Bionic Functional Surgical Suture with Hierarchical Micro–Nano Dimensions for Rotator Cuff Repair: Inducing Process‐Matching Mechanobiological and Biological Responses Adapted to the Regeneration

Surgical sutures are necessary for the rotator cuff (RC) repair to reconnect the torn and degenerating cuff tendon to its footprint. Early‐stage immunomodulation, angiogenesis, and the progressive mechanobiological response encourage complex healing processes involving micro‐nano dimensional reconstruction, including tendon‐bone interface integration and tendon remodeling. However, commercially available sutures with mismatched micrometer‐scale diameters resulted in mechanobiological and biological deficiencies, severely impeding RC regeneration. We developed a bionic functional surgical suture (SS) with helical and hierarchical micro‐nano structures using nano‐ and micro‐Poly (DL‐lactide‐co‐glycolide) (PLGA) yarns (ny), which was subsequently crosslinked in situ with a temporary chemotactic (TC) layer of physiological fibrin networks (TC‐nySS). The TC‐nySS has both mechanobiological and biological advantages: 1) biomimetic helical and hierarchical micro‐nano structures showed progressive degradation behavior, inducing the incremental mechanobiological response of the repaired tissues; 2) outer TC layer of biochemical modification by fibrin networks supplied dual‐functions of angiogenesis and immunomodulation at the early stage, subsequently resulting in timely vascularization and inflammatory regressions due to superior degradation behavior of the constructs. Consequently, TC‐nySS with structural and biochemical designs that elicit process‐matching mechanobiological and biological responses tailored to the RC regeneration successfully achieved the complex healing processes, including superior tendon‐bone interface integration and tendon remodelling.


Introduction
Rotator cuff (RC) repair using surgical sutures is a conventional treatment strategy for tendon-bone integration and tendon remodeling for RC regeneration. [1,2][5] This may be closely associated with the nonphysiological structural design, nonbiodegradability, and suboptimal biological activity of routine surgical sutures. [6]he RC regeneration involves tendonbone interface integration and cuff tendon remodeling.It is well-documented that the tendon is a dense fibrous tissue, with its primary collagen structure showing the typical triple-helix configuration in a micronano hierarchical dimension. [7,8]10][11] The inadequate cell contacts and therapeutic effects caused by the physiologically mismatched fiber morphologies and size of the current commercial surgical sutures for in vivo repair are inevitable. [6]24] The initial strength and mechanical behavior of the conventional surgical suture (SS) are limited, which may fail to provide promising repair performance throughout the healing process.Its long degradation cycle or nondegradability characteristics produce persistent sheltering and occupancy effects in the mid-to-late stage. [3,4]In prior work, we discovered that dynamic mechanical support scaffolds that accommodate regenerative requirements provided promising outcomes in tendon regeneration, enabling effective tendon-bone integration and tendon remodeling. [25]Inspired by this, RC regeneration is likely to be enhanced by creating bionic functional sutures that adapt to the mechanobiological requirements of the healing process.
Notably, the RC tendon and its enthesis physiologically lack vascularization, providing limited blood supply and nutritional support at an early stage after repair. [26,27]Conventional surgical sutures have been reported to inevitably trigger severe inflammatory responses soon after implantation in the early healing environment. [28,29]Therefore, providing appropriate vascularization and building an anti-inflammatory microenvironment at the early healing stage is another promising direction for RC regeneration. [27,30]However, it is interesting that sustained provascularization does not bring the expected superimposed healing-promoting effects for RC regeneration. [31,32]The physio-like tendon-bone integration and tendon remodeling require timely vascularization subside and regression in the mid-to-late healing stage. [33,34]This makes it possible to restore the natural avascularity of the tendon-bone interface and RC tendon, which encourages highly aligned and oriented tendon tissues.Instead, persistent chronic inflammation and continuous vascularization would result in pathological fibrosis and tendon scarring, both harmful to RC regeneration. [35,36]A favorable environment for RC regeneration will be created by designing functional sutures specifically suited to the phased-matching biological healing requirements in this situation. [37]herefore, in this study, we first used Poly(DL-lactide-coglycolide) (PLGA) to synthesize nanoyarns (nys), which were further braided into surgical sutures (nySS) with a triple helical and hierarchical micro-nano structure, mimicking the histological morphologies and micro-nano sizes of the RC tendon and interfascicular matrix (IFM) collagens.For the phased requirements of mechanobiological stimulus for RC regeneration, the progressive degradation properties of the hierarchical nys in the nySS were emphasized to adapt to the different mechanobiological support needs during the healing process.Further, we used the physiological fibrin network to construct a biochemically temporary chemotactic (TC) layer on the exterior of the nySS (TC-nySS), aiming at early cell recruitment and vascularization and regulating the anti-inflammatory macrophage polarization, thus creating a favorable immunological environment at the early healing stage.More importantly, with the progressive degradation of the sutures, the outer TC layer would timely fade away, which prevented excessive vascularization and persistent chronic inflammation, decreasing the formation of pathological fibrosis and scarring in the mid-to-late stage to facilitate tendonbone interface integration and tendon remodeling adapted to the RC regeneration processes.Based on the above design concepts, we evaluated the mechanical and degradation properties of the TC-nySS using in vitro mechanical and degradation tests.Moreover, we verified the bioactivity and propolarization effects of the TC-nySS by in vitro cytocompatibility, proliferation, and macrophage polarization tests.Finally, we observed the in vivo histological evidence in a rat RC repair model using TC-nySS.In this model, we investigated the mechanical and biological effects of the TC-nySS after repair, including the processmatching mechanobiological cues and phase-specific modulation of vascularization and macrophage polarization, facilitating tendon-bone integration and tendon remodeling adapted to the RC regeneration.

Preparation of PLGA Ny by Electrospinning
The synthesis scheme for the biomimetic functional SS is shown in Scheme 1.The ny was produced using conjugate spinning equipment (TFS-700, Beijing Xinrui Baina Technology Co., Ltd., China).The PLGA powder was dissolved in HFIP (Sigma Aldrich) and stirred for 8 h at room temperature to obtain the PLGA spinning solution (12% mass ratio).Based on our pilot study, the 12% PLGA had a suitable spinning fluid viscosity, which could form a complete nanofiber lap between the metal funnel and the core yarn for further braiding of the micro-nano structure yarn.Symmetrical conjugate spinning twisted the PLGA nanofibers around a 5-0 PLGA yarn.To obtain stable and continuous nanocore yarns during the suture preparation, we selected electrospinning parameters according to the spinning process in our pilot study and previous experience.nanofibers were vacuum-dried overnight to eliminate residual solvent before further manipulation.

Braiding of PLGA Nys to Get Ny-Braided Suture (nySS)
Different from the core-sheath structures of the nys used for sutures and scaffolds in some previous studies, [38,39] we further fabricated our suture with hierarchical, triple-helix secondary structures inspired by the native collagen structure (Scheme 1).In this context, three nys were braided on a 3-bobbin braiding machine in the Biomedical Textile Materials Research Laboratory at Donghua University, Shanghai, China.In brief, three nys of equal length were wound on three customized spindles, and then the spindles were installed on the spindle seat of the braiding machine.The three nys were passed through the guide and tension devices, respectively, then set the braiding speed of 12 m h À1 on the winding roller.After that, three yarns were twisted and wound along the axis to obtain nySS.

Temporary Chemotactic Modification of nySS by Fibrinogen
The nySS was modified with fibrin to obtain a temporary chemotactic layer on its surface.Specifically, a fibrinogen (Fg)/ thrombin (D-PBS) composite solution was used to react at 37 °C for 2 h to crosslink in situ and form a fibrin (Fb) TC layer on the surface of nySS (Scheme 1).Such an Fb network modification developed by crosslinking was similar to the physiological fibrin network, which is synthesized to regulate the early healing processes in vivo.Then, the TC-nySS was ultrasonically cleaned to remove the residual fibrinogen and inorganic salt ions and freeze-dried before characterization.

