三种快速成型制作的聚酯/钙磷盐人工骨修复兔桡骨缺损的实验研究

目的检验3种聚酯／钙磷盐植骨材料修复兔桡骨缺损的效果,筛选理想的组织工程用生长因子载体。方法分别采用复合牛骨形态发生蛋白(bBMP)及单纯快速成型工艺制作的3种聚酯／钙磷盐载体材料修复兔桡骨15mm缺损,通过量化评价、影像学、组织学、材料降解及骨密度评价3种聚酯／钙磷盐载体材料修复兔桡骨缺损的效果。结果12周时各材料实验组骨缺损均愈合,各检测指标同对照组比较差异均有统计学意义,空白对照组骨缺损未愈合,以聚乳酸．聚羟基乙酸共聚物／磷酸三钙(PLGA／TCP)材料实验组修复效果最好,聚消旋乳酸／磷酸三钙(PDLLA／TCP)材料实验组次之,最后为聚左旋乳酸／磷酸三钙(PLIA／TCP)材料实验组。结论3种聚酯／钙磷盐材料制作的仿生活性人工骨皆可以修复兔15mm长骨骨缺损,其中以PLGA／TCP材料效果最为理想。     

新型仿生活性人工骨诱导兔腰椎横突间脊柱融合研究

目的采用先进快速成形技术（RP）结合骨组织工程方法研制新型仿生活性人工骨，并探讨其在兔腰椎横突间脊柱融合的应用情况。方法首先采用RP制备薄块型聚乳酸一聚羟乙酸／磷酸三钙（PLGA／TCP）人工骨载体，进而高效复合牛骨形态发生蛋白（bBMP）以制备仿生活性人工骨。扫描电镜观察载体材料及人工骨超微结构。将健康新西兰兔28只（平均体重4.1kg）随机均分为A、B两组（每组14只）。A组：于兔腰4～5右、左侧横突间分别植入仿生活性人工骨（A1组）、自体髂骨（A2组）；B组：于兔腰4～5右、左侧横突间分别植入复合自体新鲜红骨髓的PLGA／TCP载体材料（B1组）、单纯PLGA／TCP载体材料（B2组）。于术后6周和12周，定期大体观察、手法检测、组织学[苏木素-伊红（HE）、三色法及四环素-钙黄绿素荧光检测]和影像学方法（X、CT）系统评价脊柱融合情况。结果RP制备的PLGA／TCP载体具有规则的空间支架结构、相互贯通的孔隙及材料表面微孔特征。这些均有利于bBMP的高效复合。动物实验结果显示，A1组仿生活性人工骨植入具有强的诱骨活性及骨性融合能力，不仅成骨早、新骨形成量大，而且在新骨形成及改塑的同时载体材料逐渐降解。术后12周可形成较为典型的骨小梁及骨髓结构，骨代谢活性亦接近正常。A2组自体髂骨移植能达到良好骨性融合，但术后12周所形成的新骨结构尚须进一步塑形及完善。B1组、B2组术后12周仍遗有较多的载体材料有待降解，基本无成骨能力。术后12周，A1、A2、B1、1324组横突间融合率分别为100．0％、58．3％、18．2％和0％，A1组融合率最高（P〈0．01）。结论先进RP制备的PLGA／TCP载体不仅具有良好的空间超微结构及孔隙特性，而且能高效复合bBMP以正确构建新型组织工程人工骨。该仿生活性人工骨诱导兔横突间脊柱融合获得成功．为生物制造脊柱外科所需的新型、高效人工骨移植材料奠定了重要基础。     

