同种异体间充质干细胞复合纤维蛋白修复骨缺损

[目的]探讨骨髓间充质干细胞(m esenchym al stem cells,MSCs)的体外培养,以及同种异体MSCs复合纤维蛋白修复兔股骨髁松质骨缺损的效果。[方法]在日本大耳兔双侧股骨髁制作0.6 cm×1.0 cm松质骨缺损,左侧植入同种异体MSCs纤维蛋白复合物,右侧单纯植入纤维蛋白。分别于术后2、5、8周行放射学、组织学检查,以了解骨缺损修复情况。[结果]单纯植入纤维蛋白的一侧不能自行愈合。植入复合物的一侧术后2周可见少量骨组织生成,5周可见大量模糊骨痂,术后8周骨缺损基本愈合。除少量炎性细胞浸润外,各期均未见明显免疫排斥反应。[结论]同种异体MSCs复合纤维蛋白可有效修复兔股骨髁松质骨缺损,8周内机体的免疫排斥反应较弱。     

骨髓间充质干细胞及其复合物修复兔桡骨缺损的实验研究

[目的]通过新西兰大白兔骨髓间充质干细胞离体培养后与去抗原牛松质骨复合植入新西兰大白兔桡骨缺损处，比较修复节段性桡骨缺损的效果，初步探讨治疗骨缺损的组织工程学方法。[方法]新西兰大白兔骨髓间充质干细胞（MSCs）离体培养、纯化、扩增后在等同于细胞培养的条件下与去抗原牛松质骨（BCB）复合培养，制成MSCs／BCB复合物并经电镜扫描证实复合物有细胞生长。制成同种异体新西兰大白兔桡骨干15mm节段性动物模型，MSCs／BCB复合物通过手术移植入动物模型桡骨缺损处，通过X线放射学、组织学、生物力学检测对比实验组与对照组、空白组骨缺损修复情况。[结果]术后2、4、8、12、16周实验组与实验对照组放射学检查评价新骨生成有显著性差异（P〈0．01）。术后组织学检查新骨生成速度、生成量均有显著性差异（P〈0．01）。16周生物力学三点抗弯曲实验载荷、弯曲应力检测，实验组与实验对照组新骨生成有显著性差异（P〈0．01），实验组标本与正常基本一致。空白对照组各时点均无新骨形成，最后缺损由纤维组织充填。[结论]骨髓间充质干细胞（MSCs）是骨组织工程中适宜的种子细胞。去抗原牛松质骨能够与兔骨髓间充质干细胞体外复合培养，移植入异体新西兰大白兔后未见明显免疫排斥反应，是可以选择的细胞载体。MSCs／BCB复合物植入修复兔桡骨缺损能够加速新骨形成，修复效果明显优于单纯BCB植入。     

骨髓间充质干细胞复合纤维蛋白凝胶修复大面积关节软骨缺损

目的 :用自体骨髓间充质干细胞 (MSCs)复合纤维蛋白修复关节软骨缺损。方法 :抽取兔自体骨髓并分离和培养MSCs ,扩增足够数量后与同种异体纤维蛋白复合在一起 ,植入股骨关节面上 5mm× 10mm的骨软骨缺损区 ,对侧缺损区只植入单纯纤维蛋白或留作空白对照。结果 :术后 12周时 ,植入复合物的缺损区再生出典型的透明软骨样结构 ,甲苯胺蓝染色和Ⅱ型胶原基因表达检测均为阳性。对侧缺损区表面仅为纤维组织。结论 :自体骨髓间充质干细胞与纤维蛋白复合物可用于修复关节软骨缺损 ,但长远期疗效尚需做进一步研究。     

