应用GFP标记技术示踪裸鼠体内组织工程化骨的形成

目的应用绿色荧光蛋白(GFP)标记技术,观察组织工程化骨体内形成过程中种子细胞的变化与转归.方法用GFP重组逆转录病毒载体(GFP-RV)转染犬骨髓基质干细胞(BMSCs),将其接种于β-磷酸三钙(β-TCP),形成细胞-材料复合物,移植于裸鼠皮下.术后8周,HE染色观察组织结构,碱性磷酸酶(AKP)染色和骨钙素(OCN)免疫组化检测功能蛋白,激光共聚焦显微镜下对GFP进行示踪观察.结果 8周后,大体可见组织工程化新骨形成,组织学示新生骨小梁围绕材料孔隙生成,AKP染色和OCN免疫组化结果阳性,并可见新生组织内有呈绿色的GFP标记细胞,β-TCP部分降解.结论组织工程化骨组织学结构与松质骨小梁类似.新生组织表达GFP,证实组织工程化骨组织的形成来源于供体细胞.     

全骨髓贴壁培养兔骨髓间充质干细胞的初步研究

目的：探讨兔骨髓间充质干细胞作为组织工程种子细胞修复骨缺损的可能性。方法：选取2月龄约2．5kg重健康新西兰兔8只,采用全骨髓贴壁培养法体外分离、培养兔骨髓间充质干细胞,通过倒置相差显微镜观察、MTT检测、细胞贴壁率检测观察细胞生物学特性。结果：全骨髓贴壁培养法培养出的原代骨髓间充质干细胞活力良好,细胞数量经传代后扩增,且细胞纯度提高,但细胞扩增速度较慢。结论：全骨髓贴壁培养法简便易行,能有效的在体外获得原代骨髓间充质干细胞。所提取的细胞活性良好、纯度较高,能用作组织工程种子细胞治疗骨缺损。     

应用GFP标记技术示踪裸鼠体内组织工程化骨的形成

目的 应用绿色荧光蛋白(GFP)标记技术，观察组织工程化骨体内形成过程中种子细胞的变化与转归。方法 用GFP重组逆转录病毒载体(GFP-RV)转染犬骨髓基质于细胞(BMSCs)，将其接种于β-磷酸三钙(β-TCP)，形成细胞—材料复合物，移植于裸鼠皮下。术后8周，HE染色观察组织结构，碱性磷酸酶(AKP)染色和骨钙素(OCN)免疫组化检测功能蛋白，激光共聚焦显微镜下对GFP进行示踪观察。结果 8周后，大体可见组织工程化新骨形成，组织学示新生骨小梁围绕材料孔隙生成，AKP染色和OCN免疫组化结果阳性，并可见新生组织内有呈绿色的GFP标记细胞，β-TCP部分降解。结论 组织工程化骨组织学结构与松质骨小梁类似。新生组织表达GFP，证实组织工程化骨组织的形成来源于供体细胞。     

黑色素细胞与骨髓间充质干细胞复合构建组织工程化皮肤

目的 探讨构建含有黑色素细胞组织工程化皮肤的方法.方法 从人包皮组织中获取黑色素细胞,从人骨髓中获取骨髓间充质干细胞(BMSCs),以二者为种子细胞,按照1:10的比例混和培养,并与I型胶原膜复合体外构建组织工程化皮肤,对裸鼠创面进行修复.通过大体标本观察、4,6-联脒-2-苯基吲哚(DAPI)体内示踪标记、HE染色、免疫组织化学检测及透射电镜观察等方法,观察组织工程化皮肤修复裸鼠皮肤创面缺损及黑色素细胞的分布情况.结果 裸鼠创面皮肤生长良好,DAPI体内示踪标记、免疫组织化学检测S-100蛋白及透射电镜观察可以发现黑色素细胞以正常的组织结构形式分布于创面皮肤.结论 黑色素细胞与BMSCs通过适当的比例及体外条件培养,与I型胶原膜复合可以在体外构建出具有黑色素细胞的组织工程化皮肤.     

