摘要
由于外伤、感染、肿瘤等因素使骨丧失了一些骨质,形成较大的间隙,称为骨缺损。在临床治疗上,骨缺损一直是常见的、难以治疗的疾病。目前主要采用自体骨,异体骨,人工合成替代物来进行修复,但这些方法都存在不足之处,难以满足临床上治疗的需要。随着组织工程学的发展,为治疗骨缺损提供了一条全新的道路。其基本方法是将体外培养的高浓度的功能相关的活细胞接种于天然的或人工合成的、具有良好的生物相容性和可降解性的支架材料上,在生物反应器或其它体外构建环境中形成细胞/支架复合物,然后将这种复合物移植到动物体内组织缺损部位,最终完成组织缺损的修复和再造。在骨组织工程学的研究中,种子细胞、支架材料、细胞因子是三大要素。体外构建的组织工程骨是细胞与材料的复合体,自身无营养来源,植入体内后,依靠血液-细胞间液为种子细胞提供营养及氧气。若植入区血供不良或植入物血管化过程较长,附着在支架上的种子细胞可因缺乏营养而出现代谢紊乱,细胞增殖、分化及分泌功能受损甚至死亡,使得骨沉积作用不能有效替代降解的生物材料。近年来,组织工程骨的血运构建问题的重要性日益彰显,复合细胞的支架植入体内后,必须建立血运循环后,才能存活并行使其功能,因此,血运重建已被认为是骨组织工程的第四要素。
本研究的目的是通过基因工程,细胞工程和组织工程学,构建一种能够具有这四大要素的组织工程骨,具有更高的成骨、成血管能力,以满足修复大段骨缺损的需要。利用贴壁法分离鼠的骨髓间充质干细胞,通过镜下观察细胞形态、流式细胞仪检测细胞周期、MTT法检测其生长特性、检测表面标记物、多方向诱导分化鉴定所分离细胞。构建BMP-2、VEGF基因及双基因的重组质粒,以骨髓间充质干细胞为靶细胞,进行基因转染。通过ELLISA、免疫组化等方法,证实目的基因能够在靶细胞中高表达。将骨髓间充质干细胞及转染细胞与nano-HA/PLGA支架材料复合,观察细胞与支架材料复合情况。本研究为进一步构建在体内具有成骨、成血管能力的组织工程骨提供了理论及实验数据。
Bone defect is sclerotin loss for the reasons of trauma、infection and tumor.In osteology clinic,Bone defect is the common and hard treated disease. Autogeneic bone transplantation,artifical synthesis surrogate transplantation as well as vascular anatomizes bone transplantation are the traditional strategies used at present.Following the development of tissue engineering,a new way to heal the bone defect is found.The basic method is to culture in vivo the living cells into the scaffolds,formating a compound of cells and scaffolds. Then,the compound is transplanted to the position of bone defect,to repair and reconstructi the defect.Seed cells、scaffolds and cell factors are the three critical elements of the tissue engineering bone.The compound of cells and scaffolds constructed in vitro has to get the nutrition and oxygen by blood and intracellular fluid after implantation. Poor blood supply of planting areas or long revascularization would result in nutrition deficiency of the seed cells grow on the scaffold,which leads to disorders in cell metabolism,proliferation,differentiation and secretion, at last, devitalizing of the cells. the transplanted bone would just act as a pure bone transduction material and lost the osteogenesis ability. In recent,the important of revascularization of tissue engineering bone is thought highly. Fast revascularization of combination after tissue engineering bone transplantation is the premise for keeping the vitality and activity.For this reason, the revascularization has been considered as the quartus element of tissue engineering bone.
BMSCs(bone mesenchymal stem cells)is one of good seed cells of tissue engineering bone,have the potential ability of ossific differentiation.In the specified environment, BMSCs can be induce to the osteoblast,encourage the reparative process of bone defect.For this reason,BMSCs are generally used on the field of bone tissue engineering.The utilization of many differentiation factors and inducing factor in region has the effect of promoting the osteanagenesis.The compound of BMSCs and scaffolds is not good at repairing the bone defect. The utilization of cell factors in region has the shortages such as short time leng、repeate administration、too espensive.In the view of endogenous bone growth factor,the gene treatment is gradually used on the field of bone tissue engineering,unfolding the enormous potentiality.Now we can introduce the gene of code cell factors into BMSCs,complex the scaffolds ,implant the compound to the bone defect.The cell will keep on producing the cell factors in region.With the autocrine and paracrine,the factors induce BMSCs to bone formation,promote the reparation of bone defect.
In the numerous bone growth factors, bone morphogenetic protein(BMP) is one of the most important,it is regarded as the most fortis osteoinductive factor.VEGF is not only to promote the hyperplasy of endothelial cell and vasculogenesis,but also evident osteogenesis. Therefore,BMP-2 and VEGF gene transfer into BMSCs,and use the cells to construct the tissue engineering bone,this method will promote the osteogenesis,and accelerate the vascular production in region at the same time.It is the better to meet the demand of the clinical.The nanometer materials is the new biomaterial appeared recently.The nano-HA is regarded as a kind of ideal scaffold,because the structure of nano-HA is similar with nature bone.The nano-HA can not use as scaffold alonely for it’s high fragility.The compound of nano-HA and high molecular polymer improve the high fragility of nano-HA,and compensate the descend of PH that cause by the degradation of high molecular polymer, conduce to prevent aseprtic inflammation.
This research approach the relation of BMP-2 and VEGF,and osteoplastic effect.We hope to find a way that can construct a kind of tissue engineering bone which have the ability of ossify and accomplish vessel.
This experiment was composed of four portions.The main method、consequence and conclusion decribed as follows:
1.Isolation,culture and identification of rabbit BMSCs(bone mesenchymal stem cells).
Isolation,culture of rabbit BMSCs is get from the tibia of wistar rat 5 days after birth according adherent.The BMSCs were certified by observation of morphology,determination of generation cycle,detection of surface marker.The cells have the favourable reproductive activity. With the appreciation,the cells can be induced to poly-directions.
2.Consturction of eukaryotic expression vector
The plasmids of pEGFP-N1/VEGF, pEGFP-N1/BMP-2 and pEGFP-N1/VEGF-BMP2 were constructed with gene recombinant technique by extraction of RNA. Through the sequencing ,compared with Genebank, identited by restriction enzyme,we certificated that the plasmid was right.
3.Expression of BMSCs after cells transfected BMP-2、VEGF gene in Vitro
These gene plasmids(pEGFP-N1/BMP-2、pEGFP-N1/VEGF、pEGFP-N1/BMP2-VEGF) were transfected into BMSCs by liposome in Vitro, fluorescence microscope observe eells culture after BMSCs transfected.The expression of purpose gene mRNA was detected by RT-PCR.The protein of cells transfected was detected by ELISA.
4.Construction of the gene modified tissue engineering bone in vitro
The nano-HA/PLGA scaffold was co-cultured with BMSCs and cells transfected.The compt tissue engineering bone was constructed.Cells was able to culture well with scaffold,conformed by digestion and canning electron microscope(SEM). The status that inorganic sedimentum was found in the scaffolds defferent degree. Bone salts deposited on the scoffild by the effect of cells.Compt tissue engineering bone was constructed invitro.
引文
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