颌骨骨髓基质细胞与牙周膜细胞的生物学特性比较

目的:比较颌骨骨髓基质细胞与牙周膜细胞的基本生物学特性。方法:分离颌骨来源骨髓基质细胞和牙周膜细胞,进行改良体外原代培养。倒置显微镜观察细胞生长及克隆形成情况;CCK-8检测细胞生长曲线;免疫荧光染色检测STR0-1表达;成脂诱导后检测脂滴形成,矿化诱导后检测碱性磷酸酶的变化并用RT-PCR检测7、14 d成骨相关基因OCN的表达水平。结果:两种细胞体外培养均呈成纤维样细胞外形,能克隆生长,均具有活跃的增殖能力;两种细胞STR0-1表达阳性;成脂诱导后可见脂滴形成;矿化诱导后骨髓基质细胞的碱性磷酸酶活性较强,而且OCN基因表达较早且较强。结论:颌骨来源骨髓基质细胞具有较强的增殖及成骨分化能力,具有干细胞特性,可能是有较大临床应用潜力的组织工程种子细胞。     

体外培养狗骨髓基质细胞在牙根上生长的超微结构

目的：探讨体外培养的骨髓基质细胞及其与牙根复合后的生长特性，为牙周组织工程载体材料的选择提供实验依据。方法：分离纯化的狗骨髓基质细胞与牙根复合体外培养，分别在相差显微镜和扫描电镜下动态观察细胞及其与牙根复合生长的形态变化。结果：骨髓基质细胞在牙根上贴附生长、增殖良好，功能正常。结论：牙根与骨髓基质细胞具有良好的生物相容性，可能成为牙周组织工程的载体材料选择。     

兔骨髓基质细胞的体外成骨定向诱导培养

目的:体外分离培养兔骨髓基质细胞(bonemarrowstromalcells,BMSCs),并向成骨定向诱导培养,为骨组织工程提供适宜的种子细胞。方法:抽取新西兰白兔的骨髓,体外分离纯化得到BMSCs,并利用条件培养液将其向成骨定向诱导培养、扩增。采用倒置相差显微镜观察细胞增殖分化情况;使用碱性磷酸酶(ALP)和VonKossa染色的方法,鉴定其成骨性能;用MTT法描绘标准细胞浓度-OD值曲线。结果:BMSCs在培养皿中贴壁生长、增殖;经ALP和VonKossa染色证实其有成骨潜能;标准BMSCs细胞浓度-OD值曲线显示细胞浓度和OD值呈线性正相关。结论:可以通过在体外分离纯化骨髓并诱导培养,获得足够数量、有成骨潜能的BMSCs作为骨组织工程的种子细胞。     

骨骼肌卫星细胞体外培养及分化为成骨样细胞的实验研究

目的:评估骨骼肌卫星细胞(skeletalmusclesatellitecells,SMSCs)体外分化为成骨样细胞的能力,探讨骨骼肌卫星细胞作为骨组织工程种子细胞的可能性。方法:取新生Wistar大鼠骨骼肌,采用酶消化法分离出SMSCs,体外原代及传代培养,经腺病毒介导的人骨形成蛋白2(Ad-BMP2)基因转染后,行成骨细胞标志物活性的检测及细胞化学染色,并进行细胞体外矿化能力的测定。结果:转染后细胞的碱性磷酸酶(ALP)活性增强(P<0.01)且组织化学染色呈阳性,骨钙素及I型胶原的免疫细胞化学染色呈阳性,21d后见钙结节形成。结论:体外培养的骨骼肌卫星细胞可以向成骨方向转化,有望成为骨组织工程的种子细胞。     

骨髓基质细胞软骨向分化调控的研究进展

<正>骨髓基质细胞(bone marrow stromacells或bone marrow mesenchymal stem cells,BMSCs)是指骨髓基质内含有能分化成为成骨细胞、软骨细胞、脂肪细胞或肌细胞的前体细胞群。以往,在软骨组织工程中,所用的种子细胞为成体软骨细胞,但其存在体外培养中增殖能力有限,且扩增后细胞容易老化的缺点,从而限制了该细胞的进一步应用。     

