| BackgroundSegmental bone defects caused by severe trauma, neoplasm resection, infection, revised artificial arthroplasty are common in clinical practice and it is difficult to heal by themselves and to deal with. The traditional methodology includes autologous bone graft, allogeneic bone graft, and artificial bone. There is no immunological rejection in the autologous bone graft, and it contains stem cells and growth factors that can promote osteoinduction and bone transduction, so it is still considered the gold standard of treatment of the moderate amount of bone defects. But when the bone defect is too large for autologous bone transplantation, and the surgical trauma, bleeding, surgical time increase correspondingly; meanwhile a very limited number of cells can obtain the blood supply to survive and perform normal function after autologous transplantation. Allogeneic bone graft and the artificial bone transplantation can hardly satisfy clinical needs because of immunological rejection, diseases spreading and slow creeping substitution.At present, treatment strategies for bone defects mainly include3methods:①administration of Osteogenic induction factors:for instance, bone morphogenetic proteins which are frequently delivered;②Transplantation of stem or progenitor cells that can differentiate to the osteoblast, for example, mesenchymal stem cells from various sources;③Bone tissue engineering, its basic mode is biological scaffold materials combined with cell factors or/and stem cells. BMPs were first used in animal experiments and clinical bone defects in patients. They are the most effective osteoinductive factors which can promote differentiation of mesenchymal stem cells into osteoblast or chondrocyte, so as to induce in situ and ectopic bone formation. BMP-2is one of the most effective BMPs and BMP-2,7has recently been approved by the FDA (Food and Drug Administration) in America for clinical administration. However, bone morphogenetic protein has the limitations such as preparation difficulties, low production, high price, the topical application of metabolism fast, the great consumption, lack of optimal carrier. All these problems will affect its further application. In addition, the lack of the vascular network in segmental bone defects may result in missing the opportunities for BMP-2during the bone healing process. Although BMP-2may increase the gene expression of vascular endothelial growth factor (VEGF) and induce angiogenesis indirectly; however, according to Jianjun Zhao etc, the density of new blood vessels in the transplant is higher in the periphery than that in the center; it is presumed that the vascularization induced by BMP-2depends on the blood supply around; blood supply of the central region is inadequate, thus bone repair process and vascularization induced by BMP-2are relatively slow; BMP-2exert its effect through the indirect effects of VEGF, the angiogenesis and new bone regeneration will not be affected obviously when the surrounding blood supply decreases slightly and the implants are smaller. Therefore, it is not sufficient to rely solely on the BMP-2gene therapy to induce vascularization and will also affect the final results. Although the BMP has strong osteoinductive activity, bone repair is a continuous and complex process affected by a variety of cytokines and bioinformation. Angiogenesis is a key point in the bone repair process. It is essential to rebuild vascular network in addition to the use of bone inducing factor as soon as possible in order to promote the repair of large bone defects.With the continuous development of tissue engineering technology, reserachers have also tried to apply this method to treat large bone defects, and have done considerable useful works. A variety of scaffolds and stem cells have been used in the study of tissue-engineered bone, however, it is also found that angiogenesis affect its application critically. Unsimilar with organ transplantation, which has perfect vessels network and can get blood supply shortly after anastomoses, the tissue-engineered bone has no vascularization. There is evidence that the seed cells can survive within the diffusion radius of150～200μm; the tissue fluid diffusion is not enough to support cells viability when the volume of tissue mass is greater than3mm3, and regeneration of blood vessels for the supply of oxygen and nutrients is essential; A long time of hypoxia and nutritional deprivation will lead to cell necrosis or apoptosis rapidly within3days. Neovascularization is not only transport channel for oxygen, nutrients and metabolic waste, but is an important path of cells and signaling molecules involved in the regulation of bone repair; so both angiogenesis and osteogenesis are the basic two components during the bone healing process when treated with tissue engineering technology to repair bone defects. When the tissue engineered bone constructed in vitro is implanted into the body, sufficient blood supply must also be rapidly established as soon as possible to provide adequate nutrition for the seed cells functional activity, this is the only way to ensure the survival of tissue engineered bone and ideal repair of bone defects and is particularly important in segmental bone defects repair. In other words, the consummate vascular network is the basic prerequisite for its survival and integration with the host tissue, and the rate and content of vascularization of tissue engineered bone is the key to the efficacy of repairing segmental bone defects.Angiogenesis is a complex process involved with a number of factors which interact with each other. Based on the preliminary understanding of the mechanism, researches in this area can be summarized into two lines:①Construct tissue engineered bone in vitro and at the same time construct artificial vascular network, and then implant both of them in vivo to repair the bone defects.