Abstract
BACKGROUND:Bone morphogenetic protein-2 (BMP-2) gene transfection can promote the proliferation and differentiation of bone marrow stromal stem cells and induce heterotopic bone formation. However, this method is complicated to operate and in vitro amplification needs a long time, so it is inconvenient in clinic.
OBJECTIVE: After treated with adenovirus vector carrying BMP-2 (Ad-BMP-2), fibrin gel and polylactic acid/polycaprolactone (PLA/PCL), artificial bone was used to repair bone defects.
DESIGN: A randomized control animal study.
SETTING: Experiments were performed at the Central Laboratory of China-Japan Friendship Hospital Affiliated to Jilin University from September 2003 to December 2004.
MATERIALS: PLA/PCL biodegradable materials were provided by Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, with the pore diameter of 150-250 μm and porosity of above 90%. Sixty New Zealand flap-eared white rabbits aged 3 months were selected.
METHODS: Artificial bone treated by four different methods was implanted into 1.5 cm bone defect region on the midpiece of the bilateral radial bone of rabbits. Artificial bone treated with Ad-BMP-2+ fibrin gel+ PLA/PCL was implanted into rabbits in the Ad-BMP-2 group; treated with recombinant BMP-2+ fibrin gel+ PLA/PCL was implanted in the recombinant BMP-2 group; treated with adenovirus vector carrying β-galactosidase (Ad-Lacz)+ fibrin gel+ PLA/PCL was implanted in the control gene group; treated with fibrin gel+ PLA/PCL was implanted in the PLA/PCL group.
MAIN OUTCOME MEASURES: Four, eight, twelve weeks after surgery, X-ray was used to detect callus gray (representing new bone density) on the bilateral anterior limbs. Hematoxylin-eosin (HE) staining was employed to observe the repair of bone defects. Alcian Blue staining and Vankossa staining were used to examine chondrogenesis and mineralization. Electron microscope was applied to investigate maturity of bone cells, degradation and absorption of scaffolds. Twelve weeks after surgery, biodynamic test was performed to evaluate bending strength of new bone.
RESULTS: Twelve weeks after surgery, osteogenic activity, amount of bone regeneration and structure of medullary cavity were significantly better in the defect regions in the Ad-BMP-2 group than in the recombinant BMP-2 group. Bone defects were completely repaired. Bone union was not detected in the control gene and PLA/PCL groups.
CONCLUSION: PLA/PCL combined with fibrin gel carrying BMP-2 gene can obtain good effects on repairing segmental bone defects.




INTRODUCTION

Previous studies demonstrated that bone morphogenetic protein-2 (BMP-2) gene transfection could promote the proliferation and differentiation of bone marrow stromal stem cells and induce heterotopic bone formation [1-2]. However, this method is complicated to operate and needs long-time in vitro amplification, so it is inconvenient in clinic. This study aimed to repair rabbit segmental bone defects with artificial bone after managed with adenovirus vector carrying BMP-2 (Ad-BMP-2), fibrin gel and polylactic acid/polycaprolactone (PLA/PCL), and to investigate the bone union effects after in vivo transfection with BMP-2 gene.

MATERIALS AND METHODS

Materials
Experiments were performed at the Central Laboratory of China-Japan Friendship Hospital Affiliated to Jilin University from September 2003 to December 2004. Ad-BMP-2 and adenovirus vector carrying β-galactosidase (Ad-Lacz) were presented by Dr. Oliver from Center of Molecule and Orthopaedics of Harvard Medical School and Dr. Gao from Department of Pathology of Jilin University. PLA/PCL biomaterials were prepared and donated by Institute of Applied Chemistry, Chinese Academy of Sciences, with the pore diameter of 150-250μm and porosity of above 90%, trimming into 0.2 cm×0.2 cm×1.5 cm shape.
Sixty New Zealand flap-eared white rabbits (120 sides) aged 3 months, of either sex, weighing 2.0-2.2 kg, were provided by Changchun Experimental Research Center of Medical Animal [SYXK (Ji) 2003-0007]. Rabbits were randomized into four groups, 15 in each group (30 sides).