兔骨髓基质干细胞骨向诱导实验研究

目的:研究兔骨髓基质干细胞(bone marrow stromal cells,BMSC)的体外培养以及骨向诱导分化情况,进一步探讨其作为骨组织工程种子细胞的可行性。方法:无菌条件下抽取兔的股骨骨髓,然后进行骨髓基质干细胞的分离和体外培养,并向成骨细胞定向诱导分化。结果:体外分离的骨髓基质细胞呈贴壁生长,改良MTT法测定显示骨髓基质细胞于第7.8天左右可达到增殖高峰;诱导后BMSC可向成骨细胞分化,细胞呈成骨细胞形态为梭形和多角形并可形成钙化结节。改良钙钴染色法检测诱导细胞碱性磷酸活性呈强阳性。扫描电镜观察细胞呈较典型的成骨细胞状态,并可见钙盐沉积。结论:骨髓基质细胞具有来源充足,取材方便,创伤小无明显并发症。骨髓基质干细胞分化增殖能力较强,成骨能力确定。所以采用骨髓基质细胞作为种子细胞构建组织工程骨有比较可靠的依据。     

细胞聚集体复合支撑体体外构建人鼻翼形态组织工程软骨的实验研究

目的:尝试以骨髓基质细胞(bone marrow stromal cells,BMSCs)为种子细胞,利用细胞聚集体复合聚乳乙醇酸(PLGA)内支撑体,在外支架塑形以及生物反应器环境下构建人鼻翼形态软骨.方法:全骨髓培养法收集培养兔BMSCs,转化生长因子-β1(TGF-β1)诱导,高密度连续培养构建出膜状细胞聚集体,制作人鼻翼形态PLGA内支撑体,将细胞聚集体缠绕在其表面,并用金属外支架对复合体进行塑形,进入生物反应器动态培养8周.取材从大体形态、组织学特点、蛋白多糖(glycosaminoglycans,GAG)含量以及抗压性能等方面评价新生软骨的质量.结果:体外构建的组织工程软骨保持了预先设计的外形,外观及组织学结构均类似于天然软骨,含有丰富的蛋白多糖.结论:细胞聚集体复合支撑体能形成特定形态的软骨组织,形成的组织结构均匀,细胞表型维持良好,并为软骨缺损的修复提供了新的方法.     

矿化液对骨髓基质细胞的生物学特性的影响

目的 :探讨骨髓基质细胞在矿化液的作用下生长、分化以及基质分泌情况的改变。方法 :采用矿化液 ( φ =10 ?S、10nmol/L地塞米松、5 0mg/LL 维生素C和 10mmol/Lβ 甘油磷酸钠的DMEM培养液 )诱导体外培养第 3代骨髓基质细胞 ,以含 φ =10 ?S的DMEM培养基培养第 3代骨髓基质细胞作对照。倒置相差显微镜观察细胞形态的改变 ,MTT法观察细胞诱导前后增殖情况 ,组织化学方法检测诱导前及诱导后 3、5、7d的碱性磷酸酶活性、免疫组织化学方法检测Ⅰ型胶原、Ⅲ型胶原、骨形成蛋白的合成情况 ,VonKossa方法观察矿化结节的形成情况。结果 :在矿化液的作用下骨髓基质细胞增殖性能降低 ,诱导后第 5天细胞增殖速度降低。随时间的延长碱性磷酸酶的活性增加 ,5d时Ⅰ型胶原、骨形成蛋白呈强阳性表达 ,Ⅲ型胶原逐渐呈弱阳性或阴性表达。诱导 2 0d可见矿化结节。结论 :矿化液促使骨髓基质细胞向成骨细胞转化 ,诱导第 5天的细胞可作为种子细胞与骨组织工程支架材料复合。     

