膜引导结合Adv-BMP-2局部基因治疗老年性骨缺损的实验研究
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摘要
目的
探讨在老年骨再生功能低下时应用膜引导结合Adv-BMP-2局部基因治疗对骨缺损修复的促进作用,以寻找治疗老年性骨缺损的有效方法。
1.成骨细胞的提取、体外培养和鉴定;Adv-BMP-2的扩增、纯化、滴度测定;Adv-BMP-2对成骨细胞生物学行为的影响。
2.TNF-α对于成骨细胞生长的影响和对BMP受体表达的调控作用。
3.手术建立大鼠去势后骨质疏松牙槽骨缺损动物模型,为进一步的动物实验奠定基础。
4.应用体外法(ex vivo)进行BMP-2基因转移,研究Adv-BMP-2对骨质疏松大鼠牙槽骨缺损修复的成骨效果。
5.异体冷冻骨膜与e-PTFE膜复合红骨髓对颌骨缺损成骨性能的比较分析。
6.通过老年大鼠骨质疏松骨缺损动物模型,采用体内法(in vivo)进行BMP-2基因转移结合膜引导修复老年性骨缺损并与其他不同植骨材料进行比较,以寻找治疗老年性骨缺损的有效方法。
方法
1.成骨细胞的提取、体外培养和鉴定。原代成骨细胞来自出生1周Wistar乳鼠,进行成骨细胞培养,通过形态学观察、ALP染色、体外矿化实验,鉴定成骨细胞是否培养成功。Adv-BMP-2的扩增、纯化、滴度测定。观察成骨细胞转染Adv-BMP-2后生物学行为的变化。
2.采用MTT及PNPP法检测不同浓度TNF-α对于成骨细胞增殖和分化的影响。在保证细胞量相等的前提下,利用RT-PCR技术测定不同浓度TNF-α对于成骨细胞BMP受体表达的调控作用。
3.建立大鼠骨质疏松牙槽骨缺损动物模型并通过各种检测方法证实模型建立。40只3月龄雌性大鼠随机分为4组,A组:S0组;B组:0VX组;C组:BD组;D组:0VX+BD组。术后8周、12周测定大鼠体重变化;血清中ALP、Ca、P含量及子宫重量系数变化;用双能X线吸收法(DEXA)行颌骨密度和骨矿含量测定。
4.20只健康雌性Wistar大鼠,随机分为2组。实验组:A组为Adv-BMP-2转染成骨细胞/CS组(A/C组);对照组:B组为去势牙槽骨缺损对照组(OB组)。分别在第8周、第12周处死动物,获取标本并行大体观察、X线检查、组织学检查。
5.将健康Wistar大鼠断颈法处死后取颅骨顶骨骨膜制备异体冷冻骨膜(variant deepfreeze periosteum,VDP)备用。24只成年Wistar大鼠随机分为3组。于下颌骨升支部制造约0.5cm×0.5cm的骨缺损。A组:SO组:B组:e-PTFE/RBM组;C组:VDP/RBM组。分别在第4周、第8周处死动物,获取标本行大体观察、X线检查、组织学检查。观察不同膜引导成骨性能的效果。
6.40只雌性,老年Wistar大鼠22月龄(体重330±32g),SPF级(无特定病原体动物),随机将40只Wistar大鼠分为ABCDE五组:A组为SO组;B组为VDP/Adv-BMP-2/CS组;C组为VDP/Perioglas组;D组为VDP/HPPA组;E组为VDP/Adv- GFP/CS组。于下颌骨升支部制造约0.5cm×0.5cm的的全厚下颌骨缺损模型,BCD实验各组分别植入不同材料。A组为对照组未植入任何材料,作为空白对照。分别于术后第8周、第12周处死动物。获取标本并行大体观察、X线检查、组织学检查、免疫组化检查。E组行体内绿色荧光蛋白成骨示踪,观察各组不同时期的新生骨量并进行统计学比较。
结果
1.成骨细胞体外培养形态观察:成骨细胞初期为多角形,胞核呈椭圆形;培养1周,细胞为近立方状,体积小、核圆。碱性磷酸酶(ALP)活性检测:ALP活性是成骨细胞分化成熟的重要标志之一。体外培养扩增的细胞ALP染色和钙化实验均为阳性,证实培养的细胞具有典型的成熟成骨细胞的特征。Adv-BMP-2转染后可提高成骨细胞增殖功能和分化功能。
2.本结果显示当TNF-α浓度大于50ng/ml时对于成骨细胞增殖就有明显的抑制作用。当TNF-α的浓度大于5ng/ml时对于成骨细胞分化有明显抑制作用。不同浓度TNF-α均可以抑制BMP-IA和BMPR-Ⅱ基因的表达,但对BMP-IB却有促进作用。
3.通过手术建立动物模型并进行各种指标监测显示,手术后12周已成功地制备了大鼠骨质疏松牙槽骨缺损动物模型。为进一步的动物实验奠定了基础。
4.应用体外法(ex vivo)进行BMP-2基因转移,并进行大体观察、X线检查和组织学检查后发现:Adv-BMP-2对于骨质疏松大鼠牙槽骨缺损修复有明显的促进作用。