Methods
Preparation of gene compound artificial bone
Mixture of 50 μL of Ad-BMP-2 (2×1010 pfu/mL) and 50 μL of fibrin was added with 10 U of thrombin, which were adsorbed into PLA/PCL scaffold by syphonage for storage after forming gel shape.

Establishing models and management
After intraperitoneal anesthesia with 3% pentobarbital sodium, a lateral incision (1.5 cm bone defect) was
made on forelimbs of rabbits to expose upper and middle radial shaft, and then periosteum was also removed. Try to discard individual differences. Different kinds of artificial bone were implanted into both sides, and fixed with closely suturing the sarcolemma and fascia.
Artificial bone treated with Ad-BMP-2+fibrin gel+ PLA/PCL was implanted into rabbits in the Ad-BMP-2 group; treated with recombinant BMP-2+ fibrin gel+ PLA/PCL was implanted in the recombinant BMP-2 group; treated with adenovirus vector carrying β-galactosidase (Ad-Lacz)+ fibrin gel+PLA/PCL was implanted in the control gene group; and treated with fibrin gel+ PLA/PCL was implanted in the PLA/PCL group. Five rabbits (10 sides) from each group were sacrificed 4, 8 and 12 weeks after surgery. The protocol was in accordance with animal ethical standards.

Observational indexes
X-ray examination: Four, eight and twelve weeks after surgery, callus gray (representing new bone density) of the bilateral anterior limbs was detected by X-ray. Biodynamic determination: Twelve weeks after surgery, three-point bending strength was tested, with the span of 20 mm and speed of 2 mm/min. Bending strength of new bone on bone defect regions was compared in each group. Histological observation: Hematoxylin-eosin (HE) staining was employed to observe bone defect repair. Alcian Blue staining and Vankossa staining were used to examine chondrogenesis and mineralization.
Electron microscope: Transmission electron microscope and scanning electron microscope were applied to investigate maturity of bone cells, degradation and absorption of scaffolds.

Statistical analysis
Data were analyzed with the SPSS12.0 software, and expressed by Mean±SD. Variance analysis was used to compare the difference in groups. A value of P < 0.05 was considered statistically significant.

RESULTS

Quantitative analysis of experimental animals
A total of 60 rabbits were included in the final analysis, no drop-out.

Results of X-ray and histological observation
Ad-BMP-2 group: Four weeks after surgery, punctate patchy osteogenic shadow, a large number of external callus, a large amount of hypertrophic chondrocytes and new-formed osteoid around the scaffold as well as new vessels were detected. A fraction of scaffold material was degraded and absorbed. Eight weeks after surgery, transplanted bone showed diffuse dense shadow; Cartilages were differentiated into woven bone after absorbing and mineralizing, and then changed into the shaping of lamellar bone. Twelve weeks after surgery, cortical bone was continuous, and medullary cavity was recanalized.
Recombinant BMP-2 group, four weeks after surgery, osteogenic shadow and scattered new cartilage islands were detected. Eight weeks after surgery, lamellar dense osteogenic shadow appeared, a great quantity of new cartilage and bone formed and fused, woven bone and medullary cavity were seen. Twelve weeks after surgery, bone became condensed, density was uneven and bone defects were primarily repaired. A large amount of new bone became mature lamellar bone and cortical bone formed.
Control gene and PLA/PCL groups: Four and eight weeks after surgery, osteogenesis was not detected. Low-density photic zone was found. Callus at extremities grew towards both sides of transplanted bone. Twelve weeks after surgery, extremities stiffened, medullary cavity occluded, and bone nonunion appeared.

Determination results of new bone density and biodynamics in each group 12 weeks after surgery
Examination results of bone density in each group 12 weeks after surgery are shown in Table 1.

