The Study Of Domestic Porous Tantalum Coated With Cyclic RGD Peptide In Repairing Of Segmental Bone Defects Defect In Rabbits’ Radius

Chapter 1 The preparation of Domestic Porous Tantalum Coated with Cyclic RGD PeptideObjective:To investigate the changes on the topographic characteristics and hydrophilicity of domestic porous tantalum coated with cyclic RGD peptide.Metheds:1 The preparation method of porous tantalum coated with RGD peptide:After disinfection, porous tantalum disks were soaked in a sterile filtrated 100mM solution of the RGD peptide in PBS for 24 h and subsequently washed 3 times in PBS followed by air drying in a laminar airflow chamber. All disks were sterilized using ultraviolet radiation.2 The topographic characteristics of porous tantalum disks:Three samples were taken from modified tantalum disks and non-modified tantalum disks, respectively. The topographic characteristics of the two groups of samples were tested by general observation and scanning electron microscope.3 Hydrophilicity of the two groups of samples:Six samples of modified and non-modified tantalum disks were weighed as Wl, respectively. And then they were immersed and soaked in deionized water for 24 h. These disks were taken out and weighed as W2 after the deionized water on the surface of samples was removed with filter paper. The water absorption for samples was (W2-W1)/W1× 100%.Results:1 The topographic characteristics of the two groups of porous tantalum disks:The general observation exhibited that every sample of the two groups had gray appearance, smooth surface, and uniformly honeycomb pore. Scanning electron microscope observation showed that the pore diameters of the two groups of samples ranged from 400 to 600μm and the diameters of connecting holes in inner pores ranged from 50 to 200μm. The surface of modified porous tantalum samples observed under high magnification microscope had uniform spots.2 Hydrophilicity of the two groups of samples:The water absorption of non-modified and modified porous tantalum samples were 1.98±0.37%,4.83±0.30%, respectively. The result showed that water absorption of modified tantalum was higher than that of that of non-modified porous tantalum, and the difference was statistically significant (P<0.01).Conclusions:The topographic characteristics of domestic porous tantalum coated with cyclic RGD peptide does not change significantly, and its hydrophilicity improves obviously.Chapter 2 Effect of Domestic Porous Tantalum Coated with Cyclic RGD Peptide on the Proliferation and Adhesion of Obsteoblasts in vitroObjective:To investigate the effect of porous tantalum coated with cyclic RGD peptide on the proliferation and adhesion of obsteoblasts in vitro, and provide the basis for further experiment in vivo and clinical application of domestic porous tantalum as scaffold material on bone tissue engineering.Methods:1 Isolation and culture of obsteoblasts:The craniums was taken aseptically from neonatal rabbits within 24 hours and cut into 1×1 mm. The samples were digested by 0.25% trypsin for 30 min and 0.1% collagenase II for 60 min at 37℃, respectively. And then the digested cell pellet was cultured in DMEM medium containing 10% FBS. When the cultured cells arrived a density of 105 cells/ml, they were seeded into 25cm2 culture flask at the condition of 37℃ and 5%CO2. Passage was performed after 80～90% of the cells reaching confluence, and the second generation cells were used for the experiment.2 Morphology observation:Cell growth and morphological changes at every period were observed under inverted phase contrast microscope.3 Identification of obsteoblasts:The second generation of obsteoblasts were seeded on 12-well plates. And alkaline phosphatase staining was performed when the cells reached 80～90% confluence.4 Experiment groups, morphology observation and cell growth of the obsteoblasts seeded on porous tantalums:There were three groups, Group A:porous tantalum modified with cyclic RGD peptide, Group B:porous tantalum without modification, the blank group:the obsteoblasts seeded on 24-well plates. Morphological changes and cell growth of every group were observed under inverted phase contrast microscope.5 Effect of porous tantalum coated with cyclic RGD peptide on the adhesion of obsteoblasts:The obsteoblasts suspensions with a density of 1×106/ml were seeded on the scaffolds of every group, the obsteoblasts were incubated for 2 hours and 4 hours, and the adhesion rates of every group at the two time points were measured with precipitation method and compared with statistical software. The morphological changes and growth feature of the obsteoblasts on the two groups of porous tantalum scaffolds were observed under the scanning electron microscope at 4 hours after cell cultivation. 