| Objective:Through building beagle dog condyles of femur PRF bone induction model, comparatively observe osteogenic effect of PRF, Bio-Oss, and autogenous cancellous bone, discuss microstructure and biomechanical properties of newly formed born induced by Platelet-rich fibrin, evaluate traits and advantages of PRF repairing bone defect, and offer experimental results for the clinical application of PRF induced bone osteanagenesis and jaw reconstruction.Methods:1. The establishment of animal modelAmong 13 adult male beagle dogs,12 were randomly selected. After giving them intraperitoneal anesthesia,3 tubes of 9ml venous blood were collected. Giving the tubes 10 minutes centrifugal with the speed of 3000r/m, acquire PRF and compress into membrane. To prepare three cylindrical bone defects with a diameter 6.0mm and 10mm depth in the bilateral medial femoral condyle and mark locating pins. Three defects were filled with PRF membrane, Bio-Oss substitute material and autogenous cancellous bone randomly, and Haiao biological membrane covered over Bio-Oss and autogenous cancellous bone, and apposition suture was used to treat the wound. Another beagle dog did not fill any bone graft material after making only one defect in the same way; it is used as a blank control. Make routine postoperative cared, grouping and numbering A1～A6, B1～B6 and C as random principle.2. X-ray examination and visual observation of specimenBeagle dogs were killed by air embolism after anesthesia. The bilateral femoral condyle were wrapped with normal saline gauze and put into a container, then expose the container under X-ray using the same projection angle, distance, exposure time and visual inspection. After that, store the left side of the specimen at -20℃ refrigerator and the right side immersed in NS, respectively recorded as L and R.3. Micro CT measurement and analysisRight side of the femoral condyle of A1～A6 and B1～B6 were polished outside, parallel to the inside surface, and fixed in sample cup which scanned from top to bottom using Micro CT. Scanning field was 15mm tall with 790 layers, which voxel size was 19μm, integration time of 250ms, energy of 70kVp and 114μA, the image matrix of 2048 pixels ×2048 pixels. Selected the cylindrical area with diameter of 3mm and height of 1.5mm in new bone, and repeated the process three times, using μCT V6.1 to reconstruct and analyze the image and three-dimensional Gaussian filter (sigma 1.2, support 1) to reduce the noise of the image. Then, control the threshold value, PRF and autologous cancellous bone used 220～1000, but new bone of Bio-Oss area used 220～340 and Bio-Oss granule used 341～1000. The obtained parameters of specimens were as followed:bone volume fraction, trabecular number, trabecular spacing, and the degree of anisotropy and the connection density. They are used for statistical analysis.4. Determination of insertion torquePolish the right side of the lateral femoral condyle of Group A and B to 10mm deep. Fix the surgical specimen on the bench vise and strip off the excess soft tissue, determine the every center of the bone defect and implant DIO 3.8*10mm in it with ratchet wrench until reaching the surface level of the bone. Then record the value of insertion torque with digital torque wrench and ratchet wrench in Group P, B and Z.The whole process should keep intermittent preparation and cool for statistical analysis.5. Bone compression testDue to the limitations of experimental design and conditions, the test cannot be conducted immediately. According to the requirements of biomechanical specimen preservation, the left femoral condyle of the Group A and B were wrapped in saline gauze and froze at -20℃ refrigerator. Before the experiment, they were left in the room to reach normal temperature. Cylindrical bone with height of 10mm and diameter of 5.5mm was removed by annular bone drill in the new bone area formation with PRF, Bio-Oss and autogenous bone processed. Both ends of cylindrical bone were kept in parallel and 9mm tall bone specimen was acquired by using a high-speed dental drill. During the process, the bones were drilled intermittently and flushed to cool when necessary. The specimens were recorded as the Group P, B and Z, and set aside. At room temperature and the instrument zero, a universal mechanical testing machine was used to axial compress experimental bones, the height was measured by vernier caliper and put into the system, the experimental bones were kept moist in this process and compressed with 1mm/min loading rate until they were occurred fracture. Mechanical data of new bones were obtained. Then, the load-displacement and stress-strain diagram was draw and stiffness, strength and elastic modulus of each specimen were calculated to perform the statistical