The inhibitory effect of zoledronate on early-stage osteoinduction by recombinant human bone morphogenetic protein 2 in an osteoporosis model

Abstract

This study evaluated the effect of the combined treatment of intravenous zoledronic acid (ZA, 0.08?mg/kg) and rhBMP-2 (5?µg) on osteogenesis in a calvarial defect model of ovariectomized SD rats. New bone formation was evaluated 4 or 8 weeks after calvarial defect implantation using micro-CT and histology. Micro-CT results revealed that the rhBMP-2 group showed significantly higher calvarial defect coverage ratio compared with the ZA?+?rhBMP-2 group at 4 weeks. In addition, bone formation indices were significantly lower in ZA?+?rhBMP-2 group when compared with the rhBMP-2 group after 4 weeks, which indicates a negative effect of ZA on the initial bone formation and the bone quality. At 8 weeks, the negative effect induced by ZA treatment was alleviated as time passed. Histological examination showed similar results to the micro-CT measurements. In conclusion, although ZA treatment lowered the new bone formation induced by rhBMP-2 initially, as time passed, the negative effect was decreased.     

Bone regeneration using photocrosslinked hydrogel incorporating rhBMP-2 loaded 2-N, 6-O-sulfated chitosan nanoparticles

Although rhBMP-2 has excellent ability to accelerate the repair of normal bone defects, limitations of its application exist in the high cost and potential side effects. This study aimed to develop a composite photopolymerisable hydrogel incorporating rhBMP-2 loaded 2-N, 6-O-sulfated chitosan nanoparticles (PH/rhBMP-2/NPs) as the bone substitute to realize segmental bone defect repair at a low growth factor dose. Firstly rhBMP-2 loaded 2-N, 6-O-sulfated chitosan nanoparticles (rhBMP-2/NPs) were prepared and characterized by DLS and TEM. Composite materials, PH/rhBMP-2/NPs were developed and investigated by SEM-EDS as well as a series of physical characterizations. Using hMSCs as an in vitro cell model, composite photopolymerisable hydrogels incorporating NPs (PH/NPs) showed good cell viability, cell adhesion and time dependent cell ingrowth. In vitro release kinetics of rhBMP-2 showed a significantly lower initial burst release from the composite system compared with the growth factor-loaded particles alone or encapsulated directly within the hydrogel, followed by a slow release over time. The bioactivity of released rhBMP-2 was validated by alkaline phosphatase (ALP) activity as well as a mineralization assay. In in vivo studies, the PH/rhBMP-2/NPs induced ectopic bone formation in the mouse thigh. In addition, we further investigated the in vivo effects of rhBMP-2-loaded scaffolds in a rabbit radius critical defect by three dimensional micro-computed tomographic (μCT) imaging, histological analysis, and biomechanical measurements. Animals implanted with the composite hydrogel containing rhBMP-2-loaded nanoparticles underwent gradual resorption with more pronounced replacement by new bone and induced reunion of the bone marrow cavity at 12 weeks, compared with animals implanted with hydrogel encapsulated growth factors alone. These data provided strong evidence that the composite PH/rhBMP-2/NPs are a promising substitute for bone tissue engineering.     

Improving bone formation in a rat femur segmental defect by controlling bone morphogenetic protein-2 release

Nonunion is a common complication in open fractures and other severe bone injuries. Recombinant human bone morphogenetic protein-2 (rhBMP-2) delivered on a collagen sponge enhances healing of fractures. However, the burst release of rhBMP-2 necessitates supra-physiological doses of rhBMP-2 to achieve a robust osteogenic effect, which introduces risk of ectopic bone formation and severe inflammation and increases the cost. Although the concept that the ideal pharmacokinetics for rhBMP-2 includes both a burst and sustained release is generally accepted, investigations into the effects of the release kinetics on new bone formation are limited. In the present study, biodegradable polyurethane (PUR) and PUR/microsphere [PUR/poly(lactic-co-glycolic acid)] composite scaffolds with varying rhBMP-2 release kinetics were compared to the collagen sponge delivery system in a critical-sized rat segmental defect model. Microcomputed tomography analysis indicated that a burst followed by a sustained release of rhBMP-2 from the PUR scaffolds regenerated 50% more new bone than the collagen sponge loaded with rhBMP-2, whereas a sustained release without the burst did not form significantly more bone than the scaffold without rhBMP-2. This study demonstrated that the putative optimal release profile (i.e., burst followed by sustained release) for rhBMP-2 can be achieved using PUR scaffolds, and that this enhanced pharmacokinetics regenerated more bone than the clinically available standard of care in a critical-sized defect in rat femora.     