自体骨髓间充质干细胞和软骨细胞共培养复合同种异体完全脱蛋白骨修复关节软骨全层缺损的研究

[目的]探讨自体骨髓间充质干细胞(bone m arrow-derived m esenchym al stem cells,BMSCs)与软骨细胞共培养复合同种异体完全脱蛋白骨(fu lly deprote in ized bone,FDB)修复关节软骨缺损的可行性,评价修复效果,为优化种子细胞源提供依据。[方法]取浓度为3×106/m l的第二代BMSCs和软骨细胞,按2:1比例混匀共培养作为种子细胞。FDB与共培养细胞复合接种植入修复缺损为实验A组、单纯FDB为对照B组和不处理为空白对照C组,移植8、16周后经人体观察、组织学评分和免疫组化染色评价缺损的修复。[结果]共培养的软骨细胞基质合成丰富,细胞增殖快。A组缺损修复组织呈软骨样,表面光滑平坦,与周围软骨整合的软骨细胞更为成熟。B组和C组的修复组织呈纤维组织和无修复。组织学评分表明A组优于B、C 2对照组,差异具有统计学意义(P<0.01),B组与C组差异无统计学意义(P>0.05)。免疫组化染色显示A组修复组织的细胞为透明软骨样细胞,柱状排列,Ⅱ型胶原染色阳性,与周围软骨及软骨下骨整合良好。[结论]自体BMSCs与软骨细胞共培养作为种子细胞,BMSCs能增强软骨细胞的增殖,促进软骨细胞基质合成,缩短软骨细胞培养时间和减少传代次数,节省大量的软骨细胞,与FDB复合后能有效修复关节软骨缺损。     

人脐血间充质干细胞修复颅骨缺损的实验研究

目的 应用人脐血间充质干细胞(umbilical cord blood derived mesenchymal stem cells,UCB-MSCs)复合脱钙骨材料构建组织工程化骨,修复裸大鼠颅骨标准缺损.方法 体外扩增培养、成骨诱导人UCB-MSCs,采用Alizarin Red染色和钙离子半定量的方法测定细胞成骨分化能力.将第2代细胞接种在脱钙骨支架材料上继续诱导培养,扫描电镜检测细胞在材料上的生长状况.制备裸大鼠双侧颅骨全层标准缺损(直径5 mm),一侧以细胞材料复合物修复作为实验侧(n=8);另一侧以单纯脱钙骨材料修复作为对照侧(n=8).术后6、12周取材,分别通过大体形态观察、显微CT(Micro-CT)、组织学方法检测颅骨缺损的修复效果.结果 UCB-MSCs体外能够诱导分化为成骨细胞,且在脱钙骨支架材料上生长良好.Micro-CT检测显示术后6周实验侧有部分新生骨组织形成,12周时骨缺损修复率达(78.19±6.45)%,脱钙骨材料降解完全;对照侧6周时无明显新骨生成,12周时材料完全降解,骨缺损未修复.组织学检测显示12周时实验侧有较多成熟骨生成,为骨性愈合;对照侧骨缺损为纤维愈合.结论 成骨诱导的人UCB.MSCs复合脱钙骨材料构建的组织工程化骨可修复裸大鼠颅骨全层标准缺损,有望成为新的组织工程骨种子细胞来源.     

骨形态发生蛋白2基因转染的骨髓间充质干细胞修复羊胫骨干骨缺损

目的 评价腺病毒介导的人骨形卷发生蛋白2(Adv-hBMP-2）基因转染的羊骨髓间充质干细胞(BMSCs)对长骨干骨缺损的修复效果．方法 22只羊，体重18．1～29．5kg．制造羊胫骨干骨缺损(2．1cm)．分为4组：Ⅰ组．Adv-hBMP-2转染BMSCs组(8只)；Ⅱ组．腺病毒介导的β半乳槠苷酶(Adv-βgal)转染BMSCs组(6只)；Ⅲ组，未转染BMSCs组(6只)；Ⅳ组，未治疗组（2只)。细胞自身分泌的细胞外基质作为细胞载体。分期行X线、计量组织学检查和生物力学测定。结果 X线检查示Ⅰ组的羊胫骨干骨缺损内有明显骨痂形成；24周，Ⅰ、Ⅱ、Ⅲ和Ⅳ组的愈合率分别为6/7、1／5、2／5及0／1，X线疗效评分显示Ⅰ组与Ⅱ、Ⅲ组的评分比较．差异有显著性意义。组织学检查显示，与其它组相比．Ⅰ组的新生骨量最多，并有皮质骨形成。生物力学测试示Ⅰ组的力学强度最大。结论 Adv-hBMP-2基因转染的BMSCs在缺乏骨传导性支架的条件下可修复长骨干骨缺损。     

