| BackgroundsObjectiveUtilizing the animal experiments of fibrin glue(FG)compounded withβ-tricalcium phosphate(β-TCP)/monocalcium phosphate(MCPM) artificial bone material, to explore the feasibility of FG compoundβ-TCP/MCPM artificial bone material as bone substitute material in repairing bone defects, as well as a possible guidance for clinical applications.Methods[1] Animal model of bone defect1 Preparation of the animal model: Twenty-two healthy adult New Zealand white rabbits were selected. The models of bone defects(4 mm in diameter and 8 mm in depth) were established at the bilateral femoral condyles by drilling. The composite of FG/β-TCP/ MCPM was randomly implanted in the side of the bone defect in rabbit lower limb(as experimental group), the other side of the rabbit lower limb defects inβ-TCP/ MCPM bone cement implantation(as control group).2 Implantation: Theβ—TCP/MCPM/FG samples were prepared by mixingβ—TCP/MCPM powder with the citric acid (or sodium citrate) at powder-to-liquid mass ratios (P/L) of 1g/0.3ml, Dual-chamber syringes reused at the same time add the same volume of fibrin glue solution and thrombin solution. According to the ratio of 2:1 volume ratio, calcium phosphate bone cement with fibrin glue uniformly mixed after solidification. Then one bone defect filled with composite CPC by the special injector until the cement overflowed the pore , and pressed it for 10 minutes till the cement concreted; the operating process of the control group was the same way.[2] Study of biomechanical properties of bone interface1 Preparation of biomechanical specimens: Every four rabbits were sacrificed by air embolism at postoperative 2, 4, 8, 12 weeks. After the distal femurs were sawed down, condyle specimens wrapped with double layer of physiological saline gauze were placed into polyethylene bags and stored at -20℃refrigerator. 24hours before mechanical test, the distal femoral specimens were defrosted under room temperature, and then immobilized by Tray Powder2 Biomechanical testing: Implants were evaluated mechanically (push-out test), The mechanical specimens were fixed on the test machine, then gradually compressed at the loading speed of 0.5mm / min by a 4.0mm- diameter top punch, which was vertically aligned toward the implants and parallel with the long axis of inplants. We set compression displacement in 15mm and took a record of instant loading curve. The peak of curve corresponded to the maximum shear force at interface. Then measured the thickness(h) of contact between implants and the cortical bone, and its diameter(d). And calculated with the following equation of the experimental shear strength: Maximum shear strength (MPa) = maximum shear force (N) / bone and implant contact area (mm~2), Contact area(S) =π×d×h. Each sample data were respectively recorded for statistical analysis.[3] Ultrastructural characteristics of bone interface1 Visual observation of the specimens: Before sacrificing rabbits each time at postoperative 2, 4, 8, 12 weeks, removed the soft tissue from the surface of specimens, and then observed bone growth from the surface of implants.2 Histopathology: The specimens were decalcified, dehydrated, hyalinized, and paraffin-embedded in turn, then cut the sample into 5μm slices. After conventional HE staining, observed bone and bone cement interfaces and the degradation of bone cement, as well as the surrounding inflammatory cell infiltrations, osteoblasts and osteoclasts, with optical microscope.3 Scanning electron microscope observation: Split the decalcified specimens along the direction of bone cement implanted, then trimmed into appropriate sizes (2×5×5mm~3). After handling with a series of steps, samples were placed under electron microscope to observe the interface between material and bone, material degradation, and new bone formation.Results1 A quantitative analysis of experimental animals: All the 22 rabbits survived till result analysis2 Appearance observation: After surgery no infection was observed. No obvious rejection was observed at the site of implanting site. 2 weeks, the size and appearance of implant were not significantly changed. There was no significant gap between the host bone and implant. Two group materials implants had fiber connection with the host bone, and implants were wrapped by fibrous tissue. Implant bone areas were not found obvious bone combined. The materials of experimental group became light yellow, while white was still in control group. 