| BackgroundWith the development of society and economic level,bone defect caused by various reasons quite common in clinics,reasons such as:bone tumor, trauma, has resulted in many serious consequences, and became a quite burden to the society and patients, so the repairment of bone defects has become an important issue to orthopaedic doctors. At present the main means for the repairment of bone defect is by the treatment of suitable materials filled into the bone defect place,the filling materials should contain two important characteristics,which is the bone induction and bone conduction. Bone conduction function refers to the function that is induced by providing three-dimensional scaffold, while the bone induction means the function of inducing vascular system and osteoblast functions,such as the activity of BMP VEGF and TGF- beta et al.According to the above two characteristics,three kinds of materials has already been used in both clinical and laboratory, which is Autogenous bone graft, allograft, and emerging biological materials such as:nano chitosan, coral stone et al.Autologous bone tissue has quite good compatibility with fewer complications is the golden standard for the repariment of bone defect,but no sufficant bone could be used in the clinics.New biological material has not been widely used in clinical due to its special properties.Therefore,bone allograft has become a very important solutions to the bone defect at present.Compared with the autologous bone, allograft has sufficient quantity, thus can satisfy the various types of bone defects, but just because of its immunogenicity, it can produce a variety of complications:such as allograft bone necrosis, local inflammatory reaction problem, affecting its therapeutic effect et al in the clinics. How to reduce reduce bone allograft complications without damaging the induced action has become an important issue to the scientist and otrhopaodic doctors.The reasons for the immune rejection of allogeneic bone are quite complicated,so many regular methods like improving the manufature steps of allograft and other methods could not be widely used in the clinics due to technical and economic constraints. While the use of immunosuppressants, which can not only mitigate the immune response effectively, also it can play a role in promoting the repair of bone defects, this function of immunosuppressants has offered a new direction of research for clinics in the treatment of bone defects. FTY-720 (Fingolimod) is a component which was found by the extraction of cordyceps sinensis, and in the laboratory after the change process of synthesis of side chain, in 2010 September through the FDA certification in USA,which is treated as a new immunosuppressant for diseases of the immune system in clinics. The mainly active means of this drug in the body is after being phosphorylated modified into FTY-720P function, it can modify the function of immune system by the functions such as: regulate effects of lymphocyte migration, or regulate functions of natural immune cells like dendritic cells, NK cells, NKT cells and macrophageset al. At the same time, the drug also has an important effect on bone formation, scholars in and abroad has done some animal experiments using a variety of FTY-720 composites, which achieved good therapeutic effect, but the mechanism is not clear. Therefore need further researches and exploration are needed.Objective:1.To explore the effect of the bone defect by the treatment of FTY-720P conbined with allograft, by the means of using methods of Lane-Sodihu score system, bone density examination and histological examination,then using fluorescence quantitative PCR to speculated the possible therapeutic mechanisms.2.To analysis the effect of FTY-720P on the osteoclast formation and phagocytosis by using FTY-720P in treating with 2 kinds of cells that could be induced into osteoclast, the analysis is done by counting the number of osteoclasts and osteoclast bone after phagocytosis lacunaes.Then protein chips is needed to examine the differences between the experimental and control group, which is helpful to suggest the possible mechanism, so as to lay the theoretical foundation for the experiments in the future.Methods:1.Bone allograft harvest:Allograft bone was harvested from lambs of New Zealand rabbits (mature,either sex),trimmed to a segment of 2-2.5cm long and used in the subperiosteal implantation. Grafts were stripped of soft tissue, cleaned with detergent, hydrogen peroxide and ethanol sonication washes and then allowed to fully dry. Samples were irradiated by the Cobalt-60(25kGy) for sterilization, then they were stored at -20℃ until use.2.Bone defect surgeries:All animal surgeries were performed according to an approved protocol from the Southern Medical University Animal Care and Use Committee. All animals were obtained from Southern Medical University(Guangzhou Guangdong, China). In this study,48 New Zealand rabbits (mature,either sex) with weight of 2-3 Kg were randomly assigned to four different experimental groups (n=12):blank group,allograft