Biological Characteristics And In-vitro Antibacterial Activity Of Leukocyte Platelet-rich Fibrin With Moxifloxacin

Background and Objective In 2001,leucocyte platelet-rich fibrin(L-PRF)was first proposed as a new biological material by French scientist Choukroun.L-PRF is a fiber protein component and rich in white blood cells and platelets,and which is extracted from whole blood without the addition of thrombin.It contains large amounts of cytokines,which are consistent with the proportion of cytokines in humans and play an important role in the generation of vascular endothelial cells,collagen synthesis and tissue regeneration.It is more important that the three-dimensional structure of L-PRF is formed by large amounts of fiber proteins,and which can provide the structural basis for the slow release of drugs additionally.In this study,L-PRF loaded with moxifloxacin was prepared to observe the release of moxifloxacin,whether the release of cytokines in L-PRF was affected by L-PRF loading with moxifloxacin,and the antibacterial effect of L-PRF loaded with moxifloxacin in vitro.Methods Fasting venous blood was extracted from six healthy volunteers.Blank L-PRF and L-PRF loaded with different concentrations(25μg/mL,50μg/mL,100μg/mL)of moxifloxacin were prepared using each blood sample,respectively.Buffers containing L-PRF exudate from each sample were collected during the 0-1d,2-3d,4-7d,and 8-14 d periods.We conducted the high performance liquid chromatography(HPLC)method to examine the release level of moxifloxacin in each time period,and performed the ELISA kit to test whether the release of cytokines in each period and group was affected by the moxifloxacin,and carried out the bacteriostatic ring experiment to explore the bacteriostasis ability of L-PRF in vitro after loading with moxifloxacin.Results(1)As time goes on,the L-PRF loaded with different doses of moxifloxacin was gradually degraded,and the concentration of moxifloxacin released in each group gradually decreased.The L-PRF loaded with moxifloxacin still contained a certain concentration of antibiotics during the 8-14 d period,while the concentration of moxifloxacin released by the samples collected during 4-7d and 8-14 d period tended to be stable.During the 0-1d period,there was a statistical difference among the groups(P<0.05).The higher the concentration of loaded moxifloxacin was,the higher the concentration released.The concentration of moxifloxacin released by L-PRF loaded with 100 μg/mL of moxifloxacin during the 2-3d and 4-7d periods was statistically different from that of the other two groups(P<0.05),while the concentration of moxifloxacin released by each group during the 8-14 d period was not statistically different.(2)The release of TGF-β1 in L-PRF during the 4-7d period was affected by the selected concentrations of moxifloxacin,and the release of IL-L-PRF during the 8-14 d period was affected by L-PRF loaded with 50 μg/mL and 100 μg/mL of moxifloxacin(P<0.05).However,different concentrations of moxifloxacin did not affect the release of PDGF-BB and IL-4 in L-PRF.(3)L-PRF with different concentrations of moxifloxacin during the 0-1d and 2-3d periods showed inhibitory effects on Staphylococcus aureus,Escherichia coli and Pseudomonas aeruginosa in an in vitro inhibition zone experiment.L-PRF loaded with 100μg/mL moxifloxacin during the 4-7d period showed inhibitory effect on staphylococcus aureus and pseudomonas aeruginosa.No antibacterial effect was observed in each group during 8-14 days.Conclusion L-PRF could successfully load moxifloxacin and continuously release it over a long period of time.The loaded moxifloxacin in L-PRF affected the release of cytokines for individual time,but did not affect the general trend of cytokines’ release.L-PRF loaded with moxifloxacin in vitro can effectively inhibit the common pathogens of wounds and maintain the concentration of moxifloxacin above the minimum inhibitory concentration of the three bacteria for a long time.