Table 1 demonstrates that bone density in the Ad-BMP-2 group was significantly higher than the other groups (P < 0.01). No significant difference was detected between the control gene and PLA/PCL groups (P > 0.05). Bending strength was in accordance with the results of bone density in each group.

Observational results of chondrogenesis and mineralization
Alcian Blue staining showed that BMP-2 induced a large amount of cartilage callus and chondrocytes proliferated 4 weeks after surgery in the Ad-BMP-2 group. Vankossa staining demonstrated that calcium salt granules were dense and even, and mineralization degree was high 8 weeks after surgery in the Ad-BMP-2 group. This indicated that in vivo osteoblast differentiation induced by BMP-2 gene was endochondral ossification. Cartilage proliferation, absorption and enchondral ossification occurred late in the recombinant BMP-2 group, which was associated with the speed of vascular growth.

Maturity of osteocytes, degradation and absorption of the scaffold
Scanning electron microscope showed that in the Ad-BMP-2 group material granules disaggregated, fused and became uneven, as well as new bone trabecula with low mineralization was pultaceous; chondrocytes regularly arranged and grew together with bone tissues 4 weeks after surgery. Thick bone trabecula, dense bone appeared and a large quantity of calcium salt granules deposited 8 weeks after surgery. Scaffold materials were dissolved and absorbed; porous structure was destroyed; new bone grew close to the scaffold with clear border 8 weeks after surgery in the recombinant BMP-2 group.
Transmission electron microscope demonstrated that in the Ad-BMP-2 group palisade-shape arranged osteoblasts were detected on the surface of new-formed bone matrix, most osteoblasts around new-formed small vessels; the diameter of small vessels was less than 8 μm and endothelial cells were incomplete encasement around vessels 8 weeks after surgery. Vascular endothelial cells were flat and wrapped the vascular lumen; Osteoblasts were wrapped in extracellular matrix containing many mature collagenous fibers; Mature osteocytes with long cytoplasmic process located in bone lacuna, and spread into bone canaliculus; Osteoclasts with many nuclei and many chondrosomes in the cytoplasm were examined in the medullary cavity, and ruffled border was found on the side near to the sclerotin 12 weeks after surgery. In the recombinant BMP-2 group, osteocytes tended to be mature 12 weeks after surgery. A mass of fibrocytes and mesenchymal cells, but few new vessels were seen in the control gene and PLA/PCL groups 12 weeks after surgery.