control group, autogeneous bone group and experiment group. Bone defect of 15 mm in size was created according to method of Girolamo.3.X-Ray examination:Radiographs were taken at 2,4,8 and 12 weeks after the experiments, and points were allotted according to the Lane-Sandhu methods,The points were given according to the degree of bone formation,connections and bone marrow recanalization.4.Bone density detection (BMD):Measurement of BMD was conducted using a Hologic QDR-2000/Plus DXA instrument (70 kVp/140 kVp). After carefully seperating the tibia at the site of modeling and completely removing the superficial muscles and soft tissues, BMD was expressed in grams for cm2.5.Histology:Following in vivo X-Ray scanning and ex vivo bone density detection,24 tibia samples were fixed in 10% buffered formalin for 7 days and decalcified using an HCl and EDTA decalcifying solution for 3 days at 4℃ with agitation. The bone was cut in half, centered at the defect or implant. Then sample was stored in 70% ethanol until paraffin embedding for hematoxylin and eosin (H&E) staining.6.Fluorescence quantitative PCR detection:Total mRNA was extracted from 24 tibia samples with the TRizol reagent (Invitrogen, Carlsbad, CA). PrimeScript RT Master Mix (Takara, Japan) was used to synthesize the first strand complementary DNA. Briefly,2μg of total RNA was used to synthesize cDNA, cDNA products were amplified using a SYBR Green PCR Kit (Roche). The amplification condition was consisted of incubations at 95℃ for 1min s,60℃ for 1 min, and 72℃ for 1 min for 40 cycles.7900 HT Real-Time PCR System (Applied Biosystems, Carlsbad, CA) was used to detect Real-Time PCR. Expression level was normalized against endogenous GAPDHs for related gene expression.7.Identification and formation of osteoclast induced by RAW264.7:to induce the osteoclast by RAW264.7 cell line with application of different concentrations RANKL:10,25,50, 100ng/ml, then different denerations of RAW264.7 cells were induced by RANKL, Identification of osteoclasts:Based on cell counts and tartrate resistant acid phosphatase (TRAP) staining positive identification of RAW264.7 cells induced by osteoclast situation. This was used to observe the optimal concentration of RANKL and the best experimental cell generations.8.Influence of FTY-720P on osteoclasts induced by RAW264.7 cells:cells was grouped according to the added contration of FTY-720P into 0,500,600,700,800, 900,1000,1500,2000,2500ng/ml groups, then optimum concentration of RANKL was added into the cells. All cells were put in the constant temperature box were incubated for 4 days. Then cell morphology, the number of nuclear,the effect of FTY-720P on the formation of osteoclasts induced by RAW264.7 were observed by microscope.9.Acquisition of BMMs and the identification and induction of osteoclasts by BMMs:BMMs cells come from humerus, femur, tibia trunk limbs of 1 month old SD rats, the cavity of the long bone was flushed repeatedly, then lOng/ml M-CSF was added into the culture dishes with fresh bovine bone slices, and these culture dishes were incubated about 72 hours, then 30ng/ml RANKL was added. After about 7 days of culture, the formation of osteoclast was tested by counts of cell number and TRAP enzyme staining positive.lO.Effect of FTY-720P on BMMs induced osteoclast formation and phagocytic function:according to the different concentration of FTY-720P accession, the culture dish was divided into 0,500,600,700,800,900,1000,1500ng/ml group,after 10 days of culture,TRAP enzyme and F-actin staining respectively to detect the effect of FTY-720P on osteoclast induced by BMMs, then the esorption lacunae numbers was counted after toluidine blue staining, to evaluate the FTY-720P on the phagocytosis of bone.11.Study on FTY-720P on osteoclast induced by BMMs using protein chip research:ccording to whether or not adding 700ng/ml FTY-720P, cells were divided into experimental group and control group. Then according to the note Raybiotech kit, proteins of both groups were extracted, then analyze the difference of protein expression between the two groups.Results:1.Radiological examination and Lane-Sandhu’s Score:The Lane-Sandhu radiographic score of the four groups. There were no statistical difference among the three groups 2 weeks after the surgical procedure operation (F=0.257, P=0.854). In the time point of 4 weeks(F=170.469, P<0.001), the score in the experiment (3.67±0.14)and in the autogenous group(3.97±0.17) is higher than the other two groups,and the allograft group(3.07±0.26) is higher than the blank group(0.63±0.24),but there is no statistic differences between the experiment and autogenous bone group (P=0.109). In the time point of 8 weeks (F=30.655, P<0.001),the score in the experiment group (5.73±0.77), in the autogeneous bone group(6.16±0.34) and in the allograft bone group(5.19±0.29) are higher than that in blank group(1.64±0.94),there is no there is no statistic differences among the three groups(P=0.218).In the time point of 12 weeks(F=69.152, P<0.001),score of the experiment group(7.93±0.57), the autogeneous bone group(8.76±0.64) and