骨髓间充质干细胞构建人工软骨的实验研究

目的以骨髓间充质干细胞作为种子细胞,探索组织工程化软骨的构建。方法分离、获取、扩增骨髓间充质干细胞,通过将扩增的骨髓间充质干细胞接种于聚羟基乙酸(PGA)上,在成软骨诱导因子作用下作体外诱导培养3周,将实验组和对照组的标本转植入自体兔腹腔内,进行体内培养6~12周后,再进行检测,评估工程化软骨的形成。结果骨髓间充质干细胞接种于PGA支架上,经过体外诱导培养和体内培养后,实验组获得的标本出现软骨组织样外观,进行组织学切片可见软骨陷窝,进行Ⅱ型胶原蛋白的免疫组化、Ⅱ型胶原mRNA原位杂交均为阳性。结论兔骨髓间充质干细胞在成软骨诱导剂作用下,经体外和体内培养,可生成组织工程化类软骨。     

骨髓间充质干细胞-支架复合体向软骨诱导分化的实验研究

目的:以骨髓间充质干细胞作为种子细胞,探索组织工程化软骨的构建.方法:分离、获取、扩增骨髓间充质干细胞,通过将扩增的骨髓间充质干细胞接种于聚羟基乙酸(PGA)上,在成软骨诱导因子作用下在体外诱导培养3周后,将实验组和对照组的标本转植入自体兔腹腔内,进行体内培养6～12周后,再进行检测,评估工程化软骨的形成.结果:骨髓间充质干细胞接种于PGA支架上后,经过体外诱导培养和体内培养后,获得标本(7/10)出现软骨组织外观,进行组织学切片可见软骨陷窝,进行Ⅱ型胶原蛋白的免疫组化、Ⅱ型胶原mRNA原位杂交均为阳性.结论:兔骨髓间充质干细胞在成软骨诱导剂作用下,经体外和体内培养后,可生成组织工程化类软骨.     

异种脱蛋白松质骨载体的制备及性能评价

目的研究探讨异种脱蛋白松质骨性能,以制备综合性能优异的骨组织工程载体材料。方法我们将取材于成年猪股骨远端松质骨通过理化方法制作成脱蛋白松质骨载体,并对载体形态结构、组成成分、载体与种子细胞黏附及生长增殖情况以及生物力学特性进行检测分析。同时将载体材料复合自体骨髓间充质干细胞和骨形态生长因子植入成年山羊横突间进行组织工程化成骨融合,观察成骨情况,并通过免疫酶组织染色方法检测异种脱蛋白松质骨的免疫原性。结果脱蛋白松质骨具有天然网状孔隙结构,其无机成分为羟基磷灰石,有机成分为Ⅰ型胶原,力学性能保存良好,有良好的细胞相容性,免疫原性检测阴性。异种脱蛋白松质骨复合rhBMP-2和MSCs植入体内后多点成骨,效果满意。结论异种脱蛋白松质骨是一种良好的载体材料。     

骨髓间充质干细胞成骨性能的实验研究

目的研究骨髓间充质干细咆的生物学特性以及作为组织工程骨种子细胞的特点。方法分离培养兔骨髓间充质干细胞，与纳米晶羟基磷灰石胶原材料于体外联合培养，建立兔桡骨节段性缺损模型，分为空白组（n=8，不植入材料）、对照组（n=8，植入材料）、实验组（n=8，植入复合细咆的材料）。通过大体观察、组织学分析及X线摄片了解成骨情况。结果兔骨髓间充质干细胞在体外可以大量增殖，复合细咆的材料植入16周后，X线摄片可见桡骨缺损处连接良好。结论骨髓间充质干细胞具有很强的成骨作用，是一种较好的组织工程种子细胞。     

EPCs与BMSCs干细胞联合培养体系修复机体骨缺损

目的 为解决整形外科骨缺损修复, 促进移植骨成活, 应用干细胞联合培养体系复合部分脱蛋白生物骨 (PDPBB) , 对机体胫骨缺损进行修复.方法 新西兰大耳兔18只, 分别抽取骨髓液及外周血提取骨髓间充质干细胞 (BMSCs) 及外周血内皮祖细胞 (EPCs) , 构建联合培养体系后, 复合PDPBB构建组织工程骨;于兔胫骨制造1 cm长骨缺损, 将组织工程骨植入缺损区, 分别于术后14 d、28 d及2个月观察骨缺损修复的情况.结果 BMSCs组、联合培养细胞组和空白组各个时间点及组间的吸光度值比较, 差异均有统计学意义 (P<0.001) ;组织工程骨植入体内后骨胶原蛋白含量逐渐升高, 各分组差异有统计学意义 (P<0.01) ;复合联合干细胞体系的PDPBB修复骨缺损能力最强, 修复了胫骨缺损区的结构和功能.结论 EPCs与BMSCs联合干细胞体系复合PDPBB构建的组织工程骨是良好的骨缺损修复材料.     

