Approximately 30% of general practice consultations for musculoskeletal pain are related to tendon disorders, causing substantial personal suffering and enormous related healthcare costs. Treatments are often prone to long rehabilitation times, incomplete functional recovery, and secondary complications following surgical repair. Overall, due to their hypocellular and hypovascular nature, the regenerative capacity of tendons is very poor and intrinsically a disorganized scar tissue with inferior biomechanical properties forms after injury. Therefore, advanced therapeutic modalities need to be developed to enable functional tissue regeneration within a degenerative environment, moving beyond pure mechanical repair and overcoming the natural biological limits of tendon healing. Our recent studies have focused on developing biologically augmented treatment strategies for tendon injuries, aiming at restoring a physiological microenvironment and boosting endogenous tissue repair. Along these lines, we have demonstrated that the local application of mesenchymal stromal cell-derived small extracellular vesicles (sEVs) has the potential to improve rotator cuff tendon repair by modulating local inflammation and reduce fibrotic scarring. In another approach, we investigated if the local delivery of the tendon ECM protein SPARC, which we previously demonstrated to be essential for tendon maturation and tissue homeostasis, has the potential to enhance tendon healing. Finally, I will present results demonstrating the utility of nanoparticle-delivered, chemically modified mRNAs (cmRNA) to improve tendon repair

Tendons display poor intrinsic healing properties and are difficult to treat[1]. Prior in vitro studies[2] have shown that, by targeting the Activin A receptor with magnetic nanoparticles (MNPs), it is possible to remotely induce the tenogenic differentiation of human adipose stem cells (hASCs). In this study, we investigated the tenogenic regenerative potential of remotely-activated MNPs-labelled hASCs in an in vivo rat model. We consider the potential for magnetic controlled nanoparticle mediated tendon repair strategies. hASCs were labelled with 250 nm MNPs functionalized with anti-Activin Receptor IIA antibody. Using a rapid curing fibrin gel as delivery method, the MNPs-labelled cells were delivered into a Ø2 mm rat patellar tendon defect. The receptor was then remotely stimulated by exposing the rats to a variable magnetic gradient (1.28T), using a customised magnetic box. The stimulation was performed 1 hour/day, 3 days/week up to 8 weeks. Tenogenesis, iron deposition and collagen alignment were assessed by histological staining and IHC. Inflammation mediators levels were assessed by ELISA and IHC. The presence of human cells in tendons after 4 and 8 weeks was assessed by FISH analysis. Histological staining showed a more organised collagen arrangement in animals treated with MNPs-labelled cells compared to the controls. IHC showed positive expression of tenomodulin and scleraxis in the experimental groups. Immunostaining for CD45 and CD163 did not detect leukocytes locally, which is consistent with the non-significant levels of the inflammatory cytokines analysis performed on plasma. While no iron deposition was detected in the main organs or in plasma, the FISH analysis showed the presence of human donor cells in rat tendons even after 8 weeks from surgery. Our approach demonstrates in vivo proof of concept for remote control stem cell tendon repair which could ultimately provide injectable solutions for future treatment. We are grateful for ERC Advanced Grant support ERC No.789119, ERC CoG MagTendon No.772817 and FCT grant 2020.01157.CEECIND

Macrophages play a critical role in innate immunity by promoting or inhibiting tissue inflammation and repair. Classically, macrophages can differentiate into either pro-inflammatory (M1) or pro-reparative (M2) phenotypes in response to various stimuli. Therefore, this study aimed to address how extracellular vesicles (EVs) derived from polarized macrophages can affect the inflammatory response of tendon cells. For that purpose, human THP-1 cells were stimulated with lipopolysaccharide (LPS), and interleukins -4 and -13 (IL- 4, IL-13), to induce macrophages polarization into M1, M2, and hybrid M1/M2 phenotypes. Subsequently, the EVs were isolated from the culture medium by ultracentrifugation. The impact of these nanovesicles on the inflammation and injury scenarios of human tendon-derived cells (hTDCs), which had previously been stimulated with interleukin- 1 beta (IL-1ß) to mimic an inflammatory scenario, was assessed. We were able to isolate three different nanovesicles populations, showing the typical shape, size and surface markers of EVs. By extensively analyzing the proteomic expression profiles of M1, M2, and M1/M2, distinct proteins that were upregulated in each type of macrophage-derived EVs were identified. Notably, most of the detected pro- inflammatory cytokines and chemokines had higher expression levels in M1-derived EVs and were mostly absent in M2-derived EVs. Hence, by acting as a biological cue, we observed that M2 macrophage-derived EVs increased the expression of the tendon-related marker tenomodulin (TNMD) and tended to reduce the presence of pro-inflammatory markers in hTDCs. Overall, these preliminary results show that EVs derived from polarized macrophages might be a potential tool to modulate the immune system responses becoming a valuable asset in the tendon repair and regeneration fields worthy to be further explored