负载胰岛素样生长因子-1的骨髓间充质干细胞与壳聚糖-胶原复合物修复大鼠骨缺损的实验研究

背景：目前已有较多研究表明将种子细胞复合于支架材料进行骨缺损的修复能够取得良好的效果,但仍需探索有效的生长因子以促进修复过程。 目的：探讨负载胰岛素样生长因子-1（insulin-like growth factor,IGF-1）的骨髓间充质干细胞与壳聚糖-胶原复合物对大鼠骨缺损的修复作用。 方法：分离大鼠骨髓间充质干细胞,流式细胞术鉴定细胞表面标志物;将骨髓间充质干细胞进行成骨诱导后检测碱性磷酸酶的活性以鉴定其成骨能力;IGF-1过表达载体转染骨髓间充质干细胞并筛选稳转细胞系,Real-time PCR以及Western blot鉴定稳转细胞系中IGF-1的表达。扩培细胞,并与壳聚糖-胶原进行复合培养。同时建立大鼠骨缺损模型,造模成功后将20只大鼠随机分为4组,每组5只。实验组植入负载IGF-1的骨髓间充质干细胞复合支架,支架修复组植入未经细胞复合的支架,干细胞复合支架修复组植入单纯骨髓间充质干细胞复合支架,空白对照组缺损处未植入任何物质。术后分别通过X射线和组织学切片观察比较各组大鼠的骨缺损修复情况。 结果：流式细胞术鉴定结果显示细胞CD44阳性率为98.19%,CD45阳性率为3.65%,CD90阳性率为97.62%。成骨诱导后碱性磷酸酶浓度升高至（11.57±0.48）U/L,显著高于未经成骨诱导的细胞（5.55±0.63）U/L（〈0.05）。Real-time PCR以及Western blot检测结果IGF-1稳定转染的细胞中IGF-1 mRNA以及蛋白的表达均显著升高（〈0.05）。X射线以及组织学观察结果表明空白组骨缺损不能自行修复,壳聚糖-胶原支架与骨髓间充质干细胞复合物植入大鼠后未见明显的免疫排斥反应,与其他组相比负载IGF-1的骨髓间充质干细胞与壳聚糖-胶原复合物组的成骨及修复效果均较为明显。 结论：骨髓间充质干细胞是较为理想的骨组织工程种子细胞,将IGF-1负载于骨髓间充质干细胞能够促进骨组织的修复。     

骨膜间质干细胞移植时机的实验和临床研究

目的 研究骨膜间质干细胞异体移植时机。方法 根据骨缺损内纤维组织生长时间的不同，将从大鼠骨膜分离培养的细胞分别分批植入，在植入的第2、3、4和8周时动态观察其成骨量，即骨小梁体积比；另将幼儿骨膜间质干细胞悬液，移植于26例长管状骨干骨折2周后的缺损内，观察骨折临床愈合的时间。结果 大鼠骨缺损后2周以内移植干细胞其骨小梁体积比大，愈合快，2周以后与对照组比无显著差异；临床应用9个月内病人骨折缺损愈合15例(57．7％)，骨不愈合者11例(42．3％)。结论 大鼠骨缺损2周以内移植干细胞其疗效好，大鼠实验和临床上骨缺损2周以后移植骨膜间质干细胞不能明显促进骨缺损愈合。     

兔自体骨髓基质细胞经皮移植修复骨缺损的实验研究

目的 :比较兔骨髓基质细胞 (MSC)与新鲜红骨髓经皮注射到骨缺损处后的成骨能力。方法 :1 8只新西兰兔经穿刺抽取骨髓培养 ,将扩增的MSC收集稀释成悬液 0 .5ml(细胞数约 5× 1 0 6) ,加入 1 0 0ngBMP后经皮注射到兔双侧桡骨缺损中的一侧 ( 1 8侧 ) ,对侧分别注射骨髓 0 .5ml (含BMP 1 0 0ng)( 1 3侧 )。分别于 2、 4、 8周行X线、组织病理学检查。结果 :X线、组织病理切片表明BMP MSC组多数术后 2周有明显的骨痂生成 ,术后 4～ 8周形成骨性连接 ,新骨形成、骨改建及骨髓成熟度指标均优于对照组 ,两行差异有显著性意义 (P <0 .0 5 )。结论 :在相同条件下 ,体外扩增诱导的MSC较新鲜红骨髓有更强、更快的成骨能力。经皮注射MSCs BMP能够迅速促进兔桡骨缺损处新骨形成 ,修复骨缺损。     

