| Background and objectives:Artificial bone graft was often performed in clinical treatment of repair of segmental bone defects,extremities joint reconstruction and spinal fusion and so on.Application of bone repair materials such as autologous bone and allograft bone due to inherent defects was restricted in the clinical practice greatly.Artificial bone has received tremendous attention in the field of bone repair materials in recent years because of mass-production and easy to transport and storage.At present,several types of artificial bone were applied in clinical,mainly in calcium phosphate bone cement,hydroxyapatite and biodegradable organic material that for non-structural bone graft.But repair effect of these artificial bones was limited or unsatisfied,which was far worse than autologous bone and allograft bone since lacking of osteogenic induction activities.Growth factors can promote proliferation and differentiation of mesenchymal stem cells,resulting in effectively stimulating formation of new bone.Therefore,it was expected to add effective osteogenic induction activities by applying artificial bone scaffold composite of growth factors.But because of short half-life,growth factor would be degraded or diluted in direct use,which would cause failed in forming effective and lasting concentration in local bone repair.Large dosage of drug may lead to not only the high economic costs,but also some complications as the distribution in other parts of body.Therefore,it has become a key problem need to be resolved in this field that how to construct an effective growth factor sustained release system.The rapid development of PLGA nanoparticle in pharmaceutics has brought inspiration for orthopedist.PLGA was a kind of synthetic polymer,which can be used as carrier of several drugs to realize sustained release in the body.If PLGA was used as carrier to load growth factors,which was hopeful for realizing slow release of growth factors in the body for maintaining sufficient concentrations in local bone repair to fully promote osteogenic induction function.The sustained-release system composite of main artificial bone scaffold was a effective way to prepare composite artificial bone with higher osteogenic activity to meet needs of ideal bone repair materials in clinic.Our group intends to choose a type of PLGA as main material to establish nanoparticles sustained release system.By optimizing the preparation conditions,we make it envelope protein more efficiently,protect protein biological activity effectively,and stable release of these substances for more than a month.Which can lay the foundation for preparation of highly active artificial bone material.Methods:(1)By reviewing relevant literature and consideration of actual demand of sustained-release system and nature of growth factors,PLGA with low molecular weight and 50:50 polymer ratio was finally choosen as the main material for preparation of nanoparticles.Multiple emulsion solvent evaporation method(W/O/W)was used for preparation of nanoparticles,with bovine serum albumin(BSA)as the model protein,to explore effect of PLGA concentration,quality ratio of BSA vs.PLGA and volume ratio of internal waters vs.oil phase,biphasic solvent type of internal waters on the preparation of PLGA nanoparticles,and to find the optimal preparation conditions by measuring entrapment efficiency of protein and drug-loading rate.(2)By optimizing conditions for preparing nanoparticles,sustained release kinetics of BSA,rh BMP-2,rh FGF-2 was studied respectively to evaluate sustained release effect of protein.(3)Biocompatibility of PLGA nanoparticles was confirmed by being cultivated with ectomesenchymal stem cells and CCK-8 and live&dead staining method.(4)Biological activities of rh FGF-2/PLGA nanoparticle was verified by cell proliferation experiments and biological activities of rh BMP-2/PLGA nanoparticles was verified by ALP staining,ALP activity detection and osteocalcin content,markers expression of osteogenesis.Results:(1)PLGA nanoparticles was successfully prepared by optimizing preparation conditions.Results showed the highest entrapment efficiency of BSA can be obtained when the concentration of PLGA reached to 200 mg/m L,quality ratio of BSA vs.PLGA was equal to 1:40,volume ratio of water vs.oil was equal to 1:10 and deionized water was used during the internal phase.(2)The prepared nanoparticles were spherical,size at about 300 nm,entrapment efficiency to the BSA,rh BMP-2,rh FGF-2 was(70.0±1.3)%,(68.2±1.7)%,(66.8±2.9)%,respectively.(3)Nanoparticles was contribute to obtain sustained and slow release of protein substances,but the phenomenon of sudden release was not obvious when lasted for more than a month.(4)Biocompatibility of nanoparticles was benign that cell survival was good after co-cultivated with ectomesenchymal stem cell for 3 days.(5)Cell proliferation can be promoted by rh FGF-2/PLGA nanoparticles obviously,which was stronger than that promoted by rh FGF-2 after cultivated for 7 days(p<0.05).Results of ALP staining,ALP activity detection,osteocalcin content,expression of osteogenesis markers confirmed that rh BMP-2/PLGA nanoparticles was contributed to bone differentiation of C2C12 cells,which was significantly stronger than that of the rh BMP-2 solution group(p<0.05).Conclusion: PLGA nanoparticles prepared by optimal conditions are spherical with high entrapment efficiency to the protein,which can realize sustained release more than one month with good biocompatibility.PLGA nanoparticle loaded with rh BMP-2 and rh FGF-2 both have good biological activities,and exhibit more effective than directly using the same dose of corresponding growth factor solution.PLGA nanoparticle is suitable for using as a sustained-release carrier for growth factors,which provides a new way for adding high osteogenesis induction activity for artificial bone. |
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