The Experimental Study On Repairing Bone Defect Around Prosthesis With Tissue Engineering Bone

Background:In the 1960s, Branemark proposed the conception of osseointegration that there is no soft tissue between rebuilding bone and prosthesis under microscope, that bone contacts tightly with prosthesis, and that the loading taken on by prosthesis can pass and disperse constantly into bone by the bone-prosthesis contact. Osseointegration is regarded as the ideal state after arthroplasty.Long-time satisfactory result of biological prosthesis replacement depends on the tight bone- prosthesis contact, favorable bone growth and bone growing time. Many patients who undergo an operation for prosthesis replacement or revision often suffer from bone defect around prosthesis. Bone grafts become a necessary method to recover the construction and stability of bone. According to the size and property of the bone defect, we usually adopt auto graft or allograft to treat bone defects in clinic. Due to the shape, size and quantity of bone, auto graft can not achieve the request of bone defect, especially of gigantic or special bone defect. The use of auto graft often is limited. Since Sloof etc firstly reported that bone defect of acetabular can be repaired by packing bone graft with variant granulation bone in 1984 and Ling etc used the same technique to treat bone defect of femur in 1993, the technique has been widely used in the operation for prosthesis replacement or revision. Because variant bone has no osteoblasts and much lower activity of bone induction, to achieve real creeping, substiution and remolding of new bone needs much more time. Allograft usually has many complications, such as absorption, collapse of grafting bone, and asceptic loosening of prosthesis.In the 1980s, the emergence of tissue engineering provides us a brand-new thought and method in repairing bone defect. Tissue engineering has three important elements, namely seed cell, bioactivity factor, and scaffold material. Although there are many reports on repair of segmental defect of long bone and skull bone defect by use of tissue engineering bone, untill now any reports about repairing periprosthetic bone defect with tissue engineering bone and the effects of tissue engineering bone on osseointegration of the bone-impant interface have not been found in articles at home and abroad.Aim:1. To establish method of abstraction, cultivation and bone induction of mesenchymal stem cell (MSCs).2. To construct the tissue engineering bone with MSCs and coral hydroxyapatite (CHA) in vitro, and observe the effects on repair of bone defect around implant.3. To observe the effects of tissue engineering bone on osseointegration of the bone-implant interface.4. To study the capability of BMP to boost tissue engineering bone to repair periprosthetic bone defect, promote bone growth around prosthesis and the osseointegration of the bone-prosthesis interface.Method:1. BMSCs were segregated by Ficol(l1.073g/ml)density gradient centrifugation and the adherent culture method, then cultivated and multiplied. The 4th generation MSCs were induced by bone induction culture fluid (10-8mol/L DEX+10mmol/L Sodium Glycerophosphate + 50mg/L Vitamin C).2. Osteoblasts derived from BMSCs were verified by cell morphology, cell vigor detection, cell growth curve, ALP activity examination, alizarin Bordeaux dyeing of mineralization nodes , immunohistochemistry of collagen I and Gomori dyeing of ALP.3. A penetrating transverse bone defect (0.6cm×1.2cm) was created in bilateral femoral condyles respectively in rabbits. The titanium alloy implants were implanted in centre of bone defects. Tissue engineering bone constructed with osteblasts induced from allogeneic induced BMSCs and CHA in vitro was embedded into defect around the impant in left as experimental group and only CHA was embedded into the defect in right as control group. The effects on repairing bone defect around implant were studied by X-ray examination, biomechanical examination, radionuclide bone imaging by ECT, decalcificated bone histology with HE dyeing.4. The osseointegration of the bone-implant interface was observed by X-ray examination, section observation, push-out test of implant, scanning electron microscope, spectrum analysis, undecalcified bone histology with Van Gieson carbazotic acid–solferino dyeing, bone-to-implant contact calculated by datum from image analysis.5. Total hip replacement with femoral bone defects was operated in right hip of dog. Animals were divided to three groups, called BMP group, cell group and CHA group The bone defects were imlpanted at random by the compound of rhBMP-2 and tissue engineering bone, tissue engineering bone and CHA respectively. Animals sacrificed at three months after operation. The effects of BMP on osseointegration of the bone-prosthesis interface and repairing bone defect around prosthesis were studied by X-ray examination, biomechanical examination, undecalcified bone histology with Van Gieson carbazotic acid–solferino dyeing as well as bone-to- prosthesis contact calculated by datum from image analysis.Results:1. BMSCs were segregated, cultivated and multiplied successfully by way of density gradient centrifugation and adherent culture. The round shape of cells turned to fusiform after culture for three days. Growth curve of BMSCs was divided into detention phase, logarithmic growth phase and platform phase. The surface market SH3 of BMSCs took on positive.2. The shape of cells cultivated in bone induction culture fluid turned to polygon gradually. Cells began to growt in cluster after culture for 5-8 days. ALP activity was significantly higher in experimental group than that in control group(P<0.01). Both collegan I immunohistochemistry and ALP Gomori dyeing presented positive and alizarin Bordeaux dyeing of the 3th generation BMSCs after bone induction showed typical mineral nodus .3. Cancelous bone defect was repaired by tissue engineering bone constructed in vitro. As time going by, bone repair was observed in experimental group by the way of general observation and section observation at 4th, 8th and 12th week postoperatively. Biomechanical