The Effects And Mechanisms Of Coaxial Electrospun PCL/PVA Nanofibrous Mats Incorporated With Adenosine In The Promotion Of Bone Defect Healing

Objectives:To evaluate the effects of exogenous adenosine（Ade）on osteoblast formation and osteoclast formation in vitro and explore the related molecular mechanisms,then fabricate the poly（ε-caprolactone）（PCL）/polyvinyl alcohol（PVA）coaxial nanofibers with Ade incorporated into the core to reduce the undesirable side effects and enable the sustained release of Ade for treating rat cranial bone defects.Materials and Methods:CCK8 assay was used to examine the cytotoxicity of exogenous Ade,S3I-201（a selective STAT3 antagonist）and ZM241385（a selective A2AR antagonist）with different concentrations.The effects of Ade on the osteogenic ability of rat bone mesenchymal stem cells（BMSCs）and S3I-201 on the Ade-induced osteoblast differentiation of BMSCs were evaluated by Alizarin Red staining,Real-time PCR and Western blot.Immunofluorescent staining and Western blot were taken out to observe the expression of phosphated STAT3 of Ade-treated BMSCs with or without S3I-201.TRAP staining was used to observe the effect of exogenous Ade on the osteoclasts formation of rat bone marrow macrophages（BMMs）after stimulated with M-CSF and RANKL.After the addition of Ade with or without ZM241385 in RANKL-treated RAW264.7 cells,TRAP staining and Von Kossa staining were performed to analyze the RANKL-induced osteoclasts formation and resorptive activity,respectively,and the m RNA expression of osteoclast-related genes（Ctsk,NFATc1,MMP9,and ACP5）and Ade receptor genes（A1R,A2AR,A2BR,and A3R）were measured by Real-time PCR.RNAseq genome-wide analysis was performed to explore the potential signaling involved in RANKL-treated RAW264.7 cells supplemented with or without Ade,the target signaling was then verified by Real-time PCR,Western blot and immunofluorescent staining.PCL/PVA nanofibrous mats loaded with Ade were prepared by coaxial electrospinning technology.Scanning electron microscope（SEM）and confocal laser scanning（core solution and shell solution were pre-treated with calcein and Rhodamine B,respectively）were used to observe the morphology of the composite PCL/PVA and PCL/Ade-PVA samples.The stress–strain responses of the composite mats were analyzed by a tensile tester.The drop shape analysis system was used to measure their dynamic water contact angles（WCAs）.After cultured with the seeding rat BMSCs,the cytocompatibility of the prepared composite mats was determined by fluorescent Live/Dead staining,the cell growing shape on the prepared composite mats was observed under SEM.High-performance liquid chromatography（HPLC）analysis was used to determine the drug releasing effect of the fabricated PCL/PVA nanofibrous membranes loaded with Ade.Lastly,SD rat cranial bone defects were established and implanted with the PCL/PVA and PCL/Ade-PVA nanofibrous membranes.A Power Lab system was used to evaluate the rat systemic blood pressure and heart rate.An automatic biochemical analyzer was used to examine the activity of the hepatic functional biomarkers alanine transaminase（ALT）and aspartate transaminase（AST）enzymes.The bone regeneration within the defects areas was analyzed by Micro-CT,HE,Masson’s trichrome and immunohistochemical staining after 4 and 8 weeks of healing.Results:CCK8 assay demonstrated that the exogenous Ade with different concentrations（15,30,60,or 120μg/ml）had no cytotoxicity,whereas the optimum concentration for the formation of mineralized nodules in the rat BMSCs was 60μg/ml identified by Alizarin Red staining.The Western blot results revealed that phosphated STAT3 was activated by Ade（60μg/ml）in BMSCs,which could be inhibited by the addition of S3I-201（100μM）.S3I-201 could remarkably downregulate the nuclear translocation of phosphated STAT3 observed by immunofluorescent staining,as well as the calcium deposition,induced by Ade in the BMSCs.The Real-time PCR results demonstrated that Ade enhanced the m RNA expression of the osteoblast-specific markers