Study On The Facilitation Of Tendon Stem/Progenitor Cells Tenogenesis By Bioactive Electrohydrodynamic Printed Scaffold

Repair of tendon defects remains a challenging clinical event that can cause numerous functional impairments in patients.3D electrohydrodynamic(EHD)printing technology,as a new micro/nano fabrication process strategy,can controllably manufacture biomimetic micro/nano structures.However,the hydrophobicity and biological inertness of polymers may have adverse effects on the interaction between cells and materials.This study used the self-polymerization reaction of polydopamine(PDA)as a carrier to immobilize basic fibroblast growth factor(b FGF)on the surface of the material through a secondary reaction,forming a novel bioactive composite biomaterial.The proliferation and tenogenic differentiation of TSPCs were studied in vitro by EHD printed polycaprolactone(PCL).By establishing a subcutaneous animal model,we evaluated the possible formation of tendon-like tissues and their immunomodulatory effects.We further investigated the effects of in situ implantation and immune regulation on the SD rat model of Achilles tendon defect.After immobilizing b FGF,the bioactive scaffold significantly promotes proliferation and tendon differentiation.However,PDA alone only has a proliferative effect but no promoting differentiation effect.It is interesting that b FGF immobilized on EHD printed bioactive scaffold has a synergistic promoting effect on the highest expression of tendon related genes and protein markers at 14 days,and that the promotion of tendon differentiation may occur by activating in vitro TGF-β/Smad2/3 signal pathways.Subcutaneous implantation studies had confirmed tendon structures similar to natural tendon and M2 macrophage polarization effects.In addition,the bioactive scaffold exhibited excellent new collagen formation and repair effects on Achilles tendon defects.Our work indicated that relying solely on the topological structural clues on the surface of the material is not sufficient to trigger tendon differentiation,and that appropriate chemical signals are needed.Proper immune regulation can aid in tendon formation.The tendon differentiation of TSPCs on PPF scaffolds may be related to the TGF-β/Smad2/3 signaling pathway and M2 macrophage polarization.This study is divided into 6 parts.Part 1: In vitro isolation and identification of tendon stem/progenitor cells(TSPCs)Objective: To extract TSPCs used in this study from the Achilles tendon of SD rats and identify for confirmation.Methods: Four-week-old SD rats were sacrificed and bilateral Achilles tendons were digested with collagenase and placed in preprepared culture medium for cultivation.Photographs were taken under a common microscope for observation,and gentian violet staining was used to observe proliferation effects and colony formation to evaluate clonality and self-renewal ability.At the same time,surface antigens were identified by flow cytometry,and specific culture medium was given to evaluate the osteogenic,adipogenic,and chondrogenic differentiation of TSPCs to evaluate their multidirectional differentiation potential through corresponding Alizarin Red staining,Oil Red staining,and Alcian Blue staining.Results:1.The morphology of TSPCs cells present variable spindle shape and they possess clonality like ordinary stem cells,leading to the formation of colonies through cell proliferation.2.The flow cytometry results showed that CD44 and CD90 were positive like those in BMSCs,but CD34 and CD45 in hematopoietic stem cells were not expressed.3.Alizarin red staining shows its potential for osteogenic differentiation,while oil red staining shows oil droplets and other evidence of its potential for adipogenic differentiation.After 3D cell culture,Alcian blue staining revealed that it can form a chondrocyte like morphology and turn blue.After 2D cell culture,Alcian blue staining can also be seen to turn blue.Conclusion: The tendon stem/progenitor cells(TSPCs)extracted from the Achilles tendon of SD rats exhibit significant clonality and self-renewal ability,with the potential to differentiate into multiple directions of osteogenesis,adipogenesis,and chondrogenesis.Part 2 Fabrication and characterization of 3D printed electrohydrodynamic(EHD) scaffolds with biological activityObjective: To construct a 3D printed EHD scaffold with biological activity and