The Experimental Study Of The Repair Of Large Segmental Bone Defects With Tissue-engineering Bone Vascularized By Endothelial Progenitor Cells

The bone defects, especially large segmental defects are usually caused by war high-energy injured fractures, severe open fractures complicated with infection, bone nonunion failure in several-time of internal fixation and bone graft operations, osteomyelitis pathologic fractures as well as excision of large sequestrum, congenital pseudarthrosis of tibia as well as large bone excision because of tumor etc. It is difficult for traditional treatment. Eventually in many cases, amputations have to be carried out or protective weight-bearing braces were applied. Therefore, the treatment of large bone defect has been a great difficulty and the exploration of new methods become the urgent demand of orthopedics field.In resent years, bone tissue engineering technique has been research focus for the repair of bone defects, which mainly concentrate on the selection and cultivation of seeding cells as well as the selection and modification of the scaffold. The early studies of many scholars home and abroad as well as ours have proved that the ossification in early period and bone healing in late period are obviously effective when repairing small segmental defects with tissue-engineering bone (TEB). However, when repairing large segmental bone defects with large TEB, avascular necrosis frequently occurr to the core part, leading to failure of repair. The main problem is vascularization of the TEB. The methods of vascularization of the TEB are as follows: (1) combined grafting endothelial cells, osteoblast or vascular smooth muscle cells with biological materials. However, ECs are difficult to culture and amplify in scale in vitro. Moreover, the vascularization ability of mature ECs is poor. (2) Administration of Vascular Endothelial Growth Factor (VEGF) and Angiopoietin (Ang) etc. together with biological materials to promote the vascularization. However, the angiogenic factors degrade quickly in vivo, which can not reach the effective concentration. Thus some scholars transfect various angiogenic factor genes to seeding cells and plant them to the surface of the scaffold to construct TEB, then graft the TEB to the bone defects. This is the main research direction of vascularization. The problem lies in how to improve the transfection rate and target gene high expression. (3) Implanting the osteoblast, biological materials as well as the tissue valve embedment with vessel pedicle or blood vessel bundle to reconstruct TEB blood circulation. But this method may cause ectopic injuries and has long treatment cycle. Therefore, although it is easy for thin tissue engineering transplant such as skin, cardiac valves, to re-construct the blood circulation in early period, while for large bone tissue, the vascularization in early period is the key problem, which limited the application of TEB in repairing of large segmental bone defects.Endothelial Progenitor Cells (EPCs), firstly reported by Asahara in 1997, are a group of precursor cells of ECs, which have not expressed mature ECs phenotypes. EPCs could proliferate and differentiate into ECs. They take part in the vasculogenesis of human fetal vessels and exist in bone marrow, cord blood and peripheral blood with strong ability of promoting the angiogenesis after born. This conclusion updates the theory of traditional postnatal angiogenesis and vascular, injury and repair, and offers some new thoughts for treatment of ischemic diseases. Recently, the studies on functions of EPCs on adult angiogenesis mainly focus on repairing of ischemic myocardium, ischemic limbs, injured corneal and skin, etc.With the development of isolation, cultivation, induction and amplification techniques, it is easy to obtain EPCs from peripheral blood or bone marrow. Thus in this experiment, EPCs and Bone Marrow Mesenchymal Stem Cells (BMSCs) derived from the same rabbit bone marrow, were seeded on Decalcified Bone Matrix (DBM) to be promoting vascularized TEB. The re-vascularization in early period and the effect of new born bone healing in 20% rabbit radius large segmental bone defect treated with promoting vascularied TEB were observed.The main experimental methods and results are as follows:1. Mononuclear cells were separated and purified from the same rabbit bone marrow by density gradient centrifugation. After short time cultivation and amplification under special inductive conditions, enough quantity of EPCs could be obtained in vitro, and finally EPCs could differentiate into functional mature vascular endothelial cells with endothelial cell specific markers. No immunologic rejection raised after autogenous implantation, which offered possibility for clinical applications.2. Mononuclear cells from autogenous animal bone marrow had strong proliferative ability when cultured in vitro under certain conditions and would amplified in short period, which finally were induced to BMSCs with high purity and quantity that could meet the requirement of tissue engineering seeding cells. Under some specific conditions, BMSCs could differentiate into osteocytes. So BMSCs were ideal seeding cells of bone tissue engineering. Seeded BMSCs and EPCs to DBM scaffold according to the ratio of 1:1 to be promoting vascularized TEB in vitro.3. Established the 20% rabbit radius large segmental bone defect model and autogenous implanted the EPCs promoting vascularized TEB constructed in vitro into the bone defects. The vascularization of TEB in early period was observed by ink infusion, immunohistochemistry and radionuclide bone imaging etc. The results showed that the blood flow volume in bone defect areas increased at 2 post-operation weeks (POW) and reached the peak value at 4 and 8 POW, while decreased at 12 POW. The blood flow volume in experimental group (EPCs and BMSCs group) was obviously higher than that in control group (BMSCs only group) at 2, 4, 8 POD, indicating that EPCs could promote the early re-vascularization of TEB.4. The bone healing of the large segmental defects treated with promoting vascularized TEB were observed. The results of X-ray photography showed that, the callus was much more in experimental group than that in control group at 4 POW. From the 8th week, the bone marrow cavity could be seen re-opened in experimental group but not in control group. At 12 POW, the bone mineral density (BMD) of new bone was well proportioned and the bone marrow cavity re-opened completely in experimental group, While in control group, some low density areas could be seen in X-ray and most of the bone marrow cavity re-opened. At 16 POW, the new bones had finished most remodeling in experimental group, and in control group, the bone marrow cavity re-opened completely and new bones were remodeling. The results of BMD, histological light microscope, transmission electron microscope and immunohistochemistry etc. showed that, the ossification activity and bone healing speed in experimental group were better than those in control group. The biological dynamics testing at 12 and 16 POW showed that the intensity of twist-resistance of new bone in experimental group was significantly higher than that in control group. These conclusions indicate that promoting vascularized TEB with EPCs has strong bone forming ability, and can improve bone healing, may be effective method for repair of large segmental bone defects.Conclusions:1. After short time cultivation and amplification under special inductive conditions, enough quantity of EPCs can be obtained in vitro, and finally will differentiate into functional mature vascular endothelial cells with endothelial cell specific markers. No immunologic rejection arises after autogenous implantation, which offers possibility for clinical applications.2. Mononuclear cells from autogenous animal bone marrow could be cultured and amplified in vitro under certain conditions in short period, which finally be induced to BMSCs with high purity and quantity that could meet the requirement of tissue engineering seeding cells. Under osteoplastic inductive conditions, BMSCs can differentiate into osteocytes. DBM has been one of ideal scaffolds of TEB because of its excellent histocompatibility and avirulent. Promoting vascularized TEB can be constructed in vitro by seeding BMSCs and EPCs to DBM scaffold.3. After autogenous implanted into the 20% rabbit radius large segmental bone defects, the EPCs and BMSCs can promote the vasculature of the TEB, and accelerate bone healing. These conclusions indicate that promoting vascularized TEB with EPCs has strong bone forming ability, and can improve bone healing, may be effective method for repair of large segmental bone defects.