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。     

基因修饰的组织工程化骨研究进展

利用促进成骨的蛋白多肽基因转染体外培养扩增的种子细胞,构建基因修饰的组织工程化骨。通过种子细胞表达基因产物,可有效地促进新骨的形成,为骨缺损的修复提供一种理想的方法。本文就基因修饰组织工程化骨的构建及其促进成骨的研究作一综述。     

基因修饰的组织工程化骨研究进展

利用促进成骨的蛋白多肽基因转染体外培养扩增的种子细胞，构建基因修饰的组织工程化骨，通过种子细胞表达基因产物，可有效地促进新骨的形成，为骨缺损的修复提供一种理想的方法。本文就基因修饰组织工程化骨的构建及其促进成骨的研究作一综述。     

hBMP2修饰的β-TCP/胶原支架对MC3T3-E1细胞成骨能力的影响

目的:构建含hBMP2(human bone morphogenetic proteins 2)质粒DNA的β-TCP/胶原(β-tricalcium phosphate/collagen)支架材料,并研究其对MC3T3-E1细胞成骨能力的影响.方法:制备纳米级多孔β-TCP/胶原支架并负载含hBMP2目的DNA基因及对照质粒形成基因修饰的支架材料.建立MC3T3-E1细胞株与复合支架的体外培养体系.将其分为支架组hBMP2组(Z)对照质粒组(Z0),平皿hBMP2组(M)和对照质粒组(M0).复合培养后取样通过扫描电镜观察支架表面形态,成骨诱导1、3、7、14 d检测不同浓度BMP2支架组和非支架组细胞碱性磷酸酶(ALP)的活性,并在成骨诱导的不同时间点采用实时荧光定量PCR的方法检测Runx2、OCN、ALP、OPN等成骨相关标志基因表达.检测结果进行统计学分析.结果:含hBMP2为目的基因的质粒DNA修饰纳米β-TCP/Ⅰ型胶原溶液复合材料表面呈多孔样结构;支架组和平皿组中加入hBMP2质粒DNA都能提高ALP的活性以及成骨相关标志基因的表达;支架组对MC3T3-E1细胞成骨促进能力优于平皿组.结论:负载hBMP2基因修饰的β-TC P/胶原支架材料具有良好的骨诱导性.     

实时定量PCR检测Nel样Ⅰ型分子基因对大鼠骨髓基质细胞成骨相关基因表达的影响

目的：研究Nel—like 1型分子（Nell-1）基因对体外培养大鼠骨髓基质细胞（bone marrow stromal cells,bMSCs）成骨相关基因表达的影响。方法：取6周龄雄性Fischer 344大鼠胫骨和股骨骨髓体外培养。以腺病毒为载体。进行基因转染，绘制生长曲线并测定转染效率。以转染β-半乳糖苷酶（β—Galactosidase，LacZ）基因的bMSCs为对照组，转染Nell-1的bMSCs为试验组，用实时定量PCR（real—timePCR）测定成骨相关基因碱性磷酸酶基因（alkaline phosphatase，ALP）、骨涎蛋白（bone sialoprotein，BSP）、骨钙素（osteocalcin，OC）、骨桥蛋白（osteopontin，OP）和Sox9的表达。以2^-△△CT法计算基因表达的相对比值。结果：随着Nell-1基因转染滴度的增高。细胞增殖速度减慢。基因转染效率随滴度增加而增高。Nell-1基因转染大鼠bMSCs．早期下调、中期和晚期上调ALP的表达。晚期上调OC的表达,整个成骨周期中均上调OP和BSP的表达。结论：Nell-1基因可能通过影响各成骨相关基因的表达而促进大鼠bMSCs的成骨分化。     

骨形成蛋白2基因转染的人牙周膜成纤维细胞的骨诱导作用

目的　建立表达骨形成蛋白2 (BMP-2)的人牙周膜成纤维细胞(HPDLFs),观察其体内成骨作用,为牙周缺损修复基因疗法的建立提供实验依据。方法　采用脂质体载体将人BMP-2噬菌粒表达载体pBK-B2转染至 HPDLFs。利用免疫组化ABC法检测转染细胞内BMP-2蛋白的表达,并于无菌条件下将转染细胞注射于裸鼠股部肌肉内,21 d后取材,苏木精-伊红染色观察肌肉组织内骨形成情况。结果　BMP-2基因转染后HPDLFs内有BMP- 2蛋白的表达。在注射BMP-2基因转染细胞的肌肉组织内可见明显的新骨形成。结论　BMP-2基因成功转入HP- DLFs中并得到有效表达,转染BMP-2基因的HPDLFs能够在体内诱导新骨形成。     