磷酸三钙与珊瑚羟磷灰石细胞相容性的实验研究

目的　探讨磷酸三钙 (TCP)、珊瑚羟磷灰石 (CHA)与骨髓基质细胞的生物相容性 ,为用组织工程方法修复牙周组织缺损提供依据。方法　将骨髓基质细胞分别与TCP、CHA复合体外培养 ,进行形态学观察、细胞增殖、矿化能力测定。结果　骨髓基质细胞能在TCP、CHA上贴附、生长 ,其增殖功能不受影响 ,矿化能力亦无明显变化。结论　TCP、CHA具有良好的生物相容性 ,可作为骨髓基质细胞的载体应用于牙周组织工程研究。     

骨髓基质细胞的体外培养和成骨方向的诱导分化

目的:体外分离狗的骨髓基质细胞,诱导其成骨分化并鉴定成骨活性.方法:体外分离培养狗的骨髓基质细胞,传代培养中加入10-8rnol/L地塞米松、50 μg/ml维生素C和10mmol/L β-甘油磷酸钠进行诱导分化,进行细胞形态和增殖观察,矿化结节Von Kossa染色,细胞碱性磷酸酶染色和Ⅰ型胶原免疫组化染色检测其成骨活性.结果:骨髓基质细胞经诱导后表现出明显的成骨活性,体外矿化结节Von Kossa钙染色阳性;传代细胞碱性磷酸酶染色阳性,酶活性＞80%,Ⅰ型胶原免疫组化染色强阳性.结论:体外分离培养的骨髓基质细胞中含有骨源性前体细胞,传代细胞具有较强的成骨潜能.     

人骨形成蛋白4基因修饰的兔骨髓基质细胞异位成骨试验

目的:通过人骨形成蛋白4(bonemorphogeneticprotein,BMP4)基因修饰的兔骨髓基质细胞(bonemarrowStromalcells,bMSCs)与天然型无机骨(naturalnon鄄organicbone,NNB)支架复合,进行自体皮下异位成骨试验,探索BMP4基因治疗与组织工程技术相结合的可行性。方法:贴壁法培养兔bMSCs,体外应用脂质体介导转染pEGFP鄄hBMP4及pEGFP真核表达质粒。将转染基因的细胞和未转染基因的对照组细胞,分别以5×107/ml的浓度,与NNB支架复合,构建组织工程化骨,植入6只新西兰兔自体皮下,每组6例,4周后取材,组织学观察,并进行新骨成骨面积分析。结果:空白对照NNB孔隙内无新骨形成,转染pEGFP基因组及对照细胞组,支架网孔内均有新生骨及软骨样组织形成,而pEGFP鄄hBMP4实验组形成的新生骨及软骨样组织面积更大(P<0.05)。结论:应用hBMP4基因修饰的bMSCs作为骨组织工程的种子细胞,可望取得更强的成骨能力。     

Maxillary sinus floor elevation using a tissue-engineered bone complex with β-TCP and BMP-2 gene-modified bMSCs in rabbits