中空羟基磷灰石复合rhBMP-2修复骨缺损过程的再血管化研究

目的　探讨和观察中空羟基磷灰石复合rhBMP-2在骨缺损修复过程的再血管化。  方法　将48只成年的新西兰雄性大白兔制作成桡骨骨缺损模型,随机分3组,各组分别植入以下材料：中空HA/ rhBMP-2复合人工骨、单纯中空HA人工骨、单纯rhBMP-2。植入后于4、8、12、16周分别注射99mTc-MDP进行放射性核素骨显像并监测骨缺损修复过程中再血管化情况,同时进行大体、X线、组织学观察。  结果　术后各时间段,中空HA/ rhBMP-2复合人工骨组在X线及放射性核素聚集强度明显高于单纯中空HA人工骨组(P<0.05) ,表现为成骨代谢活跃及早期的再血管化能力。  结论　中空HA/ rhBMP-2复合人工骨具有良好的骨缺损修复能力,成骨活性持久,再血管化能力强,有望成为一种理想的骨缺损修复材料。     

复合重组人骨形态发生蛋白2壳聚糖缓释水凝胶的新型HA/ZrO2多孔泡沫陶瓷人工椎体修复犬脊椎骨缺损

目的 研制复合重组人骨形态发生蛋白2(rhBMP-2)壳聚糖水凝胶的新型HA/ZrO₂多孔泡沫陶瓷人工椎体,并观察其修复beagle犬椎体骨缺损的能力。方法 离子交联法制备壳聚糖水凝胶作为rhBMP-2的缓释载体,扫描电镜下观察其微观形态,检测其载药量、包封率及缓释速率。将HA/ZrO₂多孔泡沫陶瓷人工椎体复合rhBMP-2壳聚糖水凝胶,构建新型HA/ZrO₂多孔泡沫陶瓷人工椎体。将12只beagle犬按数字表法随机分为3组,每组4只;均采用手术造成半径9 mm、高23 mm的半圆柱状L₄椎骨缺损模型,其中A组植入复合rhBMP-2壳聚糖水凝胶的新型HA/ZrO₂多孔泡沫陶瓷人工椎体,B组植入复合空白干燥壳聚糖的新型HA/ZrO₂多孔泡沫陶瓷人工椎体,C组植入实验犬自体髂骨。术后6、12、24周对实验犬行大体观察、X线影像学观察;术后24周取实验犬椎体标本行离体Micro CT新生骨量检测及生物力学检测。结果 制备所得壳聚糖水凝胶扫描电镜下呈3D网状结构,内部均匀分布壳聚糖微球,其负载rhBMP-2后包封率达91.88%±1.53%,载药量为(39.84±2.34)ng/mg;释放率第1天为28.32%±3.01%,第3天为48.92%±6.27%,第12天为74.40%±6.29%。术后6周C组动物平均活动度恢复较A组和B组快(P值均＜0.05),A、B组差异无统计学意义(P＞0.05);术后12、24周B组与C组活动度比较差异均有统计学意义(P值均＜0.05),A、C组差异均无统计学意义(P值均＞0.05)。术后6、12、24周X线影像学观察显示,A组椎体置换术后骨痂生成逐渐增多,植入材料与宿主骨之间的界线逐渐模糊,至24周时人工椎体周围新生骨与自体骨融为一体;C组在24周时出现明显非承重部位的骨吸收,出现较快的自体骨塑形;B组椎体置换术后人工椎体与自体骨的融合速度慢于A组和C组。术后24周标本Micro CT新生骨量检测结果显示,A组(145.38±18.52)mm³,B组(86.30±15.60)mm³,两组比较差异有统计学意义(t=4.879, P＜0.01)。术后24周A、B、C组手术节段椎体标本抗压强度分别为(14.03±1.67) MPa、(8.62±1.24) MPa、(13.79±1.43) MPa,A组和C组椎体极限抗压强度均高于B组(P值均＜0.01),而A组与C组比较差异无统计学意义(P＞0.05)。结论 复合rhBMP-2壳聚糖水凝胶新型HA/ZrO₂多孔泡沫陶瓷人工椎体能有效修复脊柱骨缺损,有望代替自体髂骨移植运用于临床骨缺损的修复。     

