Melatonin-loaded Electrospun Fibrous Membranes For Promoting Rotator Cuff Tendon-to-bone Healing

Objectives:To evaluate the effect of melatonin-PCL electrospun fibrous membranes on the chondrogenic differentiation of hBMSCs in vitro.To explore the repairing effect of melatonin-loaded electrospun fibrous membranes on acute rotator cuff injuries in a rat model.Methods:Melatonin-loaded aligned PCL electrospun fibrous membranes were fabricated by emulsion electrospinning technology.The morphology of PCL and melatonin-PCL electrospun membranes was observed by scanning electron microscopy(SEM).The water contact angle(WCA)of the electrospun membranes was measured by a video-enabled goniometer.The melatonin release behavior of the melatonin-PCL electrospun membranes was calculated by an automatic microplate reader.Cell Counting Kit-8 assay(CCK-8)was used to evaluate the biocompatibility of electrospun membranes.The chondroid pellets were cultured with chondrogenic medium containing the extracts of melatonin-PCL electrospun fibrous membranes or pure PCL electrospun fibrous membranes.The pellets were accessed in terms of histological analysis and immunohistochemistry analysis.The glycosaminoglycan of pellet was measured by glycosaminoglycans assay kit.The genes related to chondrogenesis were measured by Real-time quantitative polymerase chain reaction(RT-qPCR)analysis.A rat acute rotator cuff repair model was established and melatonin-loaded electrospun fibrous membranes were implanted on the surface of tendon-to-bone interface.The repair effects were accessed in terms of histological analysis,immunohistochemistry analysis and biomechanical analysis.Results:Both pure PCL and melatonin-PCL electrospun fibrous membranes presented aligned microfibers.The release of melatonin from aligned melatonin-PCL electrospun membranes was observed for about 20 days.Melatonin-PCL electrospun fibrous membranes have no effect on the proliferation of hBMSCs.Compared with the PCL and control groups,Melatonin-PCL electrospun membranes have a better effect on promoting the growth of pellets,increasing the secretion of collagen type ? and glycosaminoglycans.Meanwhile,melatonin-PCL electrospun fibrous membranes could also upregulate the expression of chondrogenesis-related genes(collagen type ?,aggrecan and sox-9).Compared with the control and PCL groups at 2 and 4 weeks after surgery,the lowest amount of macrophages infiltration was observed in the melatonin-PCL group.Melatonin-PCL electrospun membranes could effectively promote chondroid zone formation in the enthesis at 4 and 8 weeks after surgery according to the safranin O/fast green staining and collagen ? immunohistochemistry staining.The picrosirius red staining result indicated that melatonin-PCL electrospun membranes have a better effect on promoting the repair of supraspinatus tendon.The biomechanical analysis showed that the ultimate load,stress,and stiffness of the supraspinatus tendon-humerus complex in melatonin-PCL group were significantly higher than those in control and PCL groups.Conclusion:The melatonin-PCL electrospun membranes have good biocompatibility and aligned microfibers.Melatonin-PCL electrospun fibrous membranes could enhance the chondrogenic differentiation of hBMSCs.Melatonin-loaded PCL electrospun membranes could promote the reconstruction of tendon-to-bone insertion and promote tendon-to-bone healing in an acute rat rotator cuff repair model.