Morphology and Structure Characterization
Scanning electron microscope (SEM, TM-3000 Tabletop Microscope, HITACHI, Japan) was used to observe the morphology of ny and TC-nySS.The sample was fixed on the carbon tape pasted on the aluminum strip, and the gold was sputtered at 20 mA for 60 s.The representative image was used to measure the fiber diameter and orientation by Image J software (version: 1.50b, NIH, USA).The surface structure of TC-nySS was observed by Stereomicroscope (PXS8-T, Shanghai Cewei Photoelectric Technology Co., Ltd., Shanghai, China).TC-nySS diameter and yarn alignment (braiding angle) were also evaluated (n = 5).

Water Contact Angle Detection
The water contact angle (WAC) of nySS and TC-nySS was measured by the contact angle instrument (OCA15EC, Dataphysics, Germany) with a 2 μL liquid droplet.

Mechanical Properties
The mechanical properties of TC-nySS were tested through tensile mechanical testing (Figure S1, Supporting Information).Both ends of a 4 cm long sample were fixed in a metallic grip holder to get a gauge length of 2 cm.A universal testing machine [YG (B) 026 G-500] was used to measure the mechanical properties of nySS and TC-nySS at 20%/s strain rate.

Biodegradation Analysis
An accelerated in vitro degradation method at 37 °C was used to study the biodegradation of TC-nySS.A preweighed TC-nySS was immersed in 10 mL phosphate-buffered saline (PBS, pH = 7.4) and incubated dynamically (60 rpm) at 37 °C for 6 weeks.The scaffold was removed at different times, washed thrice with distilled deionized water, and lyophilized for 8 h.The scaffold was weighed again, and the degree of degradation (%) was calculated from the weight loss percentage compared with its original weight before testing.The surface morphology after degradation was observed under a SEM.Mechanical properties and crystallinity changes were also tested.The pH of the degradation solution (changed weekly) was measured using a pH meter (PHSJ-4 A, Leici, China).

Fibroblast Biocompatibility
The L929 mouse fibroblasts were employed to examine the compatibility of TC-nySS in this study.The sample was cut to 1.4 cm long and put into the 24-well plate after sterilization with ethylene oxide.L929 were seeded onto scaffolds at a density of 5 Â 10 4 cells per well with coverslips as blank controls (BCs).To ensure that the cells were exclusively seeded on the sutures, 200 μL of concentrated cell suspension was uniformly injected into the suture.After 2 h of incubation (37 °C, 5% CO 2 ), the culture medium was added to 1 mL. [40]The culture medium (containing 90% DMEM, 10% fetal bovine serum (FBS; Gibco, USA), and 1% penicillin/streptomycin (P/S; Gibco, USA)] was changed every two days if necessary.After coincubation for 4 h, the sutures were transferred to a cell-free 24-well plate, and PBS was used to wash off the nonincorporated cells.Subsequently, the CCK-8 assays were used to quantitatively evaluate early cell incorporation on the suture at 4 h on the sutures according to the manufacturer's protocol.After coincubation for 1, 3, and 7 days, the cell proliferation with time at the sutures or coverslips was similarly analyzed by CCK-8 assays quantitively. [41]The absorbance was measured at 450 nm using a microplate reader (Multiskan Spectrum Microplate Spectrophotometer).In addition, cell morphology at 7 d was observed by FITC phalloidin and DAPI staining.The cell morphology was observed using a confocal laser scanning microscope (LSM 700, Zeiss, USA).

Macrophage Functional Tests in Vitro
Extracts were prepared by soaking three prepared sutures (Blank, nySS, and TC-nySS) in Dulbecco's modified eagle medium (DMEM) at 37 °C constant temperature cell incubators for 72 h to produce the stock solution, which was centrifugated and sterilized using a 0.22 μm filter membrane (Millipore, USA) for further investigation.After culturing RAW264.7 (RAW) with these solutions for 24 h, the cells were washed thrice in PBS and fixed for 30 min. [41]Subsequently, cells were stained with anti-CD206 (M2 macrophage marker, Solarbio, China) antibody for macrophage polarization analysis according to the standard in vitro immunofluorescent staining procedures.DAPI was used to stain cell nuclei.Cells were washed thrice with PBS and photographed under a fluorescence microscope (Leica, USA).
RAW cells with different treatments were harvested and resuspended in PBS for flow cytometry analysis.Single cells were firstly pretreated with anti-CD16/32 antibody (BioLegend, China) for Fc receptors blockade.Afterward, the cells were surface stained with APC-labeled anti-CD86 antibody (BioLegend, China) for 30 min on the ice, then fixed and permeabilized with Cyto-FastTM Fix/Perm Buffer Set (BioLegend, China).The cells were then stained intracellularly with PE-labeled anti-CD206 antibody (BioLegend, China).A GuavaR easyCyte flow cytometer (Millipore, USA) and CytExpert software performed flow cytometry assays and data analysis.

Hemocompatibility Test
Blood for hemolysis was obtained from healthy New Zealand white rabbits (≈2.50 kg).Anticoagulant whole blood was prepared by mixing the anticoagulant (PBS containing 0.129 M sodium citrate) with blood at a ratio of 1:9.After centrifugation for 5 min at 5000 rpm the supernatant was discarded and cleaned with PBS (PH = 7.20-7.40)and then centrifuged again.After repeating the above operation five times, 1 mL of precipitated red blood cells (RBCs) were taken and evenly mixed with 34 mL of PBS to obtain RBC suspensions.The sutures with a length of 5 cm were placed in a test tube containing 1 mL RBCs and 4 mL PBS, incubated at 37 °C for 2 h, and centrifuged for 3 min (5000 rpm) to obtain the supernatant.The hemolysis percentage (HP) of sutures was calculated according to Equation (1). [42]1 mL RBCs was mixed with 4 mL deionized water as positive controls and 4 mL PBS as negative controls.

RC Repair in an RC Tear Rat Model
The animal experiment ethics committee of Shanghai Jiao Tong University approved animal experiments (Application No.20.1223).After being anesthetized, the Sprague-Dawley rats (n = 36; ≈12-week; 323.4 AE 12.7 g) underwent an acute RC (supraspinatus, SSP) transection through a 2 cm longitudinal incision. [43,44]After randomly assigning rats to groups, RC repair was performed in the RCT model with the TC-nySS and nySS by a modified Mason-Allen stitch technique to minimize the potential stress concentration, using the commonly-used commercial nonabsorbable suture (MERSILK, Ethicon) as the control (Con).
After debriding all the fibrocartilage and soft tissue on the footprint, two bone tunnels were created, and the cuff tendon was reattached to its footprint using the free suture ends shuttled through the tunnels, followed by a routine layer closure.All rats could do cage activities without immobilization after surgery.

