骨形态发生蛋白载体材料的研究和应用现状

骨形态发生蛋白用于骨缺损的修复需要有合适的载体，目前已经有多种材料用作BMP的载体，包括生物陶瓷，骨水泥，高分子多聚体，胶原，纤维蛋白凝块，异体骨，异种骨等，这些材料理化性能和生物学性能各不相同，因而与BMP复合后也各具特色，本介绍了不同载体与BMP复合成人体骨的各自性能特点，并回顾了各种载体材料的研究和实际应用现状。     

骨形态发生蛋白复合生物无机材料释放体系的研究进展

BMP(骨形态发生蛋白)作为一种生物活性分子,具有诱导新骨形成的能力,它与生物材料复合后作为骨替代材料以期用于临床的骨修复。理想的BMP载体材料不仅使BMP在需要修复的部位保持一定的浓度和持续的释放,而且还能成为新骨形成的支架。因此,载体材料的选择对临床应用的效果至关重要。本文将讨论BMP在骨修复中的研究情况及其释放策略,并且对BMP复合生物无机材料的特点以及当前基础实验的研究状况做了重点综述。     

体外模拟和体内植入实验分析磷酸钙骨水泥的降解活性

目的 研究CPC及CPC／BMP复合人工骨降解性能，寻找加快CPC降解的有效途径，方法将CPC作为BMP的载体制成CPC／BMP复合物，在体外模拟生理环境进行CPC和CPC／BMP复合物的溶解试验。通过植入小鼠肌袋和犬桡骨植入试验，观察材料在体内的降解情况和降解规律。结果BMP促进了CPC的体外溶解。肌肉内植入CPC／BMP可以异位诱导新骨形成。植入骨缺损后CPC／BMP可以诱导新骨形成，有效地修复骨缺损。新骨形成的同时，材料出现了较快的降解。CPC不能异位诱导新骨形成，骨缺损修复能力较弱，降解缓慢。结论CPC／BMF，生物活性人工骨具有理想的降解性能和成骨能力，可望成为新型的骨缺损修复材料。     

不同温度煅烧骨作骨形态发生蛋白载体的试验研究

采用不同温度天然煅烧骨与骨形态发生蛋白复合物进行兔桡骨骨缺损修复的动物试验。发现600℃的煅烧骨与骨形态发生蛋白的复合材料（BMP／SB）与900℃及1200℃的BMP／SB材料在诱导成骨及修复骨缺损中作用不如后两者。900℃及1200℃的BMP／SB材料在新骨形成量上要高于600℃的BMP／SB材料（P〈0．05），而后两者间无明显差别（P〉0.05）。但900℃的煅烧骨材料力学强度大，制备温度相对低，故经济效益明显。研究提示900℃的的煅烧骨与600℃、1200℃的煅烧骨相比，更适于用作骨形态发生蛋白的载体。     

生物陶瓷及其BMP和TGF复合物修复节段性骨缺损及成骨效应的研究

目的 制备生物活性陶瓷细胞外基质复合材料,并通过动物体内骨植入研究,探讨人工骨材料对骨缺损的修复作用及相互作用机理,为骨替代材料临床应用提供依据.方法 合成TCP、CHA、HA,并将其作为BMP的载体,制备成TCP/BMP、CHA/BMP复合材料,植入兔桡骨10 mm骨缺损,同时加入TGF-β1,同时以单纯的TCP、CHA、HA材料组做对照.通过X线摄片及生物力学测定,扫描电镜及组织学染色分析等手段,观察新骨形成及材料的降解规律.结果 术后动物均无不良反应.复合材料组植入2周时,骨断端部位可见软骨细胞;4周时可见新生软骨及骨组织,并向板层骨转化;8周时材料界面可见板层骨形成,TCP、CHA材料明显降解吸收,骨形成量明显增加;16周时材料界面新生骨趋向成熟,CHA材料降解区域由大量板层骨、骨髓组织填充;24周时新骨形成进一步增加,骨组织接近正常,但HA降解不明显,复合材料组新骨形成量明显高于相应的单纯材料.骨组织标本放射学及扫描电镜检测结果与组织观察结果基本一致.16周生物力学测定最大负载、弯曲及张力指标接近正常,与相应的单纯材料相比差异有显著性意义,说明细胞外基质的加入不仅有效地促进了新骨的形成,同时也加速了新骨的钙化.结论 三种生物陶瓷是细胞外基质的理想载体,生物陶瓷基质复合材料对骨缺损有较强的修复能力,其中可降解生物陶瓷将是一类较为理想人工骨替代材料.     