绿色荧光蛋白基因转染骨髓基质干细胞的瞬时表达检测

目的　检测绿色荧光蛋白基因(EGFP)的表达载体pEGFP对MSCs的转染效率及瞬时表达,为利用 pEGFP构建骨形成蛋白(BMP)表达载体并转染骨髓基质干细胞(MSCs)提供依据。方法　在体外扩增并酶切鉴定 pEGFP质粒;抽取兔骨髓,应用贴壁法培养MSCs;质粒与脂质体按照不同的比例混合,以脂质体介导法转染pEGFP, 应用荧光显微镜观察转染效率及瞬时表达情况。结果　转染效率与脂质体和质粒的比例有关,绿色荧光蛋白在基因转染24 h后开始表达,48~72 h最高,1周后表达逐渐减弱,但3~4周后仍有少量表达。结论　按照合适的质粒和脂质体比例,pEGFP转染MSCs的效率可达到30%,并能持续表达3周以上,是转染MSCs较为理想的瞬时表达载体。     

真核表达载体pIRES2-EGFP-hBMP2的构建和鉴定

目的:构建并鉴定人hBMP2基因的真核表达载体pIRES2-EGFP-hBMP2。方法:采用RT-PCR方法从人成骨肉瘤细胞中克隆hBMP2基因,连接T载体并测序正确后与真核表达载体pIRES2-EGFP连接,并进行酶切鉴定,最后将pIRES2-EGFP-hBMP2转染入HEK293细胞中,利用荧光显微镜和Western blot进行表达鉴定。结果:成功克隆了人BMP2基因,酶切鉴定和测序均正确,构建的真核表达载体pIRES2-EGFP-hBMP2能够正确表达hBMP2蛋白。结论:构建了hBMP2基因的真核表达载体pIRES2-EGFP-hBMP2,为研究该基因的功能奠定了基础。     

成骨诱导的犬骨髓间充质干细胞复合Bio-Oss构建组织工程化骨的体外研究

目的以骨髓间充质干细胞(MSCs)为种子细胞,以Bio-Oss小牛无机骨颗粒为支架材料,构建MSCs-Bio-Oss组织工程化骨。探讨犬MSCs成骨活性及与Bio-Oss复合构建组织工程化骨的可行性。方法杂种犬MSCs体外分离、培养及成骨诱导分化;取成骨诱导后的MSCs,以106个细胞/ml种植于Bio-Oss无机骨颗粒,轻度负压静置孵育培养。应用倒置相差显微镜、扫描电镜观察其形态学变化,进行细胞表面因子CD44的免疫荧光标记、钙结节染色、ALP定性和定量检测,结果以SPSS12.0统计软件包进行统计学分析。结果MSCs形态较均一,排列紧密,呈纺锤形,CD44表面抗原阳性;诱导分化后,表现出明显的成骨细胞活性,钙结节的茜素红S染色阳性,ALP的Gomori法染色阳性,ALP活性定量检测实验组在诱导分化3、7、14d时,ALP含量分别为(3.307±0.217)U/g、(5.929±0.781)U/g和(9.739±0.547)U/g,与对照组的(0.442±0.087)U/g、(0.581±0.027)U/g和(0.768±0.126)U/g比较有显著差异(P<0.01);MSCs在Bio-Oss表面贴附紧密,生长良好,构建形成组织工程化骨。结论以成骨诱导的犬MSCs作为种子细胞,复合支架材料Bio-Oss构建组织工程化骨是可行的。     

人颌骨骨膜成骨细胞复合异体部分脱钙骨的成骨实验研究

目的　研究人颌骨骨膜来源的成骨细胞接种于异体部分脱钙骨内组织工程化成骨的可行性。方法　从人颌骨骨膜分离培养成骨细胞 ,观察其体外生长情况 ,将扩增的人成骨细胞接种到异体部分脱钙松质骨支架内 ,体外和裸鼠体内培养 ,观察成骨细胞在支架孔隙内粘附、生长和成骨情况。结果　人颌骨骨膜来源的成骨细胞体外生长良好 ,细胞在异体部分脱钙松质骨支架内能正常的粘附、生长 ,裸鼠皮下培养 2月后 ,支架材料内异位形成岛状的新生骨组织。结论　人颌骨骨膜成骨细胞接种于异体部分脱钙骨内能够组织工程化新生骨组织     

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.     

