Vascular endothelial growth factor gene-activated matrix (VEGF165-GAM) enhances osteogenesis and angiogenesis in large segmental bone defects.

Healing of fractures is dependent on vascularization of bone, which is in turn promoted by VEGF. It was shown that 0.1 and 1 mg of pVEGF165-GAM led to a significant increase in vascularization and bone regeneration in defects that would otherwise have led to atrophic nonunions. INTRODUCTION: One reason for lack of bone healing in nonunions is the absence of vascularization. In skeletogenesis, which is tightly linked to angiogenesis, vascular endothelial growth factor (VEGF) promotes the vascularization of the growth plate and transformation of cartilage to bone. We postulate that a gene-activated matrix (GAM), created with a plasmid coding for human VEGF165, coated on a collagen sponge could efficiently accelerate bone healing in large segmental defects. MATERIALS AND METHODS: Sixty New Zealand white rabbits received a 15-mm critical size defect on one radius, which was filled with either 0.1 or 1 mg plasmid-DNA as GAM. Radiographs were obtained every 3 weeks. After 6 or 12 weeks, animals were killed. New bone was measured by microCT scans. Vascularity was measured using anti-CD31 staining of endothelial cells in 18 regions of interest per implant. RESULTS: Scaffold and control plasmid showed no defect healing, whereas most of the animals in the VEGF groups showed partial or total bone regeneration. Significantly more bone was found in the VEGF groups, with no significant differences between the 0.1- and 1-mg groups. Immunohistochemical staining of endothelial cells revealed that the VEGF groups showed two to three times the number of vessels and a significantly larger endothelial area after 6 weeks. Twelve weeks after surgery, the amount of vascularization decreased, whereas more new bone was detectable. CONCLUSIONS: The rabbit critical size defect was appropriate in size to produce atrophic nonunions. We showed that angiogenesis and osteogenesis can be promoted by a VEGF165-GAM that is an appropriate tool to induce bone healing in atrophic nonunions.     

Comparative performance of three ceramic bone graft substitutes.

BACKGROUND CONTEXT: A number of different synthetic calcium-based bone graft substitutes (BGS) are currently available for clinical use. There is, however, a lack of comparative performance data regarding the relative efficacy of these materials when placed in an osseous defect site. PURPOSE: To compare the rate, quality, and extent of osseous healing in a standard rabbit defect model for three commercially available BGS materials by measuring early bone formation and completion of defect healing and to identify whether rapid scaffold resorption stimulated or impaired bone healing. STUDY DESIGN: Osteochondral defects, 4.8 mm in diameter and 6 to 7 mm deep, were made through the articular surface into the subchondral bone of the femoral condyle of New Zealand White rabbits and filled with cylindrical pellets of one of three commercially available BGS materials: dense calcium sulfate (DCaS), ultraporous tricalcium phosphate (beta-TCP), and porous silicated calcium phosphate (Si-CaP). The repair response was examined at 1, 3, 6, and 12 weeks after surgery (n=4 per BGS per time point). METHOD: Qualitative histological and quantitative histomorphometric (% new bone, % bone graft substitute, capillary index, and mineral apposition rates) analysis. RESULTS: Rapid resorption of D-CaS, primarily through dissolution, elicited a mild inflammatory response that left the defect site empty before significant quantities of new bone were formed. Both beta-TCP and Si-CaP scaffolds supported early bone apposition (<1 week). However, beta-TCP degradation products subsequently provoked an inflammatory response that impaired and reversed bone apposition within the defect site. The Si-CaP scaffolds appeared to be more stable and supported further bone apposition, with the development of an adaptive bone-scaffold composite; cell-mediated resorption of scaffold and new bone were observed in response to local load and contributed to the production of a functional repair within the defect site. CONCLUSIONS: Rapid BGS resorption impaired the regenerative ability of local bone via three pathways: 1) insufficient persistence of an osteoconductive scaffold to encourage bone apposition, 2) destabilization of early bony apposition through scaffold disintegration, and 3) stimulation of an inflammatory response by elevated levels of particulate degradation products. This had a significant impact on the ultimate rate of healing. D-CaS did not stimulate early bone apposition, but bone repair was more advanced in D-CaS-treated defects at 12 weeks as compared with those treated with beta-TCP, despite the beta-TCP supporting direct bone apposition at 1 week. Si-CaP appeared to provide a more stable osteoconductive scaffold, which supported faster angiogenesis and bone apposition throughout the defect site, with the development of a functionally adaptive trabecular structure through resorption/remodelling of both scaffold and new bone. There was rapid formation of mineralized tissue at week 1 within the center of the defect and complete infiltration with dense, predominantly mature bone by weeks 3 to 6. The progressive remodeling of bone ingrowth and scaffold to reflect the distribution of local host tissue, combined with histological evidence of targeted osteoclastic resorption of both scaffold and bone, suggest that bone adaptation within the scaffold could be in response to Wolff's law. Although this model may not directly translate to a spinal fusion model and the products may vary according to the environment, these results suggest that, in patients in whom bone regeneration may be compromised, the degradation observed with some resorbable bone grafts may contribute to the decoupling of bone regeneration and resorbtion within the graft site, which may ultimately lead to incomplete bone repair.     