Summary Statement. The aim of this study was to compare patterns (aligned, random and grid) of electrospun polydioxanone scaffolds for tendon repair. The aligned design was optimal, directing cell shape, orientation and protein expression. Moreover, it naturally crimped, presenting tendon-like morphology. Introduction. Nanofibrous electrospun materials have been previously proposed as potential scaffolds for tendon repair, with emphasis on biomimetic design, postulated to encourage tissue regeneration. In this study, we characterised the interaction of primary tendon-derived cells with polydioxanone (PDO) scaffolds. PDO is a polymer with an excellent in vitro and in vivo biocompatibility, and is specifically compatible with tendon-derived cells. Here, we designed electrospun PDO scaffolds with different fibre orientations, namely aligned, random and grid-like patterns. To evaluate their potential as patches for tendon repair, we grew primary tendon derived cells on these scaffolds, and tested different aspects of cell behavior, including cell shape, proliferation and protein expression. Methods. Scaffolds with different orientations were produced using a single nozzle electrospinning set-up. Human tendon cells were extracted from rotator cuff tissue resected during surgical repair, with appropriate ethical approval. Cells were grown on different scaffolds for at least 14 days. Multiphoton microscopy (MPM) was used to image cells (green, calcein AM, and red, actin-phalloidin). Cell growth was monitored using AlamarBlue assay. Cell length, width and orientation were manually measured from images acquired by fluorescence microscopy. RNA was extracted by Trizol homogenisation in a GentleMACS (Miltenyi Biotec). RNA samples were reversibly transcribed to cDNA and RT QPCR were performed using a ViiA7 (Life Technologies) with QuantiTect primer assays (QIAGEN). Results are in relative expression to GAPDH. Results. MPM enabled the visualization of the scaffolds and viable cells grown for at least 14 days. Images demonstrate the distinct appearance of cells grown on highly aligned scaffold compared to random, grid-orientation and glass control. They also show the crimp-like appearance of the oriented scaffold as well as the cells on these crimped fibres. Interestingly, proliferation was not significantly effected by scaffold pattern. However, cell shape was clearly affected, and cells grown on oriented scaffolds showed higher anisotropy and elongated, narrower cell shape compared to all other groups, presenting a more tendon-like phenotype. This was further supported by a strong increase in the expression of β-actin on the aligned compared to the randomly oriented scaffold, correlating with observed changes in cell length and shape. Conclusion. The aim of this study was to compare patterns (aligned, random and grid) of electrospun polydioxanone scaffolds as templates for tendon repair. Aligned PDO scaffolds significantly affected cell length, width, and orientation. We also found that β-actin expression was significantly increased on aligned polydioxanone scaffolds. Moreover, PDO scaffolds fabricated as aligned/oriented were observed to present crimp-like morphology, similar to native tendon. Taken together, these findings suggest an aligned morphology of polydioxanone may hold the potential to improve tendon healing