5.VDP及e-PTFE不同引导膜修复大鼠下颌骨缺损并与空白对照组比较。术后8周结果显示:VDP及e-PTFE膜组都有新骨形成并遍布整个骨缺损区。而对照组显示:除边缘有少量骨组织外骨缺损区充满纤维结缔组织。VDP及e-PTFE膜都可引导骨缺损形成骨组织。
6.采用体内法(in vivo)进行BMP-2基因转移结合膜引导修复老年性骨缺损并与其他不同植骨材料进行比较。结果发现不同种类的生物材料直接影响界面区早期的组织反应,膜引导结合Adv-BMP-2比其它材料较早地引导了新骨形成。
6.1.X线观察:术后8周对照A组骨缺损区界线清楚,缺损边缘可见少量高密度影。实验B组、C组、D组骨缺损区域均有呈絮状或云雾状密度增高影,材料与骨界限清楚,各组差异不明显。术后12周:对照组仍有约2mm左右的骨缺损。B组缺损修复区骨密度最高,新生骨组织覆盖缺损,缺损处密度与周围骨质基本相似,交界已模糊不清。C组、D组缺损也己完全修复,交界线也模糊不清,但缺损处密度略低于周围骨组织。
6.2.组织学观察:术后8周对照A组骨缺损区周边可见成骨细胞及少量成骨,骨缺损中心区可见大量纤维结缔组织形成。实验B组、C组、D组有不同程度成骨。B组移植骨周边诱导骨明显增加,成骨细胞增生活跃;新骨自四周向中心堆积,钙盐沉积增多。C组可见部分区域Perioglas白色透明颗粒样成骨材料基本溶解,周围留下一些颗粒溶解的空腔,在其周围形成许多基骨。仅少部分材料周围有薄层的结缔组织,在颗粒间隙可见有明显的骨母细胞、骨细胞、骨样组织。D组所有材料周围均有大量类骨质生成,但类骨质的钙化程度以及基质中细胞形态都有差异。材料已牢固地与骨组织连成一片,新骨组织包绕材料,新生骨改建明显。术后12周对照A组骨缺损区边缘有少量新骨沉积,腔内被纤维组织充满。成骨细胞和类骨质仍局限在两端,缺损中央被纤维结缔组织充填,血管少,未见成骨现象。B组缺损处几乎被新生骨组织充填,仅存少许纤维组织,周边可见骨小梁形成。C组白骨缺损中心到边缘区域的新生骨均由骨桥连结起来,Perioglas降解后留下的空腔已完全被新生骨取代。骨缺损部位骨样组织、骨组织进一步增多。D组HPPA植入后材料基本由骨组织覆盖,新骨形成明显,钙化程度高,晶界模糊。
6.3.统计分析结果:术后8周B组优于C组(P＜0.05)和D组(P＜0.05);C组与D组两组差异无显著性意义(P＞0.05)。术后12周B组、C组、D组明显优于A组(P＜0.01)。
6.4.大鼠体内绿色荧光蛋白成骨示踪:E组移植术后2周冰冻切片和连续石蜡切片,在荧光显微镜下观察,均可见移植部位呈发射绿色荧光的网状支架材料并可见成骨细胞。移植后4周冰冻切片和连续石蜡切片,在荧光显微镜下观察,仍可见移植部位呈发射绿色荧光的网状支架材料及成骨细胞。
结论
1.体外实验证实:Adv-BMP2对成骨细胞增殖功能和分化功能有明显促进作用。为后续BMP-2基因治疗的应用研究奠定了基础。
2.当TNF-α浓度大于50ng/ml时对于成骨细胞增殖就有明显的抑制作用。当TNF-α的浓度大于5ng/ml时对于成骨细胞分化有明显抑制作用。不同浓度TNF-α均可以抑制BMP-IA和BMPR-Ⅱ基因的表达,但对BMP-IB却有促进作用。总体说来,TNF-α对于BMPR复合体起抑制作用。
3.可以手术制备大鼠骨质疏松牙槽骨缺损动物模型,为进一步的动物实验奠定基础。
4.体外法(ex vivo)进行BMP-2基因转移修复骨质疏松牙槽骨缺损,Adv-BMP-2对于骨质疏松大鼠牙槽骨缺损修复有明显的促进作用。
5.VDP及e-PTFE不同引导膜都可引导大鼠颌骨缺损骨组织修复。
6.体内法(in vivo)进行BMP-2基因转移结合膜引导修复老年大鼠骨缺损比其它材料较早地引导了新骨形成,是一种理想的骨组织替代材料。特别是对老年骨再生能力低下骨缺损的修复有明显的促进作用。
Objective
The purpose of this study was to evaluate weather the guided boneregeneration technique combined with Adv-BMP-2 regional gene therapycould promote the bone regenerating function of senile bone defects, andto explore a new way to treat senile bone defects.