②Reasonable use of cytokines or cells for angiogenesis locally. The first method is more difficult and complicated to operate. Reasonable application of growth factors which can promote angiogenesis such as VEGF has a good effect. VEGF is the most important blood vessel growth regulatory factors, and is widely used in the study of angiogenesis. VEGF is a specific mitogen of endothelial cells. It can mediate the process of endothelial cell migration, proliferation and constructing new blood vessels in angiogenesis. VEGF can also activate EPCs and improve their functions. Recent studies also showed that VEGF may be an important regulatory factor bone in bone development and angiogenesis, healing process of fracture. VEGF has a wide range of biological functions, including adjusting the differentiation of osteoblasts, chondrocyte, the activation of osteoblasts, promoting their proliferation as mitogen, inducing mesenchymal stem cells to differentiate into osteoblasts. VEGF-165is the most common isoform of VEGF family and has the most significantly angiogenetic effect. But disadvantages in application of VEGF include that its biological activity is greatly reduced within the composite materials; local content of VEGF is difficult to control; low levels has limited effect, while high levels of VEGF has the risk of inducing hemangioma, edema and hypotension. The application of gene therapy can solve this contradiction ingenious which can make VEGF maintain at therapeutic levels, express persistently in a period.Since Asahara discovered endothelial progenitor cells (EPCs) in1997and demonstrated their successful effect on angiogenesis, a lot of researches have been done on them. EPCs can be targeted to migrate to ischemic or thrombotic site, where they promote and participate in the formation of new blood vessels and secrete a variety of factors promoting neovascularization, such as EGF(epidermal growth factor), HGF(hepatocyte of growth factor, HGF), IL-8, Ang-1and VEGF, etc. and thus regulate the angiogenesis. They appeared as promising candidates for treatment of cardiovascular and cerebrovascular diseases, limb ischemic diseases, bio-engineering materials, plastic surgery, wound healing etc. But it has rarely been reported about angiogenetic effects in repairing segmental bone defects. The previous studies confirmed that endothelial progenitor cells can be transfected with a variety of gene carried by vectors, thereby enhancing their therapeutic effects, but there was no reports about the cells in bone tissue engineering in the literature.So in the present study, EPCs obtained from rat bone marrow were transfected with human VEGF-165gene, and the expression of target gene was detected. Then they were seeded into the nano-hydroxyapatite/Collagen/Poly(L-LacticAcid)(nHAC/PLA) scaffolds to construct tissue engineering bone. And its role in promoting angiogenesis and new bone formation in femur segmental bone defects region of rat were explored.PurposeSeed the human VEGF-165gene transfected EPCs of rat into the nHAC/PLA scaffolds to construct tissue engineered bone and to investigate its efficacy on segmental bone defects in respect with angiogenesis and new bone formation.MethodsTwo consecutive parts were included in the study:in vitro and in vivo. The experimental procedures in vitro were as the following:1. Separation, culture and identification of EPCs:The3-week-old male SD rats were employed. Their long bones of all extremities were resected. Then cancellous bone and marrow cavity were washed repeatedly with phosphate buffered saline (PBS) under sterile conditions. The bone marrow cell suspension was addad to the Percoll separating solution. After centrifugation, the cloudy cell layer between in liquid was taken and cultured into EBM-2medium which have been added with fetal calf serum (FBS). Several growth factors such as human VEGF (1ug/ml), bFGF (1ng/ml), EGF (lOng/ml) were used to facilitate their amplification. After passages, they were identified according to CD133, CD34, and VEGFR-2markers with immunohistochemistry and functional tests of low-density lipoprotein uptaking and UEA-1binding.2. Gene transfection:EPCs were transfected with Ad5-hVEGF165-EGFP, observed under a fluorescence microscope to investigate whether the transfection was successful. The expression of target gene was detected with the RT-PCR and Western-Blot.3. Combination of genetically modified EPCs with nHAC/PLA scaffolds:The scaffolds were incubated with FN for4hours at first. The EPCs were seeded. The growth and adhesion of the seeded cells within the scaffolds were observed with scanning electron microscopy (SEM) and the proliferation seed cells was detected with MTT assay. The experiments in vivo included:1. The establishment of segmental bone defect model in femora in rats and implant of tissue engineered bone:Eighty male SD rats, weighting about400g, were divided into four groups randomly, and the segmental femoral defects (5mm) were made in the middle1/3parts of the shaft with a cutting saw under constant cooling with saline. The periosteum in the defected area was removed with a scalpel. They were allo-grafted with hVEGF165/EPCs-nHAC/PLA (group A, n=20), mock EPCs-nHAC/PLA (group B, n=20), EPCs-nHAC/PLA (group C, n=20) and scaffolds only(group D, n=20), respectively.2. The detection of angioblastic effect and bone defect restoration:Radiological evaluation using Seeherman scoring system and histological observation with HE staining were employed to explore the osteogenic ability of every implant in bone defect site at different time points. And CD34immunohistochemical staining in the section of tissue was used, then the mocrovessle density (MVD) were counted microscopically to evaluate the bone regenerative effect of every implant.ResultsBy