骨形态发生蛋白-2壳聚糖微球复合组织工程支架修复兔股骨髁部骨缺损的实验研究

目的 探讨重组人骨形态发生蛋白-2(rhBMP-2)壳聚糖缓释微球复合聚乳酸-聚羟乙酸/磷酸三钙(PLGA/TCP)支架修复骨缺损的可行性和有效性.方法 取健康成年新西兰大白兔45只,在40只实验动物股骨髁部制备0.6 cm×1.0 cm骨缺损.实验分4组:A组:缺损组,B组:用PLGA/TCP空白支架修复骨缺损,C组:用等量rhBMP-2复合PLGA/TCP支架修复骨缺损,D组:用rhBMP-2壳聚糖微球复合PLGA/TCP支架修复骨缺损,每组10只动物.另5只动物为正常对照组(E组).应用X线、Micro-CT和组织病理学等方法检测术后4、12 周各实验组骨缺损修复效果.结果 术后4周X线片示:A组无新骨形成.B组有少量新生骨影像形成,C、D组骨缺损部位均有新骨影像形成.术后12周,Micro-CT结果显示:D组骨密度、骨体积分数、骨小梁厚度、骨小梁数目等指标均优于B组和C组,差异均有统计学意义(P<0.05),A组末检测到上述指标.术后4剧,D组可见大量骨组织形成,有部分成熟的骨小梁,PLGA/TCP支架大部分被降解.吸收.术后12周,D组支架和微球完全降解.骨缺损被成熟骨取代;B、C、D组骨长入率分别为5.78%±1.21%、37.26%±6.45%、74.25%±8.91%,3组之间比较差异均有统计学意义(P<0.05).结论 复合rhBMP-2壳聚糖微球的PLGA/TCP支架具有良好的骨缺损修复效果,临床应用前景较好.     

BMP复合仿生人工骨填塞犬股骨假体周围髓腔骨缺损的实验研究

目的评估仿生人工骨（PLGA／TCP）材料作为BMP缓释系统载体材料修复犬假体涂层多孔钛周围骨缺损的可行性。方法给予8只犬进行右侧股骨头置换术，并充分扩髓，周围填充BMP复合的PLGA／TCP骨移植材料，应用X线拍片、组织和荧光观察、EDX及SEM检测、骨密度定量分析等方法，观察负重情况下该材料的实际效果。结果术后X线显示，未有出现透亮带、无假体下沉，未见骨质吸收；20周时骨与假体之间紧密接触，多孔钛内骨组织明显长入，以编织骨为主。植入的活性人工骨已经完全降解，骨小梁结构成熟；假体植入肢体与非手术肢体骨密度之比，显示在术后1个月假体植入侧的骨密度值有所降低，而后逐渐升高。结论多孔钛涂层有助于假体内骨与假体的固定，BMP复合PLGA／TCP作为新的骨移植材料应用于关节外科，有广泛的潜力和价值。     

Skeletal repair in rabbits using a novel biomimetic composite based on adipose‐derived stem cells encapsulated in collagen I gel with PLGA‐β‐TCP scaffold

In bone tissue engineering, the cell distribution mode in the scaffold may affect in vivo osteogenesis. Therefore, we fabricated a novel biomimetic construct based on a combination of rabbit adipose‐derived stem cells (rASCs) encapsulated in collagen I gel with a PLGA‐β‐TCP scaffold (rASCs‐COL/PLGA‐β‐TCP, group A), the combination of rASCs and PLGA‐β‐TCP (rASCs/PLGA‐β‐TCP, group B), the combination of collagen I gel and PLGA‐β‐TCP (COL/PLGA‐β‐TCP, group C), and PLGA‐β‐TCP scaffold (group D). The composites were implanted into a 15‐mm length critical‐sized segmental radial defect. The results were assessed by histology, radiographs, bone mineral density (BMD), and mechanical testing. After 24 weeks, the medullary cavity recanalized, bone was rebuilt, and molding finished, the bone contour remodeled smoothly and the scaffold degraded completely in group A. The BMDs and mechanical properties were similar to normal. However, the bone defect remained unrepaired in groups B, C, and D. Moreover, the scaffold degradation rate in group A was significantly higher than the other groups. Thus, enhanced in vivo osteogenesis of rASCs wrapped in collagen I gel combined with PLGA‐β‐TCP was achieved, and the bone defect was repaired. We hope this study provides new insights into ASCs‐based bone tissue engineering. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:252–257, 2010     

Exogenous phytoestrogenic molecule icaritin incorporated into a porous scaffold for enhancing bone defect repair