人骨形成蛋白7荧光真核表达载体的构建及体外生物学活性检测

目的构建携带人骨形成蛋白7（hBMP-7）基因的绿色荧光蛋白真核表达载体pEGFP-hBMP-7，体外转染小鼠基质细胞系W-20-17，检测相关生物学活性的变化。方法应用亚克隆法构建真核表达载体pEGFP-hBMP-7，酶切电泳鉴定。脂质体法转染W-20-17细胞，检测瞬时转染效率及目的基因的表达，观察细胞形态及生长情况，检测碱性磷酸酶（ALP）、钙结节及骨钙素等成骨细胞表型。结果48h后，基因转染效率达到4｜D％，报告基因及免疫荧光证明目的基因的表达。目的基因转染后细胞形态未见明显变化，增殖能力无明显改变，ALP活性明显增高，钙结节增多，骨钙素表达增强。结论成功构建了具有生物学活性的pEGFP-hBMP-7，基因转染后能诱导W-20-17细胞向成骨细胞表型转化。     

hbFGF基因及hBMP-7基因修饰的组织工程化复合物联合应用促进牙槽骨再生动物实验研究

目的 观察人碱性成纤维细胞生长因子(hbFGF)基因及人骨形成蛋白-7(hBMP-7)基因修饰的组织工程化复合物联合应用对牙槽骨缺损再生的影响.方法 利用hbFGF基因转染Beagle犬牙龈成纤维细胞(GFs),并将其接种于脱细胞真皮基质(ADM)形成组织工程化复合物,同时以hBMP-7基因转染Beagle犬骨髓基质细胞(BMSCs),将其与胶原膜BME-10X复合,共同植入Beagle犬的人工牙周组织缺损区,通过组织学观察和测量分析,评价其对牙槽骨再生的影响.结果 术后6、12周,光镜下观察可见转染GFs复合ADM组和未转染GFs复合ADM组均较单纯BMSCs复合BME-10X组有更多的新生牙槽骨、新生牙周膜和新生牙骨质样组织生长;术后12周各组相对于术后6周的各组有更多的新生牙槽骨、新生牙周膜和新生牙骨质样组织.新生牙骨质与新生牙槽骨测量显示,转染hbFGF的GFs/ADM复合物的联合应用能显著提高hBMP-7基因修饰的组织工程化复合物修复牙周组织缺损的程度.结论 转染bFGF的GFs/ADM复合物有助于促进hBMP-7基因修饰的组织工程化复合物对牙周组织缺损的修复.     

人骨形成蛋白-4反转录病毒体系的构建及裸鼠肌内成骨实验

目的：建立hBMP-4反转录病毒体系，为BMP-4基因治疗在骨组织工程中的应用提供研究平台：方法：应用亚克隆方法构建pLEGFP—hBMP-4真核表达载体，脂质体介导下转染PA317细胞，包装病毒颗粒。收集病毒上清．感染NIH3T3细胞系，测定滴度。以同样方法转染、包装获得了绿色荧光蛋白LEGFP反转录病毒，作为实验对照。将实验组和对照组病毒液分别注射至4只裸鼠肌内，8周后取材，组织学检查，鉴定成骨的效果。结果：成功构建了pLEGFP-hBMP-4载体。转染包装细胞后，获得了较高滴度的LEGFP—hBMP-4和LEGFP反转录病毒颗粒。裸鼠肌内注射后8周，hBMP-4病毒注射部位有新生骨块形成，而EGFP组则未见新生骨块。结论：成功建立了LEGFP—hBMP-4反转录病毒体系．为利用BMP-4基因治疗进行骨修复的研究提供了反转录病毒工具。     

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.     