The effects of rhBMP-2 released from biodegradable polyurethane/microsphere composite scaffolds on new bone formation in rat femora

Scaffolds prepared from biodegradable polyurethanes (PUR) have been investigated as a supportive matrix and delivery system for skin, cardiovascular, and bone tissue engineering. While previous studies have suggested that PUR scaffolds are biocompatible and moderately osteoconductive, the effects of encapsulated osteoinductive molecules, such as recombinant human bone morphogenetic protein (rhBMP-2), on new bone formation have not been investigated for this class of biomaterials. The objective of this study was to investigate the effects of different rhBMP-2 release strategies on new bone formation in PUR scaffolds implanted in rat femoral plug defects. In the simplest approach, rhBMP-2 was added as a dry powder prior to the foaming reaction, which resulted in a burst release of 35% followed by a sustained release for 21 days. Encapsulation of rhBMP-2 in either 1.3-micron or 114-micron PLGA microspheres prior to the foaming reaction reduced the burst release. At 4 weeks post-implantation, all rhBMP-2 treatment groups enhanced new bone formation relative to the scaffolds without rhBMP-2. Scaffolds incorporating rhBMP-2 powder promoted the most extensive new bone formation, while scaffolds incorporating rhBMP-2 encapsulated in 1.3-micron microspheres, which exhibited the lowest burst release, promoted the least extensive new bone formation. Thus our observations suggest that an initial burst release followed by sustained release is better for promoting new bone formation.     

RhBMP-2 microspheres-loaded chitosan/collagen scaffold enhanced osseointegration: an experiment in dog

The purpose of this study is to develop a novel recombinant human bone morphogenetic protein-2 (rhBMP-2) sustained release scaffold for dental implant osseointegration, and to evaluate the effect of this scaffold on promoting bone formation. RhBMP-2 was encapsulated in the poly-D,L-lactide-co-glycolide (PLGA) biodegradable microspheres, which were subsequently dispersed in a chitosan/collagen composite scaffold. This rhBMP-2 microspheres-loaded scaffold (S-MB) was compared with a chitosan/collagen scaffold without microspheres that directly encapsulated rhBMP-2 (S-B) in vitro and in vivo. The microstructure of the new scaffold was examined with scanning electron microscopy. The release profile of rhBMP-2 in vitro was measured at interval periods. The effect of rhBMP-2 encapsulated scaffolds on enhancing bone formation through implantation in dogs' mandibles was identified by histological examination of the regenerated bone after 4 weeks of implantation. Due to PLGA microspheres being loaded, the S-MB exhibited lower values at porosity and swelling rate, as well as a higher effective release dose than that of the S-B. Bone density, bone-implant contact, and bone-fill values measured from dog experiments demonstrated that the S-MB induced bone regeneration more quickly and was timely substituted by new bone. It was concluded that this sustained carrier scaffold based on microspheres was more effective to induce implant osseointegration.     

Repair of long intercalated rib defects in dogs using recombinant human bone morphogenetic protein-2 delivered by a synthetic polymer and beta-tricalcium phosphate

