| Objective:The repairation or reconstruction of rotator cuff tear is one of the difficult problems in clinical orthopedic.However,reconstructing tendon-bone insertion and promoting tendon-bone healing in rotator cuff tears have become hot topical in both clinical and laboratory research.In recent years,so many studies have reported that PRP can promote the healing of the tendon to bone in rotator cuff tears,which has lots of active factors.However,it has been reported that a single PRP also has some limitations.The stent’s mechanical strength is low,and the active factors in PRP are released too fast,requiring repeated injections.Therefore,achieving the sustained release of PRP active factors is a hot and challenging problem for current research.In our study,Graphene oxide(GO)combined with Platelet rich plasma(PRP)was used to prepare a new type of hydrogel biological scaffold materials and to improve resistance of support to the scaffold.It also enhances the cell adhesion ability,and makes growth factor in the PRP release slowly,which will promote the osteogenesis and chondrogenesis differentiation of stem cells,and also promote the tendon-bone healing after repairation of rotator cuff tear.Moreover,the GO could provide a new tissue engineering treatment strategy for rotator cuff tear and guide the clinical treatment of rotator cuff injury.PRP’s growth factor obtains better-sustained release kinetics and promotes tendon-to-bone healing after rotator cuff tear repair.Materials and methods:(1)We collected the central artery blood of New Zealand rabbits.The PRP was prepared with two centrifugation,and a Platelet count was performed.PRP hydrogel scaffolds were obtained by mixing bovine thrombin.The graphene oxide concentration gradient was set up by 0.25mg/ml,0.5mg/ml and 1.0mg/ml respectively.The graphene oxide was mixed with PRP by physical blending and then mixed with bovine thrombin to obtain graphene oxide composite PRP hydrogel scaffold.The Experimental group was set up for sub-group: PRP hydrogel stent group,0.25 mg GO/PRP hydrogel stent group,0.5mg GO/PRP hydrogel stent group,1.0mg GO/PRP hydrogel scaffold group.We observed the scaffold’s color character and the scaffold’s internal structure by scanning an electron microscope.Moreover,we also tested the scaffold’s in vitro degradation and observed the acute toxicity test of rat’s intraperitoneal injection of scaffold extract.Then,we observed the toxic reaction,inflammatory reaction,and degradation of rat’s subcutaneous embedding scaffold materials and detect the sustained-release growth of hydrogel scaffolds with Factor TGF-β1 in each group by ELISA;(2)Each scaffold group was co-cultured with bone marrow mesenchymal stem cells in vitro to detect cell viability,cytotoxicity and proliferation ability.The best concentration of graphene oxide composite PRP hydrogel scaffold was screened,and osteogenic and chondrogenic related genes.The protein expression was detected after osteogenic and chondrogenic induction.(3)72healthy adult male New Zealand rabbits were selected.Rabbits were randomly divided into three groups: GO/PRP group,PRP group,and blank group,with 24 rabbits in each group;unilateral acute rotator cuff tear model was established in these rabbits.Then we repair rotator cuff by culturing and implantation of scaffold materials and bone marrow mesenchymal stem cells.In 8 weeks and 12 weeks respectively,MRI was performed to evaluate the tendon-bone healing and Micro-CT was assessed to the growth of new bone.Moreover,the bone’s healing strength was evaluated by biomechanical examination and the histological condition of tendon and bone healing was evaluated by histological staining.Results:(1)The preparation of graphene oxide composite PRP hydrogel scaffolds was successfully prepared.The Graphene Oxide/PRP composite hydrogel scaffolds have a three-dimensional porous structure.The degradation rate was slower,the pore structure was more compact,and hydrogel scaffolds’ strength was enhanced.Meanwhile,there was no obvious toxic side effect and good tissue compatibility.It had a certain control ability on growth factors.With the increase of GO concentration,the effect of slow-release was more effective.(2)After the co-culture of scaffolds and bone marrow mesenchymal stem cells,the proportion of the surface-active cells in the0.5mg GO/PRP hydrogel scaffold group was the highest,the cell proliferation ability was more vital,and the biocompatibility was good;PCR detection showed that the0.5mg GO/PRP hydrogel stent group was OCN,RUNX2,Sox-9,COL II.m RNA expression was higher than that of the PRP hydrogel stent group.Immunofluorescence staining showed that COL II and OCN protein expression was more potent in 0.5mg GO/PRP hydrogel stent group than in the PRP hydrogel scaffold group.(3)Using graphene oxide/PRP hydrogel composite scaffold combine BMSCs to repair rabbit rotator cuff tear showed that both collagen fibers’ growth in GO / PRP group and PRP group were better than that in the blank group at 8 and 12 weeks after the operation by the teat of histological staining,Micro-CT,MRI,and biomechanical test.Meanwhile,the Sharpey fiber growth and the number of chondrocytes in the GO/PRP group were more abundant than those in the PRP group,promoting new bone growth.The GO/PRP group had apparent new bone formation at the tendon-bone interface and marginal area.The tendon-bone healing signal-to-noise ratio was lower,which was closer to standard tendon signals.The biomechanical test results showed that the ultimate load of the PRP group and GO/PRP group was significantly higher than that of the blank group at 8 and 12 weeks after the operation(P < 0.05),the stiffness of the GO/PRP group was higher than that of PRP group and blank group(P < 0.05),and the stiffness of GO/PRP group at 12 weeks was higher than that of 8 weeks(P < 0.05).Conclusion:(1)The Graphene Oxide combine PRP hydrogel scaffold composite has a three-dimensional porous structure,which degradate slower and meanwhile enhance strength of the scaffold.It also has many advantages,such as no noticeable side effects,good biocompatibility and tissue compatibility.Moreover,it has a specific controlled release power for growth factors.The higher the concentration,the better the sustained-release effect.(2)The Graphene Oxide combine PRP hydrogel scaffoldcould promote cell proliferation,osteogenesis and chondrogenesis of stem cells and enhance PRP’s biological activity,which could be a new biomaterial scaffold material.(3)The Graphene Oxide combine PRP hydrogel scaffold composite can effectively promote osteogenesis and chondrogenesis in vivo and enhance tendon repairation.This could also enhance the strength and stiffness of the tendon bone transitional zone,improve the biomechanical properties of the tendon bone transitional zone.What,s more,it also promote tendon to bone healing for rotator cuff tear. |
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