Thrombin has many biological properties similar to those of growth factors. In a previous study, we showed that thrombin improves healing of the rat tendo Achillis. Low molecular weight heparin (LMWH) inhibits the activity and the generation of thrombin. We therefore considered that LMWH at a thromboprophylactic dose might inhibit tendon repair. Transection of the tendo Achillis was carried out in 86 rats and the healing tested mechanically. Low molecular weight heparin (dalateparin) was either injected a few minutes before the operation and then given continuously with an osmotic mini pump for seven days, or given as one injection before the operation. In another experiment ,we gave LMWH or a placebo by injection twice daily. The anti-factor Xa activity was analysed. Continuous treatment with LMWH impaired tendon healing. After seven days, this treatment caused a 33% reduction in force at failure, a 20% reduction in stiffness and a 67% reduction in energy uptake. However, if injected twice daily, LMWH had no effect on tendon healing. Anti-factor Xa activity was increased by LMWH treatment, but was normal between intermittent injections. Low molecular weight heparin delays tendon repair if given continuously, but not if injected intermittently, probably because the anti-factor Xa activity between injections returns to normal, allowing sufficient thrombin stimulation for repair. These findings indicate the need for caution in the assessment of long-acting thrombin and factor Xa inhibitors

The biomechanical evaluation of tendon repair with collagen-based scaffolds in rat model is a common method to determine the functional outcome of the tested material. We introduced a magnetic resonance imaging (MRI) approach to verify the biomechanical test data. In present study different collagen scaffolds for tendon repair were examined. Two collagen test materials: based on bovine stabilized collagen, chemically cross-linked with oriented collagenous fibres (material 1) and based on porcine dermal extracellular matrix, with no cross-linking (material 2) were compared. The animal study was approved by the local review board. Surgery was performed on male Sprague-Dawley rats with a body weight of 400 ± 19 g. Each rat underwent a 5 mm transection of the right Achilles tendon. The M. plantaris tendon was removed. The remaining tendon ends were re-joined with a 5 mm scaffold of either the material 1 or 2. Each scaffold material was sutured into place with two single stiches (Vicryl 4–0, Ethicon) each end. A total of 16 rats (n= 8 each group) were observed for 28 days follow up. The animals were sacrificed and hind limbs were transected proximal to the knee joint. MRI was performed using a 7 Tesla scanner (BioSpec 70/30, Bruker). T2-weighted TurboRARE sequences with an in-plane resolution of 0.12 mm and a slice thickness of 0.7 mm were analysed. All soft and hard tissues were removed from the Achilles tendon-calcaneus-foot complex before biomechanical testing. Subsequently, the specimens were fixed in a materials testing machine (Z1.0, Zwick, Ulm, Germany) for tensile testing. All tendons were preloaded with 1 N and subsequently stretched at a rate of 1 mm/s until complete failure was observed. Non-operated tendons were used as a control (n=4). After 28 postoperative days, MRI demonstrated that four scaffolds (material 1: n=2, material 2: n=2) were slightly dislocated in the proximal part of hind limb. In total five failures of reconstruction could be detected in the tendon repairs (material 1: n=3, material 2: n=2). Tendons augmented with the bovine material 1 showed a maximum tensile load of 57.9 ± 17.9 N and tendons with porcine scaffold material 2 of 63.1 ± 19.5 N. The native tendons demonstrated only slightly higher loads of 76.6 ± 11.6 N. Maximum failure load of the tendon-scaffold construct in both groups did not differ significantly (p < 0.05). Stiffness of the tendons treated with the bovine scaffold (9.9 ± 3.6 N/mm) and with the porcine scaffold (10.7 ± 2.7 N/mm) showed no differences. Stiffness of the native healthy tendon of the contralateral site was significantly higher (20.2 ± 6.6 N/mm, p < 0.05). No differences in the mechanical properties between samples of both scaffold groups could be detected, regardless of whether the repaired tendon defect has failed or the scaffold has been dislocated. The results show that MRI is important as an auxiliary tool to verify the biomechanical outcome of tendon repair in animal models