Objectives: To study the effects of maxillary sinus floor elevation by a tissue‐engineered bone complex with β tricalcium phosphate (β‐TCP) and bone morphogenetic protein‐2 (BMP‐2) gene‐modified bone marrow stromal cells (bMSCs) in rabbits. Material and methods: bMSCs derived from New Zealand rabbit bone marrow were cultured and transduced with the adenovirus with BMP‐2 (AdBMP‐2), adenovirus with enhanced green fluorescent protein gene (AdEGFP) in vitro. Gene transfer efficiency was detected by EGFP expression. These gene‐modified autologous bMSCs were then combined with a β‐TCP granule scaffold at a concentration of 2 × 10⁷ cells/ml and used to elevate the maxillary sinus floor in rabbits. Twenty rabbits were randomly allocated into groups and sacrificed at weeks 2 and 8. For each time point, 20 maxillary sinus floor elevation surgeries were made bilaterally in 10 rabbits for the following groups (n=5 per group): group A (β‐TCP alone), group B (untransduced bMSCs/β‐TCP), group C (AdEGFP–bMSCs/β‐TCP), and group D (AdBMP‐2–bMSCs/β‐TCP). All samples were evaluated by histology and histomorphometric analysis. The fate of implanted bMSCs was traced initially by a confocol fluorescent microscope in the AdEGFP group. Results: Gene transfer efficiency reached up to 60–80% with 50 PFU/cell transduction as demonstrated by fluorescent microscopic analysis in the AdEGFP group. The augmented maxillary sinus height was maintained for the four groups till 8 weeks post‐surgery, while new bone area increased over the time. At week 2, bone areas in groups B–D were significantly larger than those in group A, while at week 8, in group D, the BMP‐2 gene‐enhanced tissue‐engineered bone had the largest bone area among the groups (P<0.05, ANOVA). In that group, a mature bone structure was detected in the center of the elevated space. Under a confocal microscope, green fluorescence in newly formed bone was observed for the EGFP group, which suggested that those implanted bMSCs might have contributed to the new bone formation. Conclusion: bMSCs modified with the AdBMP‐2 gene can promote new bone formation and maturation in the rabbit maxillary sinus. BMP‐2 regional gene therapy and a tissue engineering technique could be effectively used in maxillary sinus elevation and bone regeneration.     

兔骨髓基质干细胞培养及鉴定

目的：对新西兰大白兔骨髓基质干细胞（BMSCs）进行体外培养及扩增,观察其原代及传代细胞的生长特点及生物学特点,获得足够量的细胞用于材料细胞相容性实验。方法：骨髓髓腔冲洗获得新西兰大白兔幼兔股骨骨髓,用全骨髓培养法进行体外培养获得BMSCs,胰酶消化传代,用条件培养基培养传代细胞。逐日倒置显微镜观察细胞生长情况,对传代细胞进行HE染色和碱性磷酸酶（ALP）染色。结果：幼兔骨髓基质干细胞体外培养生长良好,原代细胞约2周汇成单层,传代后1周左右长满瓶底。HE染色光镜下观察见兔骨髓基质干细胞为单核细胞,细胞呈梭形或不规则长多边形,传代细胞碱性磷酸酶染色呈强阳性。结论：兔骨髓基质干细胞的体外增殖能力强,可诱导为成骨细胞,可作为骨缺损材料细胞相容性研究的受试细胞。并且可以作为骨组织工程的种子细胞。     

腺病毒介导的骨形态发生蛋白-2基因转染脂肪间充质干细胞的实验研究

目的 探讨腺病毒介导的人骨形态发生蛋白-2（Ad-hBMP-2）基因转染大鼠脂肪间充质干细胞（ADSCs）的可行性及其成骨特性。方法 取4周龄SD大鼠腹股沟的脂肪组织,经体外分离培养的方法获得ADSCs,分别转染以腺病毒为载体的绿色荧光蛋白（Ad-GFP）基因和Ad-hBMP-2基因。用荧光显微镜每隔12 h观察转染Ad-GFP的细胞,并确定转染率,观察其形态变化,绘制生长曲线;利用免疫细胞化学法对转染Ad-hBMP-2的细胞作碱性磷酸酶（ALP）染色,用免疫细胞化学法作骨钙素（OC）检测确定成骨活性,Western blot法检测hBMP-2的表达。结果 12 h后荧光显微镜观察转染Ad-GFP的细胞,有52％的阳性表达细胞,48 h后转染率达95%;转染Ad-hBMP-2后倍增时间延长,ALP、OC检测为阳性表达,hBMP-2蛋白转染后48 h为阳性表达,并持续至第5代。结论 ADSCs可以作为腺病毒转染的载体,转染Ad-hBMP-2基因后有明显的成骨作用,可以作为骨组织工程的种子细胞。     

Maxillary sinus floor elevation using a tissue-engineered bone complex with OsteoBone and bMSCs in rabbits

