Preparation Of A BMP-2 Loaded MPEG-PCL Microspheres And Evaluation On Their Bone Repair Properties

It is have been shown clinically that,bone tissue often fails to repair itself,when large-area bone defects are caused by skeletal abnormalities,traumatic injuries,or tumor resection.Autologous or allogeneic bone grafts are usually methods to treat this disease.However,the clinical application of autologous and allograft bone grafts are restricted by the limited donors,the high morbidity at the donor site and the vulnerability to immune rejection.In order to overcome the defects of traditional therapies,it is particularly urgent to find and develop a repair material that can replace natural bones.It has caught people’s eye’s that the bone tissue engineering combine carrier material with the active factor to be put into the body,which can induce a generation of new bone at the bone defect site.Osteoinductive factors bone morphogenetic protein(BMP-2)can induce osteoblast differentiation and promote bone formation.However,it has several disadvantages as blow:1)short half-life in vivo;2)easily inactivated by dilution or enzymolysis;3)adverse effects caused by frequent administration and large doses.Hence,the use of microspheres to control the release of BMP-2 can effectively solve the problem mentioned above,prolong its action time at the defect site,and avoid the pain of patients caused by frequent administration.In this project,BMP-2/MPEG-PCL sustained-release microspheres encapsulated with BMP-2 were prepared by W1/O/W2 double emulsion method,which using MPEG-PCL as carrier materials.The main research contents include:preparation and evaluation of the BMP-2/MPEG-PCL microspheres;drug release and preliminary stability studies of the BMP-2/MPEG-PCL microspheres in vitro;biocompatibility and induction of ectopic osteogenesis in vivo of the BMP-2/MPEG-PCL microspheres.The main research methods and results are as follows:1.Preparation and Evaluation of the BMP-2/MPEG-PCL MicrospheresBMP-2/MPEG-PCL microspheres were prepared by the W1/O/W2 double emulsification method.The encapsulation efficiency was used as the evaluation index.The best prescription and preparation process were screened by single factor and orthogonal test.The results showed that the encapsulation efficiency of the microspheres was the highest(80.96±1.01)%when the final formulation and process were as follows:MPEG-PCL’s concentration is 100 mg/mL,PVA’s concentration is 2%,Wi:O(V/V)=1:5,O:W2(V/V)=1:12,and the shear rate is 10000 r/min..Then,the appearance and physicochemical properties of the BMP-2/MPEG-PCL microspheres were evaluated,and the results showed that the morphology of the BMP-2/MPEG-PCL microspheres was smooth and round with good fluidity.The bulk density was(0.22±0.01)g/mL.The sliding angle(a)is(23.5± 1.1)°,the angle of repose(0)is(36.84±1.84)°,the average particle size is(4.30±0.19)μm,and the Zeta potential is-(23.63±2.31)mv,The drug loading is(0.089±0.001)%,which can meet the requirements of industrial production.2.Drug release and preliminary stability of the BMP-2/MPEG-PCL microspheres in vitroIn-vitro drug release experiments were performed by semi-permeable membrane dialysis method.Phosphate buffer pH 7.4 containing 0.2%sodium azide was used as a release medium and BMP-2 solution was used as a control to determine BMP-2 solutions at different time points.After drug release profile of BMP-2/MPEG-PCL microspheres was plotted,a mathematical model was established to investigate the in vitro drug release profile of BMP-2/MPEG-PCL microspheres.Finally,the initial stability of BMP-2/MPEG-PCL microsphere preparations was studied by high temperature,high humidity,and strong light influencing factors.The results showed that the release of BMP-2 solution group in the release medium was very rapid,and the cumulative release amount was(97.33± 1.97)%for only 7.5 h,which was consistent with the Ritger-pep-pas model;and the BMP-2/MPEG-PCL microspheres was relatively stable and released slowly in the release medium.The cumulative release on the first day was(20.76± 1.43)%,and the burst release was small.The cumulative release on the 45th day was(96.61 ±2.73)%,which is significantly sustained released And meets the Higuchi equation.The results of influencing factors showed that microsphere preparations should be stored in a low temperature,dry,and relatively dark environment to ensure a constant state of their appearance,shape,size,and content.3.Biocompatibility and induction of ectopic osteogenesis in vivo of the BMP-2/MPEG-PCL microspheresFirstly,C2C12 cells were co-cultured with BMP-2,BMP-2/MPEG-PCL microspheres and blank microspheres.Cell proliferation and cytotoxicity test kit CCK-8 was used to detect cell relative proliferation(RGR).The results showed that there was no significant difference in cell relative proliferation between BMP-2 group,blank microsphere group,and BMP-2/MPEG-PCL microsphere group compared with the control group(P>0.05).The microspheres have little cytotoxicity,and accord with national standards.Meanwhile,there was a significant difference between preparation group and the blank group(P<0.05),which showed that BMP-2,BMP-2/MPEG-PCL microspheres and blank microspheres had no obvious side effects on C2C12 cells,and good biocompatibility.BMP-2/MPEG-PCL microspheres and the same dose of BMP-2 solution were then implanted into the murine muscle bags of Kunming mice.Mean bone mineral density measurements and histological examinations,ALP activity measurement,Ca2+ content determination and etc.were performed after 2 weeks,4 weeks and 8 weeks respectively..The results revealed that BMP-2/MPEG-PCL microspheres and BMP-2 alone could induce heterotopic bone tissue successfully,but the new bone size,average bone mineral density,ALP activity,and calcium content in the preparation group were higher than those in BMP-2 solution group.It was proved that the BMP-2/MPEG-PCL microspheres can not only maintain the activity of BMP-2 via the loading effect,but also exhibit more powerful ectopic bone-induction activity due to its sustained-release effect.In addition,the effect is much better than that of BMP-2 solution group.Thus,the BMP-2/MPEG-PCL microspheres achieves the purpose of maintaining the BMP-2 action time and improving the therapeutic effect.In this paper,the MPEG-PCL microspheres loaded with BMP-2 were prepared.Firstly,the BMP-2/MPEG-PCL microspheres prepared by the screening of prescription and process were well-rounded and uniform in particle size.Then,the in vitro release experiments showed significantly sustained-release effect.Cytotoxicity experiment shows that the preparation is non-cytotoxic and has good biosafety;Finally,the ectopic osteogenesis experiments in vivo indicate that the preparation has certain induced osteogenic activity.