the allograft bone group(6.99±0.37) are higher than the score in blank group(2.36±0.74),and the score of experiment group(7.93±0.57) and autogeneous bone group(8.76±0.64) is higher than the score of allograft bone group(6.99±0.37)(P=0.126),but there is no statistic difference between the experiment (7.93±0.57)and autogenous bone group(6.99±0.37)(P=0.089).2.Bone density detection:in postoperative week 2(F=98.359, P<0.001), the bone density results obtained from the autogeneous bone transplant group(0.64±0.02) were higher than in the experimental group(0.60±0.02) and in blank group(0.40±0.02). In postoperative weeks 4(F=13.370, P=0.002), the averages obtained from the experimental (1.24±0.03),autogeneous bone group(1.30±0.05) and allograft bone group(1.19±0.02)were higher than blank group(1.00±0.10), but it could not be considered that there were statistically significant difference between the averages from experimental and two control groups(P=0.138).In postoperative week 8(F=21.433, P<0.001), the averages obtained from the experimental (1.35±0.05), autogeneous bone group(1.42±0.04) and allograft bone group(1.30±0.02)were higher than blank group(1.09±0.08), but it could not be considered that there were statistically significant difference between the averages from experimental and two control groups(P=0.066).In postoperative week 12(F=33.971, P<0.001), the averages obtained from the experimental (1.51±0.07), autogeneous bone group(1.60±0.03) and allograft bone group(1.50±0.04)were higher than blank group(1.17±0.07), but it could not be considered that there were statistically significant difference between the averages from experimental and two control groups(P=0.148).3.HE staining results at all time points in the four groups:the region pointed by an arrow is necrotic bone allograft. From the HE staining,in the week 2 point,large amount of erythrocyte diapedesis occurred in the blank group,in the other groups less erythrocyte diapedesis occured; In the week 4 point,a small amount of erythrocyte diapedesis and a considerable amount of inflammatory cell infiltration were observed in the blank group, but less could be found in 2 control groups and experimental group with remaining allograft bone were observed; In the week 8 point:the grafting site presented a creeping substitution situation where the new bones and the sequestrums interwove together, the new born bone trabeculas were more ordered in blank group, autogeneous bone group and experiment group than in the allograft bone group;In the week 12 point:the recover condition of different groups were different, granulation tissue was found filled into the bone in the blank group, while in other groups, especially in allograft bone group,the new born bone trabeculas were more ordered than before, also remanining allograft bone could be found in the experiment group.4. Quantitative PCR detection:through RT-PCR detection, the results suggest that:at time point of 2 weeks after operation, the expression of BMP-2 and VEGF is higher in allograft bone transplantation control group and experimental group than in the autologous bone graft control group, while the expression of Col I alpha 1 in the autogenous bone graft control group and the experimental group were higher than that in allogeneic bone transplantation control group, no significant statistical the expression and significance of difference between the three groups of Spp-1, SIP receptor;at the time point of 4 weeks after operation, the expression of BMP-2, Spp-1 and VEGF in allograft bone transplantation control group and experimental group were higher than that of autogenous bone graft control group, while the Col I alpha 1 and SIP receptor expression showed no significant difference between the three groups;at the time point of 8 weeks, VEGF, Col I alpha 1 and SIP receptor expression of autogenous bone graft control group and the experimental group were higher than that of allogeneic bone group, but no significant differences between the expression levels of BMP-2 and Spp-1 of three groups. At the time point of 12 weeks, the expression of BMP-2 in allogeneic bone group and the experimental groups is higher than autogenous bone graft group, Spp-1 and VEGF expression showed no significant difference among the three groups, while Col I alpha 1 and SIP receptor expression of autogenous bone graft was higher than allogeneic bone control group and experimental group.5.Observation of RAW264.7 cells in the microscope:the RAW264.7 cell which was treated with no RANKL,and cells were seen to be grown in a adherent way, no multinucleated cells could be seen. the RAW264.7 cell which was treated with RANKL, and plenty of multinucleated cells with pseudopodia could be found.6.Positive rate of osteoclast induced by RAW264.7 cells in different RANKL cencentration(F=58.105,P<0.001). Positive rate of osteoclast induced by RAW264.7 cells treated with RANKL in the concentration of 50ng/ml (8.11±0.41)and 100ng/ml(7.34±0.70) was larger than that in the concentration of 10ng/ml(4.02±0.23) and 25ng/ml(5.10±0.19),and positive rate is