This study was designed to develop a bioactive scaffold to enhance bone defect repair in steroid‐associated osteonecrosis (SAON). Icaritin, a metabolite of the herb Epimedium, has been identified as an angiogenic and osteogenic phytomolecule. Icaritin was homogenized into poly lactic‐co‐glycolic acid/tricalcium phosphate (PLGA/TCP) to form an icaritin‐releasing porous composite scaffold (PLGA/TCP/icaritin) by fine‐spinning technology. In vitro, high performance liquid chromatography was used to determine the release of icaritin during degradation of PLGA/TCP/icaritin. The osteogenic effects of PLGA/TCP/icaritin were evaluated using rat bone marrow mesenchymal stem cells (BMSCs). In vivo, the osteogenic effect of PLGA/TCP/icaritin was determined within a bone tunnel after core decompression in SAON rabbits and angiography within scaffolds was examined in rabbit muscle pouch model. In vitro study confirmed the sustainable release of icaritin from PLGA/TCP/icaritin with the bioactive scaffold promoting the proliferation and osteoblastic differentiation of rat BMSCs. In vivo study showed that PLGA/TCP/icaritin significantly promoted new bone formation within the bone defect after core decompression in SAON rabbits and enhanced neovascularization in the rabbit muscle pouch experiment. In conclusion, PLGA/TCP/icaritin is an innovative local delivery system that demonstrates sustainable release of osteogenic phytomolecule icaritin enhancing bone repair in an SAON rabbit model. The supplement of scaffold materials with bioactive phytomolecule(s) might improve treatment efficiency in challenging orthopedic conditions. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:164–172, 2012     

基于陕速成型技术的可控结构多孔硅酸钙支架的制备及体外研究

目的 利用快速成型技术制备可控结构多孔硅酸钙(rapid pfototyping-calcium silicate,RP-CS)支架,并评价其特性和体外生物学表现.方法 利用间接快速成型技术,结合固态自由成型和凝胶铸模的优点.制备町控结构RP-CS支架.与采用间法制备的多孔磷酸钙(RP-tricalcium phosphate,RP-TCP)支架相对照,将其置人体外模拟体液(simulated body fluid,SBF)、体外骨髓细胞共培养进行研究.结果 所制备RP-CS支架具有相互连通的孔道结构,平均孔隙率为71%,平均轴向压缩强度为28MPa.平均孔道直径为(555.82±29.77)μm.体外SBF浸置试验发现RP-CS支架上有羟基磷灰石的沉积,说明此支架具有体外生物活性.体外细胞共培养试验表明,兔骨髓细胞可以在此支架表面贴附并分化.MTT表明共培养7 d、14 d,细胞增殖RP-CS组均明显高于RP-TCP组(P<0.05).共培养7 d时,碱性磷酸酶活性RP-CS组明显高于RP-TCP组(P<0.05),提示CS可能具有促进骨髓细胞向成骨细胞分化的能力.结论 利用快速成型技术制备的可控结构RP-CS具有良好的生物相容性,在骨组织工程领域具有广泛应用前景.     

新型生物活性陶瓷复合人工骨成骨效应的实验研究

目的　探讨新型生物活性陶瓷复合材料成骨效应 ,为人工骨替代材料临床应用提供依据。　方法　小鼠 96只 ,随机分为 4组 ,每组 2 4只。采用具有诱导活性的骨形成蛋白 (bone morphogenetic protein,BMP)分别与羟基磷灰石 (hydroxyapatite,HA)、磷酸三钙 (tricalcium phosphate,TCP)、胶原复合羟基磷灰石 (collagen HA,CHA)及氟化羟基磷灰石 (fluoridated HA,FHA)复合 ,将 4种复合材料 (HA/ BMP,TCP/ BMP,CHA/ BMP及 FHA/ BMP)分别植入 4组小鼠左侧股部肌肉内为实验侧 ,右侧分别植入 HA、TCP、CHA及脱钙牙基质 (decalcified dentin matrix,DDM)作为对照 ,在 1、3、5及 7周取材作大体观察、组织形态学、扫描电镜观察及生化测定。　结果　各组实验侧及第 4组对照侧植入后 1周软骨形成 ,第 1～ 3组对照侧为纤维结缔组织 ;3周时各组实验侧均有较多的成熟骨组织 ,组织碱性磷酸酶 (alkalinephosphatase,AL P)染色均为阳性。各组对照侧材料被结缔组织包囊 ,AL P染色阴性 ,未见骨组织形成。各组实验侧材料AL P活性及磷 (phosphrus,P)检测水平明显高于相应的对照侧材料 ,实验侧与对照侧比较具有统计学意义 (P<0 .0 5 ) ,而 TCP/ BMP复合材料明显高于另 3种复合材料 ,有统计学意义 (P<0 .0 5 )。5、7周各实验侧及对照     

