Study On A Novel Injectable, Biodegradable And Anti Osteoporotic SIM-PLLA(Simvastatin,SIM,Poly-L-Lactic Acid,PLLA)-calcium Phosphate Cement

BackgroundOsteoporotic fractures often occur in thoracic and lumbar vertebra, hip and distal radius bone. The risk of osteoporotic fracture is related with age, the prevalence of osteoporosis related fractures in China will become more and more serious with the increasing of age in China. Simvastatin not only has lipid-lowering effects, reported in the literature also has the role of anti osteoporosis, but the anti osteoporosis effect is not fully tapped, oral SIM need metabolism by the liver and kidney, long-term use in patients with inconvenient poor compliance, and the arrival of the bone tissue of the drug less, can not reach the lowest effective drug concentration, therefore, the search for a suitable drug carrier, change of orthopedic diseases single administration become a hot issue in the field of domestic and foreign scholars. Calcium phosphate bone cement has been used for the treatment of osteoporotic vertebral compression fractures due to its advantages such as self curing, biodegradable, non heat producing, bone conductivity and good tissue compatibility. However, its slow degradation rate and no anti osteoporosis effect limit its clinical application. Poly L lactic acid manufacturing drug loaded microspheres and CPC mixed, can better control release of drug, SIM target to release therapy can greatly improve the SIM biological utilization, enhance the CPC in osteoporotic fracture efficacy, in osteoporotic spinal compression fractures in the material degradation and bone formation rate matching and anti osteoporosis effect, osteoporotic vertebral compression fractures healed physiologically and broaden the application approach of the CPC material.In this experiment, SIM-PLLA-CPC material of cytological study, a rabbit model of osteoporosis in vivo, in vivo animal experiments of anti osteoporosis analysis for further clinical studies provide basic data support, to lay the foundation for clinical application.ObjectiveBy mixing the SIM-PLLA loaded microspheres with CPC, the best material composite ratio is optimized, To construct a new type of SIM-PLLA-CPC multi function bone cement material. Through the materials science research, the cytology experiment and the animal in vivo experiment and so on to evaluate the multi function bone cement material, provides another choice for the clinical treatment of osteoporosis vertebral compression fracture.Methods1、Preparation of SIM-PLLA microspheres:preparation of microspheres by rapid membrane emulsification;2、Preparation of multifunctional bone cement:Calcium phosphate cement powders containing 10%,5% and 0% mass fraction of PLLA microspheres were prepared respectively. Group was divided into control group (CPC group), SIM-PLLA-CPC low dose group and SIM-PLLA-CPC high dose group. The drug loaded SIM-PLLA microspheres were mixed with CPC solid powder by physical blending method, and the control group was not added with drug loaded microspheres.3、The characterization of SIM-PLLA microspheres was observed by SEM4、Encapsulation efficiency and drug loading rate of SIM-PLLA microspheres: The drug quality of microspheres was calculated by measuring the dissolution of microspheres. Calculation of encapsulation efficiency and drug loading rate.5、Physical and chemical properties of compound bone cement:The injection, solidification time, mechanical strength, X-ray diffraction analysis and in vitro degradation rate of the materials were determined.6、Detection of drug release in vitro of SIM-PLLA microspheres:Weighing microspheres to the dialysis bag into the PBS solution, released at 37 degrees Celsius, observation of drug release.7、Multi functional bone cement cytology test:The tissue culture method was used to culture the osteoblasts in the skull of the newborn SD rats. After inoculation on the surface of the sample, the surface of the sample was cultured for a certain time. Electron microscope was used to observe the adhesion of cells on the surface of the material. The effect of the composite material on the adhesion and proliferation of osteoblasts was detected by CCK8 method. The effect of the material on the differentiation of osteoblasts was detected by ALP kit.8、Construction of osteoporosis model:The establishment of rabbit osteoporosis model according to the method of Zhang Kun, namely castration+dexamethasone treatment.9、Animal grouping and animal experiments in vivo:New Zealand white rabbits were randomly divided into 3 groups, CPC group, SIM-PLLA-CPC low dose group and SIM-PLLA-CPC high dose group. Take the rabbit femoral condyle lateral incision, drill out cylindrical bone defect, bone cement implantation.10、Micro-CT analysis:At 4 