ALP,Col1,Osterix,and Runx2,and this effect could be inhibited by S3I-201.The expression of these proteins showed the same tendency with the gene expression profile.60μg/ml Ade obviously diminished the osteoclastic differentiation of BMMs stimulated by M-CSF and RANKL,resulting in the distinct reduction in the mature osteoclasts revealed by TRAP staining,meanwhile promoting cell proliferation examined by CCK8 assay.TRAP staining also showed that 60μg/ml Ade significantly impaired the RANKL-induced osteoclast formation of RAW264.7 cells.The RAW264.7 cells cultured in RANKL supplemented with 60μg/ml Ade showed an upregulation of A2AR expression and an downregulation of osteoclast-related markers expression,including Ctsk,NFATc1,MMP9,and ACP5 in m RNA level,both of which could be reversed by the addition of ZM241385（1μM）.Consistent with the gene expression profile,the Ade-mediated suppression of the number of RANKL-induced TRAP-positive osteoclasts and the resorptive activity of osteoclasts were attenuated in the presence of ZM241385.The DEG expression heat map and sample cluster analysis showed that the expression of Fra2 was considerably downregulated in the RANKL+Ade group compared with that in the RANKL group.The upregulated m RNA expression of Fra2 in the RANKL group was significantly decreased after treatment with Ade;the decrease in upregulation could be reversed by the addition of ZM241385.Concurrent with the gene expression profile,the expression of Fra2 both in protein level and fluorescence level showed the same tendency.SEM results revealed that the PCL/Ade-PVA(_0.3/0.4)composite nanofibrous with the 0.3 to 0.4 ratio（w/w）of Ade and PVA exhibited the most uniform distribution of fiber diameter.Under the observation of confocal laser scanning microscope,the PVA or Ade-PVA core with a green color was uniformly distributed at the center of the PCL/PVA and PCL/Ade-PVA(_0.3/0.4)nanofibers with red color.With the incorporation of Ade,the stress–strain curves showed that the stress at break of the PCL/Ade-PVA(_0.3/0.4)group significantly increased and reached 7.1±1.33 Mpa compared to PCL/PVA group,whereas there had no significant statistical difference about the WCAs between these two groups.Although the Live/Dead staining results revealed that both the PCL/PVA and PCL/Ade-PVA(_0.3/0.4)nanofibrous mats exhibited a green color after rat BMSCs seeding,cells on the PCL/PVA group were spindle shaped,different from the polygon-shaped cells on the PCL/Ade-PVA(_0.3/0.4)group by SEM analysis.Through HPLC assay,the PCL/Ade-PVA(_0.3/0.4)nanofibrous mats preformed a sustained and controlled release of Ade during a 60-days period in vitro.After implanted in the SD rat critical cranial defects sites,PCL/Ade-PVA(_0.3/0.4)nanofibrous mats showed more robust promotion in bone regeneration and higher Col1 expression level within the defect regions when compared with those of the bare PCL/PVA groups both at 4 weeks and 8 weeks.However,this promotion effect could be suppressed by intraperitoneal injection of S3I-201（10mg/kg）every two days.These results also agreed with those of Alizarin Red staining of BMSCs cultured with the PCL/Ade-PVA(_0.3/0.4)nanofibrous membranes in vitro.In addition,the decreased mean blood pressure and heart rate of rats induced by intravenous injection of Ade could be avoided by application of PCL/Ade-PVA(_0.3/0.4)nanofibrous mats.Conclusions:These findings represent that Ade could promote the osteogenic ability of rat BMSCs by activating the STAT3 signaling pathway,and exert a negative effect on the osteoclastogenesis of rat BMMs meanwhile stimulating their proliferation.Furthermore,Ade could interact with A2AR to suppress the RANKL-induced oseoclastogenesis via downregulation of Fra2 signaling in RAW264.7 cells.Lastly,the PCL/PVA coaxial electrospinning membrane could be a promising drug delivery system to release Ade for the restoration of large bone defects void of side effects,especially the undesirable hypotension and bradycardia,induced by systemic administration of Ade.