Long intercalated defects in canine ribs can be repaired successfully using porous beta-tricalcium phosphate (beta-TCP) cylinders, infused with a biodegradable polymer (poly D,L-lactic acid-polyethylene block copolymer) containing recombinant human bone morphogenetic protein-2 (rhBMP-2). We previously reported the successful regeneration of bony rib and periosteum defects using beta-TCP cylinders containing 400 microg of rhBMP-2. To reduce the amount of rhBMP-2 and decrease the time required for defect repair, we utilized a biodegradable polymer carrier, in combination with rhBMP-2 and the porous beta-TCP cylinders. An 8 cm long section of rib bone was removed and replaced with an implant comprised of the porous beta-TCP cylinders and the polymer containing 80 microg of rhBMP-2. Six weeks after surgical placement of the beta-TCP cylinder/polymer/BMP-2 implants, new rib bone with an anatomical configuration and mechanical strength similar to the original bone was regenerated at the defect site. The stiffness of the regenerated ribs at 3, 6, and 12 weeks after implantation of the composite implant was significantly higher than that of ribs regenerated by implantation of rhBMP-2/beta-TCP implants. Thus, addition of the synthetic polymer to the drug delivery system for BMP potentiated the bone-regenerating ability of the implant and enabled the formation of mechanically competent rib bone. This new method appears to be applicable to the repair of intercalated long bone defects often encountered in clinical practice.     

Comparative study between coral-mesenchymal stem cells-rhBMP-2 composite and auto-bone-graft in rabbit critical-sized cranial defect model

Tissue engineered bone has become a bone substitute for the treatment of bone defects in animal research. This study investigated the osteogenesis capacity of coral-MSCs-rhBMP-2 composite with the auto-bone-graft as control. Coral-MSCs-rhBMP-2 composite were fabricated by coral (as main scaffold), rhBMP-2 (as growth factor), and MSCs (cultured from iliac marrow as seed cells). Critical-sized defects (d = 15 mm) were made on forty rabbits crania and treated by different composite scaffolds: iliac autograft (n = 8), coral (n = 8), rhBMP-2/coral (n = 8), and MSCs/rhBMP-2/coral (n = 8). The defects were evaluated by gross observation, radiographic examination, histological examination, and histological fluorescence examinations after 8 and 16 weeks. The results showed that repair of bone defect was the least in coral group, and significant ingrowth of new bone formation and incorporation could be seen with 77.45% +/- 0.52% in radiopacity in MSCs/rhBMP-2/coral group, which was similar to that in iliac autograft group (84.61% +/- 0.56% in radiopacity). New bone formation in MSCs/rhBMP-2/coral group was more than that in rhBMP-2/coral group. And osteogenesis rate in MSCs/rhBMP-2/coral group (10.23 +/- 1.45 microm) was much faster than that in rhBMP-2/coral group (5.85 +/- 2.19 microm) according to histological fluorescence examination. Newly formed bone partly came from induced MSCs in composite scaffold according to bromodeoxyuridine immunohistochemical examination. These data implicated that MSCs could produce synergic effect with coral-rhBMP-2, and the tissue engineered bone of coral-MSCs-rhBMP-2 is comparable to auto-bone-graft for the repair of critical-sized bone defect.     

Ectopic bone formation in mice associated with a lactic acid/dioxanone/ethylene glycol copolymer-tricalcium phosphate composite with added recombinant human bone morphogenetic protein-2

A new putty-like material with bone-inducing capacity was made by combining a block copolymer of poly d,l-lactic acid with randomly inserted p-dioxanone and polyethylene glycol (PLA-DX-PEG) and beta-tricalcium phosphate (beta-TCP) powder with added recombinant human bone morphogenetic protein-2 (rhBMP-2). To optimize the material's efficacy for bone formation, we formulated the optimal composition ratio of the respective constituent that gives the greatest osteoinductive efficacy in a mouse model of ectopic bone formation. In this series of studies, we investigated the size of ectopic bone mass induced 3 and 6 weeks after implantation of the materials composed of 30 mg of PLA-DX-PEG with 2 microg of rhBMP-2 and 0, 15, 30, or 60 mg of beta-TCP powder. An additional experiment was designed to investigate how content ratios of beta-TCP powder in 30 mg-putty implants (0%, 16.7%, 33.3%, 50%, 66.7%, 83.3%, or 100%) for a fixed dose (5 microg) of the rhBMP-2 altered the size of the induced ossicle. The results from the first experiment indicated that the bone yields were linearly dependent on the amount of additional beta-TCP powder. In the second experiment, the largest ossicles induced by 5 microg of rhBMP-2 were obtained when the polymer/beta-TCP ratio was 1/2 in mice. The data provide important insights into the fabrication of implants that provide efficacious delivery of rhBMP-2. The new putty-like material may be valuable for repairing or regenerating bone in a clinical setting.     