Summary. Tissue grafts fail to recapitulate native tendon function, imposing the need for development of functional regeneration strategies. Herein, we describe advancements in tendon repair and regeneration using functionalised natural and synthetic devices and scaffold-free cell-based therapies. Introduction. Tendon and ligament injuries constitute an unmet clinical need with approximately 100,000 new cases annually in US alone. Tissue grafts are considered the gold standard in clinical practice. However, allografts and xenografts can lead to potential disease transmission, whilst the limited supply of autografts in severe injuries and degenerative conditions restricts their use. To this end, scaffold and scaffold-free therapies are under development to address the tissue grafts shortage. Herein, we describe biophysical, biochemical and biological methods to maintain tendon derived cell phenotype and/or differentiation of other cell types towards tenogenic lineage; development of tendon-equivalent facsimiles; and ultimately functional neotendon formation. Materials and Methods. Growth factor supplementation was assessed as means to either maintain tendon derived stem cell phenotype or differentiate them towards tenocytes. The influence of conditioning media was assessed as means to differentiate skin fibroblasts and stem cells towards tenogenic lineage. Biophysical and biochemical/biological features were assessed as means to maintain tendon derived cell phenotype and directional neotissue formation in rat patellar tendon model. Rich in tendon-specific extracellular matrix cell sheets were produced by appropriate modulation of the in vitro microenvironment. Structural, biophysical and biological analyses were subsequently carried out. Discussion & Future Studies. Treatment with 10 and 100 ng/mL of IGF-1 preserved tendon stem cell multipotency for up to 28 days in culture and minimised changes in marker expression and extracellular matrix molecules production enhancing that way the clinical potential of these cells. Hierarchically assembled collagen scaffolds and anisotropically ordered polymeric substrates of rigidity similar to native tendons facilitate tenocyte phenotype maintenance in vitro, whilst in vivo studies are under way to assess the extent of functional tendon regeneration. Appropriate modulation of the in vitro microenvironment of tenocytes with macromolecules enhances tendon specific extracellular matrix deposition within 6 days in culture, facilitating that way the wide acceptance of cell-sheet technology for tendon repair and regeneration

Introduction and Objective. Zone 2 flexor tendon injuries are still one of the challenges for hand surgeons. It is not always possible to achieve perfect results in hand functions after these injuries. There is no consensus in the literature regarding the treatment of zone 2 flexor tendon injuries, tendon repair and surgical technique to be applied to the A2 pulley. The narrow fibro-osseous canal structure in zone 2 can cause adhesions and loss of motion due to the increase in tendon volume due to surgical repair. Different surgical techniques have been defined to prevent this situation. In our study, in the treatment of zone 2 flexor tendon injuries; Among the surgical techniques to be performed in addition to FDP tendon repair; We aimed to compare the biomechanical results of single FDS slip repair, A2 pulley release and two different pulley plasty methods (Kapandji and V-Y pulley plasty). Materials and Methods. In our study, 12 human upper extremity cadavers preserved with modified Larssen solution (MLS) and amputated at the mid ½ level of the arm were used. A total of 36 fingers (second, third and the fourth fingers were used for each cadaver) were divided into four groups and 9 fingers were used for each group. With the finger fully flexed, the FDS and FDP tendons were cut right in the middle of the A2 pulley and repaired with the cruciate four-strand technique. The surgical techniques described above were applied to the groups. Photographs of fingers with different loads (50 – 700 gr) were taken before and after the application. Proximal interphalangeal (PIP) joint angle, PIP joint maximum flexion angle and bowstring distance were measured. The gliding coefficient was calculated by applying the PIP joint angle to the single-phase exponential association equation. Results. Gliding coefficient after repair increased by %21.46 ± 44.41, %62.71 ± 116.9, %26.8 ± 35.35 and %20.39 ± 28.78 in single FDS slip repair, A2 pulley release, V-Y pulley plasty and Kapandji plasty respectively. The gliding coefficient increased significantly in all groups after surgical applications (p<0.05). PIP joint maximum flexion angle decreased by %3.17 ± 7.92, %12.82 ± 10.94, %8.33 ± 3.29 and %7.35 ± 5.02 in single FDS slip repair, A2 pulley release, V-Y pulley plasty and Kapandji plasty respectively. PIP joint maximum flexion angle decreased significantly after surgery in all groups (p<0.05). However, there was no statistically significant difference between surgical techniques for gliding coefficient and PIP joint maximum flexion angle. Bowstring distance between single FDS slip repair, kapandji pulley plasty and V-Y pulley plasty showed no significant difference in most loads (p>0.05). Bowstring distance was significantly increased in the A2 pulley release group compared to the other three groups (p<0.05). Conclusion. Digital motion was negatively affected after flexor tendon repair. Similar results were found in terms of gliding coefficient and maximum flexion angle among different surgical methods. As single FDS slipe repair preserves the anatomical structure of the A2 pulley therefore we prefer it as an ideal method for zone 2 flexor tendon repair. However, resection of FDS slip may jeopardizes nutrition to the flexor digitorum profundus tendon which weakens the repair site. Therefore the results must be confirmed by an in vivo study before a clinical recommendation can be made. Keywords: Flexor tendon; injury; pulley plasty; cadaver;