胎兔骨髓基质细胞修复骨缺损的实验研究

为了证实体外培养的胎免骨髓基质细胞可以修复骨缺损,将培养的基质细胞吸附于明胶海绵,植入兔骨缺损处,1、2、4、6、8周作99mTc-MDP骨显像、x片、组织切片观察骨痂生长情况。结果显示,平均4周左右实验组骨缺损得到修复,提示胎儿骨髓基质细胞可以作为种子细胞来修复骨缺损。     

Tissue-engineered calcium phosphate cement in rabbit femoral condylar bone defects

Background  Calcium phosphate cement (CPC) is a favorable bone-graft substitute, with excellent biocompatibility and osteoconductivity. However, its reduced osteoinductive ability may limit the utility of CPC. To increase its osteoinductive potential, this study aimed to prepare tissue-engineered CPC and evaluate its use in the repair of bone defects. The fate of transplanted seed cells in vivo was observed at the same time.

Methods  Tissue-engineered CPC was prepared by seeding CPC with encapsulated bone mesenchymal stem cells (BMSCs) expressing recombinant human bone morphogenetic protein-2 (rhBMP-2) and green fluorescent protein (GFP). Tissue-engineered CPC and pure CPC were implanted into rabbit femoral condyle bone defects respectively. Twelve weeks later, radiographs, morphological observations, histomorphometrical evaluations, and in vivo tracing were performed.

Results  The radiographs revealed better absorption and faster new bone formation for tissue-engineered CPC than pure CPC. Morphological and histomorphometrical evaluations indicated that tissue-engineered CPC separated into numerous small blocks, with active absorption and reconstruction noted, whereas the residual CPC area was larger in the group treated with pure CPC. In the tissue-engineered CPC group, in vivo tracing revealed numerous cells expressing both GFP and rhBMP-2 that were distributed in the medullar cavity and on the surface of bony trabeculae.

Conclusion  Tissue-engineered CPC can effectively repair bone defects, with allogenic seeded cells able to grow and differentiate in vivo after transplantation.

     

Reconstruction of orbital defect in rabbits with composite of calcium phosphate cement and recombinant human bone morphogenetic protein-2

PURPOSE Calcium phosphate cement (CPC) is a biocompatible and osteoconductive bone substitute, and recombinant human bone morphogenetic protein-2 (rhBMP-2) has strong osteoinductibility, therefore we developed a composite bone substitute with CPC and rhBMP-2 and evaluate its reconstruction effect in rabbit orbital defect. METHODS Thirty-six rabbits were randomly divided into two groups and a 5mm×5mm×2mm bone defect in the infraorbital rim was induced by surgery in each orbit (72 orbits in all). The orbital defects were treated with pure CPC or composite of CPC and rhBMP-2. The osteogenesis ability of different bone substitute was evaluated by gross observation, histological examination, histomorphometrical evaluation, compressive load-to-failure testing, and scanning electron microscope (SEM). RESULTS Gross observation showed that both bone substitutes were safe and effective for reconstruction of orbital defect. However, histological examination, histomorphometrical evaluation and SEM showed that CPC/rhBMP-2 group had faster speed in new bone formation and degradation of substitute material than CPC group. Compressive load-to-failure testing showed that CPC/rhBMP-2 group had stronger compressive strength than CPC group at every stage with significant difference (P<0.05). CONCLUSIONS Composite of CPC/rhBMP-2 is an ideal bioactive material for repairing orbital defect, with good osteoconductibility and osteoinductibility.     

Reconstruction of orbital defect in rabbits with composite of calcium phosphate cement and recombinant human bone morphogenetic protein-2

Background Calcium phosphate cement （CPC） is a biocompatible and osteoconductive bone substitute, and recombinant human bone morphogenetic protein-2 （rhBMP-2） has strong osteoinductibility, therefore we developed a composite bone substitute with CPC and rhBMP-2 and evaluate its reconstruction effect in rabbit orbital defect.Methods Thirty-six rabbits were randomly divided into two groups and a 5 mmx5 mmx2 mm bone defect in the infraorbital rim was induced by surgery in each orbit （72 orbits in all）. The orbital defects were treated with pure CPC or composite of CPC and rhBMP-2. The osteogenesis ability of different bone substitute was evaluated by gross observation, histological examination, histomorphometrical evaluation, compressive load-to-failure testing, and scanning electron microscope （SEM）.Results Gross observation showed that both bone substitutes were safe and effective for reconstruction of orbital defect. However, histological examination, histomorphometrical evaluation and SEM showed that CPC/rhBMP-2 group had faster speed in new bone formation and degradation of substitute material than CPC group. Compressive load-to-failure testing showed that CPC/rhBMP-2 group had stronger compressive strength than CPC group at every stage with significant difference （P ＜0.05）.Conclusion Composite of CPC/rhBMP-2 is an ideal bioactive material for repairing orbital defect, with good osteoconductibility and osteoinductibility.     