Segmental bone regeneration using an rhBMP-2-loaded gelatin/nanohydroxyapatite/fibrin scaffold in a rabbit model

We aimed to develop a hybrid scaffold with a porous structure and similar composition as natural bone for the controlled release of bone morphogenetic protein-2 (BMP-2) to enhance bone regeneration. We fabricated a gelatin/nanohydroxypatite (nHAP) scaffold by glutaraldehyde chemical cross-linking a gelatin aqueous solution with nHAP granules at a 5:1 ratio (v/w). Then, fibrin glue (FG) mixed with recombinant human BMP-2 (rhBMP-2) was infused into the gelatin/nHAP scaffold and lyophilized to develop an rhBMP-2-loaded gelatin/nHAP/FG scaffold. On scanning electron microscopy, the composite had a 3-D porous structure. The rhBMP-2 release kinetics from the hybrid scaffold was sustained and slow, and release of rhBMP-2 was complete at 40 days. Immunohistochemistry, azo-coupling and alizarin S-red staining were used to study in vitro differentiation of human bone-marrow mesenchymal cells (hBMSCs). Strong positive staining results confirmed that rhBMP-2 released from the scaffold could improve osteocalcin (OCN) and alkaline phosphatase (ALP) expression and calcium deposition formation. RT-PCR results showed significantly high mRNA expression of ALP and OCN in hBM-MSCs cultured on the gelatin/nHAP/FG scaffold with rhBMP-2. DNA assay demonstrated that the scaffold was noncytotoxic and could promote hBMSC proliferation from the components of the hybrid scaffold, not released rhBMP-2. The hybrid scaffolds were then used to repair critical-size segmental bone defects of rabbit radius. Gross specimen, X-ray, bone histomorphology and bone mineral density assay demonstrated that the rhBMP-2-loaded gelatin/nHAP/FG scaffold had good osteogenic capability and could repair the segmental bone defect completely in 12 weeks.     

The kinetic and biological activity of different loaded rhBMP-2 calcium phosphate cement implants in rats

The healing of large bone defects can be improved by osteogenic bone graft substitutes, due to growth factor inclusion. A sustained release of these growth factors provides more efficient bioactivity when compared with burst release and might reduce the dose required for bone regeneration, which is desirable for socioeconomical and safety reasons. In this study, we compared different rhBMP-2 loadings in a sustained release system of CaP cement and PLGA-microparticles and were able to couple kinetic to biological activity data. Fifty-two rats received a critical-size cranial defect, which was left open or filled with the cement composites. The implants consisted of plain, high, and five-fold lower dose rhBMP-2 groups. Implantation time was 4 and 12 weeks. Longitudinal in vivo release was monitored by scintigraphic imaging of (131)I-labeled rhBMP-2. Quantitative analysis of the scintigraphic images revealed a sustained release of (131)I-rhBMP-2 for both doses, with different release profiles between the two loadings. However, around 70% of the initial dose was retained in both implant formulations. Although low amounts of rhBMP-2 were released (2.4 +/- 0.8 mug in 5 weeks), histology showed defect bridging in the high-dose implants. Release out of the low-dose implants was not sufficient to enhance bone formation. Implant degradation was limited in all formulations, but was mainly seen in the high-dose group. Low amounts of sustained released rhBMP-2 were sufficient to bridge critically sized defects. A substantial amount of rhBMP-2 was retained in the implants because of the slow release rate and the limited degradation.     

