Oxygen-releasing Hydrogel Microspheres Loaded With Probiotics For The Synergistic Treatment Of Periodontitis

Objective Sodium alginate(AG)hydrogel microspheres(ACL)loaded with calcium peroxide(CPO)and Lactobacillus rhamnosus(L.rha)were prepared.The physicochemical properties of the hydrogel microspheres were characterized,and the cytocompatibility and hemocompatibility of the hydrogel microspheres were evaluated.The inhibitory ability of the hydrogel microspheres against Fusobacterium nucleatum(F.nucleatum)was investigated,with the elucidation of its antibacterial mechanism.The effect of hydrogel microspheres on osteoblast differentiation and experimental periodontitis in rats was assessed.Methods In this study,the aqueous solution AG and CPO with L.rha bacterial solution were mixed evenly to occur ACL hydrogel microspheres with uniform size through the high voltage electrostatic method.The morphology and composition of ACL hydrogel microspheres were analyzed by optical microscope,scanning electron microscope(SEM),and Fourier Transform Infrared Spectroscopy(FT-IR),and the surface element distribution was observed by Energy Dispersive Spectroscope(EDS).The activity and metabolism of probiotic L.rha inside were analyzed by live bacteria staining,agar plate diffusion method,and p H value measurement.The dissolved oxygen analyzer was used to measure the oxygen release ability of ACL hydrogel microspheres.The biocompatibility of ACL hydrogel microspheres was evaluated by the CCK-8 method,live/dead cell staining,and hemolysis test.The antibacterial activity of ACL hydrogel microspheres against F.nucleatum was detected by the agar plate diffusion method.The destructive effect of ACL hydrogel microspheres to the integrity of F.nucleatum cell membrane was observed by transmission electron microscope(TEM).The antibacterial mechanism was elucidated by detecting the DNA and β-galactosidase leakage,as well as the protein synthesis.Crystal violet staining and live/dead bacteria fluorescence staining were used to further investigate the inhibitory effect of ACL hydrogel microspheres against the formation of F.nucleatum biofilm.Alkaline phosphatase(ALP)assay kit,ALP staining,and Alizarin red staining(ARS)were used to detect the ability of ACL hydrogel microspheres to promote osteoblast differentiation.A rat model of periodontitis was established,on which the ability of ACL hydrogel microspheres to treat experimental periodontitis in rats was evaluated by micro-CT,gingival bleeding index(GBI),Hematoxylin & eosin(H&E)staining,Masson staining,Tartrate-resistant acid phosphatase(TRAP)staining,and immunohistochemical(IHC)staining.Statistical analysis was performed using Graph Pad PRISM 8.0,and all data were analyzed using One-Way ANOVA and t-test to determine differences between groups.Results Under the optical microscope,the synthesized hydrogel microspheres with uniform size were observed.SEM images showed that the lyophilized hydrogel microspheres possessed a pleated porous morphology,with CPO particles observed on the surface of ACL.In addition,as revealed in EDS,a large amount of Ca elements located on the surface of ACL.The FT-IR spectrum of ACL showed the characteristic peaks of CPO and L.rha,indicating that CPO and L.rha were successfully loaded.Probiotic bioactivity analysis revealed that probiotics were evenly distributed in ACL without any debility.Along with the proliferation of probiotics,the p H value decreased continuously.As tested,the capacity of releasing oxygen can be promoted in the area of periodontitis,mainly impacted by the body temperature and p H.Moreover,the CCK-8and live/dead cell staining experiments demonstrated that ACL hydrogel microspheres had good cytocompatibility.The hemolysis rate of hydrogel microspheres in each group was lower than 2%.As indicated from the agar plate diffusion experiments,ACL had a significant inhibition effect on F.nucleatum(P < 0.05)with the inhibition rate reaching100%.TEM images displayed that the cell membrane of bacteria treated with ACL hydrogel microspheres was significantly damaged,where the cell contents flowed out and the cytoplasm became lighter since the membrane integrity was destroyed.The increase of DNA and β-galactosidase further confirmed the damage of the cell membrane,while the decrease of protein content suggested that the metabolic activities of bacteria were inhibited.The results of crystalline violet staining and live/dead bacteria staining revealed that the ACL hydrogel microspheres could effectively inhibit the formation of F.nucleatum biofilm,as evidenced by the declined biofilm amount,thinner biofilm layers,and the red dead state of biofilm.From the results of ALP activity analysis,ALP staining,and ARS,ACL could effectively promote the differentiation of osteoblasts,which was in line with the experiment of micro-CT demonstrating that ACL hydrogel microspheres promoted alveolar bone regeneration.The H&E staining,Masson staining,TRAP staining,and IHC staining indicated that ACL hydrogel microspheres effectively reduced inflammation levels in periodontal tissues,promoted collagen matrix deposition,inhibited osteoclast activity,and declined inflammatory factor expression,thus achieving the treatment of experimental periodontitis and promoting the repair of periodontal tissues in rats.Conclusion In this study,oxygen-releasing ACL hydrogel microspheres loaded with CPO and L.rha were prepared.ACL hydrogel microspheres could retain the activity of probiotic bacteria and sustainably release oxygen,with good biocompatibility.In vitro experiments revealed that ACL hydrogel microspheres selectively inhibited the growth of F.nucleatum and its biofilm formation,and promoted osteoblast differentiation.Experimental periodontitis in rats demonstrated that ACL hydrogel microspheres effectively reduced the inflammation level of periodontal tissues and promoted the formation of alveolar bone.Therefore,ACL hydrogel microspheres are expected to be used as a promising biomaterial in the synergistic treatment of periodontitis.