组织工程骨修复颅颌面骨缺损预塑形技术的研究

目的：采用快速成型技术对组织工程支架材料进行预塑形,探讨其在治疗颅颌面骨缺损中的应用前景.方法：选择1 0只成年杂交犬,均拟行组织工程骨修复颅颌面骨缺损实验研究（下颌骨节段缺损,颅骨全层缺损）,螺旋CT连续薄层容积扫描,数据传至工作站后行三维重建,按实验设计方案进行模拟手术,并转换成＊.stl文件格式,快速成型机制造骨缺损三维实体模型,据此制作阴模,填充磷酸三钙高温烧结后制成多孔支架,复合骨髓基质细胞后回植,螺旋CT检测组织工程骨和骨缺损完全匹配情况.结果：10例均按预设计制备骨缺损三维实体模型,据此制备个性化组织工程支架材料,手术回植和骨缺损完全匹配.结论：采用快速成型技术可对组织工程骨支架材料进行预塑形,对未来组织工程技术治疗复杂性颅颌面骨缺损具有重要的临床实用价值.     

Response of the rabbit metaphysis to implants of bovine bone morphogenetic protein (bBMP)

The response of protodifferentiated and differentiated bone cells to bovine bone morphogenetic protein (bBMP) was observed in implants in the adult rabbit distal femoral metaphysis. Bovine serum albumin and denatured bBMP were implanted in the contralateral femur of controls. The changes of the bone marrow reflected the reaction of protodifferentiated cells. The changes in preexisting trabecular bone tissue reflected the reaction of differentiated cells to bBMP. 45Ca radioisotope quantitative methods demonstrated that the bone morphogenetic response was superimposed upon the reaction to the injury of surgical implantation. By the end of the fourth week, roentgenograms and histologic sections showed larger deposits of intrametaphyseal cartilage and bone in bBMP than in control implanted femurs. By the end of the eighth week, bone formation was associated with remodeling of the entire distal femur and expansion of the external diameter of the metaphysis. These observations indicate the need for investigation of perisinusoid and perivascular cells of periosteum, endosteum, and marrow stroma that are undifferentiated with respect to cartilage and bone but are principal target tissues for BMP.     

An injectable composite material containing bone morphogenetic protein‐2 shortens the period of distraction osteogenesis in vivo

To investigate new methods that can decrease the duration of bone transport (BT) distraction osteogenesis, we injected composite materials containing recombinant human bone morphogenetic protein‐2 (BMP‐2) and induced the generation of a callus bridge by rapid segmental transport (4 mm/day) in a rabbit bone defect model. The composite materials consisted of BMP‐2 (0, 30, or 100 µg), β‐tricalcium phosphate powder (βTCP, 100 mg/animal; particle size, <100 µm), and polyethylene glycol (PEG; 40 mg/animal). A paste of equivalent composition was percutaneously injected at the lengthening and the docking sites after surgery and after BT, respectively. The radiographic, mechanical, and histological examinations 12 weeks post‐operative revealed that the generation of bridging callus in the presence and in the absence of BMP‐2 was significantly different. The callus mass in the bone defect region was adequately and consistently developed in the presence of 100 µg of BMP (administered for 6 weeks), and the bones were consolidated in 12 weeks. Such an adequate callus formation was not observed in the control animals without BMP‐2 treatment. The result of this experimental study suggests the potential application of BMP‐2 in accelerating callus formation and in enabling rapid bone transporting, thereby shortening the treatment period for the repair of diaphyseal bone defects by distraction osteogenesis. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:452–456, 2011     

Comparative in vivo study of injectable biomaterials combined with BMP for enhancing tendon graft osteointegration for anterior cruciate ligament reconstruction

This study was to compare effect of osteointegration of grafted tendon in bone tunnels between injected calcium phosphate cement (ICPC) and injected fibrin sealant (IFS) combined with bone morphogenetic protein (BMP) after anterior cruciate ligament (ACL) reconstruction. ACL reconstruction was performed bilaterally in 51 rabbits. ICPC‐BMP composite was injected into one knee, with the contralateral knee IFS–BMP composite. The rabbits were killed at postoperative weeks 2, 6, and 12 for testing. Histological observations showed the ICPC composite gradually increased the new bone formation during the whole healing process, while the IFS composite had a burst effect on enhancing the healing of tendon‐to‐bone at 2 and 6 weeks. By 12 weeks, there was more new cartilage and new bone in the interface in the ICPC‐bBMP group. Micro‐CT showed that the values of BMD in the ICPC‐bBMP group were lower than those in the IFS‐bBMP group at 6 weeks, while the values in the ICPC‐bBMP group were higher than those in the IFS‐bBMP group at 12 weeks (p > 0.05). Fluorescent labels showed that the rate of new bone formation of IFS–BMP composite was significantly higher than that of ICPC composite at 6 weeks (3.45 ± 0.62 µm/day vs. 2.93 ± 0.51 µm/day), but the rate was decreased compared with ICPC composite at 12 weeks (2.58 ± 0.72 µm/day vs. 3.05 ± 0.68 µm/day; p < 0.05). Biomechanically, the ultimate failure load in the ICPC‐BMP group was always higher than that in the IFS–BMP group. It is evident that the ICPC composite achieved a more prolonged osteogenic effect than that by IFS composite. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1015–1021, 2011     