Histological and Immunohistochemical Analyses
At 2 and 8 weeks after the repair, the rats were sacrificed to harvest shoulders for histological analysis. [43,44]The cuff tendonbone complexes were fixed in 10% neutral buffered formalin and decalcified using 10% ethylenediaminetetraacetic acid at 37 °C for 3-4 weeks.The SSP tendon-bone complex was subsequently dehydrated and embedded in paraffin, and multiple 5 μm coronal sections were prepared.The sections were then stained with hematoxylin and eosin (H&E), Masson trichrome (MT), and toluidine blue (TB) stainings and captured by a light microscope (DM4000B, Leica, Germany).As previous studies recommended, the early-stage immunomodulatory effects were detected at 2 weeks. [45,46]At this timepoint, accumulative inflammation effects cause more significant damage to the repaired tissues than those at the earlier timepoint, which could better reflect the immunomodulatory effects of the suture to ensure successful repair at the early healing stage.The RC regeneration was analyzed by tendon-bone healing, fibrocartilage formation at the footprint, and tendon remodeling at 8 weeks.According to the previous reports, the tendon-bone histological score system (TBSS, Table S1, Supporting Information) [47,48] for tendon-bone healing analysis and the tendon histological score system (THSS, Table S2, Supporting Information) [47,48] for tendon remodeling analysis were assessed by H&E and MT stainings.The few fibroblasts (tenocytes, specialized to tendons) that have flattened nuclei are usually oriented parallel to the tension axis in a normal tendon.Using H&E stained slides, the morphometric alterations of fibroblast nuclei were assessed to investigate the tendon differentiation and remodeling following injury or healing. [49,50]ibroblast density (number of nuclei mm À2 ) and nuclear orientation angle (between the collagen fiber axis and the major axis of the nuclear angle) were measured.After measuring five regions of interest, the average was ultimately employed. [49,50]urthermore, the area ratio of the new fibrocartilage formation at the footprint in the TB-stained sections was quantitatively determined using the metachromasia ratio using Image J software (National Institutes of Health). [43]The untreated RCs after transection and the normal ones with the sham surgery were also analyzed as histological morphology controls to better assess the RC regeneration after treatments.To further determine the gene expression of the area adjacent to the tendon-bone healing interface, RNA was isolated from the SSP reattachment site and the portion of the humeral head proximal to the growth plate using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) for quantitative polymerase chain reaction (qPCR) analysis according to the manufactural protocol. [43]Interface healing remodeling-related genes at the repaired site, including tenogenic-related genes (Scleraxis, SCX; collagen type I-alpha 1, Col1A1) and mineralized tissue-related genes (Aggrecan; runt-related transcription factor 2, Runx2) were analyzed. [43,51]Table S3, Supporting Information, provides a summary of all the primers for the target genes.After normalizing to the housekeeping gene GAPDH, the relative expression levels of each target gene were determined using the 2 ÀΔΔCt method.In addition, to evaluate the toxicity of the nanoparticles in vivo, samples of heart, liver, lung, and kidney tissue were collected after rats were sacrificed and routinely stained with H&E, with congestion grading for intraorgan hematological analysis as previously recommended. [52]ections were also stained with some antibodies using standard immunofluorescent staining procedures: (anti-CD86 (GB13585, Servicebio, China) and anti-CD206 (GB13438, Servicebio, China) for macrophage polarization analysis; anti-Yes-associated protein (YAP; bs-3605 R, Bioss, China) for mechanobiological response analysis; anti-CD31 (GB113151, Servicebio, China) for angiogenesis analysis; anti-TNMD (bs-7525 R, Bioss, China), anti-COL1 (collagen type I; GB11022, Servicebio, China), and anti-COL3 (collagen type III; GB13023, Servicebio, China) for tenogenic-related markers and tenogenic ECM synthesis analyses).Image J software was used to quantify the number of positive cells or intensity in each field of view.

Micro-Computed Tomography (Micro-CT) Scanning
The 8-week shoulder specimens were scanned using a Skyscan 1176 micro-CT imaging system, with collected data analyzed by NRecon, DataViewer, and CTAn software. [43,44,53]Each sample was scanned using the following conditions: 90 kV, 270 mA, and a 0.018 mm effective pixel size.The region of interest was selected as the trabecular bone within the humeral head near the tendon insertion and proximal to the growth plate.The bone formation on the footprint was qualitatively assessed on CT images, and the bone quality was quantitatively calculated by gray values, with the larger value indicating better bone quality.Moreover, bone morphometric parameters, including bone mineral density (BMD), bone volume to total volume (BV/TV) ratio, trabecular number (Tb.N), and trabecular thickness (Tb.Th), were quantitatively calculated.

Biomechanical Testing
The postsurgery cuff tendon-bone complexes were subjected to biomechanical testing using a uniaxial testing system.The SSP tendon was covered with a polyester cloth and woven using sutures in a whipstitch style to prevent tissue slippage before being clamped by a custom-designed jig.Furthermore, the proximal humerus was rigidly fixed into a polyvinyl chloride tube by plaster. [43,44]The distraction axis was adjusted and applied parallel to the long axis of the cuff tendon-bone complex at a 0°angle to the longitudinal axis of the humerus, in line with the physiological cuff tendon-bone complex insertion direction. [43,44]A 0.1 N preload was first used for five cycles for 5 min, and then a uniaxial tension of 1 mm min À1 was applied for failure load.The test was completed when the reattached tendon broke or failed at the tendon-to-bone repair site.The failure load and stiffness were determined from the load-deformation curves.

Statistical Analysis
All data were presented as the mean AE standard (SD) deviation.Independent samples t-tests or one-way analysis of variance (ANOVA) with Bonferroni post hoc tests were used to compare the results between groups when appropriate (Prism 8; GraphPad Software).Corresponding nonparametric tests were performed according to the normality test (Shapiro-Wilk test).Significance was set at p<0.05.*p < 0.05, **p < 0.01, ***p < 0.001.

Fabrication and Characterization of the Hierarchical Micro-Nano TC-nySS
The RC tendon has been shown to be a dense and hierarchical fibrous tissue. [7,8]The majority of tendons consist of hierarchical helical collagen I structure, tropocollagen molecules (≈1.5 nm), fibrils (≈100-500 nm), fibers (≈15-50 μm), and fascicles (≈300 μm) are typical micro-nano hierarchies. [9,10]This native ingenious triple helix structure in the crimped collagen fiber bundles exerts remarkable triphasic mechanical properties and excellent axial tensile strength. [25]Moreover, The 30% of the tendon's ECM (mainly consists of an interfascicular matrix, IFM) outside of the primary collagen I hierarchy is composed of a minor hierarchical collagen with a similar micro-nanosized structure that provides supplementary resistance to deformation primarily in the fiber direction. [8,9]At the hierarchical fibril and fiber levels, the primary collagen hierarchical framework and adjacent minor collagen ECM structure collaboratively exhibit a characteristic wavy crimp pattern and provide tendon with highly nonlinear and anisotropic mechanical properties by supporting the majority of loading in the fiber direction (axial), exhibiting a characteristic nonlinear "toe" region in which collagen uncrimps and a "linear" region in which collagen is fully uncrimped.However, commercial yarns utilized in routine surgical sutures had larger fiber diameters than collagen fibrils, showing a typical micron range (typically >10 μm). [6]In this context, the inadequate cell contacts and inferior therapeutic effects caused by the mismatched fiber morphologies and size of asfabricated biotextiles are inevitable after surgical repair. [6]It has been widely demonstrated that constructing scaffolds that mimicked the native ECM structure presented superior biocompatibility and potential repair effects, [6] such as the ECMmimicking tendon scaffold. [39]Inspired by the bionic structural design concepts and native collagen architecture of the RC tendon and its ECM, we further fabricated the suture with a triple helix construct using micro-nanosized nys, mimicking the hierarchical conformation of the native collagen fiber of the tendon and its natural micro-nanosized collagen network structure in the tendon ECM (Figure 1a1), aiming to reproduce the specific mechanical properties of tendons.First, we electrospun the coresheath nys using the PLGA microfiber as the core and the nanofiber as the sheath (Figure 1a2 and b), which was determined in the SEM analysis with the diameters of 242.89 AE 0.66 μm, matching the size of the native tendon fascicles (≈300 μm) (red dashed box, Figure 1b).Furthermore, the PLGA nanofibers of the nys sheath were detected to be orderly aligned along the longitudinal axis of nys (Figure 1c), with the mean diameters of 366.00 AE 111.85 nm that matched the size of the collagen fibrils ranging from 100 to 500 nm (Figure 1d).Hence, the nys were determined as micro-nano structures.Subsequently, the nys were braided into sutures with typically triple helix constructs to bionic hierarchical triple helix structures of the native collagen in the tendon and ECM (Figure 1a3), which differed from the conventionally singular micron dimensions of the commercial suture (Con, Figure S2, Supporting Information).Afterward, ).e) The morphology of the triple helix structure of the micro-nanosized TC-nySS.The purple stuffs were PLGA microyarns while the white on the surface were PLGA nys.f ) The braiding angle of the nys (n = 5).The blue box denotes the high magnification of the local region of the TC-nySS sheath surface treated with Fg/thrombin/FIV.g) FTIR spectra for TC-nySS (n = 3).h) N1s narrow scan XPS spectra for TC-nySS (n = 3).i) Crystallinity(n = 3).j) WCA (n = 3).***p < 0.001 between groups after statistical analysis by independent samples t-test according to the normality tests.
the hierarchical sutures were treated with the Fg/thrombin/FIV (coagulation factor IV) composite solution (Figure 1a4), generating the physiological Fb networks on the hierarchical sutures by in situ crosslink at 37 °C (diameter: 0.65 AE 0.03 mm) (Figure 1e).Compared with the clear nanomorphology in the nySS group (Figure 1c, blue dotted box), the nanomorphology of TC-nySS disappeared due to the existence of the Fb crosslinking network (Figure 1f, blue dotted box).The mean braiding angle of the ny in the sutures was 15.18 AE 3.61°along the longitudinal axis (Figure 1f and S2, Supporting Information), comparable to the crimp angle of collagen fiber ranging from 10°to 45°, which is biomimetic to match the mechanical properties of the native tendon for potentially promoting RC regeneration.
One of the essential molecules in hemostasis is Fg.The thrombin-mediated release of fibrinopeptides from Fg converts this soluble protein into a three-dimensional network of Fb fibers, which is required for cellular chemotaxis and physiological hemostasis. [54,55]This three-dimensional network of Fb fibers is where blood clots are created. [54,55]The Fb network is also determined to have immunomodulatory effects, including lowering neutrophil extracellular traps and triggering macrophage reprogramming for M2 phenotypic polarization. [55,56]e further noted that the FTIR spectra displayed the peptide bonds (-CO-NH; 1,641 cm À1 ) and -NH2 (3455 cm À1 ) groups in TC-nySS (Figure 1g), which indicated the successful chemical modification of the crosslinked fibrin networks on the fundamental micro-nano hierarchies structure.In this context, it was demonstrated that the three-dimensional network of Fb was successfully formed on the micro-nanosized nys as a TC modification of the TC-nySS biochemically.The proportions of C, O, and N were determined by XPS analysis, confirming the presence of the Fb proteins on the surface of the TC-nySS (Figure S3, Supporting Information).In addition, it can be seen from the N1s narrow spectra (Figure 1h) that the majority of the nitrogen involved in N─C═O bonds (≈399.79eV), while N─H/ N─H 2 bonds (≈397.60 eV) were lower at 0.62%, which was associated with the formation of crosslinked fibrin networks in the TC-nySS.The XRD analysis (Figure 1i) was used to gain a better understanding of the structure at the molecular level.Two highintensity diffraction peaks could be identified at 21.60°and 28.06°i n the nySS, demonstrating the semicrystalline structures of the PLGA. [25]After being treated with Fg/thrombin/FIV composite solution to form the fibrin temporary chemotactic layer, two small diffraction peaks were observed at 31.70°and 45.47°from TC-nySS, indicating the presence of fibrin structure. [57]In addition, the Fb network in the TC-nySS (41.38°AE 0.61°) significantly increased the hydrophilicity of nySS (86.60°AE 5.19°) (Figure 1j).