Tendon injuries constitute a major healthcare burden owing to the limited healing ability of these tissues and the poor clinical outcomes of surgical repair treatments. Recent advances in tendon tissue engineering (TTE) strategies, particularly through the use of biotextile technologies, hold great promise toward the generation of artificial living tendon constructs. We have previously developed a braided construct based on suture threads coated with gelMA:alginate hydrogel encapsulating human tendon cells. These cell-laden composite fibers enabled the replication of cell and tissue-level properties simultaneously. Based on this concept, in this study we explored the use of platelet lysate (PL), a pool of supra-physiological concentrations of growth factors (GFs), to generate a hydrogel layer, which is envisioned to act as a depot of therapeutic factors to induce tenogenic differentiation of encapsulated human adipose stem cells (hASCs). For this purpose, commercially available suture threads were first embedded in a thrombin solution and then incubated in PL containing hASCs. Herein, thrombin induces the gelation of PL and consequent hydrogel formation. After coating suture threads with the mixture of PL-ASCs, cells were found to be viable and homogeneously distributed along the fibers. Strikingly, hASCs encapsulated within the PL hydrogel layer around the suture thread were able to sense chemotactic factors present in PL and to establish connections between adjacent independent fibers, suggesting a tremendous potential of PL cell-laden hydrogel fibers as building blocks in the development of living constructs aimed at tendon repair applications

Digital Ventilated Cages (DVC) offer an innovative technology to obtain accurate movement data from a single mouse over time [1]. Thus, they could be used to determine the occurrence of a tendon damage event as well as inform on tissue regeneration [2,3]. Therefore, using the mouse model of tendon experimental damage, in this study it has been tested whether the recovery of tissue microarchitecture and of extracellular matrix (ECM) correlates with the motion data collected through this technology.

Mice models were used to induce acute injury in Achilles tendons (ATs), while healthy ones were used as control. During the healing process, the mice were housed in DVC cages (Tecniplast) to monitor animal welfare and to study biomechanics assessing movement activity, an indicator of the recovery of tendon tissue functionality. After 28 days, the AT were harvested and assessed for their histological and immunohistochemical properties to obtain a total histological score (TSH) that was then correlated to the movement data.

DVC cages showed the capacity to distinguish activity patterns in groups from the two different conditions. The data collected showed that the mice with access to the mouse wheel had a higher activity as compared to the blocked wheel group, which suggests that the extra movement during tendon healing improved motion ability. The histological results showed a clear difference between different analyzed groups. The bilateral free wheel group showed the best histological recovery, offering the highest TSH score, thus confirming the results of the DVC cages and the correlation between movement activity and structural recovery.

Data obtained showed a correlation between TSH and the DVC cages, displaying structural and movement differences between the tested groups. This successful correlation allows the usage of DVC type cages as a non-invasive method to predict tissue regeneration and recovery.

Acknowledgements: This research is part of the P4FIT project ESR13, funded by the H2020-ITN-EJD MSCA grant agreement No.955685.

Introduction and Objective

In the elderly population, chronic rotator cuff tears are often associated with high re-rupture rates after surgical tendon refixation. Implant materials, especially in combination with additives are supposed to positively influence healing outcome. Furthermore, adequate mechanical properties are crucial. In order to realize degradable implants with high specific surface area, polycaprolactone (PCL) was chosen as basic material and processed by electrospinning to achieve a high surface area for growth factor implementation and subsequent cell attachment.

