| With the improvement of living standards,the activities of people to participate in physical training and competitions have increased significantly,and the resulting sports injuries have increased year by year.Tendon injury is a common kind of sports injuries,and the tendon injuries of shoulder rotator cuff is the challenges in clinical treatment.According to the patient’s postoperative follow-up and imaging examination,we found that the reconstruction of rotator cuff is difficult to achieve enough mechanical strength,so quite a number of patients met rotator cuff repair failure and repture again.The reasons of rotator cuff injuries repair difficult are various,one of the most important reason is that it is difficult to achieve ideal tendon bone healing.Tendon bone healing is the key to successful repair of tendon injury.The physical tendon bone interface is classified into two types:indirect tendon bone interface and direct tendon bone interface.The indirect tendon bone interface is fixed on the periosteum through thick ligaments and is characterized by interpenetrating collagen fibers,which is usually aligned with the longitudinal axis of the bone,known as’sharpey fibers’.The direct tendon bone interface is directly through a gradual transformation area gradually deep anchor tendons in the bone,which can be divided into four layers,including ligament tissue,fibrous cartilage,calcified cartilage and bone tissue.Forming a direct tendon bone interface requires a long time after repair or reconstruction of tendon injury,which can take several months to several years.Due to the long time,this process is often difficult to complete.At present,most clinical surgeons use autologous or allograft tendon reconstruction to repair tendon injury.In order to achieve the ideal tendon bone healing,it is necessary to undergo necrosis stage,proliferative stage and ligaminization stage.The necrosis stage is also known as the inflammatory phase.In this process,the tendon grafts will undergo necrosis,and the mechanical strength of it will be significantly reduced,usually within 4 weeks after operation.The proliferative stage includes revascularization and revascularization of tendon grafts.During this period,the amount and activity of the cells in the graft determine the content of the extracellular matrix and the degree of vascularization,usually between 4 and 12 weeks after operation.The ligamentous stage refers to the reconstruction of the ideal tendon bone interface after operation,which can take several months to several years depending on the specific conditions.In the stages above,the proliferative stage is the key of the tendon bone healing.Tendon grafts could restore in time and fully vascularization in this period largely determines the latter ligamentous stage can achieve ideal tendon bone healing.However,the spatial structures of autologous or allogeneic tendons we used in hospital are dense,and the pore of them is narrow,which is difficult to meet the needs of early cells rapid growth.Due to insufficient cells,it is very difficult for tendon grafts to effectively proliferate and revascularization in time after necrosis stage,resulting in delayed repair and even failure of tendon bone healing.In recent years,the application of tissue engineering technique to repair tendon bone healing has become a hotspot.Because of the human tendon ligaments need to be subjected to a strong load during the joint movement,to meet the requirement of early rehabilitation exercise,the the scaffold materials require mechanical strength.Silk is a kind of biocompatible,strong mechanical properties and slow degradation of fibrous protein,which is applied deeply in the tendons,ligament tissue engineering.Altman and Sahoo studied the use of silk scaffolds for tendon reconstruction,found that their mechanical strength was reliable,and could obtain a good pre-intermediate effect.But they also found that there was almost no pore in the internal silk scaffold,which prevented the growth of the new tissue.The weaving of silk to a certain extent can effectively increase the internal porosity,but the inner pore will be greatly reduced when the silk is carrying a large tension.Recently,some scholars had combined the woven silk with collagen sponge to construct a more practical scaffold.Collagen sponge can provide the necessary space for the adhesion and growth of surrounding tissue cells,and the woven silk fibers can provide sufficient mechanical strength.This type of scaffold showed good effect in repair of some tendon and ligament injury,improved the structure and function of the repair tissue.However,due to the special layered structure of the tendon bone interface,we hypothesis that the spatial topological structure of collagen-silk scaffold has great influence on the repair effect of tendon bone healing.At present,there are no similar reports.In this study,collagen scaffolds and collagen-silk