provide further treatment and modification,and to evaluate the surface characteristics and changes in some physicochemical properties of the modified scaffold.Methods: Dopamine was dissolved in Tris HCI buffer with a p H of 8.5 to prepare a working solution of 2mg/ml dopamine(DA).The printed PCL scaffold was placed in the working solution and placed overnight at 37℃ in the presence of oxygen,fabricating a PDA coated PP scaffold.Then,a 500ng/ml b FGF solution was added to the PP scaffold and placed overnight at 37℃ to finally produce a scaffold PPF containing b FGF.Scanning electron microscopy,atomic force microscopy,X-ray photoelectron spectroscopy was performed to evaluate the surface structure,roughness and surface composition of scaffolds,and the in vivo simulated release,mechanical and hydrophilic testing were performed.Results: After PDA and b FGF treatment,the appearance changed,surface particles(mainly PDA and substances synthesized with b FGF)deposited,roughness changed significantly,hydrophilicity increased significantly,and the surface mechanics of scaffold fibers decreased significantly,but macroscopic mechanics did not change significantly.X-ray photoelectron spectroscopy showed that the nitrogen content significantly increased after b FGF binding,and the in vivo simulated release curve was flat.Conclusion: b FGF can be bound to the surface of PCL through PDA,and a PCL scaffold containing b FGF has been successfully prepared,which can produce a certain sustained-release effect.At the same time,the material surface has undergone changes,including roughness,hydrophilicity,and micromechanics.Part 3: TSPCs’ growth and differentiation on bioactive scaffoldsObjective: To evaluate the growth and differentiation of cells on PCL scaffolds treated with PDA and b FGF.Methods: After the prepared scaffold was disinfected with alcohol and ultraviolet radiation,TSPCs of P2-P5 were seeded onto the scaffold.Corresponding apoptosis test,viability staining,cytoskeleton staining,and scanning electron microscopy observation and evaluation were performed at a specific time interval.Then,RNA and protein were extracted from the TSPCs on the scaffold after trypsin digestion,and RT q PCR,IF and WB detection were performed.Scanning electron microscopy and transmission electron microscopy were used to evaluate the formation of collagen fibers on the surface of TSPCs in vitro and the aspect ratio of cell nuclei.Results:1.Compared with the PCL group,TSPCs viability and cytoskeletal staining were better in PP and PPF groups,but neither could inhibit cell apoptosis on the scaffold.2.Only PPF group upregulated the expression of related tendon genes and related tendon proteins,and had a higher cell nucleus aspect ratio than the other two groups.3.TSPCs can generate collagen fibers in vitro,but the fibers in the PPF group are denser.Conclusion: Both of PP and PPF group can promote the proliferation and adhesion performance of TSPCs,as well as the secretion of collagen fibers.The PPF scaffold can promote the differentiation of TSPCs towards tenogenesis,secrete more dense collagen fibers,but cannot inhibit apoptosis.Part 4: Mechanism study of tendon differentiation of TSPCs on bioactive scaffoldsObjective: To explore the signaling pathways that TSPCs may be involved in tendon differentiation.Methods: After 2 weeks of cell culture,RNA extraction was performed,followed by transcriptome sequencing analysis.Based on the results of transcriptome sequencing,potential pathway inhibitors were added to the cultured cells.The control group consisted of cells without any scaffolds,and proteins were extracted for WB detection of proteins upstream and downstream of the pathway.The effect of inhibitors on proteins upstream and downstream of the pathway was analyzed and statistically analyzed.Results: TGF-β signal pathway was obtained through sequencing of KEGG gene enrichment analysis.The pathway may be involved in tendon differentiation,while the enrichment of GO gene resulting in Smad protein complex may be involved.After the use of inhibitors,the expression of the upstream proteins p-Smad2/3,TGFβ-1 and TGFβ-2 in the pathway was downregulated,and downstream related tendon proteins such as Tnmd,Scx,and Col1 were also downregulated.Conclusion: The TGF-β/Smad2/3 signaling pathway may be involved in the promotion of tendon differentiation by TSPCs on PPF scaffolds,and blocking this