Healing of an ulnar defect using a proprietary TCP bone graft substitute, JAX, in association with autologous osteogenic cells and growth factors

Currently, available synthetic bone substitutes have adequate osteoconductive properties but have little or no osteoinductivity. Recent research has focused on using osteogenic growth factors or cells to provide this. JAX is a beta tricalcium phosphate bone graft substitute that has a novel shape and interlocking design. This study investigated delivery methods and the use of autologous cell therapy to enhance healing of a bone defect using JAX as a scaffold. Bone marrow was harvested from 24 New Zealand White rabbits. The mononuclear cell fraction was isolated and culture expanded. Bilateral 1.5 cm defects in the ulna were filled with: Group 1: JAX alone, Group 2: JAX plus 1x10(7) autologous BMSCs injected at the time of surgery, Group 3: JAX plus 8x10(6) autologous BMSCs cultured on granules for 14 days prior to surgery, Group 4: JAX plus fresh bone marrow (BMA), Group 5: cortical autograft, Group 6: JAX plus 2.5 microg VEGF. Radiographs demonstrated that there was more new bone in the BMA and VEGF groups compared to JAX alone. Groups containing autologous BMSCs were only slightly better than JAX alone in the amount of bone in the defect but did improve bridging of the osteotomy. Histomorphometry identified a significant increase in bone volume in the BMA group compared to JAX alone. BMA and VEGF enhanced healing of bone defects whereas expanded BMSCs provided little advantage over scaffold alone. There was no difference between delivery methods of autologous BMSCs. These observations suggest that the provision of osteogenic cells alone is insufficient to enhance bone healing and that additional factors are required to initiate this process in vivo.     

Coral grafting supplemented with bone marrow.

Limited success in regenerating large bone defects has been achieved by bridging them with osteoconductive materials. These substitutes lack the osteogenic and osteoinductive properties of bone autograft. A direct approach would be to stimulate osteogenesis in these biomaterials by the addition of fresh bone-marrow cells (BMC). We therefore created osteoperiosteal gaps 2 cm wide in the ulna of adult rabbits and either bridged them with coral alone (CC), coral supplemented with BMC, or left them empty. Coral was chosen as a scaffold because of its good biocompatibility and resorbability. In osteoperiosteal gaps bridged with coral only, the coral was invaded chiefly by fibrous tissue. It was insufficient to produce union after two months. In defects filled with coral and BMC an increase in osteogenesis was observed and the bone surface area was significantly higher compared with defects filled with coral alone. Bony union occurred in six out of six defects filled with coral and BMC after two months. An increase in the resorption of coral was also observed, suggesting that resorbing cells or their progenitors were present in bone marrow and survived the grafting procedure. Our findings have shown that supplementation of coral with BMC increased both the resorption of material and osteogenesis in defects of a clinical significance.     

In vivo study on the healing of bone defects treated with bone marrow stromal cells, platelet-rich plasma, and freeze-dried bone allografts, alone and in combination.