1.To obtain, culture in vitro, and identify the rat's osteoblasts; to amplify,purify and determine the concentration of Adv-BMP-2; and to investigatethe effects of Adv-BMP-2 transfection on biological characteristic ofosteoblasts.
2.To study the affection of TNF-αon the growth of osteoblasts and itsregulating function on the expression of BMP receptors.
3.To establish the animal model of alveolar bone defects withosteoporosis.
4.To carry on the BMP-2 gene transfer ex vivo, and to study the effect ofAdv-BMP-2 on repairing alveolar bone defects of osteoporosis rat.
5.To compare effectiveness of variant deepfreeze periosteum and e-PTFEmembranes with red bone marrow on bone regeneration in surgicallycreated bone defects.
6.To carry on the guided bone regeneration technique combined withAdv-BMP-2 regional gene therapy on the aged animal models (in vivo),and to compare the effect with other types of bone graft materials.
Methods
1. Osteoblast's isolation, culture in vitro and identification. The primary osteoblast-like cells was picked from the Wistar neonate rat, and cultured in vitro. The osteoblasts were determined through phase-contrast microscope's observation; tetrazolium salts chromatometry detection, alkali phosphatase activity measurement and dyeing with alizarin Bordeaux. Adenovirus vectors containing BMP-2 gene were reproduced and purified by cesium chloride density gradient ultracentrifugation. Biological activity identification was carried out using infectious dose. After the adenoviral vectors encoding BMP-2 gene were transfected into osteoblast, the biological characteristics of the osteoblast were examined.
2.The influence of TNF-αwith different concentration on the proliferation and differentiation of the osteoblasts was evaluated through MTT and PNTT methods. On the premise of that the cell quantity was equal, RT-PCR technique was used to examine the regulation function of TNF-αwith different concentration to the expressing of osteoblast BMP receptors.
3.Establishment of animal model. A total of 40 three months old female Wistar rats were randomly divided into four groups: control group (group A), ovariectomy group (group B), bone defects group (group C), and ovariectomy with bone defects group (group D). 8thW, 12thW after surgery, the weight change of rats was observed, ALP, Ca, P content in blood serum and womb weight coefficient were examined; jaw bone density and the bone mineral content were determined through DEXA technique.
4.Twenty female Wistar rats were randomly divided into two groups. Experimental group (group A): Osteoblast transfected by Adv-BMP-2 and Collagen Sponge group (group A/C); Control group (group B): Ovariectomy with bone defects group, (group OB). The animals were killed respectively at the 8th weeks and 12th week; morphological observation, radiographic examination, histological determination were performed.
5.Healthy Wistar rats were killed and periosteum of the parietal bone was got; and variant deepfreeze periosteum (VDP) was made of the periosteum. Twenty -four adult Wistar rats were randomly divided into three groups. Bone defects approximately 0.5 cm×0.5 cm was produced on the mandible ramus. Group A: control group (SO); group B: e-PTFE membrane/ red bone marrow; group C: variant deepfreeze periosteum / red bone marrow (VDP/ RBM) group. The animals were killed respectively at the 4th and 8th week; the specimens were obtained and examined by X-ray, histology.
6. Forty female aged Wistar rats, 22 months, (weight 330±32g), SPF (no specific pathogen animal) were randomly divided into five groups: group A was control group; group B was VDP / Adv-BMP-2/CS group; group C was VDP /perioglas group; group D was VDP / HPPA group; group E was VDP /Adv- GFP / CS group. Full thickness bone defects approximately 0.5 cm×0.5 cm was produced in the mandible rumus, group BCD were respectively implanted with different materials. As a blank control group, group A was not implant with any material. The animals were sacrificed respectively at the 8th week and the 12th week and the specimens were examined by X-ray, histology and immunohistochemical staining. Group E was examined with fluorescent microscopy.
Results
1.The osteoblasts obtained from the Wistar neonate rat were cultured. The biological characteristics of cultured cells were examined through morphological observation, staining of Alkaline Phosphatase and Staining of Red alizarin. That cultured cells had typical mature osteoblasts characteristics. Detecting by MTT method we found that the transfected osteoblasts proliferated with higher speed than the control group. Through detecting the alkali phosphatase activity, we found that the transfected osteoblasts generated more alkali phosphatase than the control group.
