Oriented Collagen Fibers Formed Through Counter-rotating Extrusion And Their Application In Tendon Repair

Tendon injury caused by disease,traffic accident or inappropriate physical training is one of the most common clinical problems.Since tendons are featured with low cell density,scarce blood supply and modest metabolic rate,the regeneration of injured tendons,including injured tendon bodies and tendon-bone insertions,remains a formidable challenge.Combination of stem cells and biomimetic scaffolds has been proposed as a potentially effective tissue engineering strategy for tendon repair.From the viewpoint of comprehensively mimicking the composition,topography,and mechanical properties of native extracellular matrix?ECM?,we suppose that the collagen fibers scaffolds with well-aligned orientations and those with graded orientations are ideal biomimetic alternatives for defected tendon bodies and tendon-bone insertions,respectively.Since insoluble collagen fibers?ICFs?are the aggregation of collagen fibers harboring native cross-links and molecular alignment so as to possess a self-assembly degree and mechanical properties more approprixate to native tendon ECMs,we further suppose that ICFs are more suitable materials for tendon scaffolds than soluble ones.Unfortunately,few technologies can be applicable for fabrication of oriented ICFs scaffolds.Counter-rotating extrusion technology?CRE?,which has been proved to possess advantages as adjustable fiber orientation,fast extrusion and good controllability,enables ICFs fabrication.Despite,little attention has been paid to its application in tendon tissue engineering.In this study,CRE technology was employed,for the first time,to fabricate ICFs into collagen fiber scaffolds of various orientations.And the feasibility of the obtianed ICFs-based scaffolds to repair defected tendon bodies and tendon-bone insertions was evaluated through a systematic investigation of their physiochemical propeties,in vitro biological effects and in vivo repair outcomes.The main studies and conclusions are listed as follows:?1?Fabrication of CMs with different orientations by using CREICFs were extracted and purified from bovine corium layers.By using pepsin-soluble collagen?PSC?as a control,the purity and structural integrity of ICFs were detected via kjeldahl determination,high performance liquid chromatography?HPLC?,sodium dodecyl sulfonate-polyacrylamide gel electrophoresis?SDS-PAGE?,Fourier transform infared spectroscopy?FTIR?and circular dichroism?CD?.The results indicate that the extracted ICFs and PSC are both type I collagen with preserved triple helix structures.Their purity and hydroxyproline content meet the trade standard of collagen sponge in China?YY/T 1511–2017?.Besides,the tensile mechanical properties of ICFs together with the cross-link density,thermal stability and enzymatic stability were much better than those for PSC,verifyinng that ICFs are more suitable materials for the fabrication of collagen fiber scaffolds.Further,by using ICFs as the raw materials,three types of collagen membranes?CMs?with various orietations were succesfully fabricated through adjusting the speed of the outer and inner rotating extruder cones,including CMs with aligned orientation?CM_a,0°-15°?,moderate orientation?CM_m,-15°–30°?and random orientation?CM_r,-60°-60°?.The fiber orientations of CMs were further verified by detecting the ratios of longitudal and transversal thermal shrinkage?S_l/S_t?and thermal shrink force?F_l/F_t?.Tensile mechanical results reveal that three types of CMs have good longitudinal tensile strength and present an order of CM_a?18.45±0.91 MPa?>CM_m?16.35±0.75 MPa?>CM_r?13.81±0.39 MPa?.These results suggest that the ICFs-based CMs fabricated by CRE are capable of mimicking the compositions,alignment,and mechanical properties of native ECMs of tendons.?2?The effects of CMs on the morphology and differentiation of rBMSCs.In order to explore the effects of fiber orientation on stem cells,rBMSCs were employed as model cells and the celluar morphologies on various CMs were visualized by SEM and immunofluorescence staining.Furthermore,the in vitro tenogenic,osteogenic and chondrogenic differentiations of rBMSCs in growth medium and induction medium were investigated by quantitative real-time polymerase chain reaction?qRT-PCR?and western blot?WB?.The results reveal that CM_a with aligned orientation significantly promotes the tenogenic differentiation yet inhibits osteogenic and chondrogenic differentiations of rBMSCs by inducing an elongated spindle cell shape,and even possesses the ability to resist osteogenic/chondrogenic induction media to a certain degree.This indicates that CM_a may become a promising scaffold for defected tendon bodies.On the other hand,with the fiber orientation tending to be randomized from CM_a to CM_r,the tenogenic differentiation became weakened yet the osteogenic/chondrogenic differentiation was promoted,suggesting that CMs with graded orientation may be a promising scaffold for tendon-bone repair.?3?The in-situ repair of tendon bodies by CM_a-BMSCsTo evaluate the feasibility of CM_a as scaffolds for defected tendon bodies,CM_a was co-cultured with rBMSCs to construct tissue-engineered tendon bodies?CM_a-BMSCs?.The in vivo suvival of rBMSCs was first detected via nude subcutaneous implantation of CM_a-BMSCs.rBMSCs were found to still survive at 2weeks post-implantation and CM_a could organize the alignment of rBMSCs and the deposited collagen fibers.Thereafter,a rat Achilles tendon defect model was constructed and CM_a-BMSCs were implanted to evaluate their in-situ repair efficiency for tendon bodies by using the autogenous tendon suturing as positive control,the defect without suturing as negative control and CM_r-BMSCs as experimental control.The results from macroscopic scoring,Achilles functional index?AFI?evaluation,magnetic resonance imaging?MRI?,histological analysis,biomechanical testing and detections of tendon-related genes and proteins reveal that CM_a-BMSCs promote the in vivo tenogenic differentiation of rBMSCs as well and result in significantly better repair quality than CM_r-BMSCs and almost comparable quality to the autogenous tendon suturing.This verifies that the ICFs-based CM_a by CRE is a promising scaffold for defected tendon bodies.?4?The in-situ repair of tendon-bone by CM_ar-BMSCs----a preliminary studyCM_a and CM_r were combined to construct a scaffold with graded orientation(CM_ar).The results from tensile mechanical testing,immunofluorescence and ALP stainings show that CM_ar possesses satisfactory mechanical stability and can induce graded cell morphologies and differentiation of rBMSCs.Further,a rat Achilles tendon-calcaneus defect model was constructed and CM_ar-BMSCs were implanted to evaluate the in-situ tendon-bone repair.X-ray and micro-CT visualizations indicate that CM_ar-BMSCs group produced comparable new bone formation to CM_r-BMSCs group at the calcaneus region yet CM_r-BMSCs rather than CM_ar-BMSCs saw obvious calcification at the tendon body region.On the other hand,HE and MT images show that,though both CM_ar-BMSCs group and CM_a-BMSCs group produced highly dense and aligned collagen fibers at the tendon body region,only CM_ar-BMSCs saw the fibrocartilage transition zone at the tendon-bone interface.These results indicate that CM_ar is a promising scaffold for defected tendon-bone regions.In summary,CRE has been successfully employed,for the first time,to fabricate three types of ICFs-based scaffolds;the CM_a with a well-aligned orientation and the CM_ar with a graded orientation have been proved as promising scaffolds for tendon bodies repair and tendon-bone repair,respectively due to their simultaneous biomimetry of the composition,topography and mechanical properties of tendon bodies/tendon-bone.These findings provide a new approach to construct scaffolds for tendon bodies/tendon-bone tissue engineering and may help to advance the clinical application of tendon tissue engineering.