The repair of confined trabecular bone defects in rabbits treated by autologous bone marrow stromal cells (BMSC), platelet-rich plasma (PRP), freeze-dried bone allografts (FDBA) alone and in combination (BMSC + PRP; FDBA + BMSC; FDBA + PRP; FDBA + PRP + BMSC) was compared. A critical size defect was created in the distal part of the femurs of 48 adult rabbits. Histology and histomorphometry were used in the evaluation of healing at 2, 4, and 12 weeks after surgery. The healing rate (%) was calculated by measuring the residual bone defect area. Architecture of the newly formed bone was compared with that of bone at the same distal femur area of healthy rabbits. The defect healing rate was higher in PRP + BMSC, FDBA + PRP, FDBA + BMSC, and FDBA + PRP + BMSC treatments, while lower values were achieved with PRP treatment at all experimental times. The highest bone-healing rate at 2 weeks was achieved with FDBA + PRP + BMSC treatment, which resulted significantly different from PRP (p < 0.05) and BMSC (p < 0.05) treatments. At 4 weeks, the bone-healing rate increased except for PRP treatment. Finally, the bone-healing rate of FDBA + PRP, FDBA + BMSC, and FDBA + PRP + BMSC was significantly higher than that of PRP at 12 weeks (p < 0.05). At 12 weeks, significant differences still existed between PRP, BMSC, and FDBA groups and normal bone (p < 0.05). These results showed that the combination of FDBA, BMSC and PRP permitted an acceleration in bone healing and bone remodeling processes.     

BMSCs来源成骨细胞和内皮细胞复合壳聚糖-羟基磷灰石多孔支架构建血管化组织工程骨研究

目的探讨大鼠BMSCs来源的成骨细胞和内皮细胞复合壳聚糖-羟基磷灰石多孔支架植入大鼠桡骨缺损处的成骨作用和成血管作用。方法取分离培养至第3代的SD大鼠BMSCs行成骨和成内皮细胞诱导并鉴定。分别将内皮细胞(A组)、成骨细胞(B组)、混合细胞(成骨细胞和内皮细胞比例为1∶1,C组)均匀滴加于壳聚糖-羟基磷灰石多孔支架上制备3组细胞-支架复合物,MTT检测支架内细胞增殖活性。取2月龄雄性SD大鼠30只,制作大鼠桡骨5 mm长缺损模型并分别植入3组细胞-支架复合物(n=10)。术后4、8、12周分别取移植物行HE染色观察,CD34免疫组织化学染色计数微血管密度,RT-PCR法检测骨桥蛋白(osteopontin,OPN)和骨保护素(osteoprotegrin,OPG)mRNA表达。结果 BMSCs成骨诱导7 d后ALP染色可见细胞质内蓝染颗粒,细胞核呈红染;内皮细胞诱导14 d后,CD34免疫细胞化学染色可见细胞内棕色颗粒。MTT检测示3组细胞活性随时间延长逐渐升高。HE染色示,术后12周A组未见明显类骨质形成,而有较密集的微血管结构及较多纤维组织形成;B、C组可见均质的类骨质,呈条索状和岛状分布,可见大量成骨样细胞存在。术后各时间点A、C组微血管密度均显著高于B组(P<0.05);A组术后12周微血管密度高于C组(P<0.05),其余2个时间点A、C组间差异无统计学意义(P>0.05)。A组3个时间点OPN和OPG mRNA表达水平均较低,与B、C组比较差异有统计学意义(P<0.05);B、C组分别于术后8、12周OPN mRNA表达达峰值,4周时OPG mRNA表达达峰值。结论 BMSCs来源的成骨细胞和内皮细胞按1∶1比例共培养于壳聚糖-羟基磷灰石多孔支架作为组织工程骨移植物,可以促进大鼠桡骨缺损部位骨的形成和血管化,促进骨缺损愈合。     

Bone transport using hydroxyapatite loaded with bone morphogenetic protein in rabbits

The feasibility of bone transport with bone substitute and the factors which are essential for a successful bone transport are unknown. We studied six groups of 12 Japanese white rabbits. Groups A to D received cylindrical autologous bone segments and groups E and F hydroxyapatite prostheses. The periosteum was preserved in group A so that its segments had a blood supply, cells, proteins and scaffold. Group B had no blood supply. Group C had proteins and scaffold and group D had only scaffold. Group E received hydroxyapatite loaded with recombinant human bone morphogenetic protein-2 and group F had hydroxyapatite alone. Distraction osteogenesis occurred in groups A to C and E which had osteo-conductive transport segments loaded with osteo-inductive proteins. We conclude that scaffold and proteins are essential for successful bone transport, and that bone substitute can be used to regenerate bone.     

