Studies On The Vascularization And Bone Healing Of Tissue Engineering Bone Promoted By VEGF-alginate Beads Under Stress Stimulus

Background and Objective: It was poor even failure that segmental defect of bone was repaired with tissue engineering bone(TEB). The main causes included that enough nourishment wasn't absorbed at early stage of bone healing and long time needed for remodeling of callus at middle and late stage of bone healing. Vascularization could be strengthened by vascular endothelial growth factor(VEGF) whose half life was short and effects was poor if it was used directly. In addition, it was showed that intermittent stress could promote remolding of new callus. So aims of this study included: (1). The VEGF-alginate beads were co-cultured with endothelial cells(ECs) and goat mesenchymal stem cells(MSCs) respectively after the beads was formulated. The biological effects on the ECs and proliferation or differentiation on the MSCs were both observed. (2) A new micro-motion locking nail was designed and segmental bone defect model of femur was prepared with the new interlocking bone nail basing on the anatomical parameters of the femur in goat. (3) Vascularization and bone healing of TEB promoted by VEGF-alginate beads under stress stimulus were studied in the new model.Materials and Methods: (1). VEGF-alginate beads were formulated by using the needle extrusion/external gelation method and it's encapsulation yield, burst release yield and release time were assayed with the hVEGF ELSA kit. (2). The effects on the proliferation of ECs or MSCs and differentiation of MSCs were observed with MTT colorimetric assay, flow cytometer, immunohistochemistry of type-â… collagen, reverse transcription-polymerase chain raction(RT-PCR). (3). Cyclic stress stimulus machine was designed and the release characteristics of VEGF-alginate was measured under cyclic stress stilmulus. (4). Segmental bone defect model of femur were prepared with A new micro-motion locking nail designed basing on the anatomical parameter of the femur in goat. Meanwhile the micro-motion gap and mechanical strength were measured with X-ray, biomechanics in vivo or vitro. (5). The segmental bone defect model of femur was prepared with the new interlocking bone nail, which were evaluated with X-ray, in nake eyes, biomechanics and HE staining. (6). Completely decalcified bone matrix (DBM)was formulated and it's physical characters and biomechanical properties were also measured with biophysical technique and scanning electron microscope(SEM). (7). TEB was fabricated with DBM and MSCs which was labed and traced with enhanced green fluorescent protein(EGFP) with gene transfection method and observed under SEM or Laser scanning confocal mircroscope(LSCM). (8). Vascularization of TEB in early stage promoted by VEGF-alginate beads under stress stimulus were studied in the new model with radionuclide bone imaging, perfusion of ink, HE staining and immunohistochemistry ofâ…§factor related antigon. (9). Bone healing of TEB in the middle and late stage promoted by the beads under stress stimulus were studied in the new model with X-ray score, HE staining, Dual Energy X- ray Absorption (DERA)and biomechanical methods.Results: (1). VEGF-alginates beads were successfully formulated by using the needle extrusion/external gelation method and the encapsulation yield, burst release yield and release time were (8.70±0.63)ng/mg, (87.0±3.3)% and (16.1±1.3)d respectively. (2). OD value detected with MTT method in two groups whose densities of beads were 0.2mg/ml or 2mg/ml were both higher than the blank cell in control group. The analysis of cell cycle showed that the rates of S stage cells in alginate solution were more than the control group. (3). Cyclic stress stimulator whose frequency and moving range of the punch were60～150/min, 0～3mm respectively was manufactured basing on the step frequency and weight loading pressure (4). The beads release could be slightly advanced under the stress stimulus in the five days. (5). The positive express rate of CD₄₄＋CD₂₉,CD₄₅ and CD₆₂were 93.6%, 1.58%, 1.0% respectively in the third generation MSCs. OD value detected with MTT method in two groups whose densities of beads were 0.2mg/ml or 2mg/ml were both similar to blank cell in control group. The analysis of cell cycle showed that the rates of S stage cells in alginate solution were both equal to the control group. Calcium tubercle could be found and osteocalcin and cbfa—1 gene of MSC could be assay in co-culture groups. (6). The contours of femur were similar to the ones of human and Length and front curvature of femur were(15.2±0.83)cm and (9±1.22)°respectively. The proximal, middle and distal diameters of fumer were (12.7±0.35) mm,(12.3±0.21) mm,(12.7±0.33)mm respectively. There weren't significant difference between them. The length and diameter of new micro-motion locking nail were 12.5cm and 9mm. There were two locking hole in the proximal and distal nail, which diameters were 4.0mm and 5.5mm and diameter of locking nail was 4mm. (7). The maximal anti-pressing strength was more than 5000N, which was over 20 times of goat weight,and the length of micro-motion was 0.7～1.4mm under 50～800N axial compression. (8). The average diameter of microporosities of DBM was (413.3±41.7)μm and the maximum of absorption time and rate of DBM were 12 hours and (419±44.4)%. (9).There were no failure in the model with segmental bone defect of femur and the defect were filled by fibrous tissue, not bone-like tissue at 24 weeks postoperatively. (10). The adhesive rates of MSCs labed by EGFP in the DBM was (91±8.2)% and the labed cells could be found in the DBM at 28d after transplanted into bone defect of the goat. (11). The ratio of T/NT of experimental group was 4.33±0.25,11.09±0.50, higher than the one in the control group(p＜0.05). Immunohistochemistry ofâ…§factor related antigen, HE staining and ink perfusion all showed that the vessels in experimental group reticulately distributed, regularly aligned and it's diameters were unity. (12). The X-ray scores were 4.95±0.43 and 5.36±0.51, the value of bone mineral desities(BMD) were (1.37±0.08), (1.68±0.18)g/cm²respectively at 12 or 16 weeks and the limitied torsion strength was 83% of normal femur at 16 weeks postoperatively in experimental group, which were higher than the ones of every control group(p＜0.05).Conclusion (1). Different densities VEGF-alginate beads with satisfied encapsulation yield, burst release yield and release time could promote the proliferation of ECs. (2). Cyclic stress stimulator was prepared and VEGF-alginate release could be slightly advanced under the stress stimulus at early stage. (3). 0.2mg/ml and 2mg/ml of beads didn't effect the proliferation and differentiation of MSCs. (4). The new micro-motion locking nail could produce micro-motion under axial compression,which can be used to fix thefemur with bone defect in goat and observe vascularization and bone healing of TEB under stress stimulus. (5). The adhesive rate of MSCs labed by EGFP in the DBM with larger diameter, higher rate of microporosities and better hydrophilicity was (91±8.2)%, and the labed cells could be found in the DBM at 28d after transplanted into bone defect of the goat. (6). Vascularization of TEB was promoted by VEGF-alginate beads under stress stimulus in the new model. (7). Bone healing could also be advanced by VEGF-alginate beads under stress stimulus.