Injectable rhBMP-2-loaded chitosan hydrogel composite: osteoinduction at ectopic site and in segmental long bone defect

Carriers for bone morphogenetic protein-2 (BMP-2) used in clinical practice still suffer from limitations such as insufficient protein retention. In addition, there is a clinical need for injectable carriers. The main objective of this study was to assess bone forming ability of rhBMP-2 combined either with chitosan hydrogel (rhBMP-2/CH) or chitosan hydrogel containing β-tricalcium phosphate (β-TCP) (rhBMP-2/CH/TCP). Formulations were first compared in a rat ectopic intramuscular bone formation model, and the optimal formulation was further evaluated in healing of 15-mm critical size defect in the radius of a rabbit. Three weeks after injection ectopically formed bone was analyzed by microcomputerized tomography (micro-CT) and histology. Significantly higher (4.7-fold) mineralized bone formation was observed in the rhBMP-2/CH/TCP group compared to rhBMP-2/CH group. In a pilot study, defect in a rabbit radius treated with rhBMP-2/CH/TCP showed incomplete regeneration at 8 weeks with composite leakage from the defect, indicating the need for formulation refinement when segmental defect repair is foreseen.     

重组人骨形态发生蛋白2/骨修复材料的制备与性能

BACKGROUND: It has become a hotspot to prepare the bone repair material that exhibits natural bone structure and is used in combination with biological factors. OBJECTIVE: To prepare the recombinant human bone morphogenetic protein-2 (rhBMP-2)/bone repair material, and to evaluate its capacities of release, activity and ectopic osteoinduction. METHODS: A collagen-binding domain was added to the N-terminal of native rhBMP-2 that allowed bind to collagens in the bone repair material. Then, rhBMP-2/bone repair material was obtained through freeze-dried method. The releasing ability of rhBMP-2 in vitro was assayed by ELISA. C2C12 cell lines were loaded to the composite material with 0.25, 0.5 and 1 µg rhBMP-2, respectively. Afterwards, alkaline phosphatase activity was detected at 72 hours. The composite materials with 0, 2, 5 and 10 µg rhBMP-2 were implanted into the quadriceps of Sprague-Dawley rats, respectively. Alkaline phosphatase activity and the newly formed bone were detected at 2 and 4 weeks after implantation. The CY-7-labeled composite material was implanted into the quadriceps of Sprague-Dawley rats to observe its stability. RESULTS AND CONCLUSION: Substantially no rhBMP-2 from the rhBMP-2/bone repair material was released within 45 days. The alkaline phosphatase activity of C2C12 was in a rise with the increased concentration of rhBMP-2. The stability of the composite material in vivo was better, the alkaline phosphatase activity and ectopic bone formation increased as the concentration of rhBMP-2 rose. To conclude, the rhBMP-2/bone repair material preserves the stability of rhBMP-2, and improves ectopic osteoinduction ability.     

负载骨形态发生蛋白新型骨填充材料应用于椎体成形

背景：磷酸钙骨水泥克服了聚甲基丙烯酸甲酯的诸多缺点并具有良好的生物相容性。而负载复合重组人类骨形态发生蛋白2的磷酸钙骨水泥经固化后可具有微孔结构,可提高经皮椎体成形充填材料的临床价值。 目的：探讨以可注射型磷酸钙骨水泥和纤维蛋白胶作为共同载体,复合重组人类骨形态发生蛋白2,替代聚甲基丙烯酸甲酯应用于新西兰大白兔椎体成形的可行性。 方法：制备磷酸钙骨水泥/纤维蛋白胶/复合重组人类骨形态发生蛋白2新型复合材料。采用小鼠肌袋异位诱导成骨模型对不同植入材料进行骨诱导活性评价;模仿椎体成形观察新型复合材料和聚甲基丙烯酸甲酯植入兔椎体后的生物力学改变。 结果与结论：新型复合材料植入后2,4周碱性磷酸酶水平最高,植入后4周软骨细胞逐渐成熟,新骨形成,抗压强度和抗扭转强度明显低于正常椎体和聚甲基丙烯酸甲酯植入后(P < 0.05),8周后材料被进一步降解,抗压强度和抗扭转强度均有所上升,扛扭转强度与正常椎体相比无显著差别,但仍明显低于聚甲基丙烯酸甲酯(P < 0.05)。microCT提示其新生骨形成多而早,但聚甲基丙烯酸甲酯未见材料吸收及周围骨质长入。说明新型复合材料植入椎体后能够获得良好的骨诱导和骨传导功能,材料降解和新骨替代同步,接近于正常椎体的骨愈合,可望替代聚甲基丙烯酸甲酯应用于椎体成形。     

Potential of porous poly-D,L-lactide-co-glycolide particles as a carrier for recombinant human bone morphogenetic protein-2 during osteoinduction in vivo.