2.The results showed that when the concentration of TNF-αwas greater than 50 ng / ml, the proliferation of osteoblasts were significantly depressed. When the concentration of TNF-αwas higher than 5 ng / ml,the osteoblast differentiation was significantly depressed. All kinds ofconcentration of TNF-αcould inhibit BMP-IA and BMPR-II geneexpression, at the mean time could promote the BMP-IB gene expression.In a word,TNF-αplays an inhibitory role to the BMPR complex.
3.All signals showed that twelve weeks after the surgery, the alveolarbone defect animal model of osteoporosis rat had been successfullyestablished.
4.We found that Adv-BMP-2 could promote the repairing process of theosteoporosis rat alveolar bone defects.
5.Eight weeks after surgery, we compared the effects of variantdeepfreeze periosteum and the e-PTFE with the control group, the resultsshowed that variant deepfreeze periosteum and the e-PTFE may bothguide the bone defects to form the bone tissue.
6.Different types of biological material influenced the response of frontalzone organization at early time. The guided bone regeneration techniquecombined with Adv-BMP-2 regional gene therapy guided the new boneformation earlier than other methods.
6.1.Radiographic observations: Eight weeks after surgery, demarcationline in the bone defect area of the control group was clear, small amountsof high-density image were shown at the edge of the defect area. Cottonor fog- like high-densisy image was shown in the bone defect regions ofgroup B, C and D, with clear boundaries between material and bone.Differences between each group were not obvious. 12 weeks after surgery:the control group still had approximately 2mm bone defect. Bone densityof repair area of group B was the highest, which was similar withperiphery bone tissue and the border was blurred. Bone defects of group C,group D were also completely repaired, and the demarcation line was alsoambiguous, but the density of defect area was slightly lower than that ofthe periphery bone tissue.
6.2.Histological observation: 8 weeks after surgery, osteoblasts and small amounts of new bones were detected in the peripheral areas of defected bone in group A, the central area of bone defect showed massive fiber formation. Group group B, C, and D all had some new bone formed in different degrees. In group B, newly-formed bone increased significantly surrounding the transplanted bone area; the osteoblast proliferation was active; the new bone form from the circumference to the center; calcium deposition increased. In group C, the white transparent particles of Perioglas material were almost completely dissolved, leaving holes behind. Surrounding the holes, basal bone formed. This could be the microscopic mechanism of its bone induction function. Thin layer of connective tissue could be found around small amounts of materials, and obvious bone metrocytes, the bone cells, the osteoid tissue could be detected in the particle gap. In group D, all materials were surrounded by large amounts of osteoid, but the calcification degree of the osteoid and cell appearance in the matrix were different. The material had connected with the bone tissue into an entirety; the new bone tissue surrounded the material; the newborn bone's reconstruction was obvious. 12 weeks after surgery in group A, the osteoblast and ossein were still limited in the two sides, the central was filled with the fibrous connective tissue, just small amouts of blood vessels were seen and the bone formation had not been detected .In group B, the defect area was almost filled up with new bone tissue, left just a little fibrous tissue, and the trabecular bone could be seen in peripheral area. In group C, bony bridge connected the centre and the fringe of the defected area; holes left by Perioglas degradation were completely substituted by new bone. The osteoid tissue and the bone tissue in bone defect increased. In group D, the material of implanted HPPA was almost covered by the bone tissue, and there were large amount of new bone formation and a high degree of calcification, with unclear boundaries.
6.3.Statistical analysis result: 8 weeks after surgery, the effect of group B was better than group C(P<0.05) and better than group D ( P<0.05); there was no significant difference between group C and group D (P>0/05); 12weeks after surgery, the new bone formation of group B, C, D weresignificantly better than group A(P<0.01).
6.4.Osteoblasts were traced with Green fluorescent protein in vivo of rat:In group E, two weeks after transplantation, frozen section and continualparaffin section were observed under the fluorescence microscope,net-framed material could be seen with green fluorescent light, andosteoblats could be detected in the transplanted area at fourth week aftertransplantation, the same scene were found.
Conclusion
1.As one of the seeded cells of bone tissue engineering, osteoblast havemany advantages and could be used in many ways. After infectedefficiently by Adv-BMP-2, osteoblasts showed stronger capability ofosteogenesis. The study demonstrated the potentiality of BMP-2 genetherapy in the future clinical applications.
2.TNF-αplays an inhibitory role to the BMPR complex.
3.The alveolar bone defect animal model of osteoporosis rat wassuccessfully established.
4.The BMP-2 gene transfer mediated by adenovirus ex vivo could inducebone formation of osteoporosis rat alveolar bone defect.
5.Variant deepfreeze periosteum and the e-PTFE may both guide the bonedefect to form the bone tissue. This study demonstrates favorableregenerative outcomes by the use of two different types of membranesthat could be used as alternatives for guided tissue regeneration (GTR).
6.The guided bone regeneration technique combined with Adv-BMP-2regional gene therapy (in vivo) in aged rats guided the new boneformation earlier than other materials. It was an ideal bone substitutematerials, especially for the senile bone defects.