DISCUSSION

Presently, gene therapy for bone defects has been a hot study [3]. Scholars had obtained good outcomes in repairing segmental bone defects by direct injection of various genetic carriers (in vivo gene therapy) or by inducing osteogenesis after indirect transfection target cells (in vitro gene therapy) [4-8]. Indirect gene therapy with target cells had high transfection efficiency, but the operation was complicated and the cost was high. Lee et al [9] determined that a small fraction of transplanted cells (5%) differentiated into osteocytes; 95% cells served as carriers delivering BMP-2; induced host mesenchymal cells participated in osteogenesis. Taken together, this study repaired bone defects by direct metastasis of Ad-BMP-2.
X-ray and histology demonstrated that in vivo osteoblast differentiation induced by BMP-2 gene was endochondral ossification. At week 4, a mass of hypertrophic chondrocytes grew into PLA/PCL pores, forming cartilage islands, and additive osteoblasts proliferated. With the prolongation of time, blood capillary grew, chondrocytes absorbed, mineralized, and transformed into woven bone, simultaneously, scaffolds disaggregated, fused and replaced by new bone. Subsequently, the callus was remoulded, and lamellar bone formation and enlarged medullary cavity were present. Scanning electron microscope showed that mineralization degree of new bone was higher than controls. X-ray determination of bone density obtained the same results as above. At week 12, significant differences in three-point bending test were confirmed between Ad-BMP-2 and recombinant BMP-2 groups. These results indicated that BMP-2 direct gene therapy for repairing bone defects could obtain better osteogenic speed, mass and mechanic strength compared to the BMP-2 exogenous combination. This method is simple to operate and fits for clinical application.
After implanting adenovirus containing BMP-2 gene into bone defect regions, the adenovirus infects myocytes in surrounding muscle tissues and mesenchymal cells in the medullary cavity of two broken ends. Subsequently, mature bone tissues were present by inducing the differentiation of mesenchymal cells into chondrocytes via entochondrostosis. Baltzer et al [10] injected Ad-BMP-2 into rabbit femoral defect regions wrapped with muscles, whereas injected firefly luciferase gene into the control group. High expression of BMP gene was present in muscle, broken end of bone and scar tissues. Twelve weeks later, complete callus filling was seen in the experimental groups, but only fibrous joint was detected in the control group.
BMP-2 direct gene therapy uses direct injection or biomaterial compound methods. Direct injection is simple and easy, but genetic carrier extention and deliquation with blood cannot be easily controlled. Without biological scaffolds, bone guidance effects are weakened and healing time is prolonged. In this study, we used two kinds of genetic carriers: ①PLA/PCL provides supporting structure and surface chemical environment which fits for cell proliferation, and offers suitable degradable speed and mechanic strength. Three-dimensional porous structure can smooth the way for the growth of cells and vessels. ②Fibrin gel keeps even distribution of genetic carrier and ensures its local osteogenesis. Fibrin gel, the main component of local blood clot, possesses low antigenicity, is easy to be degraded and absorbed, and characterized by promoting vascularization and bone conduction. Moreover, gel with pore is convenient for nutrition infiltration, chemotaxis and aggregation of host mesenchymal cells, which provides basis for gene transfection.
Adenovirus vector mediated BMP-2 gene transfection successively secreted high-performance bioactive BMP-2 for 6 weeks, induced chemotaxis, aggregation and osteogenic transformation of host mesenchymal cells, as well as amplified inductive signal and spread all over the transplanted bone [11]. The advantages of using adenovirus vector resided in the fact that transfection efficiency was high and transfection was safe, which did not conform in host genome; Expression time of exogenous gene (4-6 weeks) fitted for the feature of bone repair, so it could not stimulate bone overgrowth [12]. In addition, BMP activity was regulated at multiple levels after gene transfection. Some negative regulatory factors affected by BMP inside and outside of nuclei severely control bone induction, showing self-limitation. Taken together, bone induction was limited in BMP released regions, and played effects only when BMP was present [13]. Wang et al [14] confirmed that good bone union and no new bone compressed the spinal cord, which showed the safety of Ad-BMP-2 gene transfection to induce spinal column confluence. In this study, we did not find bone overgrowth.
In summary, BMP-2 direct gene therapy could induce the repair of bone defects. PLA/PCL and fibrin gel carrying BMP-2 gene in repair of segmental bone defects can achieve good effects[15]. However, whether direct application of adenovirus can induce more immunogenicity, what about the cytotoxicity of many adenoviral particles, and how to choose the optimal dose of adenovirus require further studies.

REFERENCES

1 Li JJ,Han D,Liu JG,et al. Effects of Adenovirus mediated BMP-2 gene transfection on bone induction and vascularization in vitro. Zhonghua Xianwei Waike Zazhi 2004;27(4): 284-285
2 Li JJ, Wang WJ, Han D, et al. Heterotopic osteogenesis induced by xenogeneic bone delivery of human mesenchymal stem cells transfected by bone morphogenetic protein-2 gene in nude mice. Zhonghua Chuangshang Zazhi 2004;20(6):347-350
3 Bonadio J, Smiley E, Patil P, et al. Localized, direct plasmid gene delivery in vivo: Prolonged therapy results in reproducible tissue regeneration. Nat Med 1999;5(7):753-759
4 Sugiyama O, Orimo H, Suzuki S, et al. Bone formation following transplantation of genetically modified primary bone marrow stromal cells. J Orthop Res 2003;21(4):630-637
5 Lee JY, Musgrave D, Pelinkovic D, et al. Effect of morphogenetic protein-2-expressing muscle-derived cells on healing of critical-sized bone defects in mice. J Bone Joint Surg Am 2001;83-A (7):1032-1039
6 Baltzer AW, Lattermann C, Whalen JD, et al. Gene enhancement of fracture repair: healing of an experimental segmental defect by adenoviral transfer of the BMP-2 gene. Gene Ther 2000;7(9):734-739
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8 Gugala Z, Olmsted-Davis EA, Gannon FH, et al. Osteoinduction by ex vivo adenovirus mediated BMP2 delivery is independent of cell type. Gene Ther 2003;10(16):1289-1296
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骨形态发生蛋白2基因复合纤维蛋白凝胶/ 聚乳酸-聚己内酯双相支架修复节段性骨缺损★