Nell-1基因修饰骨髓基质细胞复合β-磷酸三钙提升兔上颌窦底的实验研究

目的：评价Nel样I型分子（Nell-1）基因修饰骨髓基质细胞（bone marrow stromal cells,bMSCs）复合β-磷酸三钙提升兔上颌窦底的效果。方法：抽取兔骨髓进行bMSCs培养,体外采用腺病毒载体携带Nell-1基因（AdNell-1）及绿色荧光蛋白EGFP基因（AdEGFP）转染bMSCs,GFP表达检测转染效率、Nell-1免疫细胞化学检测目的基因的表达。碱性磷酸酶（ALP）染色、半定量检测及钙结节茜素红染色检测细胞成骨分化。将基因修饰bMSCs与β-磷酸三钙颗粒复合用于兔上颌窦底提升,分别在术后2周和8周取材,HE染色,测量成骨面积,并采用SPSS11.0软件包对2组间数据进行t检验。结果：AdEGFP基因修饰组GFP表达效率可达60%～80%,Nell-1细胞化学染色显示,AdNell-1基因修饰组呈阳性表达。AdNell-1基因修饰组ALP染色及钙结节茜素红染色均高于AdEGFP基因修饰组,ALP半定量检测具有统计学差异（P〈0.05）。体内实验研究中,AdNell-1基因修饰组新骨形成面积在8周时显著高于AdEGFP基因修饰组（P〈0.05）。结论：采用AdNell-1基因转染兔bMSCs可促进其成骨分化,体内可促进上颌窦底提升的效果。     

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.     

Maxillary sinus floor elevation using a tissue engineered bone complex with BMP-2 gene modified bMSCs and a novel porous ceramic scaffold in rabbits

Objectives

To study the effects of maxillary sinus floor elevation by a tissue engineered bone complex with bone morphogenetic protein-2 (BMP-2) gene modified bone marrow stromal cells (bMSCs) and a novel porous ceramic scaffold (OsteoBone™) in rabbits.

Materials and methods

bMSCs derived from New Zealand rabbit bone marrow were cultured and transduced with adenovirus AdBMP-2 and with AdEGFP gene (without BMP-2 gene sequence) as a control, respectively, in vitro. These bMSCs were then combined with OsteoBone™ scaffold at a concentration of 2 × 10⁷ cells/ml and used to elevate the maxillary sinus floor in rabbits. Eight rabbits were randomly allocated into groups and sacrificed at weeks 2 and 4. For each time point, 8 maxillary sinus floor elevation surgeries were made bilaterally in 4 rabbits for the two groups (n = 4 per group): group A (AdBMP-2-bMSCs/material) and group B (AdEGFP-bMSCs/material). All samples were evaluated by histologic and histomorphometric analysis.

Results

The augmented maxillary sinus height was maintained for both groups over the entire experimental period, while new bone area increased over time for group A. At week 4 after operation, bone area in group A was significantly more than that in group B (P < 0.05), and was more obviously detected in the center of the elevated space. Under a confocal microscope, green fluorescence in newly formed bone was observed in the EGFP group, which suggests that those implanted bMSCs had contributed to the new bone formation.

Conclusion

bMSCs modified with AdBMP-2 gene can promote new bone formation in elevating the rabbit maxillary sinus. OsteoBone™ scaffold could be an ideal carrier for gene enhanced bone tissue engineering.     

hBMP-7基因修饰犬骨髓基质干细胞复合珊瑚羟基磷灰石修复下颌骨缺损

目的:构建携带人骨形态发生蛋白7(hBMP-7)基因的重组腺病毒载体,体外转染犬骨髓基质干细胞(dMSCs)复合珊瑚羟基磷灰石(coral hydroxyapatite, CHA),观察其修复下颌骨缺损的效果。方法:利用AdEasy腺病毒表达系统,体外构建高效表达hBMP-7重组腺病毒载体,转染dMSCs 与珊瑚羟基磷灰石支架材料复合,再分别植入取材动物的自体下颌骨缺损区,分别于4周和8周取材观察成骨情况。结果:大体观察、X线检查及组织学观察均发现构建组织工程骨在祼鼠皮下及骨缺损处均有明显新骨组织形成。结论:构建携带人骨形态发生蛋白7(hBMP-7)基因的重组腺病毒载体,体外转染dMSCs后复合珊瑚羟基磷灰石有较多量骨组织形成,可有效修复下颌骨缺损。[关键词] 人骨形态发生蛋白7 骨髓基质干细胞 腺病毒 组织工程