The study of the feasibility of segmental bone defect repair with tissue- engineered bone membrane: a qualitative observation

The objective of the study was to investigate the feasibility of intramembranous osteogenesis from tissue-engineered bone membrane in vivo. Bone marrow mesenchymal stem cells (MSCs) of rabbits were harvested, expanded and some of them were induced into osteoblasts. Porcine small intestinal submucosa (SIS) was converted by a series of physical and chemical procedures into a scaffold. MSCs and induced osteoblasts were seeded separately onto the scaffold, thus fabricating two kinds of tissue-engineered bone membrane. A total of 12 New Zealand rabbits were subjected to a surgical operation; a 15 mm bone segment, including the periosteum, was resected from the radius on both sides of each rabbit to create critical bone defects. The two kinds of tissue-engineered bone membrane and SIS (as control) were implanted randomly into the site of bone defect. The animals had radiographs and were killed after 4 weeks. The specimens were harvested and histological examination performed for evidence of osteogenesis. Bone tissue had formed in defects treated by the two kinds of tissue-engineered bone membrane at 4 weeks. This was supported by the X-ray and histological examination, which confirmed the segmental gap bridged by bone. There was no attempt to bridge in the bone defect treated by SIS. Tissue-engineered bone membrane, constructed by seeding allogeneic cells on an xenogeneic and bio-derived scaffold, can repair critical bone defects successfully.     

The combination of mesenchymal stem cells and a bone scaffold in the treatment of vertebral body defects

Purpose

Vertebral body defects represent one of the most common orthopedic challenges. In order to advance the transfer of stem cell therapies into orthopedic clinical practice, we performed this study to evaluate the safety and efficacy of a composite bioartificial graft based on a hydroxyapatite bone scaffold (CEM-OSTETIC^®) combined with human mesenchymal stem cells (MSCs) in a rat model of vertebral body defects.

Methods

Under general isoflurane anesthesia, a defect in the body of the L₂ vertebra was prepared and left to heal spontaneously (group 1), implanted with scaffold material alone (group 2), or implanted with a scaffold together with 0.5 million MSCs (group 3) or 5 million MSCs (group 4). The rats were killed 8 weeks after surgery. Histological and histomorphometrical evaluation of the implant as well as micro-CT imaging of the vertebrae were performed.

Results

We observed a significant effect on the formation of new bone tissue in the defect in group 4 when compared to the other groups and a reduced inflammatory reaction in both groups receiving a scaffold and MSCs. We did not detect any substantial pathological changes or tumor formation after graft implantation.

Conclusions

MSCs in combination with a hydroxyapatite scaffold improved the repair of a model bone defect and might represent a safe and effective alternative in the treatment of vertebral bone defects.     

Fluorescence molecular tomography enables in vivo visualization and quantification of nonunion fracture repair induced by genetically engineered mesenchymal stem cells

Fluorescence molecular tomography (FMT) is a novel tomographic near‐infrared (NIR) imaging modality that enables 3D quantitative determination of fluorochrome distribution in tissues of live small animals at any depth. This study demonstrates a noninvasive, quantitative method of monitoring engineered bone remodeling via FMT. Murine mesenchymal stem cells overexpressing the osteogenic gene BMP2 (mMSCs‐BMP2) were implanted into the thigh muscle and into a radial nonunion bone defect model in C3H/HeN mice. Real‐time imaging of bone formation was performed following systemic administration of the fluorescent bisphosphonate imaging agent OsteoSense?, an hydroxyapatite‐directed bone‐imaging probe. The mice underwent imaging on days 7, 14, and 21 postimplantation. New bone formation at the implantation sites was quantified using micro‐computed tomography (micro‐CT) imaging. A higher fluorescent signal occurred at the site of the mMSC‐BMP2 implants than that found in controls. Micro‐CT imaging revealed a mass of mature bone formed in the implantation sites on day 21, a finding also confirmed by histology. These findings highlight the effectiveness of FMT as a functional platform for molecular imaging in the field of bone regeneration and tissue engineering. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:522–530, 2008     