探讨在老年骨再生功能低下时应用膜引导结合Adv-BMP-2局部基因治疗对骨缺损修复的促进作用,以寻找治疗老年性骨缺损的有效方法。
1.成骨细胞的提取、体外培养和鉴定;Adv-BMP-2的扩增、纯化、滴度测定;Adv-BMP-2对成骨细胞生物学行为的影响。
2.TNF-α对于成骨细胞生长的影响和对BMP受体表达的调控作用。
3.手术建立大鼠去势后骨质疏松牙槽骨缺损动物模型,为进一步的动物实验奠定基础。
4.应用体外法(ex vivo)进行BMP-2基因转移,研究Adv-BMP-2对骨质疏松大鼠牙槽骨缺损修复的成骨效果。
5.异体冷冻骨膜与e-PTFE膜复合红骨髓对颌骨缺损成骨性能的比较分析。
6.通过老年大鼠骨质疏松骨缺损动物模型,采用体内法(in vivo)进行BMP-2基因转移结合膜引导修复老年性骨缺损并与其他不同植骨材料进行比较,以寻找治疗老年性骨缺损的有效方法。
方法
1.成骨细胞的提取、体外培养和鉴定。原代成骨细胞来自出生1周Wistar乳鼠,进行成骨细胞培养,通过形态学观察、ALP染色、体外矿化实验,鉴定成骨细胞是否培养成功。Adv-BMP-2的扩增、纯化、滴度测定。观察成骨细胞转染Adv-BMP-2后生物学行为的变化。
2.采用MTT及PNPP法检测不同浓度TNF-α对于成骨细胞增殖和分化的影响。在保证细胞量相等的前提下,利用RT-PCR技术测定不同浓度TNF-α对于成骨细胞BMP受体表达的调控作用。
3.建立大鼠骨质疏松牙槽骨缺损动物模型并通过各种检测方法证实模型建立。40只3月龄雌性大鼠随机分为4组,A组:S0组;B组:0VX组;C组:BD组;D组:0VX+BD组。术后8周、12周测定大鼠体重变化;血清中ALP、Ca、P含量及子宫重量系数变化;用双能X线吸收法(DEXA)行颌骨密度和骨矿含量测定。
4.20只健康雌性Wistar大鼠,随机分为2组。实验组:A组为Adv-BMP-2转染成骨细胞/CS组(A/C组);对照组:B组为去势牙槽骨缺损对照组(OB组)。分别在第8周、第12周处死动物,获取标本并行大体观察、X线检查、组织学检查。
5.将健康Wistar大鼠断颈法处死后取颅骨顶骨骨膜制备异体冷冻骨膜(variant deepfreeze periosteum,VDP)备用。24只成年Wistar大鼠随机分为3组。于下颌骨升支部制造约0.5cm×0.5cm的骨缺损。A组:SO组:B组:e-PTFE/RBM组;C组:VDP/RBM组。分别在第4周、第8周处死动物,获取标本行大体观察、X线检查、组织学检查。观察不同膜引导成骨性能的效果。
6.40只雌性,老年Wistar大鼠22月龄(体重330±32g),SPF级(无特定病原体动物),随机将40只Wistar大鼠分为ABCDE五组:A组为SO组;B组为VDP/Adv-BMP-2/CS组;C组为VDP/Perioglas组;D组为VDP/HPPA组;E组为VDP/Adv- GFP/CS组。于下颌骨升支部制造约0.5cm×0.5cm的的全厚下颌骨缺损模型,BCD实验各组分别植入不同材料。A组为对照组未植入任何材料,作为空白对照。分别于术后第8周、第12周处死动物。获取标本并行大体观察、X线检查、组织学检查、免疫组化检查。E组行体内绿色荧光蛋白成骨示踪,观察各组不同时期的新生骨量并进行统计学比较。
结果
1.成骨细胞体外培养形态观察:成骨细胞初期为多角形,胞核呈椭圆形;培养1周,细胞为近立方状,体积小、核圆。碱性磷酸酶(ALP)活性检测:ALP活性是成骨细胞分化成熟的重要标志之一。体外培养扩增的细胞ALP染色和钙化实验均为阳性,证实培养的细胞具有典型的成熟成骨细胞的特征。Adv-BMP-2转染后可提高成骨细胞增殖功能和分化功能。
2.本结果显示当TNF-α浓度大于50ng/ml时对于成骨细胞增殖就有明显的抑制作用。当TNF-α的浓度大于5ng/ml时对于成骨细胞分化有明显抑制作用。不同浓度TNF-α均可以抑制BMP-IA和BMPR-Ⅱ基因的表达,但对BMP-IB却有促进作用。
3.通过手术建立动物模型并进行各种指标监测显示,手术后12周已成功地制备了大鼠骨质疏松牙槽骨缺损动物模型。为进一步的动物实验奠定了基础。
4.应用体外法(ex vivo)进行BMP-2基因转移,并进行大体观察、X线检查和组织学检查后发现:Adv-BMP-2对于骨质疏松大鼠牙槽骨缺损修复有明显的促进作用。