骨形成蛋白局部基因治疗在骨修复中的应用

骨形成蛋白(bonemorphogeneticproteins熏BMPs)是一组多功能的分泌性蛋白质,最主要的作用是在体内诱导异位和原位新骨、软骨及与骨组织有关的结缔组织如肌腱及韧带的形成,故可用于骨修复的局部基因治疗。本文简要介绍了目前BMP局部基因治疗在骨修复中的应用情况,包括所用的方法、载体、载体细胞及在动物模型中的疗效。另外,还简述了两个新发现的与BMP用于骨修复有关的转录因子:Cbfal和Osterix。     

NAC-PCCs载骨形态发生蛋白2基因纳米微球促大鼠骨髓间充质干细胞成骨分化

目的　构建半胱氨酸及磷酸胆碱共接枝的仿生壳聚糖载体（NAC-PCCs），并包封骨形态发生蛋白2（BMP2）基因进行诱导骨髓间充质干细胞成骨分化的研究。 方法　制备pBMP2/NAC-PCCs材料，检测其粒径、形态，绘制DNA缓释曲线，研究微球抗DNA酶降解能力。在骨髓间充质干细胞与材料共培养过程中，检测材料的转染效能、ROS清除能力、BMP2蛋白分泌水平、成骨相关基因RUNX2、OC表达、碱性磷酸酶活性等指标，以研究其对骨髓间充质干细胞成骨分化的影响。 结果　微球材料能有效避免DNA被生物酶降解，其转染效率为23.1%，ROS清除率为(36.13±0.47)%。同时实验结果显示与NAC-PCCs共培养的细胞， 其细胞内BMP2表达水平高于其余各组且成骨分化效果最佳。 结论　NAC-PCCs包封BMP2基因形成的纳米微球材料具有促进BMSC成骨分化作用。     

Cem-OsteticTM人工骨浆复合骨形态发生蛋白对山羊椎体骨缺损修复作用研究*

目的 观察人工骨浆复合骨形态发生蛋白（BMP）对成年山羊椎体骨缺损的修复作用,并探讨其临床应用的可行性。方法将健康成年山羊24只随即分成单纯Cem-Ostefic^TM人工骨浆组（A组）和Cem-OsteticrM／BMP人工骨浆复合骨形态发生蛋白组（B组）,每组12只动物。在山羊胸腰段3处不相邻椎体分别建立椎体压缩性骨折撑开复位后骨缺损模型。将Cem-Ostetic^TM/BMP人工骨浆复合材料填充于缺损处,同时设立单纯人工骨浆对照组。术后4周、8周、12周分别处死动物,每组每次处死4只。通过大体观察,影像学检查,HE染色和生物力学测试。结果术后4周及8周时B组成骨情况、材料降解速度及生物力学强度和刚度测试明显优于A组（P〈0．05）,但仍未达到正常椎体的力学性能,差异有显著性（P〈0．01）。第12周时,两组X线及CT下均可见大量新骨生成,基本完整填充空隙,填充材料均基本完全降解,生物力学测试两组强度和刚度无显著性差异,基本达到了正常椎体的力学性能。结论Cem-Ostetic^TM人工骨浆是BMP的良好载体,Cem—Ostetic^TM／BMP复合材料具有较强的传导成骨和诱导成骨活性,生成的新骨有良好的强度和刚度。     