Several different biodegradable bone graft materials are in clinical or preclinical use for the repair of bone defects in orthopedics, maxillofacial surgery, and periodontics. This study tested the hypothesis that poly-D,L-lactide-co-glycolide copolymer (PLG) can be used as an effective carrier of recombinant human bone morphogenetic protein-2 (rhBMP-2) and that the composite has osteoinductive ability. Porous PLG rods were shredded to a particle size ranging from 250 to 850 microm. Active and inactive demineralized freeze-dried bone allografts (DFDBA) with a comparable particle size were used as positive and negative controls, respectively. PLG particles were treated with vehicle or with 5 or 20 microg rhBMP-2. DFDBA and PLG particles were placed in gelatin capsules, mixed with vehicle or rhBMP-2, and implanted at intramuscular sites in male Nu/Nu (nude) mice. Each mouse underwent bilateral implantation with implants of the same formulation, resulting in five groups of four mice per group: active DFDBA, inactive DFDBA, PLG, PLG + 5 microg rhBMP-2, and PLG + 20 microg rhBMP-2. After 56 days, the implants were recovered and processed for histology. Bone induction was assessed by use of a semiquantitative scoring system based on the amount of new bone formed in representative histological sections. Histomorphometry was also used to measure the area of new bone formed and the area of residual implant material. The results showed that active DFDBA induced the formation of ossicles containing new bone with bone marrowlike tissue, whereas inactive DFDBA or PLG particles alone did not induce new bone. The addition of rhBMP-2 to PLG particles resulted in new bone formation that had a greater bone induction score than active DFDBA. Moreover, the histomorphometric analysis showed that the addition of rhBMP-2 to PLG particles induced the formation of a greater area of new bone and bone marrowlike tissue than active DFDBA. The resorption of the PLG particles was markedly increased with the addition of rhBMP-2, suggesting that rhBMP-2 may attract and regulate resorptive cells at the implantation site. The results of the present study indicate that PLG copolymers are good carriers for BMP and promote the induction of new bone formation. Further, the PLG copolymers with rhBMP-2 had a greater effect in inducing new bone formation and resorbing the implanted material than active DFDBA alone.     

重组人骨形态发生蛋白2-肝素-人工骨复合材料的骨诱导活性

背景：已有将重组人骨形态发生蛋白2应用于骨再生及修复的报道,但由于其在生物体内半衰期短而导致诱导骨形成的能力受到限制。 目的：制备具有较好缓释效果的重组人骨形态发生蛋白2-肝素-人工骨复合材料,并检测其缓释性能及骨诱导活性。 方法：通过高效液相色谱法检测重组人骨形态发生蛋白2-肝素复合物对于酶解的保护作用。将重组人骨形态发生蛋白2与肝素溶液混匀后复合于人工骨材料表面,ELISA方法检测其体外释药性质,茜素红染色法检测其诱导成骨细胞的能力,应用小鼠体内实验评价其异位骨诱导能力。 结果与结论：成功制备了具有良好缓释效果的重组人骨形态发生蛋白2-肝素-人工骨复合材料,具有较强的诱导骨钙蛋白及异位骨形成能力。     

Orthotopic bone formation by implantation of apatite-coated poly(lactide-co-glycolide)/hydroxyapatite composite particulates and bone morphogenetic protein-2