BMP2重组慢病毒转染兔BMSCs黏附DBM支架的生物矿化研究

目的 探究BMP2重组慢病毒转染兔骨髓间充质干细胞(BMSCs)黏附脱钙骨(DBM)支架体外构建转基因组织工程骨的生物矿化能力。方法 密度梯度离心及贴壁培养法获取第5代兔BMSCs,用BMP2重组慢病毒转染细胞，通过RT-PCR检测目的基因的表达，利用倒置荧光显微镜观察细胞在DBM支架上的生长情况，借助扫描电镜和能谱分析观测细胞在DBM支架上的表面微观形貌及生物矿化能力。结果 BMP2重组慢病毒成功转染兔BMSCs, RT-PCR显示转染后细胞能高效表达BMP2目的基因，在倒置荧光显微镜下观察见转染后细胞黏附在DBM支架上呈满天星样，并沿DBM支架孔隙内壁贴壁叠加生长、分裂增殖，扫描电镜下见细胞填满整个DBM支架孔隙，分泌大量细胞外基质，并在DBM支架表面形成凹凸不平的光泽结晶矿化物，能谱分析显示其钙磷比(Ca/P)为1.89±0.31,与正常骨组织成分相近(P>0.05)。结论 BMP2重组慢病毒转染兔BMSCs黏附DBM支架构建的转基因组织工程骨成功完成了生物矿化过程，体现出较好的生物矿化能力。     

Rabbit Tibial Periosteum and Saphenous Arteriovenous Vascular Bundle as an In Vivo Bioreactor to Construct Vascularized Tissue‐Engineered Bone: A Feasibility Study

The aim of this project was to construct vascularized tissue‐engineered living bone with an autologous vascular network by means of a rabbit bioreactor in vivo. The key components of the in vivo bioreactor for bone formation were the vascularized tibial periosteum and the saphenous vascular bundle. Beta‐tricalcium phosphate (β‐TCP) scaffolds were implanted into the in vivo bioreactor (vascular pedicle implantation and vascularized periosteum encapsulation). At 4 weeks postsurgery, new bone formation was mainly “cartilage‐bone inducing” in the inner periosteum, and was primarily seen in the outer aspects of the scaffold with some amount in the middle part as well. Microvascular infusion showed that direct revascularization of β‐TCP was obtained by means of vascular implantation. Triple staining results showed a large amount of blue collagen fibers. Vascular endothelial growth factor immunohistochemical staining displayed endothelial cells of new blood vessels in bone tissue. The bioreactor established in this study can be used to prepare tissue‐engineered bone with a vascular network.     

The effect of different mineral frames on ectopic bone formation in mouse hind leg muscles induced by native reindeer bone morphogenetic protein

Introduction Bone morphogenetic proteins (BMPs) require carrier material for slow release and framing material for osteoconduction.Materials and methods The effect of a frame on early bone formation induced by partially purified native reindeer BMP in composite implants containing 3 mg of BMP, type IV collagen and tricalcium phosphate (TCP/Col/BMP) or hydroxyapatite (HA/Col/BMP) or biphasic tricalcium phosphate-hydroxyapatite (TCP/HA/Col/BMP) or biocoral (NC/Col/BMP) was evaluated using a mouse hind leg muscle pouch model. Collagen with native reindeer BMP (Col/BMP) and corresponding implants without native reindeer BMP served as controls. Evaluation was done by incorporation of ⁴⁵Ca, radiographically and histologically 3 weeks after the implantation.Results None of the implants without native reindeer BMP were able to induce new bone visible on radiographs. The area of new bone formation in the Col/BMP (p=0.026) and TCP/HA/Col/BMP (p=0.012) groups was significantly greater than in the TCP/Col/BMP group. The optical density of the new bone area was significantly greater in the TCP/HA/Col/BMP group than in the TCP/Col/BMP (p=0.036) or Col/BMP (p=0.02) groups. ⁴⁵Ca incorporation was many times greater in all the groups containing native reindeer BMP than in the corresponding groups without BMP. In the Col/BMP (p=0.046) and TCP/HA/Col/BMP (p=0.046) groups, ⁴⁵Ca incorporation was significantly greater than in the TCP/Col/BMP group. No significant differences were found in any parameters between HA/Col/BMP and NC/Col/BMP groups and the other BMP-containing groups.Conclusions Hydroxyapatite, biocoral and biphasic tricalciumphosphate-hydroxyapatite are equally good as framing material for native reindeer BMP, while tricalciumphosphate is somewhat worse. Osteoinduction of native reindeer BMP works well with collagen alone.     