results showed that both the maximum pressure load and ratio of load to straining in experimental group were significantly higher than that in control group; The maximum strain- displacement in experimental group was lower than that in control group(P<0.01). Radionuclide bone imaging showed that mean of ROI in experimental group increased significantly higher than that in control group at different time points. Mean of ROI increased at quicker epacme within 8 weeks and began to increase at slower epacme at 8th week and achieved peak amplitude at 12th week postoperately. Histology study demonstrated that little new bone around CHA granulation in experimental group was found at 4th week; Scaffold materials were absorbed partially and replaced by new bone tissue, and bone defects were repaired basically by some new bone which rebuild maturely in partial domain in experimental group at 8th week; The obvious woven bone, lamellar bone and bone trabecula with blood sinus were found, but unabsorbed CHA granulation still existed in experimental group at 12th week. Little new bone around CHA granulation in controll group was found at 8th and12th week.4. The effects of tissue engneering bone on osseointegration of the bone-implant interface were observed. As time going by, the implants were embedded well by new bone in experimental group in general observation at 8th and 12th week postoperatively. X-ray showed that CHA granulations were absorbed and replaced by new bone and that the position of implant surrounded by new bone was good in experimental group; Gaps around implants in control group were found. Biomechanical examination showed that the shear-strength of bone-implant intreface in experimental group was significantly higher than that in control group at different time points. Scanning electron microscope notified that amount of amorphous material on pores of the implant surface increased gradually with time going by in experimental group. The percent content of calcium and phosphate was significantly higher in experimental group than that in control group at 4th week postoperatively; The percent content of calcium was significantely higher in experimental group than that in control group, but the percent content of phosphate increased unsignificantely at 8th, 12th week; The ratio calcium of and phosphate increased gradually with time going by and reached to 2.02 at 12th week postoperatively(P<0.01). Histology study: (1) At 4th week postoperatively, the thick and lax soft tissue on the implant surface and CHA granulation without new bone were found in control group; In experimental group the connective tissue membrane with little new bone on the implant surface was presented. (2) At 8th week postoperatively, uneven loosening connective tissue membrane with little new bone on the implant surface was found in control group; New bone formation with point bone-implant contact at interface existed in experimental group. (3) At 12th week postoperatively, the connective tissue fibrous membrane with little point bone-implant contact was presented in control group; In experimental group bone tissue with continuous and encircled bone-implant contact was found at the interface. The bone-to-implant contact (BIC) was zero both in experimental group and control group at 4th week postoperatively. The bone-to-implant contact in experimental group and control group was 15.36±3.57%, 6.09±1.46% at 8th week, and 46.54±6.89%, 17.70±6.32% at 12th week respectively. The difference between two groups had statistical sense.5. Repair of bone defect around prosthesis and the effects of rhBMP-2 and tissue engineering bone on osseointegration of the bone-prosthesis interface were observed. X-ray study: Lots of new bone with stable femoral prosthesis was found and CHA granulation absorbed completely in BMP group. Although massive new bone formed with stable prosthesis, the shape of CHA granulation still was presented in cell group; New bone formation was not obvious with radiolucent zone around prosthesis in CHA group. Biomechanical study: The shear-strength of bone-implant interface was 1.868±0.351, 1.269±0.432, 0.588±0.226 respectively in BMP group, cell group and CHA group; The difference had no statistical significance. Histology study: New bone tissue mainly with mature lamellar bone and little fibrous tissue and continuous bone-prosthesis contact at the interface were found in BMP group; New bone tissue partially with fibrous connective tissue and point bone-prosthesis contact at the interface were presented in cell group; Fibrous connective tissue with little lamellar bone and verge and slight bone- prosthesis contact were presented in HA group. Image analysis: The bone-to-implant contact(BIC) was 67.23±18.36,38.78±19.40,15.47±8.05 respectively in BMP group, cell group and control group(P<0.01).Conclusion:1. BMSCs can be segregated and cultivated successfully. Cells express high level of ALP activity and collagen I and capability of mineralizing intracellular matrix after bone induction. This demonstrates that BMSCs have differentiated to mature osteoblasts.2. Cancellous bone defect around the implant can be repaired by tissue engineering bone constructed with BMSCs and CHA.Vascularization and bone metabolic activity of bone graft is at stage of quick development within 8 weeks and enter into stage of ripeness and slow development at period of 8th to 12th week. CHA granulation still chould be found at 12th week in histology.3. Tissue engineering bone constructed with BMSCs andCHA can repair the bone defect around implant and promote the osseointegration on the bone-implant interface.4. Microanalysis of implant surface shows that percent contents of calcium and phosphate have the trend to increase and the ratio of calcium to phosphate gradually approaches the data of normal bone tissue with time going by in experimental period. This demonstrates that earlier period is the stage of new bone formation and gradually turns into stage of bone rebuilding and ripeness.5. BMP improves the efects of tissue engineering bone on the repair of bone defect around prosthesis and the osseointegration on the bone-prosthesis interface.