Biomimetic Mechanical Performance
Most repair failures involving synthetic sutures are brought on by their subpar mechanical qualities and incompatible mechanical patterns with native tissues.The triphasic mechanical qualities of native tendons, which are brought on by their crimped collagen fibers, account for their superior mechanical performance in large part. [9,10]This suggests that surgical sutures that closely resemble the mechanical characteristics of the native tendon can aid the tendon in adapting to its stress pattern in response to physiological mechanical stimuli and achieve uniform stress transmission.Therefore, using commercially available sutures as controls, we investigated the mechanical performance of TC-nySS and nySS sutures with triple helix micro-nanosized constructions.Interestingly, parallel to the biomimetic structural properties, both TC-nySS and nySS sutures showed triphasic mechanical behavior identical to the native tendon based on the typical load-elongation curves (Figure 2a and S4, Supporting Information).The stress-strain curve similarly demonstrated this mechanical behavior (Figure 2b), attributed to the braided structure of TC nySS and nySS sutures.This mechanical behavior could potentially create a similar load-bearing environment in vivo to the tissue being repaired, minimizing the high localized stress on the tissue or suture that can be associated with repair in traditional sutures, which can lead to a cutting effect on the tissue or suture breakage.In addition, it can be found that the failure load (Figure 2c) and ultimate tensile strength (UTS) (Figure 2d) of TC-nySS and nySS sutures were significantly better than those of the commercial suture fabricated using silks (Con).The higher failure load and UTS could effectively avoid suture breakage during intraoperative manipulation due to the increased stress when pulling and fixation.Moreover, the TC-nySS and nySS had a larger stiffness and smaller elongation (Figure 2e,f ), indicating lower brittleness and higher flexibility, which could possess better penetration through the tissue and increased fit to the soft tissues.These superior mechanical properties of the TC-nySS and nySS mainly resulted from the inherent advantages of PLGA compared to those of the silk regarding some particular mechanical parameters (Figure S5, Supporting Information).We further focused on the effect of TC coating on the mechanical properties of micro-nano sutures.TC modification by the Fb network had a slightly increasing effect on breaking load and UTS, while it had no significant effect on stiffness and breaking elongation.This may be due to the increased frictional adhesion between fibers caused by the Fb sealing network on the surface, which required a greater tensile load for fiber slip, resulting in an increase in the failure load and UTS.In short, the prepared hierarchical micro-nano TC-nySS had superior failure load and UTS for high-stress pulling and fixation, as well as flexible operability, benefiting intraoperative applications as most surgical procedures involve knotting, wrapping, and twisting scenarios when using sutures.More importantly, the observed triphasic mechanical behavior of the TC-nySS that is identical to the native tendon can potentially minimize the high localized stress on the tissue or suture compared to the commercial suture, especially providing relatively even protections of the reconnected cuff tendon at zero-time and early-healing stage after repair.

Easy Application and Superior Coating Stability
The excellent biomimetic mechanical properties are essential factors that guarantee the sutures can be used conveniently in surgery.Therefore, we detected the practical application of TC-nySS in the surgical repair procedures in a rat model of RCT (Figure 2g).The suture was utilized to repair the torn cuff tendon to the bony footprint, passing through the dense tendon and rigid bone structures in this procedure.In the rat model, we exposed the cuff tendon (Figure 2h①) and discovered that the TC modification was still present even after the TC-nySS passed through the thick tendon (yellow triangles, Figure 2h②).Moreover, the detached cuff tendon could be tensioned by the TC-nySS (Figure 2h③) for reattaching the torn tendon stump to the bony footprint after passing the TC-nySS through the bone f ) The elongation.g) Surgical application for RC repair in a rat RC tear model (n = 5).h) Detailed surgical procedures to validate the easy application of TC-nySS.i) The testing apparatus to evaluate the stability of the Fb network nanofiber coating in the TC-nySS.The SEM analysis (100Â) showed the reserved coating nanofiber after penetrating the cuff tendon tissues j) 100 and k) 500 times (n = 6).The cross-sectional observations (150Â) indicated the stability of the core-sheath structure in the TC-nySS (yellow boxes).**p < 0.01, ***p < 0.001 between groups after statistical analysis by ANOVA according to the normality tests.tunnels (Figure 2h④).Smooth intraoperative manipulation indicated the easy application of TC-nySS in surgical repair procedures.During surgery, sutures often need to travel between biological tissues and bear intra-tissue friction due to tissue slippage, which poses a significant challenge to the stability of the coating.To further test the stability of the Fb network nanofiber coating, the TC-nySS was passed through fresh rat cuff tendon tissues 100 and 500 times (Figure 2i).We observed that the coated nanofiber was reserved even though it penetrated the tendon tissues hundreds of times (Figure 2j,k), showing the excellent stability and wear resistance of the Fb network coating on the surface of the TC-nySS.Additionally, we also detected the crosssectional views of the TC-nySS after it penetrated the cuff tendon tissues 100 and 500 times (yellow boxes, Figure 2j,k), which further indicated the stability of the core-sheath structure.

Hierarchical Micro-Nano TC-nySS-Induced Incremental Mechanobiological Response by Progressive Degradation
RC tissues are physiologically stressed in vivo and the repair after tear requires sutures to provide appropriate strengths at different stages to persistently maintain tissue continuity between the cuff tendon and the bony footprint during the healing period. [3,4]In the early healing stage, surgical sutures should provide highstrength mechanical support to ensure effective structural continuity, which is the prerequisite of the following histological RC regeneration. [58,59]Instead, it is necessary to gradually reduce the mechanical sheltering effects of the sutures in the mid-to-late healing stage, which exerts effective mechanobiological stimulus to accelerate tendon-bone integration and tendon remodeling close to the physiological conditions. [3,4]Notably, sustained stress sheltering by sutures at the mid-to-late healing stage might substantially hinder the positive mechanical effects on RC regeneration since the superior tissue healing and ECM remodeling processes are highly sensitive to mechanical stimulus. [25,35,60,61]herefore, the incremental mechanobiological response of the healing tissue induced by progressive suture degradation may facilitate tissue healing and ECM remodeling for improving RC regeneration (Figure 3a).