绿色荧光蛋白标记骨髓基质干细胞在恒河猴体内构建组织工程骨的示踪作用

目的 用绿色荧光蛋白(GFP)标记恒河猴骨髓基质干细胞(rBMSC),以示踪其在体内参与组织工程骨形成的情况.方法 用OBI-293A细胞对腺病毒Ad5.CMV-GFP进行扩增,用Ad5.CMV-GFP转染rBMSc,将转染成功的第三代BMSc在转染后48 h消化制成细胞悬液,接种至块状可吸收HA上,体外培养3 d后,将其植入恒河猴(同体移植)背阔肌的肌袋内,以未转染GFP的BMSC用同样的方法接种块状可吸收HA上,作为对照,术后6周取材,4%的中性多聚甲醛固定,塑料包埋,制作骨磨片PI染色在激光共聚焦显微镜下进行观测.结果 转染GFP后,rBMSc仍贴壁生长,呈梭形或多角形,仍分裂增殖,但增殖速度有所降低.48 h可见细胞发出强烈的荧光,呈全细胞分布,计数转染率达80%.在激光共聚焦显微镜下观察骨磨片,可见材料内有发出较强荧光的细胞结构,能同时被PI染液着色.结论 绿色荧光蛋白能有效示踪组织工程骨种子细胞,种人体内的BMSC是组织工程骨骨组织形成的主要细胞来源.     

组织工程骨修复山羊负重骨大段骨缺损的长期观察

目的 探讨组织工程骨修复山羊大段负重骨骨缺损的长期效果及所用支架材料珊瑚羟基磷灰石的体内最终转归情况。方法 中国青山羊15只,制备单侧胫骨2cm的骨膜与骨缺损,缺损内植入组织工程骨（珊瑚羟基磷灰石＋经诱导分化的骨髓基质干细胞）。术后早期行ECT、X线、组织学等手段检测,评价骨缺损修复情况。远期在术后6、12、18、24月行X线及组织学检查,评价骨缺损修复情况及珊瑚羟基磷灰石的体内转归。结果 早期ECT显示在术后2个月内骨再生和再血管化进展顺利,X线和组织学显示术后组织工程骨成骨呈渐进性和偏心性：远期X线和组织学显示组织工程骨与山羊胫骨牢固愈合,并开始塑形且出现髓腔再通,珊瑚羟基磷灰石在体内逐渐成为骨基质的组成成分,自身架构消失。结论 组织工程骨可以完全修复山羊大段负重骨骨缺损,形成正常骨组织并发挥功能;珊瑚羟基磷灰石最终被降解转化成骨基质。     

组织工程骨预制及其骨缺损修复作用的研究

目的 探索组织工程化骨预制的方法,研究组织工程化预制骨修复骨缺损的可行性及实用性。方法 实验1：应用骨髓基质成骨细胞(MSO)和脱细胞骨基质(ABM)形成MSO／ABM复合人工骨(MACAB),并将其植入肌瓣内。实验2：将预制8周后的MACAB移植修复骨缺损。术后用X线摄片、组织学检查评价MACAB成骨、血管化情况及其骨缺损的修复作用。结果 MACAB肌瓣内移植成骨效果优于对照组,术后8周已完全血管化。预制骨的骨缺损修复作用优于对照组。结论 在肌瓣内成功地预制了组织工程化骨组织,预制骨(MACAB)修复了骨缺损。     