Bone morphogenetic proteins (BMPs) are the most potent osteoinductive growth factors. However, a delivery system is essential to take advantage of the osteoinductive effect of BMPs. In the present study, we tested the suitability of apatite-coated poly(D,L-lactide-co-glycolide)/nanohydroxyapatite (PLGA/HA) particulates as carriers for the controlled release of BMP-2. The release of BMP-2 from apatite-coated PLGA/HA particulates was sustained for at least 4 weeks in vitro. A delivery system of apatite-coated PLGA/HA particulates suspended in fibrin gel further slowed the BMP-2 release rate. In vivo implantation of either Fibrin gel + BMP-2 or Fibrin gel + apatite-coated PLGA/HA particulates showed enhanced new bone formation in critical-sized calvarial defects of rats 8 weeks after implantation, compared to implantation of fibrin gel only. Importantly, new bone formation was much higher in the defects treated with BMP-2 delivery using apatite-coated PLGA/HA particulates in fibrin gel (Fibrin gel + PLGA/HA + BMP-2 group) than in the defects treated either with apatite-coated PLGA/HA particulates in fibrin gel (Fibrin gel + BMP-2 group) or with BMP-2 delivery using fibrin gel alone (Fibrin gel + BMP-2 group). BMP-2 and osteoinductive HA had an additive effect on orthotopic bone formation. In conclusion, the apatite-coated PLGA/HA particulates showed good results as carriers for BMP-2. The BMP-2 delivery system showed high osteogenic capability in a rat calvarial bone defect model. The local and sustained delivery system for BMP-2 developed in this study may be useful as a carrier for BMP-2 and would enhance bone regeneration efficacy for the treatment of large bone defects.     

Repair of a proximal femoral bone defect in dogs using a porous surfaced prosthesis in combination with recombinant BMP-2 and a synthetic polymer carrier

Total hip arthroplasty (THA) has become an almost standard procedure for the treatment of various hip lesions. However, one of the limitations has been the mechanical loosening of the prosthesis, a condition termed peri-prosthetic osteolysis. Consequently, at revision surgery, various grades of bone defect are often noted. Alternative approaches aimed at overcoming this problem have included a special design of the revision prosthesis and allo- or autogeneic bone grafting in combination with or without biomaterials. In a further attempt to address the loosening of the prosthesis, we have combined human bone morphogenetic protein-2, produced by DNA recombination (rhBMP-2) with a new synthetic biodegradable polymer (poly-D,L-lactic-acid-para-dioxanone-polyethyleneglycol block co-polymer; PLA-DX-PEG). We present data on the efficacy of the rhBMP-2 laden prosthesis to reconstruct a bone defect in a canine model. In this model, medial half of the proximal femur was surgically resected to create a bone defect that was repaired with the rhBMP-2/PLA-DX-PEG composite. Twelve weeks after implantation, the original bone defects in the rhBMP-2 treatment groups had been repaired. Thus, this type of 'hybrid' prosthesis may provide a new modality to repair bone defects or restore lost bone mass encountered in revision arthroplasty.     

缓释型重组人骨形态发生蛋白2/壳聚糖生物骨修复材料诱导骨形成

背景：重组人骨形态发生蛋白2在体内半衰期短、易降解代谢,达不到理想的骨再生效果。 目的：制备缓释型重组人骨形态发生蛋白2/壳聚糖生物骨修复材料,并观察其缓释性能、骨诱导活性。 方法：将重组人骨形态发生蛋白2与壳聚糖混合制备壳聚糖膜,涂覆于生物骨修复材料表面,ELISA方法检测其体外释药性能。茜素红染色检测缓释型人骨形态发生蛋白2/壳聚糖生物骨材料、重组人骨形态发生蛋白2生物骨材料、单纯骨填充材料诱导C2C12细胞骨钙蛋白的形成,观察其诱导成骨细胞能力。同时将3种骨修复材料植入清洁级KM小鼠股部肌袋内,2周后检测新生骨Ca2+离子含量,评价其异位骨诱导能力。 结果与结论：材料表面的壳聚糖膜分布均匀,负载的重组人骨形态发生蛋白2呈团簇状。重组人骨形态发生蛋白2/壳聚糖生物骨修复材料体外释药存在突释,前4 d释放量达总药量的50%,持续至12 d,释药量达到90%,第18天时释放完全。与单纯骨填充材料、重组人骨形态发生蛋白2生物骨材料相比,缓释型人骨形态发生蛋白2/壳聚糖生物骨修复材料诱导C2C12细胞向成骨晚期分化能力与异位骨形成能力显著增强(P < 0.05)。结果提示缓释型人骨形态发生蛋白2/壳聚糖生物骨修复材料缓释性能好,促进骨形成能力强。