于 威1，李建军2
1阜新市第二人民医院骨科，辽宁省阜新市 123000；2中国医科大学附属盛京医院骨科，辽宁省沈阳市 110003
于 威★，男，1973年生，黑龙江省哈尔滨市人，汉族，在读硕士，主治医师，主要从事骨创伤和骨缺损修复的研究。
摘要
背景：骨形态发生蛋白2基因转染骨髓基质干细胞后，可促进其增殖分化并诱导异位骨形成。但这种方法操作复杂，体外细胞扩增需时较长，不便于临床应用。
目的：将人工骨经骨形态发生蛋白2腺病毒载体与纤维蛋白凝胶和聚乳酸/聚己内酯处理，移植修复骨缺损。
设计：随机对照动物实验。
单位：实验于2003-09/2004-12在吉林大学中日联谊医院中心实验室完成。
材料：聚乳酸/聚己内酯生物可降解材料块由中国科学院长春应用化学研究所提供，孔隙直径150~250 μm，孔隙率90%以上；实验动物为3月龄新西兰大耳白兔60只。
方法：于新西兰大耳白兔双侧桡骨中段造成1.5 cm骨缺损，分别植入4种经不同方法处理的人工骨：①Ad-BMP-2组：骨形态发生蛋白2腺病毒载体＋纤维蛋白凝胶＋聚乳酸/聚己内酯。②重组BMP-2组：重组骨形态发生蛋白2＋纤维蛋白凝胶＋聚乳酸/聚己内酯；③对照基因组：β-半乳糖酐酶基因腺病毒载体＋纤维蛋白凝胶＋聚乳酸/聚己内酯。④PLA/PCL组：纤维蛋白凝胶＋聚乳酸/ 聚己内酯。
主要观察指标：术后4，8，12组周摄双侧前肢正位X射线片测定骨痂灰度（代表新骨密度）；苏木精-伊红染色观察骨缺损修复情况，爱辛蓝和Vankossa染色观察软骨形成及矿化；电镜观察骨细胞成熟、支架降解吸收情况；术后12周行生物力学检测评价新骨抗弯强度。
结果：术后12周Ad-BMP-2组缺损区在成骨活跃程度、骨再生量和再生髓腔结构等方面均显著优于重组BMP-2组，其骨缺损得到了较彻底的修复。对照基因组和PLA/PCL组均不能产生骨性愈合。
结论：聚乳酸/聚己内酯协同纤维蛋白凝胶运载骨形态发生蛋白2基因修复节段性骨缺损可达到较好的效果。
关键词：骨形态发生蛋白2；骨组织工程；骨缺损；生物材料
中图分类号: R318.08 文献标识码: A 文章编号: 1673-8225(2008)14-02761-04
于威，李建军.骨形态发生蛋白2基因复合纤维蛋白凝胶/聚乳酸-聚己内酯双相支架修复节段性骨缺损[J].中国组织工程研究与临床康复，2008，12(14):2761-2764
[www.zglckf.com/zglckf/ejournal/upfiles/12-14/14k-2761(ps).pdf]
(Edited by Li ZH/Qiu Y/Wang L)