复合血管内皮祖细胞的组织工程骨在修复大段骨缺损实验中的血管化评价

目的 评价复合了血管内皮祖细胞(EPCs)的组织工程骨在修复兔桡骨大段骨缺损实验中的血管化情况.方法 自体骨髓通过不同方法的体外培养,获得EPCs及经成骨诱导的骨髓基质干细胞(BMSCs),与脱钙骨基质(DBM)构建组织工程骨修复兔桡骨大段骨缺损.用墨汁灌注、放射性核素骨显像、血管内皮细胞生长因子和Ⅷ因子相关抗原的免疫组化方法观察术后不同时期实验组(EPCs+BMSCs+DBM)、自身对照组(BMSCs+DBM)、阴性对照组(DBM)的骨组织血管化情况.结果 各组手术后2周骨缺损区新生血管数量增加,血流量开始升高,4周和8周时达到峰值,12周后开始下降.实验绀的新生血管数量和局部血流量在术后2、4、8周明显高于自身对照组和阴性对照组,血管排列更整齐.结论 复合EPCs的组织工程骨在修复大段骨缺损时,能促进早期血管化,从而进一步促进成骨.     

Repair of bone defect with vascularized tissue engineered bone graft seeded with mesenchymal stem cells in rabbits

This study evaluated the results of repair of the radius defect with a vascularized tissue engineered bone graft composed by implanting mesenchymal stem cells (MSCs) and a vascular bundle into the xenogeneic deproteinized cancellous bone (XDCB) scaffold in a rabbit model. Sixty-four rabbits were used in the study. Among them, four rabbits were used as the MSCs donor. Other 57 rabbits were divided into five groups. In group one (n = 9), a 1.5 cm bone defect was created with no repair. In group two (n = 12), the bone defect was repaired by a XDCB graft alone. In group three (n = 12), the defect was repaired by a XDCB graft that included a vascular bundle. In group four (n = 12), the defect was repaired by a XDCB graft seeded with MSCs. In group five (n = 12), the defect was repaired by a XDCB graft including a vascular bundle and MSCs implantation. The rest three rabbits were used as the normal control for the biomechanical test. The results of X-ray and histology at postoperative intervals (4, 8, and 12 weeks) and biomechanical examinations at 12 weeks showed that combining MSCs and a vascular bundle implantation resulted in promoting vascularization and osteogenesis in the XDCB graft, and improving new bone formation and mechanical property in repair of radius defect with this tissue engineered bone graft. These findings suggested that the vascularized tissue engineered bone graft may be a valuable alternative for repair of large bone defect and deserves further investigations.     

长段同种异体骨与自体带血管组织组合治疗大段骨缺损的总结

目的 通过研究血管化及促进骨愈合的方法,解决大段异体骨移植骨吸收、骨不连及再骨折等并发症.方法 对四肢10 cm以上长段骨缺损21例,采用钢板、外固定架等常规方法将大段异体骨与自体骨固定,同时采用异体骨与自体血管束、带血供骨或骨膜组合的方法,使异体骨血管化,达到促进骨愈合、防止异体骨溶解、吸收等并发症作用.其中4例采用局部血管束植入一侧断端;4例采用带血管髂骨块嵌入长段骨中部;2例带血管髂骨植入一侧骨断端;2例带血管腓骨嵌入中部;6例带血管骨膜植入一侧断端;另外血管束或骨膜加骨块组合植入3例.结果 21例中经一次治疗骨性愈合14例,经二次手术痊愈6例,失败1例.出现骨吸收、不愈合及再骨折等并发症7例,合并感染4例.经随访功能、外观满意.结论 采用自体带血管骨(膜)与同种异体骨组合移植治疗长段骨缺损,可改善大段异体骨移植的骨吸收、骨不连等并发症,有利于骨折的早期愈合.为治疗长段骨缺损较为理想的方法.     