Bony defect repair in rabbit using hybrid rapid prototyping polylactic-co-glycolic acid/β-tricalciumphosphate collagen I/apatite scaffold and bone marrow mesenchymal stem cells

Background:

In bone tissue engineering, extracellular matrix exerts critical influence on cellular interaction with porous biomaterial and the apatite playing an important role in the bonding process of biomaterial to bone tissue. The aim of this study was to observe the therapeutic effects of hybrid rapid prototyping (RP) scaffolds comprising polylactic-co-glycolic acid (PLGA), β-tricalciumphosphate (β-TCP), collagen I and apatite (PLGA/β-TCP-collagen I/apatite) on segmental bone defects in conjunction with combination with bone marrow mesenchymal stem cells (BMSCs).

Materials and Methods:

BMSCs were seeded into the hybrid RP scaffolds to repair 15 mm defect in the radius of rabbits. Radiograph, microcomputed tomography and histology were used to evaluate new bone formation.

Results:

Radiographic analysis done from 12 to 36 weeks postoperative period demonstrated that new bone formed at the radial defect site and continues to increase until the medullary cavity is recanalized and remodelling is complete. The bone defect remained unconnected in the original RP scaffolds (PLGA/β-TCP) during the whole study. Histological observations conformed to the radiographic images. In hybrid RP scaffold group, woven bone united the radial defect at 12 weeks and consecutively remodeled into lamellar bone 24 weeks postoperation and finally matured into cortical bone with normal marrow cavity after another 12 weeks. No bone formation but connective tissue has been detected in RP scaffold at the same time.

Conclusion:

Collagen I/apatite sponge composite coating could improve new bone formation in vivo. The hybrid RP scaffold of PLGA/β-TCP skeleton with collagen I/apatite sponge composite coating is a promising candidate for bone tissue engineering.     

Repair of radius defect with bone-morphogenetic-protein loaded hydroxyapatite／collagen-poly（ L-lactic acid）composite

Objective: To explore the method to repair bone defect with bone-morphogenetic-protein loaded hydroxyapatite/collagen-poly ( L-lactic acid) composite. Methods: 18 adult beagle dogs were randomly divided into 3 groups. In Group A, bone-morphogeneticprotein ( BMP ) loaded hydroxyapatite/collagen-poly ( L-lactic acid) (HAC-PLA) scaffold was implanted in a 2 cm diaphyseal defect in the radius. In Group B, unloaded pure HAC-PLA scaffold was implanted in the defects. No material was implanted in Group C (control group). The dogs were sacrificed 6 months postoperatively. Features of biocompatibility, biodegradability and osteoinduction were evaluated with histological, radiological examinations and bone mineral density (BMD) measurements. Results: In Group A, the radius defect healed after the treatment with BMP loaded HAC-PLA. BMD at the site of the defect was higher than that of the contralateral radius. Fibrous union developed in the animals of the control group. Conclusions: BMP not only promotes osteogenesis but also accelerates degradation of the biomaterials.Optimized design parameters of a three-dimensional porous biomaterial would give full scope to the role of BMP as an osteoinductive growth factor.     