自体外周血及自体红骨髓干细胞与脱钙骨复合移植治疗骨缺损的比较研究

目的 对比观察自体外周血干细胞（APBSC）／脱钙骨（DB）复合移植与自体外红骨髓（ARBM）／DB复合移植治疗骨缺损的疗效。方法 36只家兔双侧桡骨造成1cm骨缺损,随机分为DB组、ARBM／DM级和APBSC／DB组,每组12只,分别进行X线片、生物力学和组织学检查,然后作对比分析。结果 术后第2、4、8、14周,APBSC／DB组和ARBM／DB组X线片、改进的GaryX线评分和光镜观察结果,以及术后第14周整骨破坏载荷和骨缺损修复形态学评分均明显优于DB组;但APBSC／DB组与ARBM／DB组间差异无显著性。结论 APBSC与ARBM都能在DB的骨形态蛋白诱导下促进成骨细胞的形成,复合移植疗效明显优于单纯DB移植。     

大鼠椎间盘巢源性干细胞的体外分离、培养和特性鉴定

目的:对大鼠椎间盘干细胞巢来源的干细胞(ISN-SCs)进行体外分离、培养和特性鉴定。方法:以10周龄雄性SD大鼠作为研究对象,按照解剖区域分离椎间盘干细胞巢组织(骺板外周软骨膜部分),用Ⅱ型胶原酶消化获取细胞后进行体外培养,选用第四代细胞进行干细胞相关特性的鉴定:采用流式细胞技术测定细胞周期;MTT法测定增殖曲线;流式细胞技术检测干细胞相关表型;q PCR检测干细胞相关基因的表达水平;细胞三系诱导分化培养后采用茜素红染色及钙钴染色检测成骨分化能力,阿利新蓝染色检测成软骨分化能力,油红O染色检测成脂肪分化能力,q PCR检测多向分化相关基因的表达水平。结果:大鼠ISN-SCs呈成纤维样细胞,多触角,具有贴壁能力,是一种慢周期细胞,高表达干细胞相关阳性表面抗原分子CD29、CD90、CD44,低表达干细胞相关阴性表面抗原分子CD34、CD45、CD19、CD11b;成骨诱导分化后茜素红染色和钙钴染色均为阳性,成软骨分化后阿利新蓝染色阳性,成脂肪分化后油红O染色阳性,且各分化相关基因(成骨:Runx2、OPN、OCN;成软骨:SOX-9、COL2a1、ACAN;成脂肪:PPARγ、C/EBPα)表达水平较对照组显著增高,其与骨髓间充质干细胞(BMSCs)具有相似的干细胞相关基因(NANOG、SOX-2、OCT-4)表达水平。结论:ISN-SCs具备干细胞的生物学特性,在体外经诱导后可以向软骨细胞及脂肪细胞转化,可为椎间盘的自体生物学修复研究提供良好的细胞来源。     

间充质干细胞治疗缺血性胆道损伤的研究进展

目前,缺血性胆道损伤（ITBL）的临床治疗仍以外科手术、内镜治疗等方法为主,具有治疗策略单一、治疗方法难度大和医疗费用高的缺点,亟需寻找一种新的治疗策略。间充质干细胞（MSC）具有高度的自我更新能力、多向分化潜能、低免疫原性及免疫调节作用等特性,已然成为再生医学中组织和器官修复的潜在种子细胞。近年来众多研究发现,MSC移植到ITBL动物模型后,不但可归巢到损伤区域,而且可通过抗凋亡、促血管新生等作用促进损伤胆道组织的修复,这表明MSC移植有望成为治疗ITBL的新策略。本文对MSC的生物学特性、MSC移植治疗ITBL的机制及其临床应用做一综述。     