聚乳酸作为骨形态发生蛋白载体修复骨缺损的实验研究

目的 探讨聚乳酸（ｐｏｌｙｌｄａｃ ａｃｉｄ,ＰＬＡ）作为骨形态发生蛋白（ｂｏｎ ｅ ｍｏｒｐｈｏｇｅｎｅｔｉｃ ｐｒｏ－ｔｅｉｎ,ＢＭＰ）载体的可行性及观察其诱导成骨能力。方法 手术造成日本大耳白兔左尺骨中上段１２ｍｍ骨缺损实验模型。随机分为实验。对照及空白组,实验组植入以ＰＬＡ为载体的ＢＭＰ１０ｍｇ、对照组植入以牛松质骨基质为载体的ＢＭＰ１０ｍｇ、空白组不做任何处理,术后摄Ｘ线片观察各组不同时相骨缺损修复情况,并于术后第４、８、１２周观察各组缺损内组织学变化。图像分析骨小梁的生成量。结果 实验修复情况优于对照组,无论是骨连接发生时间还是骨成熟时间,实验组均较对照组提前２周左右,同期骨生成量也明显多于对照组,而空白组缺损内主要形成纤维组织。结论 ＰＬＡ可以作为ＢＭＰ的载体修复骨缺损,它比异种松质骨基质载体的成骨效     

Circulating endothelial/skeletal progenitor cells for bone regeneration and healing

An emerging strategy in the regeneration and repair of bone is to use stem cells, including bone marrow mesenchymal stem cells, which are the most investigated and reliable source for tissue engineering, as well as circulating skeletal stem/progenitor cells, which are receiving abundant attention in regenerative medicine due to their ease of isolation and high osteogenic potential. Because failures in fracture healing are largely due to poor vascularization among many environmental factors, we highlight the first proof-of-principle experiments that elucidated the collaborative multi-lineage differentiation of circulating CD34 positive cells - a cell-enriched population of endothelial/hematopoietic progenitor cells - into not only endothelial cells but also osteoblasts. These cells develop a favorable environment for fracture healing via vasculogenesis/angiogenesis and osteogenesis, ultimately leading to functional recovery from fracture. This review will also highlight current concepts of circulating stem/progenitor cell-based therapy and their potential application for bone repair.     

VEGF165基因修饰脂肪干细胞对糖尿病大鼠骨缺损的修复研究

目的 :探讨采用基因转染大鼠脂肪干细胞构建血管化组织工程的方法对糖尿病骨质疏松性骨缺损的修复效果。方法:选取雄性Wistar大鼠78只,体重180~220 g,其中72只通过化学药物(STZ)诱导法建立糖尿病动物模型,成模大鼠血糖值均≥16.7 mmol/L。将实验动物随机分为5组,正常对照组6只,其他实验组各18只。正常对照组:在正常大鼠骨缺损内植入经VEGF165基因修饰的脂肪干细胞;糖尿病组:单纯糖尿病骨缺损大鼠;生长因子组:在糖尿病大鼠骨缺损内单纯植入VEGF生长因子;干细胞组:在糖尿病大鼠骨缺损内单纯植入脂肪干细胞;实验组:在糖尿病大鼠骨缺损内植入经VEGF165基因修饰的脂肪干细胞。将5×106个VEGF165-ADSCs细胞与凝胶海绵结合后,植入到糖尿病大鼠骨缺损模型中,在植入后第4周时,采用光学显微镜观察缺损修复组织大体形态;采用免疫组化SP法测定骨缺损区修复后局部微血管密度;应用美国IRIS IntrepidⅡXSP电感耦合等离子体发射光谱仪对修复骨痂内钙/磷含量和碱性磷酸酶(ALP)含量测定;统计分析上述测量结果验证VEGF165-ADSCs对糖尿病大鼠骨缺损的修复作用。结果:荧光染色结果显示,VEGF165表达定位于ADSCs的细胞浆,表达率在87﹪以上;大体组织学观察结果显示:实验组修复区内骨痂生成范围和质量接近正常组,糖尿病组、生长因子组、干细胞组修复效果欠佳。植入后第4周,实验组单位体积的修复组织钙、磷含量和ALP含量明显高于生长因子组、干细胞组(P0.05),与正常对照组组比较差异无统计学意义(P0.05);第4周时,实验组修复局部的血管密度低于正常对照组(P0.05),而显著高于其他组(P0.05)。结论 :VEGF165基因修饰的脂肪干细胞在糖尿病大鼠体内具有良好的成骨及成血管作用,有望成为修复糖尿病特定骨质条件下骨缺损的一种有效手段。     