山羊胫骨大段骨缺损的组织工程骨修复及放射性核素显像监测

目的探讨组织工程骨对山羊大段骨缺损的修复能力及放射性核素骨显像技术在此过程中的监测效果。方法将27只中国青山羊,分为组织工程骨组、人工骨组和对照组(每组9只)并分别造成左侧胫骨2cm骨缺损。对组织工程骨组缺损区植入珊瑚羟基磷灰石(CHAP)和骨髓基质干细胞(BMSc)复合体,人工骨组植入CHAP,对照组则不植入任何填充物。采用放射性核素骨显像于术后2、4、8周监测各组骨修复情况。结果通过放射性核素骨显像测定的感兴趣区(ROI)计数和T/NT比值显示:对照组在各时间点均未见再血管化的表现及明显的成骨活动;人工骨组随着时间的延长其血管再生的数量和成骨的质量呈现出上升的趋势;组织工程骨组上升趋势更为显著。结论组织工程骨较人工骨具有更佳的修复大动物大段骨缺损的能力,放射性核素骨显像在修复过程中有比较准确的预测效果。     

复合富血小板血浆的可注射型组织工程骨体外培养观察

目的 以纤维蛋白胶(FG)、富血小板血浆(PRP)及骨髓基质干细胞(BMSCs)构建一种可注射型组织工程骨,体外培养并研究其体外生物学特性及超微结构.方法 从兔髂骨处抽取骨髓体外培养BMSCs并诱导向成骨细胞分化,抽取兔自体动脉血提取PRP,以FG、BMSCs、PRP共同构建可注射型组织工程骨并体外培养.观察其生物学特性如凝胶形成时间,组织学特点、细胞存活情况及其超微结构特征等.结果 构建的可注射型组织工程骨可在短时间内形成凝胶,体外培养1周时其中细胞生长良好,电镜下见纤维蛋白网格结构致密,种子细胞及血小板颗粒分布广泛.结论 以FG、BMSCs、PRP构建可注射型组织工程骨操作简单,其生物活性良好,可塑形性好,种子细胞在其中生长增殖较佳,具有较好的临床实用价值.     

COA:Effect of impaction on gene modified cells seeded on granular bone allograft in vitro and in vivo

Background Impaction bone grafting during revision joint surgery to restore bone stock may result in slow and limited allograft incorporation into host bone. A new approach has been proposed by gene modified bone marrow stromal cells (BMSCs) in combination with impaction bone grafting may effectively restore bone stock and improve allograft incorporation. This study aims to investigate the effect of impaction on gene modified BMSCs seeded on granular bone allograft in vitro and in vivo. Methods Deep frozen granular cancellous bone allograft from canine was prepared to serve as cell delivery scaffold, which was seeded with green fluorescent protein (GFP) genetically modified BMSCs to construct cell allograft composites. The composites were impacted in a simulative impactor model and treated with successive incubation under the standard condition after impaction in vitro. Four Beagle dogs, treated with bilateral uncemented proximal tibial joint hemiarthroplasty with prosthesises, were implanted with autologous GFP gene modified cell allograft composites to repair the bone cavity around the prothesis to mimic impaction bone grafting in vivo. Results Significant reduction of viable cells was observed after impaction under fluorescence microscope in vitro, but a proportion of GFP positive cells remained alive and functionally active to secret GFP protein in vitro and in vivo. Conclusions Gene modified bone marrow MSCs seeded on granular allograft will withstand the forces of impaction and maintain their normal functions in vitro and in vivo in spite of partial loss.     

Effect of impaction on gene-modified cells seeded on granular bone allografts in vitro and in vivo

Background While attempting to restore bone stock, impaction bone grafting employed during revision joint surgery may result in slow and limited allograft incorporation into host bone. A new approach including gene-modified bone marrow stromal cells （BMSCs） in combination with impaction bone grafting may effectively restore bone stock and improve allograft incorporation. This study aimed to investigate the effect of impaction on gene-modified BMSCs seeded on granular bone allografts in vitro and in vivo.Methods Deep-frozen, granular, cancellous bone allografts from canines were prepared to serve as cell delivery scaffolds and were seeded with green fluorescent protein （GFP） genetically-modified BMSCs to construct cell-allograft composites. The composites were impacted in a simulative, in vitro impaction model and cultured for further analysis under standard conditions. Four Beagle dogs, treated with bilateral, uncemented proximal tibial joint hemiarthroplasty with a prosthesis, were implanted with autologous GFP gene-modified cell-allograft composites to repair the bone cavity around each prosthesis.Results A significant reduction in cell viability was observed after impaction by fluorescence microscopy in vitro.However, there remained a proportion of GFP-positive cells that were viable and functionally active, as evidenced by the secretion of GFP protein in vitro and in vivo.Conclusions Gene-modified BMSCs seeded on granular allografts were able to withstand the impaction forces and to maintain their normal functions in vitro and in vivo, in spite of a partial loss in cell viability.