TC-nySS Displayed Progressive Degradation Behavior In Vitro and In Vivo
In vitro degradation experiment (pH = 7.4; 37 °C), the whole micro-nano constructs of the TC-nySS exhibited no detectable morphological changes within 3 weeks only with few porous surface structures observed (Figure 3b1), showing a minor mass loss rate of less than 5% in the TC-nySS and nySS groups (Figure 3b2).In parallel, the ultimate failure load only decreased by less than 30% in both sutures (Figure 3b3).In general, the degradation analysis revealed that the inner PLGA nanofibers were observed to disintegrate after the outside PLGA nanofibers (Figure 3b).Notably, there was a relatively steeper but smooth degeneration curve of mass loss from 4 to 6 weeks, showing a mass loss rate of 30-40%, indicating the substantially partial degradation with some broken fragments observed at 6 weeks (Figure 3b1).This primary degradation occurred at 4-6 weeks and was further confirmed by the apparent pH change of the degradation solution at 4-6 weeks (Figure 3b4).Correspondingly, the ultimate failure load in TC-nySS or nySS was intuitively reduced to 36% or 23% compared to 0 day (Figure 3b5).The XRD analysis was performed to detect the crystalline structure.It could be observed that the characteristic peak of Fb (31.70°and 45.47°) is no longer visible at 6 weeks compared to 0 days, indicating the degradation of Fb components (Figure 3b6). [57]In addition, its crystallinity decreased from 59.57% at 0 days to 49.12% at 6 weeks, which further explained the relatively considerable decrease in ultimate failure load at 6 weeks. [57]Moreover, the initial degradation behavior of TC-nySS could be bulk degradation at the early stage of 3 weeks, and the water molecules started to invade the amorphous region of the material with the amorphous region degraded.Notably, these detected characteristics of progressive degeneration of the hierarchical micro-nano TC-nySS exerted a gradually decreasing suture strength at the late stage over 3 weeks, potentially resulting in a progressively increasing healing tissue stress in vivo correspondingly (Figure 3b7), which could be the prerequisite of the incremental mechanobiological response within the healing tissue.
In the H&E staining, the green arrows indicated empty areas (no tissue infiltration areas) caused by the occupancy of undegraded sutures (Figure 3c).In general, the surrounding tissues of the nySS and TC-nySS had good cellular infiltration, showing no detectable cell death and heterogeneity at 2 weeks after partial degradation.At 8 weeks, the newly-formed tissues have fully grown into the former suture-occupied positions in the nySS and TC-nySS groups after further degradation, indicating their good long-term biocompatibilities in vivo (Figure 3c).Furthermore, the histological staining of the internal organs showed good in vivo biocompatibility of the nySS and TC-nySS, with no apparent tissue damage, inflammation, toxicological effects in the organs, or abnormal intraorgan hematological accumulations at 8 weeks after implantation (Figure S6, Supporting Information).Additionally, the degeneration properties of the TC-nySS and nySS were further quantitatively confirmed.We found that there were slightly fewer empty regions at the early stage of 2 weeks in the TC-nySS (13.00 AE 3.61%) and nySS (15.67 AE 4.16%) than in the Con (32.67 AE 9.29%) (Figure S7, Supporting Information).Notably, these empty regions remained in the Con group (24.33 AE 3.06%) at 8 weeks, indicating sustained occupancy effects of commercial sutures.Interestingly, the tendon-bone interface and cuff tendon in the TC-nySS and nySS groups rarely had any discernible empty regions, implying complete suture degradation.The substantial continuity between the cuff tendon and tendon-bone interface during the whole healing stage indicated that the progressive degenerative properties of TC-nySS and nySS were appropriate for RC repair since the commonly assumed repair failure due to breakage of biodegradable sutures was avoided.More importantly, when compared to the commercial sutures, the progressive degenerative behavior of the TC-nySS showed more potentials and probabilities to match the characteristic regeneration rate of the complicated tendonbone interface with several distinct continuous tissue regions, which warrants more investigations into a controllable degradation rate of the SS to be more similar to the rate of tendon-bone tissue regeneration.The immunofluorescent staining analyzing the incremental mechanobiological response via the expression of YAP at the cuff tendon d1,d2) and tendonbone interface (e1,e2) from 2 to 8 weeks (n = 3).The dotted lines denote the tendon-bone interface at the repaired sites.*p < 0.05, **p < 0.01, ***p < 0.001 between groups after statistical analysis by independent samples t-tests/ANOVA according to the normality tests.

TC-nySS-Induced Incremental Mechanobiological Response at the Cuff Tendon and Tendon-Bone Interface
Biological activities are always accompanied by mechanical stimulation, and biomechanical microenvironmental homeostasis plays a vital role in tissue repair, remodeling, and regeneration. [62]YAP is a core transcriptional coactivator in the Hippo signaling pathway, a critical mechanotransduction protein that significantly promotes cell proliferation and differentiation. [63]ell behaviors could be affected by mechanobiological signals like the stress stimulation of the repair tissue, the ECM's stiffness, the fluid's shear stress, and the tension of the actin cytoskeleton. [63]Therefore, we detected the YAP expression for mechanobiological response in vivo at the cuff tendon and tendon-bone interface by immunofluorescent stainings.At the cuff tendon, the YAP started to be detectable in the TC-nySS (20.22 AE 3.26) or nySS (22.09AE 1.80) at 2 weeks (Figure 3d1), with a significantly higher intensity than the Con group (12.07 AE 1.28) (Figure 3d3).More importantly, this difference in YAP was substantially intuitive at 8 weeks (Figure 3d2), showing more YAP expression at the cuff tendon with higher intensity (TC-nySS, 59.62 AE 6.24; nySS, 54.87 AE 3.55; Con, 19.47 AE 3.86), which indicated the more robust mechanobiological response in the TC-nySS or nySS.Notably, the TC-nySS or nySS group displayed a significantly higher intensity of YAP at 8 weeks compared to that at 2 weeks, whereas the Con group did not.This implied that the TC-nySS or nySS had an incremental mechanobiological response during the healing stage, which might result from the progressive degradation of the hierarchical micro-nano structures of these sutures, as we demonstrated in the previous sections.Similar findings were also determined at the tendonbone interface (Figure 3e1-e3).Combining these results, we confirmed that the continuous mechanical sheltering effects derived from the nonabsorbable commercial suture were probably detrimental to stimulating the mechanobiological response at the healing tissues.In contrast, the specially-designed structure of progressive degradation in the TC-nySS or nySS group was more conducive to gradually activating the mechanobiological signaling.