5.VDP及e-PTFE不同引导膜修复大鼠下颌骨缺损并与空白对照组比较。术后8周结果显示:VDP及e-PTFE膜组都有新骨形成并遍布整个骨缺损区。而对照组显示:除边缘有少量骨组织外骨缺损区充满纤维结缔组织。VDP及e-PTFE膜都可引导骨缺损形成骨组织。
6.采用体内法(in vivo)进行BMP-2基因转移结合膜引导修复老年性骨缺损并与其他不同植骨材料进行比较。结果发现不同种类的生物材料直接影响界面区早期的组织反应,膜引导结合Adv-BMP-2比其它材料较早地引导了新骨形成。
6.1.X线观察:术后8周对照A组骨缺损区界线清楚,缺损边缘可见少量高密度影。实验B组、C组、D组骨缺损区域均有呈絮状或云雾状密度增高影,材料与骨界限清楚,各组差异不明显。术后12周:对照组仍有约2mm左右的骨缺损。B组缺损修复区骨密度最高,新生骨组织覆盖缺损,缺损处密度与周围骨质基本相似,交界已模糊不清。C组、D组缺损也己完全修复,交界线也模糊不清,但缺损处密度略低于周围骨组织。
6.2.组织学观察:术后8周对照A组骨缺损区周边可见成骨细胞及少量成骨,骨缺损中心区可见大量纤维结缔组织形成。实验B组、C组、D组有不同程度成骨。B组移植骨周边诱导骨明显增加,成骨细胞增生活跃;新骨自四周向中心堆积,钙盐沉积增多。C组可见部分区域Perioglas白色透明颗粒样成骨材料基本溶解,周围留下一些颗粒溶解的空腔,在其周围形成许多基骨。仅少部分材料周围有薄层的结缔组织,在颗粒间隙可见有明显的骨母细胞、骨细胞、骨样组织。D组所有材料周围均有大量类骨质生成,但类骨质的钙化程度以及基质中细胞形态都有差异。材料已牢固地与骨组织连成一片,新骨组织包绕材料,新生骨改建明显。术后12周对照A组骨缺损区边缘有少量新骨沉积,腔内被纤维组织充满。成骨细胞和类骨质仍局限在两端,缺损中央被纤维结缔组织充填,血管少,未见成骨现象。B组缺损处几乎被新生骨组织充填,仅存少许纤维组织,周边可见骨小梁形成。C组白骨缺损中心到边缘区域的新生骨均由骨桥连结起来,Perioglas降解后留下的空腔已完全被新生骨取代。骨缺损部位骨样组织、骨组织进一步增多。D组HPPA植入后材料基本由骨组织覆盖,新骨形成明显,钙化程度高,晶界模糊。
6.3.统计分析结果:术后8周B组优于C组(P＜0.05)和D组(P＜0.05);C组与D组两组差异无显著性意义(P＞0.05)。术后12周B组、C组、D组明显优于A组(P＜0.01)。
6.4.大鼠体内绿色荧光蛋白成骨示踪:E组移植术后2周冰冻切片和连续石蜡切片,在荧光显微镜下观察,均可见移植部位呈发射绿色荧光的网状支架材料并可见成骨细胞。移植后4周冰冻切片和连续石蜡切片,在荧光显微镜下观察,仍可见移植部位呈发射绿色荧光的网状支架材料及成骨细胞。
结论
1.体外实验证实:Adv-BMP2对成骨细胞增殖功能和分化功能有明显促进作用。为后续BMP-2基因治疗的应用研究奠定了基础。
2.当TNF-α浓度大于50ng/ml时对于成骨细胞增殖就有明显的抑制作用。当TNF-α的浓度大于5ng/ml时对于成骨细胞分化有明显抑制作用。不同浓度TNF-α均可以抑制BMP-IA和BMPR-Ⅱ基因的表达,但对BMP-IB却有促进作用。总体说来,TNF-α对于BMPR复合体起抑制作用。
3.可以手术制备大鼠骨质疏松牙槽骨缺损动物模型,为进一步的动物实验奠定基础。
4.体外法(ex vivo)进行BMP-2基因转移修复骨质疏松牙槽骨缺损,Adv-BMP-2对于骨质疏松大鼠牙槽骨缺损修复有明显的促进作用。
5.VDP及e-PTFE不同引导膜都可引导大鼠颌骨缺损骨组织修复。
6.体内法(in vivo)进行BMP-2基因转移结合膜引导修复老年大鼠骨缺损比其它材料较早地引导了新骨形成,是一种理想的骨组织替代材料。特别是对老年骨再生能力低下骨缺损的修复有明显的促进作用。
Objective
The purpose of this study was to evaluate weather the guided boneregeneration technique combined with Adv-BMP-2 regional gene therapycould promote the bone regenerating function of senile bone defects, andto explore a new way to treat senile bone defects.