组织工程骨软骨复合体修复犬膝关节负重区骨软骨缺损的实验研究

目的 观察以骨软骨支架复合骨髓基质干细胞(bone-fflarrow mesenchymal stem cells,BMSCs)修复犬膝关节负重区骨软骨缺损的疗效.方法 利用软骨细胞外基质作为软骨支架部分,以脱细胞骨作为骨支架部分,采用相分离技术制备骨软骨双相支架,将成软骨诱导的BMSCs种植到双相支架上构建组织工程骨软骨复合体,并以此复合体修复犬膝关节股骨髁负重区骨软骨缺损,分为细胞-双相支架组(实验组)和单纯支架组(对照组).分别在术后3和6个月时取材,根据大体、组织学、Micro-CT等检测结果进行半定量或定量评估.结果 大体及组织学评价表明:同一时间点实验组的修复效果优于对照组,且实验组在术后6个月时的修复效果优于其术后3个月时,两项差异均有统计学意义;而对照组小同时间点修复效果的差异无统计学意义.Micro-CT检测结果表明实验组与对照组软骨下骨均得到重建,两者的差异尤统计学意义.结论 骨软骨双相支架复合成软骨诱导的BMSCs能成功修复犬膝关节负重区的骨软骨缺损,其修复效果明显优于单纯支架植入组.     

自体外周血干细胞与脱钙骨复合移植修复骨缺损的临床应用

目的：观察自体外周血干细胞(APBSC)与脱钙骨(DBM)复合移植治疗良性骨肿瘤术后骨缺损的疗效。方法：对12例骨肿瘤患者，手术切除骨肿瘤脱钙骨填充骨缺损，术后第7天行骨髓动员，每天皮下注射特尔立3．0μg／kg，连续3d，第4天静脉滴注10mg地塞米松后采集APBSC。在X线电视透视下将一枚骨穿针准确穿入骨缺损部位，然后抽取自体外周血干细胞即可注入骨缺损部位。通过术后连续X线片，了解其骨缺损修复能力。结果：12例病人术后得到随访，时间为4～16个月，在2～4个月以内均开始有不同程度的骨化，成骨效应满意。结论：自体外周血干细胞(APBSC)／脱钙骨(DBM)复合移植是一种治疗骨缺损有效的新方法。     

The influence of 18F-fluorodeoxyglucose positron emission tomography/computed tomography on the N- and M-staging and subsequent clinical management of intrahepatic cholangiocarcinoma

BackgroundIntrahepatic cholangiocarcinoma (ICC) is a highly metastatic cancer. ¹⁸F-fluorodeoxyglucose positron emission tomography/computed tomography (¹⁸F-FDG PET/CT) enables sensitive tumor and metastasis detection. Our aim is to evaluate the influence of pre-treatment PET/CT on the N- and M-staging and subsequent clinical management in ICC patients.MethodsBetween August 2010 and August 2018, 660 consecutive ICC patients, without prior anti-tumor treatments nor other malignancies, were enrolled. The diagnostic performance of PET/CT on the N- and M-staging was compared with conventional imaging, and the preoperative staging accuracy and treatment re-allocation by PET/CT were retrospectively calculated. Survival difference was compared between patients receiving PET/CT or not after propensity score matching.ResultsPatients were divided into group A (n=291) and group B (n=369) according to whether PET/CT was performed. Among 291 patients with both PET/CT and conventional imaging for staging in group A, PET/CT showed significantly higher sensitivity (83.0% vs. 70.5%, P=0.001), specificity (88.3% vs. 74.9%, P<0.001) and accuracy (86.3% vs. 73.2%, P<0.001) than conventional imaging in diagnosing regional lymph node metastasis, as well as higher sensitivity (87.8% vs. 67.6%, P<0.001) and accuracy (93.5% vs. 89.3%, P=0.023) in diagnosing distant metastasis. Overall, PET/CT improved the accuracy of preoperative staging from 60.1% to 71.8% (P<0.001), and modified clinical treatment strategy in 5.8% (17/291) of ICC patients, with unique roles in different tumor-node-metastasis (TNM) stages. High tumor-to-non-tumor ratio (TNR) predicted poor overall survival [hazard ratio (HR) = 2.17; 95% confidence interval (CI): 1.49–3.15; P<0.001]. Furthermore, patients performing PET/CT had longer overall survival compared with those without PET/CT (HR =0.74; 95% CI: 0.58–0.93; P=0.011) after propensity score matching.ConclusionsPET/CT was valuable for diagnosing regional lymph node metastasis and distant metastasis in ICC patients, and facilitated accurate tumor staging and optimal treatment allocation.