TC-nySS Exhibited Good Biocompatibility for Cell Adhesion and Proliferation
Two key biological factors have been reported to dramatically influence RC regeneration at the early healing stage after RC repair.On the one hand, the acute inflammatory response considerably impairs the healing and regeneration processes, resulting in undesirable scar tissue development and repair failure. [29,30]On the other hand, the tendon and tendon-bone interface physiologically lack vascularization, providing limited blood supply and nutritional support earlier after repair. [26,27]herefore, immunomodulation and angiogenesis induced by the TC-nySS in vivo are critical to build up a favorable healing microenvironment (Figure 4a).Notably, these proangiogenic and immunomodulatory dual functions were premised on good biocompatibility of the TC-nySS.Fibroblasts and macrophages, the representative cell types in which significant changes were observed in the microenvironment, were chosen to discern the biological effects of the TC-nySS on cells in vitro.The biocompatibility was first investigated since the composite nanofiber suture must be nontoxic to normal cells.The synthetic TC-nySS and nySS were, therefore, coincubated with fibroblasts.The cell adhesion on the sutures at the early 4 h after incubation was quantitively determined by the CCK-8, with the coverslip as a BC (Figure 4b1).We observed that both TC-nySS (0.12 AE 0.01) and nySS (0.13 AE 0.01) had significantly superior early cell adhesions than the blank (0.10 AE 0.01).Similar cell incorporation was observed in the nySS and TC-nySS groups after culturing for 4 h, possibly due to the similar cell recruitment effects of nanocues in the nySS group and TC coatings in the TC-nySS group in the short term.As shown in Figure 4b2, although the nySS and TC-nySS showed no difference in the cell proliferation at 1 and 3 days, the TC-nySS had a significantly higher cell proliferation than the nySS group at 7 days, suggesting the continued recruitment and promotion of cell proliferation by the TC coating in the long term.Figure 4b3 shows the fluorescence staining of the fibroblast nucleus by DAPI and the microfilament skeletons by phalloidin.The morphologies of the fibroblasts incubated with TC-nySS and nySS for 7 days showed similar spindle morphology to that of the blank, which implied that the sutures had no significant cytotoxicity to normal cells.Moreover, the cells recruited around the sutures in both nySS and TC-nySS groups, with the latter showing a more significant cell accumulation, suggesting that sutures facilitated cell adhesion, proliferation, and recruitment for a long time in vitro.

TC-nySS Had Good Hemocompatibility
Hemocompatibility is one of the important indexes to characterize the compatibility of biomaterials: high absorbance and HP mean the destruction of RBCs. [42]Figure S8a, Supporting Information, shows the hemocompatibility of the TC-nySS.The positive control (deionized water) appeared red color due to the destruction of the RBCs, while the negative control (PBS buffer) presented transparent color.The color of the TC-nySS was almost the same as the negative control.Additionally, TC-nySS showed a similar HP (0.05 AE 0.06%) to the negative control (0.00 AE 0.06%), with a significantly lower one than the positive control (100.00AE 0.95%) (Figure S8b, Supporting Information).According to ASTM standard F756-13, materials with a hemolytic percentage of 5.0% can be considered nonhemolytic, indicating that TC-nySS was not hematotoxic. [64]

TC-nySS-Induced M2 Macrophage Polarization for Favorable Immunomodulation
After determining the direct biocompatibility of cell adhesion and proliferation by coincubating the cells with sutures, the extract solutions from the TC-nySS were further collected to detect the immunomodulatory effects of TC modification on the macrophage polarization in vitro and in vivo, using the one from nySS as control.During the acute inflammatory stage after repair, M1 proinflammatory phenotypes are predominant at the tendon-bone interface and cuff tendon, substantially promoting inflammatory cytokine secretion. [65]In contrast, the accumulation of M2 anti-inflammatory phenotypes facilitates tissue repair and regeneration by secreting beneficial cytokines, such as IL-4, IL-13, and TGF-β. [65]Therefore, modulating macrophage phenotypes from M1 to M2 can potentially eliminate undesirable inflammatory responses at the early healing stage for mid-to-late stage RC regeneration.Some reports have demonstrated that the physiological formation of fibrin networks in the early healing stage, known as the fibrin clot, substantially facilitates cellular chemotaxis and the M2 macrophages polarization in vivo, which could similarly provide a favorable healing environment after RC repair for tendon-bone integration and cuff tendon remodeling. [24,66,67]t was initially established that the TC-nySS promoted macrophage polarization toward the M2 phenotype.Following various treatments, RAW macrophages showed altered expression of CD 86 (an M1 marker) and CD206 (an M2 marker), as demonstrated by flow cytometry analyses.M2 phenotype proportion was equivalent to CD206þ/CD86-cell proportion.As shown in Figure 4c1, the nySS (5.91 AE 0.71%) showed a similar M2 phenotype proportion to the blank group (Figure S9, Supporting Information, and 4.63 AE 1.39%), indicating that no macrophage polarization occurred after the ny-SS intervention.Notably, the TC-nySS (40.43 AE 4.30%) group presented significantly higher M2 phenotype proportions than the nySS and blank, indicating TC modification of the nySS substantially elevated the M2 phenotype macrophage polarization.Additionally, immunofluorescent staining demonstrated a considerably larger intensity of M2 phenotype macrophages in the TC-nySS (13.76 AE 2.00) than that in the nySS (3.25 AE 1.41) (Figure 4c2).Notably, the antiinflammatory effects were further confirmed in vivo at the early healing of 2 weeks.H&E staining demonstrated that the tendon and tendon-bone interface of the nySS group was filled with granulation tissues, showing substantially detectable cellularity and more inflammatory cell infiltration (Figure 4c3).The quantitative analysis further indicated that the nySS group had a substantially higher inflammatory cell intensity at either the tendon (16.76 AE 2.86 vs 3.66 AE 0.34) or tendon-bone interface (19.36 AE 1.40 vs 8.01 AE 2.80) (Figure 4c4).In parallel to in vitro findings, the immunofluorescent staining in vivo also confirmed that the TC-nySS group displayed visibly higher expression of the CD206þ cells (M2 macrophages) accompanied by the lower one of the CD86þ cells (M1 macrophages) (Figure 4c5), with a significantly higher ratio of M2/M1 at the tendon (13.66 AE 0.34% vs 0.62 AE 0.29%) and tendon-bone interface (14.68 AE 4.03% vs 0.43 AE 0.29%) (Figure 4c6), thereby indicating that the TC-nySS group promoted the macrophage polarization toward the M2 phenotype.

TC-nySS Promoted in Vivo Early-Stage Angiogenesis and Timely Induced Vascularity Regression
Due to the physiological lack of vascularity at both the tendonbone interface and cuff tendon, the effective angiogenesis at the early stage for adequate nutrition supply is a considerable challenge, thus hardly building up a favorable microenvironment for further RC regeneration. [26,27]Some studies have recognized the superiorities of early-stage angiogenesis in further mid-to-late stage healing and remodeling. [26,68]However, it has been demonstrated that continuous pro-vascularization would not persistently promote RC regeneration; instead, it may result in excessive fibrosis and scarring, inferior interface mineralization, and inadequate biomechanical strengths of the repair tissues. [31,34]Therefore, realizing the early-stage angiogenesis and timely vascularity regression by suture structure design guarantees promising healing and remodeling patterns similar to the native RC postoperatively.As a proof-of-concept to delineate that the outer TC modification of the TC-nySS could induce the chronologically periodic vascular formation and fading that matched the typical healing progression of RC regeneration, the immunofluorescent staining for CD31 was employed at 2 and 8 weeks to assess the early-stage angiogenesis and late-stage vascularity regression.We observed that the TC-nySS group presented more CD31-positive neovascularization at the early-stage of 2 weeks at both tendon (382.61AE 32.74% vs 162.90 AE 18.90%) (Figure 4d1) and tendon-bone interface (334.76 AE 22.88% vs 98.32 AE 21.55%) (Figure 4d2), with significantly higher vessel intensity compared to the nySS group.It was worth noting that the marked and mature vessels with larger vessel thickness in the nySS group were sustained at the late-stage of 8 weeks, t, which on the contrary, turned undetectable in the TC-nySS group at both tendon (202.09AE 23.33% vs 40.53 AE 8.48%) (Figure 4d1) and tendon-bone interface (126.65 AE 14.25% vs 32.15 AE 8.16%) (Figure 4d2).The significant decrease in vessel intensity also proved that the TC-nySS could induce progressive vascularity regression at 8 weeks.The progressive degradation properties and the particular modification of the outer TC layer of fibrin could explain this chronologically periodic modulation of vascularity.Such a combined structural and biochemical design, at least, has provided the possibility to build a phased vascularization microenvironment via surgical sutures to match the typical healing and regenerative progression for RC repair, which preliminarily validated the superiorities in the TC-nySS.

Validation of the Therapeutic Effects of the TC-nySS on In Vivo RC Regeneration in a Rat Model
In this section, we determined the beneficial effects of the TC-nySS on RC regeneration by histological cues in a rat model, including the enhancements of tendon-bone interface integration and cuff tendon remodeling (Figure 5a).