analysis.Results:1.13 experimental animal surgery completed, healing well, PRF bone induction model of beagle dogs’femoral condyle was successfully established.2. Gross observationThe Group P, B and Z of 3 months after operation:Anatomical level was unclear and scar was obvious in soft tissue of the operation area. In joint capsule, there was a little exudate and part of the biofilm which was not degraded but wrapped by connective tissue and fixed in place. Periosteum healed well. After periosteum stripped, slight sunken were seen with unclear border and none residual Bio-Oss particles in the three places of new bone surface.The Group P, B and Z of 6 months after operation:The surgical scar still existed. In joint capsule, exudate decreased and residual biofilm was not significant. Bone sunken still existed in the P, B, Z places but was shallower with unclear border compare to 3 months’situation.The blank group of 3 months after operation:there was no significant difference in the healing of soft tissue, but significant depression in the bone defects.3. X-ray examinationThe Group P, B and Z of same day after operation:compared with surrounding normal bone tissue, The Group P was significantly lower density; The Group Z was slightly lower density; The Group B showed a high density image; all had clear interface.The Group P,B and Z of a month after operation:The Group P gradually increased density, lower than the surrounding bone tissue; The Group Z had increased density proximity to surrounding normal bone tissue; The Group B, which density decreased, still slightly above the surrounding bone tissue, no significant narrowing range. All three groups illustrated fuzzy interface with the surrounding bone tissue.The Group P, B and Z of 3 month after operation:The density of Group P was further increased compare to 2 months ago with unclear interface; The Group Z had higher density approximately to the surrounding bone without obvious interface; The Group B density dropped sharply, but still higher than the surrounding bone tissue, where high-density images region narrow to the center and the interface had visible low density around.The Group P, B and Z of 6 month after operation:The Group P and Z were close to the density of the surrounding normal bone tissue without obvious interface; The Group B had high-density images region of irregular shape further narrow to the center, and its interface was still visible narrowing circle of low density around.The blank group of 3 month after operation:bone defect area was clear round low density image, dropped gradually into the center, and the interface was clear.4. Micro-CT measurement and analysisTomography:3 months after surgery, the Group P and Z were similar and the density slightly lower than the surrounding normal bone. The regular arrangement of visible bone trabecular was shown and no obvious interface was illustrated. However, the Group B showed a high density with clear interface.6 months after surgery, interfaces of the three groups were unclear. Quantity of bone trabecular of Group P and Z gradually approaching the surround bone tissue, while the Group B high density area narrowed. The blank group had few bone trabecular with low density images and clear interface, which was fundamentally different from surrounding normal bone tissue.Three-dimensional reconstruction image:bone trabecular of natural area was continuous, structured clearly and orderly shown as strip or plate. 3 months after surgery, the Group P showed a few new bone trabecular of large thick plate and good continuity; the width, thickness, quantity and intensity of new born trabecular of Group Z were between Group P and B with good continuity; in Group B, new bone tightly wrapped Bio-Oss particles and grow on the surface, bone trabecular had cord-like, thin, plentiful and concentrated; The blank group failed to repair defect, without continuous bone trabecular but spotty callus distribution. 6 months after surgery, the number of bone trabecular, dense and thickness slightly increased within both Group P and Z compare to 3 months ago.While the bone trabecular of Group B became slightly thinner, and the arrangement gradually decreased to the form of normal bone tissue.The dual color interface image of Group B, from three months to six months, the red part of Bio-Oss particles narrowed significantly towards the center with the volume reduced and was replaced gradually by new bone, with interface moved to the center. It means that osteogenesis showed concentric manner. The Group P interface image, due to the same thresholds between surrounding bone and new bone of PRF, the interface was not obvious. But with time passes, the bone trabecular dense gradually increased from the middle to the surrounding area, while the