一种具有骨诱导活性的羟基磷灰石结晶的制备与研究

目的将骨形成蛋白2（BMP2）活性多肽与Ⅰ型胶原复合煅烧牛松质骨以制备出具有骨诱导活性的矿化羟基磷灰石结晶,并进一步探讨其生物学性能,为组织工程化人工骨提供实验基础。方法将BMP2活性多肽与Ⅰ型胶原复合煅烧牛松质骨作为实验组,以Ⅰ型胶原复合煅烧牛松质骨作为对照组。通过环境扫描电镜和能谱法以及x射线衍射实验观察能否生成羟基磷灰石结晶,同时对2组材料进行体外细胞培养,计算2组材料细胞黏附率,初步评价BMP2活性多肽引导Ⅰ型胶原复合煅烧牛松质骨表面矿化生成羟基磷灰石结晶的作用与能力。结果环境扫描电镜下可见实验组煅烧骨表面有矿化羟基磷灰石结晶生成,而对照组没有：能谱法对实验组矿化骨部分进行钙磷元素检测,质量比分别为16．23％、7．76％．原子百分数分别为6．34％、3．88％,X射线衍射检测证实矿化物的成分为磷灰石。大鼠骨髓基质干细胞分别与2组材料体外复合培养24h,实验组细胞黏附率明显高于对照组（a〈0．05）。结论BMP2活性多肽能引导Ⅰ型胶原复合煅烧骨表面矿化生成羟基磷灰石结晶,可以改善煅烧骨的骨诱导活性,提高细胞黏附性能,与煅烧骨复合后是一种理想的骨组织支架复合材料。     

在具有几何形态适宜和不适宜结构的羟基磷灰石表面BMP诱导骨生成:骨生成的局部记载

在机体内,骨形成蛋白(BMP)需要一个合适的载体来诱导异位骨形成。作为BMP诱导骨形成的载体,羟基磷灰石(HA)陶瓷有些形式是有效的,而另一些形式则是无效的。本研究比较了三种几何形态不同的HA以检查它们作为BMP诱导骨形成载体的作用。含有所有活性BMPs(BMP混合物)的物质成份是用3步层析法(chromatographicprocedure)从牛骨中提取物的4mol/L胍中部分纯化而获得。将BMP混合物和三种形式的HA分别组合:固态颗粒(SPHAP)、多孔颗粒(PPHAP)、珊瑚复制的多孔小丸(珊瑚HAP),将其植入鼠皮下,采用形态学和生物化学方法观察。植入后2周,…     

Bovine BMP osteoinductive potential enhanced by functionalized dextran-derived hydrogels

This study evaluated functionalized dextran-derived hydrogels as BMP carriers using both in vitro and in vivo models. In vitro release kinetics indicated that dextran-derived hydrogels could retain rhBMP-2 growth factor in a variable manner depending on their functionalization ratio. The potential of these hydrogels when combined with extracted bovine BMP to enhance the bone formation was evaluated in a rat ectopic model. The largest osteoinduction was found when using hydrogels exhibiting the highest growth factor retention capacity. In addition, some implanted hydrogels demonstrated a capacity to induce an in-vivo calcification certainly related to their chemical composition. These properties make these materials interesting osteoconductive BMP carriers, allowing to decrease the amount of implanted factor required for bone regeneration.     

Two Distinctive BMP-Carriers Induce Zonal Chondrogenesis and Membranous Ossification,Respectively; Geometrical Factors of Matrices for Cell-Differentiation

A partially purified BMP preparation was combined with a fibrous glass membrane (FGM) or porous particles of hydroxyapatite (PPHAP), and then implanted subcutaneously into the backs of rats. As a control of these new carriers, a conventional carrier of insoluble bone matrix (IBM) was also used. These new geometrically different solid-state carriers induced tissues in quite different manners. FGM/BMP implants induced cartilage formation within the entire inner area of the membrane accompanied by a small amount of bone formation on the surface of the membrane. In contrast, PPHAP/ BMP implants induced only bone within the pores of PPHAP without any detectable cartilage formation. Enzyme-linked immunosorbent assay revealed that the type II collagen content in FGM/BMP was six times higher than that in IBM/BMP, while there was no detectable type II collagen in PPHAP/BMP. The results were explained by the geometric properties of the two distinctive carriers.     