TC-nySS-Induced Tendon-Bone Interface Integration and Cuff Tendon Remodeling
Compared to the untreated tears with the unconnected cuff-bone interface (Figure S10, Supporting Information), three groups after repair achieved substantial continuities between the repaired cuff tendons and bones by the H&E stainings (Figure 5b1).Notably, the TC-nySS group showed the formation of native-like collagen networks with the densest and most organized collagen fibers at the tendon and interface among the three groups, which was closer to the native histological characteristics of thr normal cuff tendon-bone interface.In contrast, the Con group displayed maximum scar formation with the most sparse fibrous tissues.Regarding cell behaviors, both Con and nySS groups presented higher cellularities with fewer cell arrangements at the tendon and interface than the TC-nySS.Further, it should be noted that normal tendons have few fibroblasts with flattened nuclei that are typically aligned parallel to the tensile axis.Quantitative analysis of the cellularity showed that the TC-nySS had the most similar fibroblast density and the nuclear orientation angle to the normal tendon among the three groups, while the nySS and Con groups were significantly different from the normal (Figure S11, Supporting Information).These findings indicated the superiorities of the TC-nySS in general morphologies and cell behaviors after repair, close to the normal tendon condition.Furthermore, the MT staining showed that normal tendons were characterized by highly packaged and .TC-nySS facilitated tendon-bone interface integration and cuff tendon remodeling in vivo for improving RC regeneration, thus enhancing the biomechanical performance of the repaired cuff (n = 3).a) A schematic illustration shows the promising RC regeneration required tendon-bone interface integration and cuff tendon remodeling.b1-b4) The H&E and Masson stainings showing the tendon-bone interface integration and cuff remodeling in vivo: b1) the H&E staining.b2) The Masson staining.b3) The THSS scores for cuff tendon remodeling analysis.b4) The TBSS scores for tendon-bone interface integration analysis.c1) The toluidine blue staining for fibrocartilage formation analysis.The yellow dotted lines denote the regions of metachromasia.c2) The micro-CT images for bone formation analysis.d1-d4) The immunofluorescent staining showing the tenogenic-related marker (TNMD; d1,d3) and tenogenic-rich ECM synthesis (COL1 and COL3; d2,d4) for cuff tendon remodeling analysis.e1,e2) The mechanical testing apparatus of the tendon-bone complex (e1) for analyzing the biomechanical performance of the repaired cuff tendon (e2).*p < 0.05, **p < 0.01, ***p < 0.001 between groups after statistical analysis by independent ANOVA according to the normality tests.※ p < 0.05 versus the normal group.
perfectly aligned collagen bundles (Figure S10, Supporting Information).The TC-nySS group had the optimal collagen fiber continuity, orientation, and density at the interface and tendon among the three groups, followed by the nySS group (Figure 5b2), similar to the native tendon.Parallel to the morphological findings, the TC-nySS had significantly higher THSS (25.67 AE 2.08 vs 16.33 AE 3.51 vs 9.00 AE 1.00) and TBSS (27.67 AE 5.86 vs 18.33 AE 2.08 vs 8.67 AE 1.53) scores than nySS and Con groups, indicating the best tendon-bone interface integration and tendon remodeling, which was comparable to the normal cuff tendon and interface (Figure 5b3,b4).Moreover, the scores in nySS were greater than those in the Con group.Notably, further qPCR analysis indicated that the TC-nySS induced significantly higher expression of cuff-bone healingrelated genes than the Con and nySS groups, including the tenogenic-related genes (Scleraxis; Col1A1) for tendon remodeling and mineralized tissue-related genes (Aggrecan; Runx2) for tendon-bone interface integration (Figure S12, Supporting Information).

TC-nySS-Enhanced Fibrocartilage Formation and Bone Regeneration
Inspired by the composition of the native tendon-bone interface, most earlier studies have concentrated on using biomaterials for RC regeneration to imitate the typical structures of nonmineralized, mineralized fibrocartilage and bone in enthesis for improving tendon-bone healing. [69,70]Figure 5c1 indicates more fibrocartilage formed at the tendon-bone interface in the TC-nySS group by the TB staining, followed by the nySS group.Further quantitative analysis showed that the TC-nySS group had a significantly higher metachromasia (fibrocartilage) ratio than the other groups (60.66 AE 6.29 vs 15.82 AE 0.78 vs 5.34 AE 2.76).Moreover, it is noteworthy that more bone regenerated at the tendon-bone interface in the TC-nySS group (Figure 5c2), with the most considerable gray value representing the best bone quality (177.70AE 21.51 vs 129.40 AE 10.63 vs 76.24 AE 14.14).The nySS group was superior in bone regeneration to the Con group.Moreover, the quantitative bone microarchitecture assessments at the tendon-bone interface also indicated that the TC-nySS achieved the best BMD, BV/TV, and Tb parameters.N and Tb.Th (Figure S13, Supporting Information).

TC-nySS Promoted the Expressions of Tenogenic-Related Markers and Tenogenic-Rich ECM Synthesis
The potential of TC-nySS to support the tenogenic commitment of encapsulated tendon cells was assessed through the protein expression analysis of recognized tendon-related markers.A well-known marker of mature tendon/ligament lineage, TNMD is a type II transmembrane glycoprotein strongly expressed by tenocytes as a regulator of tendon matrix remodeling. [71,72]The blue represented the nucleus, and the red represented the positive presentation of the TNMD (Figure 5d1).It can be seen that the repaired cuff tendon in the TC-nySS group exhibited more red fluorescence, with significantly higher TNMD intensity than the other two groups (22.19 AE 1.45 vs 13.32 AE 2.24 vs 3.95 AE 1.62) (Figure 5d3), suggesting higher tenogenic-related marker expressions with more increased tendon matrix remodeling and less fibrotic-like ECM formation.Additionally, the nySS group presented substantially higher TNMD intensity than the Con group.
Additionally, the quality of the de novo ECM deposited within the cuff tendon was assessed.We evaluated the expression of tendon-related matrix components, specifically the COL1 and COL3.The tendon ECM primarily consists of COL1 (86 wt%) and small amounts of proteoglycans, assembled into a hierarchical structure of fibrils.The COL3 synthesis was favored instead of the COL1 in the degenerated tendon, while the opposite was verified in the regenerated one. [71]Moreover, the differences in the deposition of these two types of collagen resulted in a significantly lower COL1/COL3 ratio in degenerated tendons, an effect related to the formation of fibrotic-like tissues. [71,73]Similar observations also occur in pathologic tendons, which show microscopic irregular fibrillar collagen alignment and molecular changes in collagen composition involving a decreased ratio of COL1/COL3. [71,73]Notably, in the later stages of tendon remodeling phase, there is a denser connective tissue dominated by COL1 produced by tendon cells and a small amount of COL3.Therefore, we observed the ratio of COL1/COL3 to assess the tendon remodeling, with the higher ratio of COL1/COL3 indicating a better remodeling process.The expressions of COL1 (the red in Figure 5d2) in the TC-nySS and nySS groups were considerably more evident than in the Con group, indicating the tenogenic-rich ECM depositions and collage maturities in the repaired tendons.More importantly, the red fluorescence of COL1 in the TC-nySS was more regular and arranged than the nySS, indicating better fibrillar collagen alignment and organization.In contrast, we observed that both TC-nySS and nySS groups showed lower COL3-positive expressions (the green Figure 5d2) than the Con group, especially at the tendon portion near the tendon-bone interface, which proved that the TC-nySS and nySS groups indeed inhibited tendon degeneration and pathologic changes significantly.The differences in the depositions of these two types of collagen resulted in the highest COL1/COL3 ratio in the TC-nySS in all groups, followed by the nySS (3.88 AE 0.33 vs 2.60 AE 0.28 vs 0.68 AE 0.19) (Figure 5d4), indicating that the TC-nySS had the most regenerative tenogenic tissues.