肝素壳聚糖涂层促进脱细胞骨基质材料修复兔阶段性骨缺损血液灌注及血管化的实验研究

[目的]观察复合肝素壳聚糖脱细胞骨基质材料植入骨缺损早期血液供应的变化及血管化的情况.[方法]利用层层自组装方法将带有不同电荷的肝素、壳聚糖聚电解质组装到脱细胞骨基质材料上,通过超微结构及XPS等观察特性并在体外测定抗凝活性.取健康新西兰大白兔30只随机分成6组(1、3、7、14、21、28 d),制作桡骨中段骨缺损模型.一侧植入复合材料,另一侧植入单纯ACBM作为自身对照.在上述时间点行CT灌注成像、组织学等观察.[结果]时间密度曲线显示复合材料呈Ⅱ型,而对照侧呈平滑的Ⅳ型渗透模式;术后1d开始复合材料侧血容量( blood volume BV)、血流量(blood flow BF)显著高于对照侧;术后1～3d组织学观察见复合材料中有大量红细胞和有核细胞,而对照侧以液体为主,偶见细胞;3～7d在复合材料网孔中有血管形成,对照侧在血管材料外周出现且数量显著少于复合材料侧(P＜0.05).[结论]材料表面改性后体内、外均有可控缓释的抗凝效果,桡骨骨缺损植入小体积复合材料后血液呈现灌注模式并加快材料的早期血管化.     

生物衍生骨复合骨髓基质干细胞修复山羊胫骨缺损的血管化研究

目的研究生物衍生骨与骨髓基质干细胞(marrow stromal stem cells, MSCs)复合修复山羊胫骨缺损的血管化过程,了解其修复长段管状负重骨缺损的血管化情况. 方法制备生物衍生骨作为支架材料,培养、诱导MSCs作为种子细胞,二者在体外复合构建组织工程骨.20只山羊双侧胫骨中段制备成20 mm长的骨-骨膜缺损模型,采取自身左右侧对照,实验侧(右侧)缺损处植入组织工程骨,对照侧(左侧)植入单纯支架材料,采用钢板内固定.术后2、4、6及8周用墨汁灌注透明标本及血管面积图像分析方法观察血管化过程,组织学观察血管形成及成骨情况. 结果术后2、4周,实验侧血管形成较对照侧少(P<0.05);术后8周,两侧均完全血管化,差异无统计学意义(P>0.05).实验侧于术后6、8周新骨形成逐渐增加,材料降解吸收较对照组快;对照侧术后8周材料孔隙内仍无明显新骨形成. 结论生物衍生骨作为骨组织工程的支架材料,能够较快发生血管化;组织工程骨成骨能力较单纯支架材料强.     