identification, the cells isolated and cultured from rat bone marrow were EPCs, they expressed marker molecules CD133, CD34and VEGFR-2. They could uptake Dil-LDL into cells and combine FITC marked UEA-1to their membranes, and they were double-stained with red in plasma and green attached to membranes.The target gene expression could be detected through RT-PCR and Western Blot which reached to a high level at ten days after gene transfection. SEM showed that after the EPCs combined with nHAC/PLA scaffold, their adherence to the inner wall of scaffolds and growth were in good condition. The EPCs began to reach out filopodia at2hours after seeding and gradually exhibited spindle-shaped appearance. And they are also connected to each other in some visions by increasing filopodia.MTT assay showed that they proliferated well in the scaffolds. The average absorbances in the scaffolds seeded with hVEGF-165gene modified EPCs on2th,4th,6th day were0.73±0.04vs0.52±0.04vs0.36±0.03respectively, with each P<0.001; They were0.32±0.13vs0.49±0.08vs0.71±0.05respectively, in the scaffolds seeded with mock transfected EPCs, with each P<0.001. But the average absorbance was not statistically significant between the two groups at the same time points (P>0.05). These suggested that all the seed cells continuously proliferated well in the scaffolds.Radiological analysis revealed that at the6th week after implantation, new bone formation was more and in high density and with some bridging ossification in bone defect areas in group A; in group B&C, it was much and in low density and without bridging ossification; while in group D, it was less. At the12th weeks, the bone defected regions were completely repaired with bridging ossification and medullary cavity was recanalized in most rats in group A; in group B&C, new bone formation was more and in higher density and some bridging ossification could be seen; while in group D, it became more but without bridging ossification. The average radiological score by Seeherman system in bone defect sites demonstrated the highest in group A, both group B and group C higher than group D. The differences were statistically significant.Histological analysis with HE staining showed that in group A at3th week after operation, hyperplasia of fibrous tissue and a small quantity of inflammatory cell infiltration could be seen between host bones and implants, a small amount of chondrocytes appeared; at6th week, new bone was more mature, and there was a large amount of new bone formation in pores of scaffolds with obvious scaffold degradation, but there was no bone marrow cavity recanalization; at12th week, bone defected areas were almost completely restored, with trabecular bone maturation, cortical continuous, more of lamellar bone, mostly visible marrow recanalization, scaffolds almost complete degradation. In all control groups, the new bone formation was poorer compared with group A at the same time points. The new bone formation was faster in group B&C than in group D. And scaffold degradation was the same as the above accordingly.At week three postoperatively, The average microvascular density in each group was:Group A14.99±1.52, Group B7.39±0.69, Group C7.16±0.79, Group D5.53±0.59; At week six, they were:Group A10.36±1.21, Group B6.02±0.61, Group C5.85±0.93, Group D4.44±0.67, respectively; At week nine, they were:Group A9.32±0.84, Group B5.09±0.62, Group C4.83±0.54, Group D3.77±0.50. The differences were statistically significant at each time points.ConclusionThe new type of tissue engineered bone constructed with the hVEGF165gene-modified EPCs seeded into the nHAC/PLA scaffolds can promote angiogenesis and osteogenesis in the bone defected areas in rat femora and the hVEGF165/EPCs-nHAC/PLA composites may have potential application in repair of segmental bone defects. In the present study, a new type of tissue-engineering bone was constructed by seeding human VEGF165gene-modified endothelial progenitor cells (EPCs) of rats into the nano-hydroxyapatite/collagen/Poly(L-Lactic Acid)(nHAC/PLA) scaffolds, and its role was explored in promoting angiogenesis and osteogenesis in segmental femoral defects of rats. In vitro study, EPCs isolated from the bone marrow of SD rats were cultured, proliferated and identified. Then, EPCs were transfected with Ad5-hVEGF165-EGFP, and the expression of the target gene was detected with RT-PCR and Western Blot. Finally, the gene-modified EPCs were seeded into the nHAC/PLA scaffolds and their growth was observed through scanning electronic microscope (SEM) and their proliferation was evaluated by MTT assay. For in vivo study,80SD rats were divided randomly into four groups and the segmental femoral defects (5mm) were made. They were allo-grafted with hVEGF165/EPCs-nHAC/PLA (group A, n=20), mock EPCs-nHAC/PLA(group B, n=20), EPCs-nHAC/PLA (group C, n=20) and scaffolds only(group D, n=20), respectively. Radiographic, histological and microvessle density tests were performed to evaluate their angiogenic and osteogenic ability. The isolated cells were EPCs. RT-PCR and western blot results showed that the target gene was expressed by EPCs. The SEM findings and MTT assay revealed that EPCs attached, grew and proliferated well within the nHAC/PLA scaffolds. The study in vivo demonstrated that the average radiographic score and capillary density were the highest in group A, and those in group B&C were higher than group D (each P<0.05). The histological test showed that osteogenesis and scaffolds degradation were the most obvious in group A, and those in group B&C were better than group D. The hVEGF165gene-modified EPCs can promote angiogenesis and osteogenesis in bone defected areas and the hVEGF165/EPCs-nHAC/PLA composites may have potential application in repair of segmental bone defects.
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