scaffolds of aligned and random were produced,and the effect of their repair on tendon bone healing was studied.It was used to provide theoretical basis for the application of this type of scaffold.Our research was divided into three main parts:(1)The fabrication and detection of scaffolds;the culture,identification and differentiation of bone marrow mesenchymal stem cells in New Zealand rabbits(RBMSCs);(2)Compare the effect of aligned and random collagen scaffolds on RBMSCs cell proliferation,morphology,migration and the expression of tendons and osteogenesis;(3)Compare the effect of aligned and random collagen-silk scaffolds on the tendon bone healing of the rotator cuff.Part 1 The fabrication and detection of scaffolds;the culture,identification and differentiation of bone marrow mesenchymal stem cells in New Zealand rabbits(RBMSCs)Objective:Production of collagen scaffold,collagen-silk scaffold,detection of its basic characteristics.Extraction and cultivation of RBMSCs,identification RBMSCs by flow cytometry and three lines differentiation.Methods:The hamstring tendon was peeled off in aseptic condition,and then the protease was used.The pure collagen was obtained by centrifugation,filtration,salting,centrifugation,acetic acid dissolution and collagen dialysis.Woven silk was purchased from Zhejiang Cathaya International Co.Ltd,placed 1%(w/v)collagen solution in acrylic mold,used liquid nitrogen as a frozen source,we produced aligned and random collagen semi-finished scaffolds by using conventional and one-way freezing technology.After freeze-drying and heat crosslinking,the aligned and random collagen scaffolds were obtained.Before freezing,put woven silk into collagen solution,the aligned and random collagen-silk scaffolds were obtained by the similar steps.The shape scaffolds were observed by general and microscope.Making scaffolds with different collagen content(6mg/ml、10mg/ml、18mg/ml),The scanning electron microscope(SEM)was used to evaluate the pore size in the scaffolds.After implanting the scaffolds under the skin of the rabbit for 2 weeks,the internal pore of the scaffold was assessed by H&E staining.Mechanical detection the maximum tension of aligned,random collagen scaffolds and collagen-silk scaffolds,compared the mechanical properties with the normal rabbit rotator cuff.Taking red bone marrow from 3 months old female New Zealand rabbit femoral marrow cavity.RBMSCs were extracted from the original generation,and then cultured and stored.The RBMSCs were identified by flow cytometry.RBMSCs were induced to be osteogenic,chondrogenic,and adipogenic differentiation.Alizarin red staining,alixin blue staining and oil red staining were used respectively,observation of mineralized calcium deposits,cartilage particles and fat particles.Results:The aligned collagen scaffold presented a smooth,bright white appearance,similar to the tendon,the random collagen scaffold was spongy,with relatively rough surfaces,similar to the cancellous bone.The collagen fibers of aligned collagen scaffold were arranged in a parallel arrangement,while the collagen fibers of the random collagen scaffold were arranged in an irregular arrangement.Collagen fibers and woven silk can be packed together tightly.In the same collagen concentration.The pores of the aligned collagen scaffold were slightly smaller than that of the random collagen scaffold.The space topological structure of 10 mg/ml collagen scaffold was stable,with more cells growth than the 6 mg/ml and 18 mg/ml collagen scaffolds.After packed with silk,the mechanical strength of collagen-silk scaffolds mainly depended on the woven silk.There was no obvious difference in mechanical properties of collagen-silk scaffolds with parallel and random.The maximum tensions of the collagen-silk scaffolds were slightly stronger than the mechanical strength of normal rabbit shoulder rotator cuff.Osteoinduction of RBMSCs,the red mineralized calcium deposits were observed in alizarin red staining.Chondrogenic induction of RBMSCs,the blue and green cartilage granules were found in alixin blue staining.Adipogenic induction of RBMSCs,the red fatty particles were seen in oil red staining.Conclusions:Aligned,random collagen scaffolds and collagen-silk scaffolds can be successfully produced.10 mg/ml collagen scaffold had a larger inner pore and the spatial topological structure was stable.The mechanical properties of aligned and random collagen-silk scaffolds mainly depended on woven silk.The RBMSCs extracted and cultured from the original generation had the potential of osteogenesis,cartilage and fat.Its stem cell characteristics gradually decreased with the times of generations.Part 2 Compare the effect of aligned and random collagen scaffolds on RBMSCs cell proliferation,morphology,migration and the expression of tendons and osteogenesisObjective:Compared the biocompatibility of aligned and random collagen scaffolds with RBMSCs,observed