Adenovirus BMP2-induced osteogenesis in combination with collagen carriers

Adenovirus BMP2 gene therapy has potential of a robust endogenous BMP2 production, while circumventing many of the problems currently associated with recombinant BMP2. The study objective was to determine and compare the ability of adenovirus BMP2 ex vivo gene therapy in combination with three types of collagen carriers to release BMP2 in vitro and to induce heterotopic bone formation in vivo. Human CD45-negative bone marrow cells were ex vivo transduced with a chimeric Ad5F35BMP2. The bioactivity of BMP2 produced by the transduced cells without a carrier, or in combination with three types of collagen carriers (injectable gel, microporous sponge, collagen-mineral composite) was measured and compared to rhBMP2. The heterotopic osteoinductivity assay was performed in immunocompromised NOD/SCID mice. A statistically significant decrease in the amount of rhBMP2 and adenoviral BMP2 released in vitro from the collagen-mineral composite carrier was noted (21% and 12%, respectively), whereas the amounts of rhBMP2 and adenoviral BMP2 released from the gel or sponge carriers were comparable. In vivo, 14 days post-implantation, no bone was formed consistently in groups with the empty Ad5F35HM4 control vector. New bone formation was evident radiographically and histologically in all groups with the Ad5F35BMP2-transduced cells irrespective of the presence or absence of a carrier. The presence of a carrier resulted in osteogenesis limited to the implantation site, and was most pronounced for solid (sponge, composite) carriers. The physical characteristics of the carrier determined the new bone spatial distribution at the site. Solid carriers reduced the clearance of AD5F35-transduced cells by the host immune cells. Adenoviral ex vivo BMP2 gene therapy in combination with collagen carriers with distinct physical characteristics offers the prospects of adjusting this approach to optimally match the specific therapeutic requirements.     

四种BMP载体修复骨缺损的形态学观察

目的：研究带血供肌瓣、带血供肌瓣复合纤维蛋白粘合剂（FS）、无血供肌瓣和同种异体脱钙骨（AAA）等四种BMP载体修复骨缺损的形态变化。方法：取新西兰大白兔32只,采用双前肢桡骨中下段12mm缺损实验模型,分别在3、6、9、12周观察带血供肌瓣、带血供肌瓣复合FS、无血供肌瓣和同种异体脱钙骨等4种载体修复骨缺损的大体解剖学、X线和组织学。结果：带血供肌瓣和FS作为BMP复合载体修复骨缺损3周时有大量的软骨细胞生成,呈弹性软骨样特征。6周时大量编织骨、骨小梁形成。9周时出现哈佛氏系统。肌纤维萎缩消失。12周时出现哈费氏骨板和少量的间骨板,哈佛氏管内表面衬有成骨细胞。该复合载体比同时期单用带血供肌瓣成熟程度好,相当于AAA骨载体的骨修复能力。无血供肌瓣复合BMP形成“壳”状新骨。结论：带血供肌瓣复合纤维蛋白粘合剂是BMP的良好载体。     