TC-nySS Improved Biomechanical Properties of Regenerated RC in Vivo
The native RC has dense and well-organized tendon tissues that attach to the mineralized interface with a gradual gradient of fibrocartilage and bone, thus providing robust mechanical strengths to adapt to the intense mechanical environment in vivo. [1,32,74]The regenerated RC after surgical repair is required to restore superior mechanical properties in the cases of sound histological healing and remodeling between the cuff tendon and the footprint. [23,26,59]After discovering the histological evidence in the TC-nySS, likely paralleled to the native RC, the failure mode analysis was required to further investigate the potential biomechanical advantages (Figure 5e1).Notably, the highest failure load was observed in the TC-nySS group, followed by the nySS group.They both showed significant increases in the failure load compared to the Con group (27.03AE 2.22 vs 18.81 AE 0.34 vs 8.67 AE 1.53) (Figure 5e2).Moreover, significant increases in stiffness were also observed in the TC-nySS group compared to that in the nySS group and Con groups postoperatively (15.23 AE 1.11 vs 9.10 AE 0.59 vs 4.65 AE 2.25), displaying the largest stiffness among the three groups.

Conclusion
We fabricated the functional SS (TC-nySS) with the biomimetic micro-nano and hierarchical triple helix structure to promote tendon-bone interface integration and cuff tendon remodeling for improving RC regeneration, thus substantially enhancing biomechanical properties of the repaired tissues.Notably, the progressive degradation behavior of the TC-nySS correspondingly stimulated the incremental mechanobiological response of the repaired tissues during the healing stage.We further deciphered the proangiogenic and immunomodulatory dual-function of the in situ crosslinked TC layer of the fibrin network, which enabled the TC-nySS to promote vascularization and M2 macrophage polarization at the early healing stage.Such an outer biochemical modification accompanied by the structurally progressive degradation of the micro-nano construct timely induced vascularization regression, avoiding excessive scars and fibrosis due to uncontrolled vascularization.In summary, for the first time, we successfully fabricated a novel biomimetic micro-nano and triple-helix hierarchical SS with superior progressive degradation behavior coupled with biochemically temporary chemotactic modification by the physiological fibrin network.Additionally, extensive in vitro and in vivo testing demonstrated that this functional suture exerted time-series mechanobiological stimulation and biological effects for tailoring the process-matching healing needs, thus matching the complicated processes for RC regeneration (Scheme 1).This off-the-shelf combined structural and biochemical fabrication strategy may provide novel perspectives on SS design and deep theoretical foundations, which inspire biotextile or scaffold preparations for tissue repair and regeneration.

Figure 1 .
Figure 1.Fabrication and characterization of the TC-nySS.a1-a4) The fabrication scheme of TC-nySS with biomimetic micro-nano hierarchies structure and Fb network.b) The SEM image of the core-sheath ny in the top and cross-sectional view (red dashed box).The blue box denotes the high magnification of local region of the surrounding sheath.c) The SEM image of PLGA nanofiber as the surrounding sheath of the ny.d) Diameter distribution of the nanofibers (n = 5).e) The morphology of the triple helix structure of the micro-nanosized TC-nySS.The purple stuffs were PLGA microyarns while the white on the surface were PLGA nys.f ) The braiding angle of the nys (n = 5).The blue box denotes the high magnification of the local region of the TC-nySS sheath surface treated with Fg/thrombin/FIV.g) FTIR spectra for TC-nySS (n = 3).h) N1s narrow scan XPS spectra for TC-nySS (n = 3).i) Crystallinity(n = 3).j) WCA (n = 3).***p < 0.001 between groups after statistical analysis by independent samples t-test according to the normality tests.

Figure 2 .
Figure 2. Mechanical properties of the TC-nySS and its surgical application in an in vivo rat model.a) The load-elongation curve.b) The stress-strain curve.c) The failure load.d) The UTS. e) The stiffness.f ) The elongation.g) Surgical application for RC repair in a rat RC tear model (n = 5).h) Detailed surgical procedures to validate the easy application of TC-nySS.i) The testing apparatus to evaluate the stability of the Fb network nanofiber coating in the TC-nySS.The SEM analysis (100Â) showed the reserved coating nanofiber after penetrating the cuff tendon tissues j) 100 and k) 500 times (n = 6).The cross-sectional observations (150Â) indicated the stability of the core-sheath structure in the TC-nySS (yellow boxes).**p < 0.01, ***p < 0.001 between groups after statistical analysis by ANOVA according to the normality tests.

Figure 3 .
Figure 3. Progressive degradation of the TC-nySS accelerated incremental mechanobiological response in vivo.a) Schematic illustration shows the progressive degradation process of the TC-nySS and the correspondingly incremental mechanobiological response of the repaired tissues.b1-b6) The progressive degradation behavior of the TC-nySS in vitro experiment (n = 6): b1) the general and SEM morphologies of TC-nySS in a 6-week in vitro experiment.b2) The mass loss (%).b3) The failure load during four consecutive weeks.b4) The pH changes.b5) The failure load loss after a 6-week in vitro degradation.b6) The crystallinity change after a 6-week in vitro degradation.b7) Schematic analysis indicating the progressively degraded TC-nySS could correspondingly induce increasing healing tissue stress.c) The progressive degradation process of the TC-nySS in vivo experiment (n = 3).d1-e2)The immunofluorescent staining analyzing the incremental mechanobiological response via the expression of YAP at the cuff tendon d1,d2) and tendonbone interface (e1,e2) from 2 to 8 weeks (n = 3).The dotted lines denote the tendon-bone interface at the repaired sites.*p < 0.05, **p < 0.01, ***p < 0.001 between groups after statistical analysis by independent samples t-tests/ANOVA according to the normality tests.

Figure 4 .
Figure 4. Temporary chemotactic modification by fibrin networks in the TC-nySS promoted immunomodulatory and angiogenesis effects in vivo at the early healing stage.a) A schematic illustration shows the immunomodulation by inducing M2 macrophage polarization and the improvement of vascularity by the TC-nySS.b1-b3) The biocompatibility analysis of the TC-nySS using fibroblasts in vitro experiment (n = 3): b1) The cell incorporation on the TC-nySS after 4-hour coincabulation.b2) The cell proliferation on the TC-nySS after 1, 3, and 7-day coincabulation.b3) The fluorescence staining showed that the cells were recruited around the TC-nySS after 7-day coincabulation.c1-c6) The in vitro and in vivo immunomodulatory effects of the TC-nySS by M2 macrophage polarization (n = 3): c1-c2) the flow cytometry (C1) and immunofluorescent staining (C2) analyses demonstrating the M2 macrophage polarization induced by the TC-nySS in vitro.c3-c6) The H&E (C3 and C5) and immunofluorescent (C4 and C6) stainings showing less inflammatory cell infiltration and M2 macrophage polarization by the TC-nySS in vivo (n = 3).d1,d2) The immunofluorescent staining assessing the early-stage angiogenesis and late-stage vascularity regression via the expression of CD31 at the cuff tendon (d1) and tendon-bone interface (d2) at 2 or 8 weeks (n = 3).The dotted lines denote the tendon-bone interface at the repaired sites.*p < 0.05, **p < 0.01, ***p < 0.001 between groups after statistical analysis by independent samples t-tests/ANOVA according to the normality tests.※ p < 0.05 versus the blank group.

Figure 5
Figure5.TC-nySS facilitated tendon-bone interface integration and cuff tendon remodeling in vivo for improving RC regeneration, thus enhancing the biomechanical performance of the repaired cuff (n = 3).a) A schematic illustration shows the promising RC regeneration required tendon-bone interface integration and cuff tendon remodeling.b1-b4) The H&E and Masson stainings showing the tendon-bone interface integration and cuff remodeling in vivo: b1) the H&E staining.b2) The Masson staining.b3) The THSS scores for cuff tendon remodeling analysis.b4) The TBSS scores for tendon-bone interface integration analysis.c1) The toluidine blue staining for fibrocartilage formation analysis.The yellow dotted lines denote the regions of metachromasia.c2) The micro-CT images for bone formation analysis.d1-d4) The immunofluorescent staining showing the tenogenic-related marker (TNMD; d1,d3) and tenogenic-rich ECM synthesis (COL1 and COL3; d2,d4) for cuff tendon remodeling analysis.e1,e2) The mechanical testing apparatus of the tendon-bone complex (e1) for analyzing the biomechanical performance of the repaired cuff tendon (e2).*p < 0.05, **p < 0.01, ***p < 0.001 between groups after statistical analysis by independent ANOVA according to the normality tests.※ p < 0.05 versus the normal group.