and 12 weeks after operation,6 rabbits were randomly executed at two time points. Distal femur was separated and then Micro-CT scan. The volume fraction of the remaining material (RMVF) and the new bone volume fraction (BVF) were obtained by analyzing the software. As well as the measurement of the thickness, the number and the density of the trabecular bone, such as the thickness, the quantity and the density of the trabecular bone.Results1、Electron microscopy showed the microspheres were uniform spherical structure, good dispersion, no adhesion between the microspheres. Uniform particle size, surface more folds.2、Encapsulation efficiency and drug loading rate of SIM-PLLA microspheres: The drug loading rate of SIM-PLLA microspheres was 9.3%, and the encapsulation efficiency was 36.1%.3、Determination of physical and chemical properties of in vitro composite materials:3.1、Detection of injection performance, solidification time and mechanical strength:Injection:before 4min, simple CPC group and containing injection properties of PLLA microspheres group had no significant difference. After 8 min, containing PLLA microspheres group can be injection was better than simple CPC group and injection has improved, no bone cement composite material solid-liquid separation phenomenon, three groups of materials than the difference has statistical significance (P< 0.05) than with microspheres was high and low dose group of no statistical difference(P> 0.05). Setting time:a comparison between CPC group and two PLLA group containing microspheres, microspheres group coagulation time is prolonged, but the difference was not statistically significant (P> 0.05). The mechanical strength of the mechanical strength of each group:the materials in 1D,7d time point, no significant difference between groups (P> 0.05).3.2、 X-ray diffraction analysis (XRD):simple CPC group and SIM-PLLA-CPC group samples were analyzed by means of XRD spectral line and the standard hydroxyapatite spectral lines of the same, two groups of material XRD peak is located near 30 degrees and found no other diffraction peaks.3.3、 Characterization of composite SIM-PLLA bone cement:Electron microscopy results see composite surface is compact, at a magnification of 1000 times, partial visible 70 to 80 m size of aperture, visible on the surface of a large amount of crystal body, visible portion of the microspheres distribution, in 5000 times magnification conditions, visible folds more microspheres embedded inside the material, uniformly distributed in the material, microspheres structure maintain integrity in the view of particle size is about 1-3 m in.4, Detection of drug release, drug release observation time period of 25 days, the microspheres SIM-PLLA in 5 days before the time has obvious burst release phenomenon, the 10 days, the release rate of 51.25+4.35%,10 days after SIM-PLLA microspheres stationary, until day 25 to 69.75+4.79%.5、 CPC and SIM-PLLA-CPC material surface osteoblast cell adhesion test:After 4 hours of culture, adhesion of each group had no difference (P> 0.05), with prolongation of culture time, improve the adhesion between the low dose and high dose SIM-PLLA-CPC groups of cells in the and and simple CPC compared with statistical difference (P< 0.05, high-dose group, the OD value of the SIM-PLLA-CPC than in the low dose group, but the difference of no were statistically significant (P> 0.05, cells in material surface after 12h culture, low and high dose SIM-PLLA-CPC group, the adhesion index more high, and simple CPC materials compared with statistical difference (P< 0.05).6、 CPC and SIM-PLLA-CPC material surface into bone cell proliferation assay: at 1,3,5,7 days of CCK-8 method measured the absorbance values, on day 1, the low and high dose SIM-PLLA-CPC group than simple CPC group OD value higher, but only the high dose group and simple CPC group compared with P< 0.05. On day 3 of culture, two groups of SIM-PLLA-CPC were better than CPC high and statistically significant, the OD value increases with the increase SIM-PLLA-CPC microspheres content and that loaded microspheres of cells and has no toxic side effect, good biocompatibility, and exhibits and sim dose related activity.7、 Bone cell differentiation experiment CPC and SIM-PLLA-CPC material on the surface of:simple CPC and the low dose and high dose SIM-PLLA-CPC surface was seeded cells 7 days after detection of ALP activity results, visible with high dose in the SIM group, ALP activity is higher than that of the simple CPC and low dose group, the difference has statistical significance P< 0.05, low dose SIM-PLLA-CPC enzyme activity than pure CPC, but no statistical difference (P> 0.05).8、 Micro CT analysis results:simple CPC group RMVF from 4W 91.6+0.13% to 12W 84.32+2.03, and high dose groups of materials