1.To obtain, culture in vitro, and identify the rat's osteoblasts; to amplify,purify and determine the concentration of Adv-BMP-2; and to investigatethe effects of Adv-BMP-2 transfection on biological characteristic ofosteoblasts.
2.To study the affection of TNF-αon the growth of osteoblasts and itsregulating function on the expression of BMP receptors.
3.To establish the animal model of alveolar bone defects withosteoporosis.
4.To carry on the BMP-2 gene transfer ex vivo, and to study the effect ofAdv-BMP-2 on repairing alveolar bone defects of osteoporosis rat.
5.To compare effectiveness of variant deepfreeze periosteum and e-PTFEmembranes with red bone marrow on bone regeneration in surgicallycreated bone defects.
6.To carry on the guided bone regeneration technique combined withAdv-BMP-2 regional gene therapy on the aged animal models (in vivo),and to compare the effect with other types of bone graft materials.
Methods
1. Osteoblast's isolation, culture in vitro and identification. The primary osteoblast-like cells was picked from the Wistar neonate rat, and cultured in vitro. The osteoblasts were determined through phase-contrast microscope's observation; tetrazolium salts chromatometry detection, alkali phosphatase activity measurement and dyeing with alizarin Bordeaux. Adenovirus vectors containing BMP-2 gene were reproduced and purified by cesium chloride density gradient ultracentrifugation. Biological activity identification was carried out using infectious dose. After the adenoviral vectors encoding BMP-2 gene were transfected into osteoblast, the biological characteristics of the osteoblast were examined.
2.The influence of TNF-αwith different concentration on the proliferation and differentiation of the osteoblasts was evaluated through MTT and PNTT methods. On the premise of that the cell quantity was equal, RT-PCR technique was used to examine the regulation function of TNF-αwith different concentration to the expressing of osteoblast BMP receptors.
3.Establishment of animal model. A total of 40 three months old female Wistar rats were randomly divided into four groups: control group (group A), ovariectomy group (group B), bone defects group (group C), and ovariectomy with bone defects group (group D). 8thW, 12thW after surgery, the weight change of rats was observed, ALP, Ca, P content in blood serum and womb weight coefficient were examined; jaw bone density and the bone mineral content were determined through DEXA technique.
4.Twenty female Wistar rats were randomly divided into two groups. Experimental group (group A): Osteoblast transfected by Adv-BMP-2 and Collagen Sponge group (group A/C); Control group (group B): Ovariectomy with bone defects group, (group OB). The animals were killed respectively at the 8th weeks and 12th week; morphological observation, radiographic examination, histological determination were performed.
5.Healthy Wistar rats were killed and periosteum of the parietal bone was got; and variant deepfreeze periosteum (VDP) was made of the periosteum. Twenty -four adult Wistar rats were randomly divided into three groups. Bone defects approximately 0.5 cm×0.5 cm was produced on the mandible ramus. Group A: control group (SO); group B: e-PTFE membrane/ red bone marrow; group C: variant deepfreeze periosteum / red bone marrow (VDP/ RBM) group. The animals were killed respectively at the 4th and 8th week; the specimens were obtained and examined by X-ray, histology.
6. Forty female aged Wistar rats, 22 months, (weight 330±32g), SPF (no specific pathogen animal) were randomly divided into five groups: group A was control group; group B was VDP / Adv-BMP-2/CS group; group C was VDP /perioglas group; group D was VDP / HPPA group; group E was VDP /Adv- GFP / CS group. Full thickness bone defects approximately 0.5 cm×0.5 cm was produced in the mandible rumus, group BCD were respectively implanted with different materials. As a blank control group, group A was not implant with any material. The animals were sacrificed respectively at the 8th week and the 12th week and the specimens were examined by X-ray, histology and immunohistochemical staining. Group E was examined with fluorescent microscopy.
Results
1.The osteoblasts obtained from the Wistar neonate rat were cultured. The biological characteristics of cultured cells were examined through morphological observation, staining of Alkaline Phosphatase and Staining of Red alizarin. That cultured cells had typical mature osteoblasts characteristics. Detecting by MTT method we found that the transfected osteoblasts proliferated with higher speed than the control group. Through detecting the alkali phosphatase activity, we found that the transfected osteoblasts generated more alkali phosphatase than the control group.
2.The results showed that when the concentration of TNF-αwas greater than 50 ng / ml, the proliferation of osteoblasts were significantly depressed. When the concentration of TNF-αwas higher than 5 ng / ml,the osteoblast differentiation was significantly depressed. All kinds ofconcentration of TNF-αcould inhibit BMP-IA and BMPR-II geneexpression, at the mean time could promote the BMP-IB gene expression.In a word,TNF-αplays an inhibitory role to the BMPR complex.