Prefabrication of a Vascularized Bone Graft With Beta Tricalcium Phosphate Using an In Vivo Bioreactor

We aimed to introduce an in vivo bioreactor‐vascular pedicle threaded through the central portion of a scaffold in which a vascularized bone graft was prefabricated using adenoviral human BMP‐2 gene (AdBMP2)‐modified bone marrow mesenchymal stem cells (BMSCs), beta tricalcium phosphate (β‐TCP), a vessel bundle, and muscularis membrane(group A). As controls, Adβgal‐BMSCs/β‐TCP granules, vessel bundle, and the muscularis membrane (group B); BMSCs/β‐TCP granules, vessel bundle, and muscularis membrane (group C); and β‐TCP granules, vessel bundle, and muscularis membrane (group D) were prepared. Formation of bone tissue and a vascular network was assessed by microangiography and histological methods 4 weeks after prefabrication. New cartilage and bone tissue in the space between β‐TCP granules (mainly endochondral bone) were confirmed by histology, and a de novo vascular network circulating from the vessel bundle through newly formed bone tissue was observed in group A. Formation of bone or cartilage was not observed in the control groups. We concluded that the in vivo bioreactor is a promising method for prefabrication of vascularized functional bone.     

Collagen-hydroxyapatite/tricalcium phosphate microspheres as a delivery system for recombinant human transforming growth factor-beta 1

The purpose of this study is to evaluate the carrier capability of collagen-hydroxyapatite/tricalcium phosphate (Col-HA/TCP) microspheres to the rhTGF-beta 1 (recombinant human transforming growth factor-beta 1). After anesthesia, a bone defect (7.0 mm in diameter and 10.0 mm in depth) was created at the distal femoral condyles of New Zealand white rabbits. These defects were then completely filled with the implant materials. After 5, 7, 9, 11, 13, and 15 weeks, the animals were sacrificed and histological evaluations were performed. The results showed that when the defects were treated with Col-HA/TCP microspheres without rhTGF-beta 1, there was only spotty new bone formation during the 15 week experimental period and most of the defect was filled with fibrous tissue and inflammatory cells, whereas active bone formation with mature marrow tissue formation was evident in the defect treated with Col-HA/TCP microspheres containing rhTGF-beta 1. Collagen-hydroxyapatite/tricalcium phosphate microspheres were expected to be replaced by the regenerated bone structure as the bone reconstruction and bone-remodeling process occurred. It was apparent that bone regeneration was influenced by the addition of rhTGF-beta 1. Collagen-hydroxyapatite/tricalcium phosphate microspheres were a good carrier for rhTGF-beta 1.     

Autogeneic cancellous bone grafts in extensive segmental ulnar defects in dogs. Effects of xenogeneic bovine bone morphogenetic protein without and with interposition of soft tissues and interruption of blood supply

Xenogeneic (bovine) bone morphogenetic protein (bBMP) and associated insoluble noncollagenous proteins (NCP) were implanted in inbred adult beagle dogs with 3-4 cm diaphyseal defects in the ulna. Defects were stabilized with internal plate fixation, and the control defects were not stabilized. The defects were implanted with either autogeneic cancellous bone grafts (ACG), bBMP/NCP, or a composite of ACG and bBMP/NCP. Of the plated ulnae, 18 of 19 ACG controls restored bone continuity; six of seven defects healed under the influence of bBMP/NCP plus ACG. Two of four defects with bBMP/NCP plus ACG healed and two were filled with osseous tissue, but fibrous tissue developed at one or both bone ends. Eight of nine defects implanted with bBMP/NCP capsules alone were repaired with fibrous tissue only. Of the nonplated defects, four were implanted with bBMP/NCP plus ACG and only one regenerated; three of four showed hypertrophic bone growth around a pseudarthrosis. Of six nonplated defects implanted with bBMP/NCP without ACG, all developed atrophic bone ends and fibrous tissue repair. Thus, to restore continuity of large segmental defects three times greater than the critical size for spontaneous regeneration, xenogeneic bBMP/NCP failed to induce bone regeneration in dogs. To exclude cell-mediated immune reactions and soft-tissue ingrowth, one defect was bridged with a polytetrafluoroethylene semipermeable tube (pore size 0.45 micron) containing implants of bBMP/NCP. In response to bBMP/NCP, cells from the host bone ends produced ossicles of induced woven bone formation. The observation that bBMP/NCP induced bone formation across the defect inside of semipermeable cylindrical chambers suggests that the experiments on bone defects larger than the critical size for spontaneous repair should be repeated with: (1) allogeneic dog BMP/NCP; (2) semipermeable cylinders to protect against muscle interposition; (3) compartment angiograms to evaluate blood supply; (4) treatment of the recipient with immunosuppressants and immunostaining to observe the concentration gradient of BMP; and (5) histologic observations on the first three days after implantation to evaluate cell-mediated immune barriers to the response of BMP.