纳米级羟基磷灰石/骨形态发生蛋白复合物修复兔桡骨大段缺损及局部血管内皮细胞生长因子的表达

背景：研究发现,在小段骨缺损中植入骨或仿生骨组织,坏死组织逐渐被替换,移植骨中会长入有活性的血管肉芽组织,移植骨被吸收,新骨主动形成。但在大段骨缺损,这一过程发生较慢且不完全。 目的：观察纳米级羟基磷灰石材料复合骨形态发生蛋白后大段骨缺损修复能力及诱导生成血管能力。 方法：制作兔桡骨大段骨缺损模型,抽签随机分2组,选择一侧分别植入纳米级羟基磷灰石/骨形态发生蛋白、纳米级羟基磷灰石修复,均以另一侧为空白对照。植入后6个月行大体观察、X射线、组织形态学检查、组织切片碱性磷酸酶染色、成骨量分析、血管内皮细胞生长因子阳性细胞率及阳性血管数检测。 结果与结论：空白对照组基本无骨组织生成。纳米级羟基磷灰石植入后被新生骨组织分割成小块,材料原有结构破坏。纳米级羟基磷灰石/骨形态发生蛋白组较纳米级羟基磷灰石组残存材料更少,材料降解更为彻底。纳米级羟基磷灰石/骨形态发生蛋白组成骨量、血管内皮细胞生长因子表达及血管内皮细胞生长因子阳性血管数目均明显高于纳米级羟基磷灰石组(P < 0.001),且血管内皮细胞生长因子表达与血管内皮细胞生长因子阳性血管数目成正比关系。说明纳米级羟基磷灰石与骨形态发生蛋白复合后,骨修复能力进一步增强,诱导血管生成能力明显提高。     

Pediatric pulmonary arterial hypertension due to a novel homozygous GDF2 missense variant affecting BMP9 processing and activity

Pulmonary arterial hypertension (PAH) is a rare and severe disorder characterized by progressive pulmonary vasculopathy. Growth differentiation factor (GDF)2 encodes the pro-protein bone morphogenetic protein (BMP) 9, activated after cleavage by endoproteases into an active mature form. BMP9, together with BMP10, are high-affinity ligands of activin receptor-like kinase 1 (ALK1) and BMP receptor type II (BMPR2). GDF2 mutations have been reported in idiopathic PAH with most patients being heterozygous carriers although rare homozygous cases have been described. The link between PAH occurrence and BMP9 or 10 expression level is still unclear. In this study, we describe a pediatric case of PAH also presenting with telangiectasias and epistaxis. The patient carries the novel homozygous GDF2 c.946A > G mutation, replacing the first arginine of BMP9's cleavage site (R316) by a glycine. We show that this mutation leads to an absence of circulating mature BMP9 and mature BMP9-10 heterodimers in the patient's plasma although pro-BMP9 is still detected at a similar level as controls. In vitro functional studies further demonstrated that the mutation R316G hampers the correct processing of BMP9, leading to the secretion of inactive pro-BMP9. The heterozygous carriers of the variant were asymptomatic, similarly to previous reports, reinforcing the hypothesis of modifiers preventing/driving PAH development in heterozygous carriers.     

Amino-modified silica surfaces efficiently immobilize bone morphogenetic protein 2 (BMP2) for medical purposes

Due to its ability to induce de novo bone formation the differentiation factor bone morphogenetic protein 2 (BMP2) is often used to enhance the integration of bone implants. With the aim of reducing possible high dose side-effects and to lower the costs, in order to produce affordable implants, we developed a simple and fast method for the immobilization of BMP2 on silica-based surfaces using silane linkers which carry amino or epoxy functions. We put an especial emphasis on the influence of the nanoscale surface topography of the silica layer. Therefore, we chose glass (for control experiments) and Bioverit? II (as a typical implant base material) as support materials and coated these substrates with unstructured or nanoporous amorphous silica layers for comparison. Immobilized BMP2 was quantified by two different methods: by ELISA and by a cell-based assay for active BMP2. These tests probe for immunologically and biologically active BMP2, respectively. The results show that the amino functionalization is better suited for immobilizing the protein. Strikingly, a considerably higher amount of BMP2 could be immobilized on coated Bioverit? II surfaces compared with coated glass substrates, which was presumably due to the macroscopic roughness of the Bioverit? II substrates. In addition, it was found that the nanoporous silica coatings on Bioverit? II substrates were able to bind more BMP2 than the unstructured ones.     

