometric analysis including new bone area and bone density. Statistical comparisons between treatments were made using a mixed effect generalized linear model using least squares estimation. RESULTS: The carrier systems without rhBMP-2 provided a modest ridge augmentation. The addition of rhBMP-2 resulted in an almost 2-fold increase in alveolar ridge width, including a greater percentage of trabecular bone and a higher bone density compared to controls (P < or =0.05) without significant differences between the two rhBMP-2 protocols. CONCLUSIONS: TCP/HA/ACS and alphaBSM appear to be suitable carrier technologies for rhBMP-2. Alveolar augmentation procedures using either technology combined with rhBMP-2, rather than stand-alone therapies, may provide clinically relevant augmentation of alveolar ridge defects for placement of endosseous dental implants.     

Influence of bone morphogenetic protein and proportion of hydroxyapatite on new bone formation in biphasic calcium phosphate graft: Two pilot studies in animal bony defect model

PurposeThe purpose of these two pilot studies using animal bony defect models was to evaluate the influence of bone morphogenetic protein (BMP) and proportion of hydroxyapatite (HA)/beta-tricalcium phosphate (β-TCP) in biphasic calcium phosphate (BCP) graft on new bone formation.MethodsIn this study, four kinds of synthetic osteoconductive bone materials known for bone growth scaffold, OSTEON™II(HA:β-TCP 30:70), OSTEON™III (HA:β-TCP 20:80), OSTEON™II Collagen, and OSTEON™III Collagen, were prepared as BCP graft materials. In pilot study 1, three BCP materials (OSTEON™II, OSTEON™III, and OSTEON™II Collagen) were grafted in rabbit calvarial defects after impregnating in rhBMP-2. OSTEON™II without the rhBMP-2 impregnation was included in the study as the control. The amount of new bone was examined and measured histologically at 2, 4, and 8 weeks. In pilot study 2, four BCP materials (OSTEON™II, OSTEON™III, OSTEON™II Collagen, and OSTEON™III Collagen) were grafted in beagle dog mandibular defects after soaking in the rhBMP-2. The amount of total bone and new bone were measured three-dimensionally using microCT and healing process was examined histologically at 2, 4, and 8 weeks.ResultsIn pilot study 1, rhBMP-2 impregnated groups showed more new bone formation than the rhBMP-2 free group. In pilot study 2, increased new bone formation was observed in time-dependent manner after graft of BCP and BCP-collagen (OSTEON™II, OSTEON™III, OSTEON™II Collagen, and OSTEON™III Collagen) impregnated with rhBMP-2. Also, BCP with a higher proportion of HA (30% HA) showed more favorable result in new bone formation and space maintenance, especially at the 8 weeks.ConclusionFrom the results of the pilot studies, rhBMP-2 played positive roles in new bone formation and BCP could become a scaffold candidate for rhBMP-2 impregnation to induce new bone formation. Moreover, BCP with a higher proportion of HA (30% HA) could be considered more appropriate for rhBMP-2 carrier.