| Calcium phosphate cement (CPC) has been used in clinic as bone fillers because of its excellent biocompatibility, mouldability and biodegradability. Additionally, CPC is also an attractive candidate as the local delivery of skeletal drug, because its preparing procedure avoids sintering and heating release, and thus allows incorporation of drug. To date, many researchers have studied incorporation of one drug in CPC.However, CPC containing only one drug is difficult to meet the requirements in case of complicated clinical situations. For example, analgesic and antibiotics may be required in the initial stage of implantation. Subsequently, osteogenic or the remedial drug for bone disease may need to be continuously released for a long time to enhance bone growth and treat the associated bone diseases. In addition, a porous structure should be developed in time to benefit bony growth. In response to these requirements, the sequential delivery of multiple drugs is necessary to be developed.In present study, an antibiotic (gentamicin sulfate, GS) and osteogenic drug (Salbianic acid B, Sal B) were chosen as the model drugs. Firstly, poly DL-lactic-co-glycolic acid (PLGA) microspheres containing Salbianic acid B (Sal B) were prepared. Modification of the calcium phosphate cement (CPC) with PLGA microspheres (MS) and an antibiotic (gentamicin sulfate, GS) to achieve sequential release of drugs was investigated. It aimed to inhibit the bacterial growth in the initial stage and increase cell activities for a long period for the treatment of bone defects.The double emulsion solvent evaporation technique (W1/0/W2) was used to prepare microspheres containing Salbianic acid B (Sal B). Orthogonal experimental method was used to investigate process parameters of the double emulsion solvent evaporation technique. Four different kinds of PLGA microspheres, varied in sizes, were prepared and compared to encapsulate Salbianic acid B (Sal B). Microsphere properties such as surface morphology, particle size and drug release profiles were characterized.Biocement D cements was used as drug carrier and three groups of gentamicin sulfate (GS) and/or PLGA microspheres (MS) loaded CPC composites were prepared:(1). CPC loaded with GS in a weight ratio of0.5wt.%,1wt.%and1.5wt.%;(2). CPC loaded with MS in a weight ratio of5wt.%,10wt.%and15wt.%;(3). CPC loaded with GS and MS in the weight ratio of GS0.5wt.%and MS5wt.%, GS1wt.%and MS10wt.%, GS1.5wt.%and MS15wt.%, respectively. Drug-free CPC powder was also mixed in the mixer as the control group. The influence of the drugs and microspheres on setting time, phase composition of CPC, volumetric porosity, drug release behaviors, compressive strength, were characterized by Gilmore Needle, XRD, SEM, UV-vis and mechanical property tester etc. In addition, Osteoblasts (MC3T3-E1) and Staphylococcus epidermidis were used to evaluate the biological behaviors of the CPC composites loaded with GS and/or MS.In orthogonal experiment, results of statistical analysis showed that the effect degree of all impact factors on entrapment efficiency of microspheres was external phase> internal water> Sodium Chloride> Span80, and significant influences were observed for the concentration of external phase on entrapment efficiency of microspheres.The SEM results and the size distribution results showed that PLGA microspheres had the smooth surface and spherical morphology with monomodal particle size distributions. And the mean particle sizes and the drug loadings of microspheres were increased with the decreasing of polyvinyl acetate (PVA, W2) concentration and rotate speed. In vitro drug release results indicated that there were two remarkable stages, the initial rapid release and the following slow release. In addition, the burst release and release rate was obviously reduced with the increasing of the particle sizes.Gilmore Needle results showed that the setting time was significantly increased with the increasing of the loaded amount of GS and MS. Mechanical results showed that the compressive strength of CPC was significantly increased with the increasing of the loaded amount of MS.The sequential drug release from CPC loaded with MS and GS composites was achieved for all batches varying in loaded amount. Sal B, encapsulated in MS, yielded nearly zero order release profiles for over60d at least. Meanwhile, GS release followed the matrix diffusion Higuchi's law and mainly presented in the initial rapid release during the first week with an obvious burst release. Comparing to the single drug loaded CPC composites groups, the initial burst release of drugs for the CPC composites loaded with GS and Sal B increased, but the followed slow release times for two kinds of drugs were reduced, respectively. After100d immersion in SBF, The XRD results surgested that the reaction products of the CPC and the three groups of CPC composites were mainly poorly crystalline hydroxyapatite (HA). However, the diffraction peaks of starting α-TCP of the CPC-GS composites group were obviously observed and markedly increased with the increasing of the weight fractions of GS. GS might be a stabilizing agent of TCP and could inhabite the hydration reaction of TCP. And the quantified results showed that the crystallinity of all the three groups of CPC composites decreased gradually with the increasing of weight percentage of GS and/or MS. The volumetric porosity results indicated that the three groups of CPC composites loaded with GS and/or MS had a higher total porosity than that of the CPC, and increased with the increasing of the loaded drug amount, in addition, the total porosity and macro-porosity increased in the order of CPC-GS, CPC-MS and CPC-GS-MS composites group.Alamar Blue assay and ALP assay showed that the effect of the three groups of CPC composites on cell activity of Mouse osteoblasts (MC3T3-E1) varied in the time and dosage dependent manner. The total metabolic activity and ALP activity of cells was promoted by the release of Sal B, whereas, the ALP activity of cells was obviously inhibited by the addition of GS. However, the effect of Sal B was not affected by the combined release of GS from the CPC-GS/MS group, particularly for the proliferation of the cells.Cell attachment to the specimens was even and uniform in all cases. The clear-cut live cells, stained green, have adhered and spread a polygonal morphology with strong stereoscopic feelings on the CPC and the three groups of CPC composites. Also many cell-cell junctions were formed on all of the samples.The antimicrobial results showed that marked antimicrobial activity was exhibited by the diffusion of GS, Sal B exhibited in some degree as well. In addition, the antimicrobial activities were increased with the increasing of the loaded drug amount in all cases. And the antimicrobial activity of these two drugs was enlarged through combination.Sequential release of drugs was achieved by mixing CPC with MS and GS, GS release followed the matrix diffusion Higuchi's law and mainly presented in the initial rapid release during the first week. Meanwhile, Sal B, encapsulated in MS, yielded nearly zero order release profiles for over60days at least. The total metabolic activity and ALP activity of cells was promoted by the release of Sal B, and the ALP activity of cells was obviously inhibited by the addition of GS. In addition, the antimicrobial activity of these two drugs was enlarged through combination. |
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