Study On Porous Calcium Phosphate Cements Loaded With Trebling Drugs

Calcium phosphate cement (CPC) has been widely used as bone filling material and orthopedic drug carrier due to its self-setting ability, moulding ability, good biocompatibility, biodegradability, osteoconduction and osteoinduction. Moreover, the moderate preparation method of CPC allows its wide applicability for drug or bioactive molecule loading, and the drug or bioactive molecule loaded in CPC can maintain its property or activity.The reparation of bone defects, which often needs to be treated with several drugs to enhance curative effect and to reduce toxic and side effect, is a long-term and complicated process. Besides, CPC has compact structure with high crystallinity and stability when compared with porous natural bone. For the above reason, it is hard for bone tissue and blood vessel to penetrate into CPC interior and it is hard for CPC to degrade in vivo. Consequently, bone regeneration and the whole bone repairation process are retarded. In order to solve this problem, porous CPC with micropores>100μm is prepared to provide space for cell adhesion and growth, furthermore, to accelerate the replacement of CPC by bone tissue.In the study, porous CPC loaded with trebling drugs was prepared. The prepared CPC could achieve analgesia and anti-infection in earlier stage, promote bone growth over a long period of time, and provide micropores>100μm for osteocytes and bone tissue to grow.Multiple emulsion solvent evaporation method was used to prepare salbianic acid B (Sal B) loaded poly d,1-lactic-co-glycolic acid (PLGA) microspheres with the average size over100μm. The prepared PLGA microspheres were spheres with smooth surface and the average size was112.7+5.6μm. The in vitro drug release results showed two stages:one was burst release in earlier stage (before3days) and the other was slow release, thus, the drug loaded PLGA microspheres could achieve the controlled release of drug.Biocement D CPC was chosen as drug carrier. The preparation of porous CPC loaded with trebling drugs was described as follows. Typically, antibiotic gentamicin sulfate (GS), analgesic paracetamol (PC), and the powder of Sal B loaded PLGA microspheres were directly mixed with CPC powder, phosphate buffer solution (PBS) was used as liquid phase and the liquid-solid ratio was set as0.35mL/g. Experiment were divided into three different groups according to the drug loading capacity of CPC, respectively were CPC-5%MS-1%PC-0.5%GS, CPC-10%MS-3%PC-1%GS, and CPC-15%MS-5%PC-1.5%GS, and blank CPC was set as control. Subsequently, the coagulation time, the in vitro drug release behavior, and the porosity and mechanical strength after the degradation of microspheres were evaluated through Gillmore Needle, XRD, UV-vis, SEM, and mechanical property tester, respectively. The three drugs loaded porous CPC was co-cultured with MC3T3-E1osteoblasts in vitro and the activity and morphology of cells were observed through fluorescence microscope. Cell proliferation and cell differentiation were determined by Alamar Blue assay and ALP test, respectively. Besides, the antimicrobial activity of the prepared CPC was evaluated through agar plate diffusion test.Compared with blank CPC, it could be found that the encapsulation of drugs and microspheres after hydration for24h had no effect on the position and shape of the diffraction peak of the prepared drug loaded CPC. The HA transform production of drug loaded CPC was less than blank CPC, and the HA production decreased with the increasing of drug loading capacity and the amount of encapsulated microspheres. Besides, initial setting time and final setting time were both significantly extended at the same time. Drug loaded CPC possessed even lower mechanical strength than blank CPC and the mechanical strength showed decreasing trend as the drug loading capacity and the amount of encapsulated microspheres increased. In vitro drug release also exhibited as burst release and slow release stages and the amount of released drug increased with the increasing of drug loading capacity. Anti-infection drug GS and analgesic PC were mainly released during the first week. The release of Sal B used for bone tissue growth was sustained for75days. After release for90days, the diffraction peak of a-TCP in drug loaded CPC samples was disappeared. This indicated that the main component was HA and the crystallinity decreased with the increasing of drug loading capacity and the amount of encapsulated microspheres. Furthermore, interconnected spherical pores were generated after90days and the total porosity of drug loaded CPC exhibited higher porosity than blank CPC.The results of fluorescence staining revealed that abundant living cells were adhered on the surface of drug loaded CPC. The osteoblasts presented polygon shape and plentiful cell junctions were formed, which indicated good cell activity. The Almar Blue assay and ALP testing results showed that the effect of drug on osteoblasts exhibited dose-and time-dependent. CPC-5%MS-1%PC-0.5%GS sample and CPC-10%MS-3%PC-1%GS sample significantly encouraged cell proliferation while CPC-15%MS-5%PC-1.5%GS sample inhibited cell proliferation due to the excessive concentration of drug. The ALP activity of all the three experimental groups decreased. The evaluation of antimicrobial activity presented that blank CPC and PC didn’t have antimicrobial activity while GS possessed strong antimicrobial activity and Sal B showed to some extent antimicrobial activity. The united release behavior of the three drugs had no effect on the properties of single drug, but enhanced the antimicrobial activity of drug loaded CPC instead.In this study, GS, PC, and Sal B loaded PLGA microspheres were directly mixed with CPC powder to prepare porous CPC loaded with trebling drugs. GS and PC were released through burst release and lasted for1week. Sal B was slowly released along with the degradation of PLGA microspheres and sustained over75days. Thus, the prepared CPC could be used in clinical bone repairation trials in which different drugs were needed at different stages. Furthermore, the porous structure generated from the degradation of microspheres was suitable for osteroblasts and bone tissue to grow.