Objectives: To evaluate the effects of maxillary sinus floor elevation by a tissue-engineered bone complex with OsteoBone^™ and bone marrow stromal cells (bMSCs) in rabbits.
Material and methods: Autologous bMSCs from adult New Zealand rabbits were cultured and combined with OsteoBone^™ at a concentration of 20 × 10⁶ cells/ml in vitro . Twenty-four animals were used and randomly allocated into groups. For each time point, 16 maxillary sinus floor elevation surgeries were made bilaterally in eight animals and randomly repaired by bMSCs/material (i.e. OsteoBone^™), material, autogenous bone and blood clot ( n =4 per group). A polychrome sequential fluorescent labeling was also performed post-operatively. The animals were sacrificed 2, 4 and 8 weeks after the procedure and evaluated histologically as well as histomorphometrically.
Results: New bone area significantly decreased from weeks 2 to 8 in the blood clot group, while bone area in the autologous bone reduced from weeks 4 to 8. In both groups, a significant amount of fatty tissue appeared at week 8. Accordingly, augmented height in both groups was also significantly decreased from weeks 2 to 8. The bone area in the material-alone group as well as in the bMSCs/material group, on the other hand, increased over time. Significantly more newly formed bone area and mineralization was observed in the center of the raised space in the bMSCs/material group than in the material-alone group. The augmented height was maintained in these two groups throughout the course of this study.
Conclusion: These results suggest that OsteoBone^™ can successfully be used as a bone graft substitute and that the combination of this material with bMSCs can effectively promote new bone formation in sinus elevation.     

骨保护素修饰的Beagle犬骨髓基质细胞表达体系的建立与鉴定

目的利用pSecTag2/B-OPG真核分泌表达穿梭载体,建立经骨保护素基因（OPG）修饰的Beagle犬骨髓基质细胞（BMSC）瞬时表达体系并检测其表达能力,为基因工程与组织工程联合治疗牙周病提供细胞基础。方法体外分离、培养Beagle犬BMSC,通过脂质体法将已鉴定的目的基因瞬时转染至BMSC,并用RT-PCR鉴定OPG是否有录,同时通过Western blot方法检测OPG在6周内的表达水平。结果重组质粒pSecTag2/B-OPG经HindⅢ单酶切及EcoRⅠ、BamHⅠ双酶切,电泳显示切下的片段均与预期大小相符,经测序证实此基因与GeneBank中[gi：33878056]提供的序列一致;鉴定正确的重组质粒在BMSC中有转录,并且在39 d内都明显有OPG表达。结论建立了骨保护素基因修饰的骨髓基质细胞瞬时表达体系。     

The use of tissue-engineered bone with human bone morphogenetic protein-4-modified bone-marrow stromal cells in repairing mandibular defects in rabbits

In this study, the capacity of hBMP-4 gene therapy combined with tissue-engineering techniques to improve the repair of mandibular osseous defects in rabbits was explored. A mammalian plasmid vector expressing enhanced green fluorescent protein-human bone morphogenetic protein-4 (pEGFP-hBMP-4) was initially constructed through subcloning techniques. Bone-marrow stromal cells (bMSCs) from New Zealand White rabbits were cultured and either transfected with pEGFP-hBMP-4 or pEGFP, or left untransfected in vitro. Once the transfer efficiency was determined through the expression of EGFP, cells from the three groups were combined with natural non-organic bone (NNB) at a concentration of 50 x 10(6)cells/ml and placed in 15 mm x 6 mm bilateral, full-thickness, mandibular defects surgically made in 12 rabbits. Together with NNB control, there were six samples per group. Four weeks after surgery, the implants were harvested and evaluated histomorphologically. Under optimal experimental conditions, gene transfer efficiency reached a maximum of 38.2+/-9.4%. While the percentage of new bone area in the NNB control group was 8.8+/-3.1%, in the untransfected bMSC group 22.5+/-8.2%, and in the pEGFP group 18.1+/-9.0%, a significantly higher amount of 32.5+/-6.1% was observed in the pEGFP-hBMP-4 group. These results suggest that transfection of bMSCs with hBMP-4 enhances their inherent osteogenic capacity for maxillofacial bone tissue-engineering applications.