3.All signals showed that twelve weeks after the surgery, the alveolarbone defect animal model of osteoporosis rat had been successfullyestablished.
4.We found that Adv-BMP-2 could promote the repairing process of theosteoporosis rat alveolar bone defects.
5.Eight weeks after surgery, we compared the effects of variantdeepfreeze periosteum and the e-PTFE with the control group, the resultsshowed that variant deepfreeze periosteum and the e-PTFE may bothguide the bone defects to form the bone tissue.
6.Different types of biological material influenced the response of frontalzone organization at early time. The guided bone regeneration techniquecombined with Adv-BMP-2 regional gene therapy guided the new boneformation earlier than other methods.
6.1.Radiographic observations: Eight weeks after surgery, demarcationline in the bone defect area of the control group was clear, small amountsof high-density image were shown at the edge of the defect area. Cottonor fog- like high-densisy image was shown in the bone defect regions ofgroup B, C and D, with clear boundaries between material and bone.Differences between each group were not obvious. 12 weeks after surgery:the control group still had approximately 2mm bone defect. Bone densityof repair area of group B was the highest, which was similar withperiphery bone tissue and the border was blurred. Bone defects of group C,group D were also completely repaired, and the demarcation line was alsoambiguous, but the density of defect area was slightly lower than that ofthe periphery bone tissue.
6.2.Histological observation: 8 weeks after surgery, osteoblasts and small amounts of new bones were detected in the peripheral areas of defected bone in group A, the central area of bone defect showed massive fiber formation. Group group B, C, and D all had some new bone formed in different degrees. In group B, newly-formed bone increased significantly surrounding the transplanted bone area; the osteoblast proliferation was active; the new bone form from the circumference to the center; calcium deposition increased. In group C, the white transparent particles of Perioglas material were almost completely dissolved, leaving holes behind. Surrounding the holes, basal bone formed. This could be the microscopic mechanism of its bone induction function. Thin layer of connective tissue could be found around small amounts of materials, and obvious bone metrocytes, the bone cells, the osteoid tissue could be detected in the particle gap. In group D, all materials were surrounded by large amounts of osteoid, but the calcification degree of the osteoid and cell appearance in the matrix were different. The material had connected with the bone tissue into an entirety; the new bone tissue surrounded the material; the newborn bone's reconstruction was obvious. 12 weeks after surgery in group A, the osteoblast and ossein were still limited in the two sides, the central was filled with the fibrous connective tissue, just small amouts of blood vessels were seen and the bone formation had not been detected .In group B, the defect area was almost filled up with new bone tissue, left just a little fibrous tissue, and the trabecular bone could be seen in peripheral area. In group C, bony bridge connected the centre and the fringe of the defected area; holes left by Perioglas degradation were completely substituted by new bone. The osteoid tissue and the bone tissue in bone defect increased. In group D, the material of implanted HPPA was almost covered by the bone tissue, and there were large amount of new bone formation and a high degree of calcification, with unclear boundaries.
6.3.Statistical analysis result: 8 weeks after surgery, the effect of group B was better than group C(P<0.05) and better than group D ( P<0.05); there was no significant difference between group C and group D (P>0/05); 12weeks after surgery, the new bone formation of group B, C, D weresignificantly better than group A(P<0.01).
6.4.Osteoblasts were traced with Green fluorescent protein in vivo of rat:In group E, two weeks after transplantation, frozen section and continualparaffin section were observed under the fluorescence microscope,net-framed material could be seen with green fluorescent light, andosteoblats could be detected in the transplanted area at fourth week aftertransplantation, the same scene were found.
Conclusion
1.As one of the seeded cells of bone tissue engineering, osteoblast havemany advantages and could be used in many ways. After infectedefficiently by Adv-BMP-2, osteoblasts showed stronger capability ofosteogenesis. The study demonstrated the potentiality of BMP-2 genetherapy in the future clinical applications.
2.TNF-αplays an inhibitory role to the BMPR complex.
3.The alveolar bone defect animal model of osteoporosis rat wassuccessfully established.
4.The BMP-2 gene transfer mediated by adenovirus ex vivo could inducebone formation of osteoporosis rat alveolar bone defect.
5.Variant deepfreeze periosteum and the e-PTFE may both guide the bonedefect to form the bone tissue. This study demonstrates favorableregenerative outcomes by the use of two different types of membranesthat could be used as alternatives for guided tissue regeneration (GTR).
6.The guided bone regeneration technique combined with Adv-BMP-2regional gene therapy (in vivo) in aged rats guided the new boneformation earlier than other materials. It was an ideal bone substitutematerials, especially for the senile bone defects.
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