6 Effect of porous tantalum coated with cyclic RGD peptide on the proliferation of obsteoblasts:The obsteoblasts suspensions with a density of 2.5×105/ml were seeded on the scaffolds of every group, the obsteoblasts were cultured for 1,3,5, and 7 days. At each time points, MTT solution was added to the medium, and the value of optical density in every group was measured with BIO-RAD enzyme-linked immunometric meter. The proliferation curves of obsteoblasts in three group were drawn and the comparisons between groups were made at the four time points. And then, the morphology and growth feature of the obsteoblasts on the two groups of porous tantalum scaffolds were recorded under the scanning electron microscope at 3 days after cell cultivation.Results:1 Morphological observation and identification of obsteoblasts:After inoculation, the primary obsteoblasts suspended in the culture medium, which were all transparent and round-shaped with the same size. Some cells adhered to wall at 6 hours, and the inoculated cells had completely adhered and extended to the wall for 24 hours. At 3 days, the number and volume of obsteoblasts increased and the cells connected each other with the aid of pseudopodia. The obsteoblasts almost covered the bottom of the culture bottle at 6-7 days. The second generation cells had the same morphology with spindle and polygonal shape. The result of alkaline phosphatase staining confirmed that the cultural cells were the obsteoblasts.2 Observation of morphological changes and cell growth of the obsteoblasts cultured under inverted phase contrast microscope:The results showed that the numbers of obsteoblasts in the edge of the two groups of porous tantalum all gradually increased with dense cluster and good shape. And the number and denseness in Group A were higher than Group B.3 The adhesion of osteoblasts:After 2 hours and 4 hours culture, the adhesion rates of osteoblasts in Group A were all higher than those in Group B and the blank group, and the differences were statistically significant (P<0.05). Meanwhile, the adhesion rates of Group B were similar to those of the blank group and there was no significant difference (P>0.05). Furthermore, the adhesion rates in every group after 4 hours culture were higher than those after 2 hours culture and the differences were statistically significant (P<0.05).4 The proliferation of obsteoblasts: After 1,3,5, and 7 days culture, the values of optical density in Group A were higher than those in Group B and the blank group (P< 0.05), and there was no significant difference between Group B and the blank group (p>0.05).5 The morphological characteristics and growth of obsteoblasts on the tantalum scaffolds under the scanning electron microscope:After 4 hours and 3 days culture, the obsteoblasts in Group A and B were all well grown, and the cell extension in Group A was faster than that in Group B. At the same time, the cell density and extracellular matrix on the surface of porous tantalum in Group A were better than those in Group B.Conclusion:1 Domestic porous tantalum has good biocompatibility, and no effect on the proliferation and adhesion of obsteoblasts without cytotoxicity.2 The porous tantalum modified with cyclic RGD peptide promotes the proliferation and adhesion of obsteoblasts, and maybe an ideal scaffold material of bone tissue engineering.Chapter 3 Repair of Segmental Bone Defects in Rabbits’Radius with Domestic Porous Tantalum Coated with Cyclic RGD PeptideObjective:A segmental bone defect in a rabbit radius model was repaired using domestic porous tantalum coated with cyclic RGD peptide. The purpose of this charper was to evaluated the biocompatibility and the effect in repairing bone defect of porous tantalum coated with cyclic RGD peptide by imaging, histologic and biomechanical evaluation, and to provide the experimental basis for domestic porous tantalum in clinic.Metheds:1 Preparation of the materials and experimental groups:The porous tantalum material which was fabricated to long cylinder with 3.5 mm in diameter and 15 mm in length, was coated with cyclic RGD peptide according to Chapter 1, and sterilized by ultraviolet radiation. A total of 105 New Zealand white rabbits (age,6-8 months) were randomly divided into Group A, B, C, D and E. Every group contained 24 rabbits except Group E, which included 9 rabbits. Group A:porous tantalum coated with cyclic RGD peptide, Group B:porous tantalum wrapped by pedical fascial flap, Group C:only porous tantalum, Group D:xenogeneic cancellous bone, Group E:the blank control group.2 Surgical procedures:After successful anesthesia and routine disinfection, a longitudinal incision about 3.5 cm in length was made in the middle radial side of the right forearm. A 1.5 cm segmental bone defect in right radius was established as the animal model using the micro oscillating saw at a lower speed. After irrigation with saline, four kinds of the implants in Group A, B, C and D were positioned in the created bone defect respectively. Osteotomy was only made without implants in Group E as the blank group. The pedical fascial flap about 30 x 25 mm in size was made at the subcutaneous tissue of the middle right forearm, which contained rich capillary network and wrapped the implants in Group B. The muscles were carefully apposed, and the fascia and skin were closed in a routine fashion. To avoid. infection, penicillin was administered 3 days after surgery.3 General observation after surgery:The diet, daily activities and wound healing of the experiment rabbits were observed.4 Radiological examination:At the day of the surgery and 4,8,16 weeks after surgery, each rabbit was examined by the digital X-ray to observe bone connection of the interface between implant materials and the