pathway will downregulate the expression of downstream tendon proteins.Part 5: Study on subcutaneous transplantation model of bioactive scaffolds loaded with TSPCsObjective: To evaluate the tendon formation and immune regulatory phenotype of bioactive scaffolds loaded with TSPCs when transplanted subcutaneously.Methods: TSPCs were cultured on scaffolds for 2 weeks and transplanted subcutaneously in nude mice.After 8 weeks,samples were taken and subjected to histopathological examination,including HE staining,Sirius red staining,and Masson staining,as well as transmission electron microscopy to evaluate collagen fibers and immunofluorescence detection of tendon proteins.At the same time,another batch of scaffolds loaded with TSPCs were transplanted subcutaneously in SD rats,and samples were taken 1 week and 2 weeks after surgery.Immunofluorescence staining was performed on markers of macrophage differentiation to evaluate their behavior,and related statistical analysis was performed.Results: At 8 weeks after subcutaneous transplantation in nude mice,compared with the PCL and PP groups,the specimens in the PPF group were significantly heavier.HE staining,Sirius red staining,and Masson staining showed more generation of type I collagen fibers.Immunofluorescence of the specimens showed that the PPF group expressed higher levels of tendon protein,and the diameter of type I collagen fibers was significantly thicker.At 1 and 2 weeks after subcutaneous transplantation in SD rats,the immunofluorescence of the specimens showed that CD86 and i Nos were expressed higher in the PCL group,while CD206 and Arg-1 were expressed higher in the PP and PPF groups.Conclusion: Compared with the PCL and PP groups,PPF scaffolds loaded with TSPCs had stronger tendon forming ability.Dopamine may be beneficial for M2 polarization of macrophages,thereby reducing the inflammatory response produced by grafts in the body.Part 6: In vivo animal study of bioactive scaffolds loaded with TSPCs for repairing SD rats Achilles tendon defectsObjective: To investigate the biological effects of the prepared scaffolds in repairing tendon defects in SD rats.Methods: A model of Achilles tendon defect in SD rat was prepared.TSPCs were seeded on PCL,PP,and PPF scaffold and cultured for 2 weeks.They were then transplanted into the fabricated model of Achilles tendon defect in SD rats and fixed with sutures to prevent graft detachment.The control group had a simple defect without any treatment,and samples were taken at 2,4,and 8 weeks postoperatively.HE staining,Sirius red staining,and Masson staining were performed to evaluate histology.Footprints of SD rats were taken to calculate the Achilles tendon index of each group.We performed immunofluorescence detection of tendon related proteins on the specimens and evaluated the condition of newly formed type I collagen fibers at the repair site using transmission electron microscopy,and evaluated the three-dimensional morphology and surface roughness of newly formed collagen fibers using atomic force microscopy.In addition,the samples from 2 weeks after surgery will be given immunofluorescence staining of macrophage differentiation for immunomodulation markers to evaluate their polarization,and relevant statistical analysis will be conducted.Results:1.Postoperative histology showed that the control group had significantly fewer newly formed type I collagen fibers compared to other scaffold groups,and the collagen fibers were sparser,with a significant inflammatory response.2.Immunofluorescence about tenogenesis showed that compared with other groups,the PPF group had significantly higher expression of tendon protein.3.Transmission electron microscopy showed that compared with other groups,the diameter of newly formed type I collagen fibers in the PPF group was significantly thicker.4.Compared with other groups,PPF group had a higher postoperative Achilles tendon index,approaching normal at 8 weeks.5.At 2 weeks post-surgery,immunofluorescence regarding macrophages showed higher expression of CD86 and i Nos in the PCL group,while CD206 and Arg-1 were expressed more highly in PP and PPF groups.Conclusion: EHD printed scaffolds can be used for the repair and reconstruction of Achilles tendon defects in SD rats.Dopamine treatment improvement is beneficial for reducing inflammatory response,and PPF group scaffold can enhance the repair effect.