BMP2活性多肽/PLGA复合物植入异位成骨的实验研究

目的用动物实验的方法评价自行研制合成的BMP2活性多肽生物因子与可降解PLGA复合物的异位诱导成骨能力。方法实验分3组。A组：BMP2活性多肽／PLGA复合物组；B组：单纯PLGA组；C组：明胶海绵组。分别于Wistar大鼠背部两侧骶棘肌下包埋植入。术后1、4、8和12周取材。经组织学观察和CT三维成像比较成骨情况，western blot检测Ⅰ型胶原及骨桥蛋白的蛋白表达，了解异位成骨的情况。结果植入块周围初期均表现为急性炎症反应，后期均为淋巴细胞、巨噬细胞浸润为主的非特异性炎症反应。A组植入4周时植入区有软骨生成，8周时有活跃的成骨细胞出现，并有非编织骨结构。12周可见大量新骨形成，有典型的骨小梁新生血管结构。B组和C组12周时仅见纤维组织形成，未见成骨。结论人工合成的BMP2活性多肽体内能启动软骨化骨过程，具有较强的异位诱导成骨能力。有与天然BMP2类似的骨诱导活性，具有广阔的应用前景。     

猪源性骨支架材料的制备及性能研究

目的:制备猪源性骨支架材料,并与人源性骨支架材料对比检测其理化性能和组织相容性。方法:经低温深冻、超声清洗、H2O2、酒精浸泡、冻干、辐照制备猪源性骨支架材料和人源性骨支架材料。扫描电镜观察,测定材料孔隙率、蛋白质含量、钙磷含量及弹性模量。2种材料的浸提液与脂肪源间充质干细胞复合培养,观察细胞一般形态,流式细胞仪PI法检测细胞生命周期。皮下植入2种材料,在植入4、8、12、16周取材做病理切片观察和扫描电镜观察。结果:2种材料均具有骨本身的天然网状三维支架系统。猪源性骨支架材料的孔隙率高于人源性骨支架材料,蛋白含量低于人源性骨支架材料,弹性模量分别为无显著差异。材料浸提液组及空白对照组的细胞生长状态良好。流式细胞仪PI法检测细胞周期见G1期、G2期细胞百分率接近。皮下植入试验表明,随着植入时间的延长,炎症反应逐步减轻,材料降解增加,新生软骨样结构逐渐增多。结论:猪源性异种骨支架材料在理化性能和材料毒性等方面与同种异体骨支架材料接近,具有良好的应用前景。     

Calcium phosphates compounds in conjunction with hydrogel as carrier for BMP-2: a study on ectopic bone formation in rats

Current treatment of fractures often involves the use of bone graft or bone morphogenetic proteins (BMP) to induce fracture healing, especially in patients with a compromised healing capacity. BMP has to be delivered in conjunction with a carrier. Unfortunately, there are drawbacks and limitations with current carriers, including their bovine origin which carries the risk of an immunological response. The physical properties also limit the use to open surgical procedures, as it cannot be injected. New carriers with improved properties are therefore needed. The aim of this study was to assess the ectopic bone forming capability of various calcium phosphate compounds when used in conjunction with a hydrogel as the carrier for BMP-2. Five different ceramic additives were tested, including β-tricalcium phosphate and four types of hydroxyapatite (HAP) (nanoHAP, HAP, clods of HAP >100 μm, and the biomimetic HAP Ostim35?). The compounds were injected into the thigh muscle of rats, where it formed a gel in situ. After 4 weeks bone formation was evaluated by peripheral quantitative computed tomography and histology. The major finding was that the 20 nm nanoHAP yielded a higher bone density than the other additives (P=0.0008, ANOVA with Tukey's multiple comparison test). We hypothesize that the higher bone density induced by nanoHAP might be due to nanocrystals of calcium phosphate acting as direct building blocks for biomineralization.