host bone.5 Gross anatomy observation:At 4,8,16 weeks after surgery, the animals were sacrificed and the repair of bone defects soft tissue distribution were observed.6 Histological examination:At the three time points of 4,8,16 weeks after surgery, some samples of every group were fixed in 4% paraformaldehyde solution and decalcified in 5% diluted acid. After conventional dehydration, transparency, waxing, embedding and slicing, the sections were stained with hematoxylin-eosin(HE). The growth of new bone was observed at the interface under optical microscope. The other samples of every group were sliced by hard tissue slicing machine without decalcification and stained with toluidine blue. The growth of new bone at the interface and inside the implant materials were observed.7 Scanning electron microscopic observation:At the above three time points, the samples of Group A-D were fixed in 2.5% glutaraldehyde solution. After dehydration, drying, spraying, new osteoid tissue was observed at the interface, the surface of the materials and inside the pores under scanning electron microscope.8 Biomechanical testing:At 16 weeks after surgery, three point bending test was performed to measure mechanical properties of the radial specimens in Group A, B, C, D.9 Micro-CT analysis:The animals were sacrificed at 16 weeks after surgery in Group A, B, C and D. The specimens which included the radius and adjoining ulna with 2.5cm in size were centered on the defect sites, and scanned using a micro-computed tomographic imaging system.3D Micro-CT reconstructed images and new bone metrology analysis were also performed in the four groups.Results:1 General observation of the experiment animals after surgery:The diet and mental status of the rabbits recovered gradually 7 days after surgery. And the wound healed primarily without flare, effusion and suppuration. Daily activities and body weight regained to normal level without lameness.2 Radiological examination: At the day of the surgery, the X-ray images showed that the implants were well maintained without apparent displacement. As followed with time, the combination between the implants and host bone became more and more closely, and the fracture line gradually disappeared. The callus grew well and marrow cavity shaped incompletely 16 weeks after surgery in Group D. And the callus formation in Group A was best among Group A, B, C. For Group E, the radial defect was still visible, the bone density of the stump increased and marrow cavity closed.3 Gross specimen observation:The implants in Group A had been gradually covered by osteoid tissue for 16 weeks after surgery. And the combination between the implants and host bone were well firm. For Group B and C, the pore of implant surface were also partially filled by new bone tissue. The implants close to the interfaces were also covered by osteoid tissue and the interface also combined closely. The implant materials had been gradually degraded and replaced by new bone tissue with the growth of the capillaries and good solid interface fusion in Group D. The defect in Group E was still clear at 16 week after surgery with closed medullary cavity at the radial stump.4 Histological examination:At the three time points of 4,8,16 weeks after surgery, new bone mass and maturity gradually increased at the interface and inside materials in Group A-C. And the new bone gradually growed from the interface to internal pore. The new bones in Group A were better than B group C. The xenograft bones were gradually replaced by new bone tissue in Group D, and the bone defects in the rabbit radius were always visible and the walls were covered by thin layer fiber membranes in Group E.5 Scanning electron microscopic observation:At the above three time points, new bone collagen and lamellar bone gradually increased at the interface, the surface of the materials and inside the pores among Group A, B, C, and the interfaces between tantalum and the host bone gradually combined closely. Meanwhiles, new bones gradually increased and differentiated into mature trabecular bone in Group D. 6 Biomechanical testing:At 16 weeks after surgery, the results of three point bending test showed that maximum load power and bending stiffness in normal radius were higher than those of Group A, B, C and D. And the differences were statistically significant (P<0.01). Among Group A, B, C and D, mechanical properties in Group A were highest and the lowest in Group D. The differences between groups were also statistically significant (P<0.01).7 Micro-CT analysis:At 16 weeks after surgery, there were a large amount of new bone at the interface, the surface of implant materials and inside the materials. Quantitative analysis of new bone volume fraction revealed that the result in Group D was superior compared with Group A, B, C. The difference between Group A and D was not statistically significant (P>0.05), while there was significantly different in Group B, C and D (P< 0.01). There were also significantly different in Group A, B and C (P< 0.01).Conclusions:Domestic porous tantalum has a good biocompatibility. Its ability of bone conduction is stronger with surface modification of RGD peptide. The effect on repairing segmental bone defect in rabbit radius is positive.