larger in the concentration of 25ng/ml(5.10±0.19) than in the concentration of 10ng/ml(4.02±0.23), no statistic differences could be seen in the the concentration of 50ng/ml (8.11±0.41) and 100ng/ml(8.11±0.41)(P=0.061).7.Positive rate of osteoclast induced by RAW264.7 cells in different generation of RAW264.7 cells(F=61.064, P<0.001).Positive rate of osteoclast induced by RAW264.7 cells in generation of 8th(9.32±0.62),12th(8.76±0.51),16th(7.92±0.32) RAW264.7cells was the largest, and no statistics differences could be seen among these three groups(P=0.069),the positive rate of osteoclast induced by RAW264.7 cells in the 4th generation (4.94±1.02)was smaller than that in generation of 8th,12th,16th, but larger than that in the 24th generation(2.07±0.70).8.Effects of different concentration of FTY-720P on the positive rate of osteoclast induced by RAW264.7 cells(F=250.638, P<0.001).The positive rate of 0(9.37±0.54),500(9.02±0.49),600ng/ml(8.89±0.60) FTY-720P is higher than that rate in concentration of 700ng/ml (7.72±0.41)groups.Positive rate of 700ng/ml (7.72±0.41)FTY-720P is higher than in the concentration of 800(3.30±0.48), 900ng/ml(2.80±0.52)FTY-720P groups, and the positive rate in the 1000(1.22±0.39), 1500(0.47±0.16),2000(0.42±0.19),2500ng/ml(0.56±0.22) FTY-720P groups is the lowest(P=0.377).9.Osteoclast induced by BMMs by using M-CSF and RANKL.After M-CSF and RANKL was added into the cell, those cells which is not from BMMs cell line suffered from death.After 7 days after M-CSF and RANKL was added into the cell, and many huge cells with 3 or more nuclears could be found.when they were stained by the TRAP enzyme staining and F-actin staining of osteoclast induced by BMMs, the osteoclast is stained into red,with huge size and many nuclear,also with pseudopodia could be found.10.Effects of the FTY-720P on the number of lacuna absorption formed by osteoclast(F=68.173, P<0.001). Number of absorption lacuna in the 0 (147.58±13.27),500(136.21±12.71) and 600ng/ml(129.93±10.68) group is higher than that of 700ng/ml(96.47±13.11),and number of absorption lacuna in the 700ng/ml (96.47±13.11)group is higher than that in 800 (47.32±14.59)and 900ng/ml (31.13±9.08) group, no statistic differences can be found in the 800(47.32±14.59),900(31.13±9.08),1000(19.79±8.41)and 1500ng/ml (19.03±8.95) group(P=0.180).11.The protein chip result of the effects of FTY-720P on the expression of proteins of osteoclast induced by BMMs cells.Effects of FTY-720P on the proteins expression of TGFs of osteoclast induced by BMMs cells,which suggest that MMP-2,VEGF-C and GFR alpha-1 express higher in the experimental group;Activin A and other 8 kinds of proteins express higher in the control group; Effects of FTY-720P on the proteins expression of TGFs of osteoclast induced by BMMs cells,which suggest that MMP-2,VEGF-C and GFR alpha-1 express higher in the experimental group;Activin A and other 8 kinds of proteins express higher in the control group;Effects of FTY-720P on the proteins expression of Fas,Toll,insulin like proteins and chemokines of osteoclast induced by BMMs cells, which suggest that in the Fas protein familly, Fas ligand and Fas express higher in the control group; in the Toll like protein familly, TLR4 express higher in the control group; in the insulin like protein familly, bFGF and insulin express higher in experimental group, while in control group, IDE and Growth Hormone R express higher; in the chemokines familly, MIP-2 express higher in the experimental group, while MIF and other 5 proteins express higher in the control group.Conclusion1.By the results of imaging examination,Lane-Sodihu score, bone density examination and HE staining, it suggests that allogeneic bone combined with FTY-720P can have the same treatment effect with autogenous bone treatment group.2.By the results of fluorescence quantitative PCR experiments, it shows that BMP-2, VEGF and Spp-1 expression maybe related with the treatment mechanism of FTY720P combined with allogeneic bone graft for the treatment of bone defect, therefore further cell experiments are needed to clarify the specific mechanism of FTY720P.3.By counting the TRAP enzyme staining positive rate, the best RAW264.7 cell generations and the best concentration of RANKL was clarified in the osteoclast induced by RAW264.7 cells, and results suggest that FTY-720P can effectively inhibit the RAW264.7 inducing into osteoclasts.4.By stainning of osteoclast induced by BMMs with methods of TRAP enzyme and F-actin staining, the results suggest that FTY-720P has the function of inhibiting BMMs cells inducing into osteoclast.By counting the lacuna after toluidine blue staining,the results suggest that FTY-720P can reduce the phagocytosis function of osteoclast.5.Through the protein chip experiment, the results suggest that FTY-720P may control the formation of osteoclast by high expression of IL-4,and reduce the express of IL-1,3, and the mechanism can not be proved in this experiment and need further osteoblast and further protein test to clarify the mechanisms. |
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