from 4W 84.5+0.73% at 12W 75.6+2.11% that the degradation rate of the material in the high dose group was significantly faster than simple CPC group (P< 0.05), and low dose group material degradation rate is simple in the range between the CPC and the high dose group (P< 0.05) that different doses of loaded microspheres of material degradation rate was dose dependent. The ingrowth of new bone volume fraction (BVF), the material in each group were with the passage of time, trend of increase in the rate of new bone formation and the new bone formation in the group of high dose SIM-PLLA-CPC speed most quickly and at 12W reached the 17.8+1.49%, far more than simple CPC 9.72+0.75%(P< 0.05). And low dose group of materials in new bone growth speed between pure between the CPC and the high dose group (P< 0.05) and low dose group and high dose group compared with statistical difference (P< 0.05, indicating different doses of the drug loaded microspheres for material to promote bone growth rate was found to be dose related.Micro-CT 3D reconstruction and cross section reconstruction:three groups of materials were found to be 4W,12W time lapse of the materials for further degradation, the new bone gradually grow into the situation. Simple CPC group material 3D reconstruction and cross-section 4W and 12W image has no obvious change, material and 4 weeks in the low dose SIM-PLLA-CPC for12weeks group comparison, the material time degradation obviously with the increase of,4W visible new bone is star shaped growth and 12W composite further degradation, degradation of material out of the space with new bone ingrowth into the interior. High dose group and low dose group of materials, the time-dependent trend more obvious,3D reconstruction of visible material 12W is loose and porous structure, visible in the high dose group of materials and the surrounding bone tissue to meet tight, newly formed bone tissue from the periphery to the deep layer of the material growth, new bone ingrowth into the scope is wider.After material implantation 4W material around the trabecular bone parameters can be seen there was no significant difference between groups (P> 0.05), only the high dose SIM-PLLA-CPC group of trabecular bone volume 1.95+0.18 mm-1, higher than the other two group (P< 0.05); table x2 showed 12 weeks,12W high dose group (BV/TV), TB. N, TB. SP were than simple CPC group have been improved (P< 0.05), the 12th CPC group parameters bv/TV, TB. N also than 4W index declined, but no statistical difference (P>0.05). The high dose group was significantly improved than 4w.Conclusion1、By rapid membrane emulsification method into the preparation of drug loaded microspheres SIM-PLLA, particle size of(1.63+0.54) nm, surface folds, ball type full.2、More closely combined SIM-PLLA microspheres SIM-PLLA-CPC material surface, SIM-PLLA microspheres were uniform distribution within the material, reflect the XRD crystalline material is still mainly for hydroxyapatite and microspheres surface SIM-PLLA of self curing process without significant interference and composite materials in the injection performance, coagulation time, mechanical properties without significant difference.3、In vitro release and sustained-release drug observation time period of 25 days, the microspheres SIM-PLLA in 5 days before the time has obvious burst release phenomenon, the 10 days, the release rate of 51.25+4.35%,10 days after SIM-PLLA microspheres stationary,until day 25 to 69.75+4.79%.4、SIM-PLLA-CPC composite can promote bone cell adhesion, proliferation and differentiation, there are dose related, in vitro also confirmed comparable to composite and pure CPC or PLLA biocompatibility, and has better promote bone metabolism.5、SIM-PLLA-CPC composite material 1 months in vitro pH change stability, compared with the simple CPC group no difference, indicating that PLLA and CPC compound can inhibit PLLA degradation of acid production caused by the pH value is obviously reduced.6、SIM-PLLA-CPC composite material has a good mechanical strength, a higher degradation rate and new bone formation rate of the femoral condyle defect in rabbits with osteoporosis;7、SIM-PLLA-CPC composite has the effect of improving the local bone in the rabbit model of osteoporosis, which can realize the combination of immediate mechanical support and local osteoporosis;In summary, this experiment by SIM-PLLA microspheres and CPC composite construction SIM-PLLA-CPC composite bone cement material has good biological compatibility,injection performance and suitable mechanical strength, have obvious into active bone and anti osteoporosis effect on rabbit osteoporosis model, is expected to become a new biological composite material for the treatment of osteoporotic vertebral compression fractures and irregular bone defect.