central cavity shrink and sparse degree reduced with the increased density.There were significant differences between the three groups in BV/TV, Tb.Th, Tb.N, Tb.Sp, DA, Conn.D; LSD showed that both the Group P and Z were significantly different with B (P<0.05), the Group P and Z had no significant difference (P> 0.05);within group comparison of two time nodes showed no significant difference (P> 0.05).BV/TV, Tb.N, Tb.Sp, Conn.D reflected that in the Group P and Z the new bone volume, the number of trabecular, separation and connection density were close to natural bone, but not as good as it; the main difference were the smaller volume of new bone, a little sparser spatial arrangement, lower connection density. The value of the Group B was much higher than the natural bone, it showed the high volume and quantity of new bone with closer spatial arrangement and higher connection density. Tb.Th,DA reflected that:the Group P and Z were close to the natural bone with high maturity level; the Group B was inferior to natural bone, with fine new bone trabecular, low degree of anisotropy and poor bone maturity. All the indexes of the three groups illustrated the trend towards natural bone.5. Determination of insertion torque36 implants were used successfully in the Group P, B and Z according to the requirements.Insertion torque values of digital torque wrench and manual ratchet wrench of three groups showed significant differences. LSD showed that 3 months after surgery both Group P and Z were significantly different with B (P<0.05), the Group P and Z had no significant difference (P> 0.05); 6 months after surgery, the Group P and B were significantly different with Z(P<0.05); within group comparison of two time nodes showed no significant difference (P> 0.05).Characteristics of each group and the change overtime:in 3 months, insertion torque values of the Group P and Z were close,both less than the Group B; in 6 months, IT values of the Group P were less than the Group Z, but near to the Group B. IT values of the Group P and Z increased with time, but the Group B were opposite.6. Compression testCylindrical specimens of new bone were completely removed in the Group P, B and Z. There were 36 specimens totally and compression tests were completed successfully.Stiffness, elastic modulus and strength of three groups showed no significant difference (P> 0.05). In the same group, there were also no significant differences at two time node (P> 0.05).But the means of them displayed that the Group P were closer to the Group B and Z after 3 months; both the Group P and B were not as good as the Group Z after 6 months. Stiffness, elastic modulus and strength of the three groups increased with healing time.Biomechanical properties of new bone in the Group P, was similar with the Group Z at 3 months after surgery; while not as the Group Z but similar with the Group B at 6 months.Conclusion:1. This study is scientific and rational throughout the design, the postoperative care and record and operation of surgery, it serves its purpose well. Beagle dogs’model of PRF induced bone had been established successfully on femoral condyle.2. The bone defect that prepared in our experiment is unable to complete the bone regeneration by itself, in line with standard of critical size defect.3. During the process of repairing bone defects of PRF, X-ray and Micro-CT images showed new bone regeneration to the center.4. PRF can induce formation of new bone. Its microstructure is similar to physiological healing as the Group Z and is differ from the Group B. The quality and quantity of new born induced by PRF have no significant difference between two time nodes 3 months and 6 months, but the means of the indicators become gradually close to the normal bone over time.5. IT of the Group P increases with time, reflecting the enhancing bone in the quality and quantity. The Group P is similar to the Group Z with contrary to the Group B. At 6 months after surgery, new bone of the three groups is able to satisfy the condition of surgery. In addition, it requires special attention to shorten the waiting time of repairing bone defects.6. Guided by Bio-Oss and autogenous bone, compression abilities of new bone induced by PRF continues to grow with time. However, there was no significant difference. Therefore, the theory mechanical properties grow over time remains undecided.7. Stiffness, strength, elastic modulus of new bone was close among three groups after 3 months’operation, while the Group P and B were not as good as Z at 6 months’time. Due to no significant difference between groups, which material has better biomechanical properties remain unknown. Therefore, it is necessary to prolong observation period. |
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