4-8 weeks, the implants were coated dense fibrous tissue and there was new bone protruding which was irregular and roughness in implant surface. Implants block turns red, the different appearance of the two group was not obvious. 12 weeks, the implants combine evenly with the surrounding bone. Appearance of bone defects was similar to autogenous bone.3 Statistical analyses: The results of the push-out test showed that the difference of biomechanics had statistical significance between experimental and control groups(F=198.274, P=0.000). After 2 weeks the shear strength of the CPC/FG was 0.511+/-0.080MPa (average+/-sd), which increased to 5.367+/-0.182MPa at 8 weeks and finally resulted in 6.432+/-0.119MPa at 12 weeks. The shear stress of experimental group was lower than that of the control group in 2 weeks and 4 weeks. However, from 4 weeks to 8 weeks, the shear stress of experimental group became gradually higher than that of the control group, especially during the 8 to 12 weeks. There were significant differences in every weeks in the experimental group.4 Histological analysis under light microscope: Experimental group: Histological evaluation revealed that there were some more collagen fibers in the materials and bone cement was porous at 2 weeks. Meanwhile more fibroblasts and mesenchymal cells could be seen in the bone defect area. At 4 weeks of engraftment, we observed a region of absorption at the junction of bone - bone cement. Collagen fibers had been partially absorbed, newborn trabecular bone and newly formed vessels could be seen. In all sections, what remained of the graft appeared actively to be in the process of being resorbed by osteoclasts. 8 weeks after engraftment, we observed extensive recruitment of activated osteoblastic cells. Most of the CPC was degradation, many trabeculae and plastic osteoids were detected . Trabecular bone was disorder and irregular. 12 weeks after engraftment, bone cement basically completed degradation, defects are basically full of trabecular bone, and new lamellar bones grew together. New bone gradually converted into a lamellar bone structure. Control group: Substantial CPC residued in the bone defect at 2 weeks. 4-8 weeks after engraftment bone cement begun degradation, especially at the junction of the bone - bone cement site, there is cartilage proliferation and new bone formation in this period. New trabecular bone formation and ossification in part could observed, calcium phosphate cement was still residual at 12 weeks.5 Evaluation of scanning electron microscope: Experimental group: SEM examination showed that the implant completely embedded in new bone at 8 weeks. There was different density in host bone and graft bone. The degradation rate of the material was relatively high but compatible with the ingrowth of bone trabeculae within the resorbing material, newly-formed trabeculae were observed around and on the surface of the implant, the trabeculae grew on to the material surface from the edges of the implantation site. Lamellar new bone pass through the gap of implant, and implant particles are separated in groups. The FG was dramatically degradated after 8 weeks with a few FG microspheres, and new bone replaced the degradated FG. The Harvard’s System was seen in woven bone structures. At 12 weeks lamellar bone tissue was more mature. Control group: The implants were absorbed slowly compared with experimental group, the trabeculae formation was still observed and implant was still degrading. So SEM examination showed that bone formation in the experimental group was significantly higher than in the control group after the 12 week of implantation.Conclusion:1 It is revealed in the experiment thatβ—TCP/MCPM/FG is more effective thanβ—TCP/MCPM in repairing bone defect. Biomechanical test showed that there is statistical significance between experimental group and control group (P<0.05). With the elongation of healing time, the effect of anti-shearing force inβ—TCP/MCPM/FG group was shown to be stronger thanβ-TCP/MCPM group.2 Histology microscopic studies indicated that the ossification capacity of experimental group is superior to that of control group. The material ofβ—TCP/MCPM/FG rendered favorable properties of biocompatibility, osteoinductivity, degradability, as well as plasticity.3 In this study, this novel injectable material ofβ—TCP/MCPM/FG performed satisfactory osteoacusis ablity, mechanical property and biocompatibility , which is a promising material in repairing bone defect in osseous tissue engineering. |
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