the effect of aligned and random collagen scaffolds on the morphology and migration of RBMSCs,assessed the influence of aligned and random collagen scaffolds on the tendons and osteoblasts differentiation of RBMSCs.Methods:RBMSCs were planted on aligned and random collagen scaffolds,cultured 1 day,3 days,5 days and 7 days.The growth curve of RBMSCs was detected by CCK-8 detection,and the biocompatibility of RBMSCs with different space topological structures was evaluated.After cultured 3 days of RBMSCs,The effects of aligned and random collagen scaffolds on the morphology of RBMSCs were observed by optical microscope and SEM.Making the aligned and random collagen scaffolds with 1 cm x 1 cm,and planted the pellet cells ball formed by RBMSCs in the center of the scaffolds.After 1 day and 3 days,the migration of RBMSCs on aligned and random collagen scaffolds was observed by SEM.After induction RBMSCs 1 week and 2 weeks,used RT-PCR detected the expression of tendon related genes(Col Ⅰ,Col Ⅲ and TNC)and bone related genes(Col Ⅰ,RUNX-2 and BMP-2).After induction of RBMSCs 2 weeks,used Western blotting detected the expression of tendon related protein(Col Ⅲ and TNC)and bone related protein(RUNX-2 and BMP-2).Results:RBMSCs were found to grow well in both aligned and random collagen scaffolds,there was no significant difference between them.The morphology of RBMSCs in aligned collagen scaffolds was long fusiform,and the long axis of most cells were parallel to the collagen fibers.On the cont:rary,RBMSCs in random collagen scaffolds were irregular and disordered.SEM observed the migration of RBMSCs in the pellets,found that RBMSCs migrated mainly along with collagen fibers in the aligned collagen scaffolds,and radiated in the random collagen scaffolds.After tendon induction,RBMSCs in the aligned collagen scaffold group expressed higher levels of Col Ⅰ,ColⅢ and TNC genes than RBMSCs in the random collagen scaffold group.After osteoblasts induction,RBMSCs in the random collagen scaffold group expressed higher levels of Col Ⅰ、RUNX-2 and BMP-2 genes than RBMSCs in the aligned collagen scaffold group.After tendon induction,RBMSCs in the aligned collagen scaffold group expressed higher levels of Col Ⅲ and TNC protein than RBMSCs in the random collagen scaffold group.After osteoblasts induction,RBMSCs in the random collagen scaffold group expressed higher level of RUNX-2 and BMP-2 protein than RBMSCs in the aligned collagen scaffold group.Conclusions:The biological compatibility of aligned and random collagen scaffolds were both good.The spatial topological structure of collagen scaffolds had an obvious effect on the morphological changes and migration of RBMSCs.RBMSCs tended to be tendons in the aligned collagen scaffolds and tended to be osteogenesis in the random collagen scaffolds.Part 3 Compare the effect of aligned and random collagen-silk scaffolds on the tendon bone healing of the rotator cuffObjective:To evaluate the repair effect of aligned and random collagen-silk scaffolds for the tendon bone healing of the rotator cuff.Methods:Twenty female New Zealand rabbits were randomly divided into aligned collagen-silk scaffold group and random collagen-silk scaffold group.Each group was set 5 rabbits,the rabbits were sacrificed 8 weeks and 12 weeks after operation.The proximal end of the scaffold was connected by the bone marrow canal and the upper muscle of the ganglia,and the distal end of the scaffold was fixed by the silk fibers in the bone marrow canal outlet.After 8 weeks and 12 weeks,collected the shoulder rotator cuff samples,evaluated the repair effect of collagen-silk scaffolds for the tendon bone healing of the rotator cuff by histology H&E,Safrain-O staining,immunohistochemistry,collagen content detection and mechanical detection.Results:H&E,Safrain-O staining and immunohistochemistry indicated that the new tissue of the random collagen-silk scaffold group was significantly denser than that of the aligned collagen-silk scaffold group,and large cartilaginous tissue was seen in the random collagen-silk scaffold group(12 weeks after surgery).The maximum tensions in the random collagen-silk scaffold group was greater than that in the aligned collagen-silk scaffold group.The collagen content in the random collagen-silk scaffold group was more than that in the aligned collagen-silk scaffold group,and the difference became larger with time go on.Conclusions:Compared with the aligned collagen-silk scaffolds,the random collagen-silk scaffolds were more conducive to the adhesion migration of the cells from bone marrow wall,and efficiently induced the adherent cells differentiating into osteoblasts and chondrocytes.Thus,more new tissue was formed at the tendon bone interface between the graft and bone marrow canal,the repair of the tendon bone healing in the random collagen-silk scaffolds group was better. |
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