慢病毒介导BMP-2过表达质粒转染骨髓间充质干细胞联合丝素蛋白支架向成骨细胞转化的实验研究

目的：探究慢病毒介导BMP-2过表达质粒转染骨髓间充质干细胞联合丝素蛋白支架向成骨细胞转化的作用效果。方法：构建慢病毒BMP-2过表达载体,培养骨髓间充质干细胞,构建细胞核支架的联合培养体系,体外实验利用茜素红染色和碱性磷酸酶染色检测骨髓间充质干细胞的成骨转化。选择10只新西兰大白兔,体重3.2～4.5 kg,平均3.9 kg;年龄（2.89±0.45）岁;使用口腔钻在兔子胫骨钻孔（长度5 mm、宽度2 mm、深度3 mm的锥形胫骨缺损）构建兔子胫骨骨缺损模型,HE染色观察动物模型内骨缺损的修复。实验组造模后植入丝素蛋白支架+转染BMP-2过表达载体骨髓间充质干细胞复合物,阴性对照组造模后植入丝素蛋白支架+未转染骨髓间充质干细胞复合物。结果：实验组（丝素蛋白支架+转染BMP-2过表达载体骨髓间充质干细胞复合物）中支架表面黏附的细胞与对照组（丝素蛋白支架+未转染骨髓间充质干细胞）相比,细胞数明显增多。实验组细胞外基质分泌与对照组相比,支架间细胞外基质含量明显增多。对照组支架表面元素EDX分析显示钙离子含量为0.22%,实验组支架表面元素EDX分析显示钙离子含量为0.86%,可见实验组诱导钙离子形成的能力要比对照组强。钙结节茜素红染色结果显示,对照组肉眼观无明显变化,镜下观察可见少量钙结节点。实验组肉眼观可见明显红色区域染色,镜下观察可见大量钙结节点。碱性磷酸酶染色结果显示,对照组肉眼观无明显变化,镜下观察未见明显变化。实验组肉眼观可见紫色区域染色,镜下观察可见ALP染色呈强阳性。丝素蛋白支架与骨髓间充质干细胞联合培养体系可以对软骨缺损有较好的修复作用,转染BMP-2骨髓间充质干细胞后修复作用明显优于未转染组。HE染色结果显示,对照组炎性细胞减少,支架略有消失。实验组炎性细胞明显减少,支架消失,血管生成。结论：慢病毒介导BMP-2过表达质粒可以促进BMSC向骨细胞的分化作用,并且分泌更多的含Ca²⁺成分的细胞外基质,从而发挥其促进骨缺损修复的作用。     

The early phase influence of bone marrow concentrate on metaphyseal bone healing

Bone marrow concentrate (BMC) contains high densities of progenitor cells. Therefore, in critical size defects BMC may have the potency to support bone healing. The aim of this study was to investigate the effect of BMC in combination with calcium phosphate granules (CPG) on bone defect healing in a metaphyseal long bone defect in mini-pigs. A metaphyseal critical-size bone defect at the proximal tibia of 24 mini-pigs was filled with CPG combined with BMC, CPG solely (control group) or with an autograft. Radiological and histomorphometrical evaluations after 6 weeks (42 days) showed significantly more bone formation in the BMC group in the central area of the defect zone and the cortical defect zone compared to the CPG group. At the same time the resorption rate of CPG increased significantly in the BMC group. Nevertheless, compared to the BMC group the autograft group showed a significantly higher new bone formation radiologically and histomorphometrically. In BMC the count of mononuclear cells was significantly higher compared to the bone marrow aspirate (3.5-fold). The mesenchymal progenitor cell characteristics of the cells in BMC were confirmed by flow cytometry. Cells from BMC created significantly larger colonies of alkaline phosphatase-positive colony forming units (CFU-ALP) (4.4-fold) compared to cells from bone marrow aspirate. Nevertheless, even in the BMC group complete osseous bridging was only detectable in isolated instances of the bone defects. Within the limitations of this study the BMC + CPG composite promotes bone regeneration in the early phase of bone healing significantly better than the isolated application of CPG. However, the addition of BMC does not lead to a solid fusion of the defect in the early phase of bone healing an still does not represent an equal alternative to autologous bone.     

骨髓间充质干细胞联合血管内皮生长因子基因治疗肢体缺血的实验研究

目的 观察骨髓间充质干细胞(bone mesenchymal stem cell,BMSC)联合血管内皮生长因子(vascular endothelial growth factor,VEGF)基因治疗对家兔肢体缺血模型的疗效.方法 切除新西兰兔右后肢全长股浅动脉并结扎股深动脉以建立兔后肢缺血模型,随机分为空质粒对照组(EP组)、骨髓间充质干细胞组(BMSC组)、VEGF基因治疗组(VEGF组)及联合治疗组(BV组),每组各8只.分别于治疗后28 d及30 d进行动脉造影及VEGF免疫组化染色.结果 EP组、BMSC组及VEGF组的新生血管计数组间比较差异无统计学意义(P＞0.05).BV组的新生血管计数较其余3组明显增加,差异有统计学意义(F=35.47,P＜O.01).BMSC组及VEGF组的VEGF免疫组化染色呈阳性表达,与EP组比较差异有统计学意义(F=764.32,P＜0.01).BV组的VEGF免疫组化染色呈强阳性表达,与其余3组比较差异有统计学意义(F =764.32,P＜0.01).结论 BMSC联合VEGF基因治疗兔肢体缺血可使VEGF获得稳定而有效的表达,从而改善肢体缺血.     

Dual growth factor delivery from biofunctionalized allografts: Sequential VEGF and BMP‐2 release to stimulate allograft remodeling

Autografts have been shown to stimulate osteogenesis, osteoclastogenesis, and angiogenesis, and subsequent rapid graft incorporation. Large structural allografts, however, suffer from limited new bone formation and remodeling, both of which are directly associated with clinical failure due to non‐unions, late graft fractures, and infections, making it a priority to improve large structural allograft healing. We have previously shown the osteogenic ability of a polymer‐coated allograft that delivers bone morphogenetic protein‐2 both in vitro and in vivo through both burst release and sustained release kinetics. In this study, we have demonstrated largely sequential delivery of bone morphogenetic protein‐2 and vascular endothelial growth factor from the same coated allograft. Release data showed that loading both growth factors onto a polymeric coating with two different techniques resulted in short‐term (95% release within 2 weeks) and long‐term (95% release within 5 weeks) delivery kinetics. We have also demonstrated how released VEGF, traditionally associated with angiogenesis, can also provide a stimulus for allograft remodeling via resorption. Bone marrow derived mononuclear cells were co‐cultured with VEGF released from the coated allograft and showed a statistically significant (p < 0.05) and dose dependent increase in the number of tartrate‐resistant acid phosphatase‐positive multinucleated osteoclasts. Functionality of these osteoclasts was assessed quantitatively and qualitatively by evaluating resorption pit area from both osteo‐assay plates and harvested bone. Data indicated a statistically significant higher resorption area from the cells exposed to VEGF released from the allografts over controls (p < 0.05). These results indicate that by using different loading protocols temporal control can be achieved when delivering multiple growth factors from a polymer‐coated allograft. Further, released VEGF can also stimulate osteoclastogenesis that may enhance allograft incorporation, and thus mitigate long‐term clinical complications. © 2017 Orthopedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1086–1095, 2017.

     

Combined angiogenic and osteogenic factor delivery enhances bone marrow stromal cell-driven bone regeneration.

Bone formation is a coordinated process involving various biological factors. We have developed a scaffold system capable of sustained and localized presentation of osteogenic (BMP-4) and angiogenic (VEGF) growth factors and human bone marrow stromal cells to promote bone formation at an ectopic site. Combined delivery of these factors significantly enhanced bone formation compared with other conditions. INTRODUCTION: Tissue regeneration entails complex interactions between multiple signals and materials platforms. Orchestrating the presentation of these signals may greatly enhance the regeneration of lost tissue mass. Bone formation, for example, is dependent on the signaling of BMPs, molecules initiating vascularization (e.g., vascular endothelial growth factor [VEGF]), and osteogenic precursor cells capable of responding to these cues and forming bone tissue. It was hypothesized that combined and concerted delivery of these factors from biodegradable scaffolds would lead to enhanced bone formation. MATERIALS AND METHODS: Poly(lactic-co-glycolic acid) scaffolds containing combinations of condensed plasmid DNA encoding for BMP-4, VEGF, and human bone marrow stromal cells (hBMSCs) were implanted into the subcutaneous tissue of SCID mice. Implants (n = 6) were retrieved at 3, 8, and 15 weeks after implantation. Bone and blood vessel formation was determined qualitatively and quantitatively by methods including histology, immmunostaining, and muCT. RESULTS: Scaffolds delivering VEGF resulted in a prominent increase in blood vessel formation relative to the conditions without VEGF. BMP-4 expression in scaffolds encapsulating condensed DNA was also confirmed at the 15-week time-point, showing the characteristic of long-term delivery in this system. Combined delivery of all three types of factors resulted in a significant increase in the quantity of regenerated bone compared with any factor alone or any two factors combined, as measured with DXA, X-ray, and histomorphometric analysis. Furthermore, bone formed with all three factors had elastic moduli significantly higher than any other condition. CONCLUSIONS: Concerted delivery of BMP-4, VEGF, and hBMSCs promoted greater bone formation relative to any single factor or combination of two factors. Materials systems that allows multifactorial presentation more closely mimic natural developmental processes, and these results may have important implications for bone regeneration therapeutics.