Extensor mechanism and abductor reconstructions in total joint arthroplasty are problematic. Growing tendon into a metallic implant would have great reconstructive advantages. With the introduction of porous metal implants, it was hoped that tendons could be directly attached to implants. However, the effects of the porous metal structure on tissue growth and pore penetration is unknown. In this rat model, we investigated the effect of pore size on tendon repair fixation using printed titanium implants with differing pore sizes. There were four groups of six Sprague Dawley rats (n = 28) plus control (n=4). Implants had pore sizes of 400µm (n=8), 700µm (n=8), and 1000µm (n=8). An Achilles tendon defect was created, and the implant positioned and sutured between the cut ends. Harvest occurred at 12-weeks. Half the specimens underwent tensile load to failure testing, the other half fixed and processed for hard tissue analysis. Average load to failure was 72.6N for controls (SD 10.04), 29.95N for 400µm (SD 17.95), 55.08N for 700µm (SD 13.47), and 63.08N for 1000µm (SD 1.87). The load to failure was generally better in the larger pore sizes. Histological evaluation showed that there was fibrous tendon tissue within and around the implant material, with collagen fibers organized in bundles. This increases as the pore diameter increases. Printing titanium implants allows for precise determination of pore size and structure. Our results showed that tendon repair utilizing implants with 700µm and 1000µm pores exhibited similar load to failure as controls. Using a defined pore structure at the attachment points of tendons to implants may allow predictable tendon to implant reconstruction at the time of revision arthroplasty

Tendon tissue equilibrium very heavily depends on appropriate mechanical loading within a narrow, and still poorly defined, physiological range. We will present an overview of our recent work on the tendon cell-matrix interactions that drive tissue homeostasis, matrix remodelling and eventual tissue degeneration, and discuss a roadmap for unravelling these mechanically regulated signalling pathways for the development of effective treatment strategies. Our data suggest that tissue damage accumulates in the tendon until “intrinsic repair mechanisms” are overwhelmed. At this point, the metabolic cost of extracellular matrix remodeling exceeds the locally available nutrient supply. We hypothesize that upon reach S43.1 ing this “Metabolic Tipping Point”, the vascular system is recruited along with accompanying nerve supply (and pain) and the tissue enters into a chronic disease state characterized by high matrix turnover and increasingly poor tissue quality. In this paradigm, a delicate mechanically regulated balance exists between recruitment and suppression of the extrinsic vascular system by the resident tendon core cells. Upon injury or damage, this regulation in turn steers the tissue towards either functional remodeling or chronic tendon disease.

Tendon regeneration is complex since the scaffold has to bear high loads and stress concentrations, while providing suitable deformability. Previous studies demonstrated a physiological orientation of the fibers and good cell adhesion on electrospun polymeric scaffolds [1]. The aims of this work were to: (i) prepare and characterize electrospun resorbable scaffolds with different compositions and (ii) develop a process to produce a multiscale bundle assembly to mimic the hierarchical structure and biomechanical properties of a real tendon.

We produced fibrous scaffolds made of blends of poly-L-lactic acid (PLLA) and collagen (Coll):

Pure PLLA;

In order to prepare 3D bundles made of aligned fibres, we used a high-speed rotating collector. The electrospun nanofibers were deposited tangentially onto the drum, the electrospun layer was manually rolled transversely along the drum and then removed. The bundles were approximately 150 mm long and 300–450 mm in diameter. Five specimens were prepared and tested for each blend.

To evaluate the mechanical properties of the bundles a tension test was applied with capstan grips on a testing machine with a 100N load cell, under the following conditions:

Gauge length: 20 mm.

For all the bundles, the stress-strain curve showed an initial non-linear part (toe region), similar to the laxity of the tendon at rest. The mechanical analysis confirmed the outstanding ductility and toughness of pure PLLA. Increasing the percentage of collagen resulted in a reduction of ductility. The PLLA/Coll 50/50 had a rather brittle behaviour.

The values of mechanical properties found for the different compositions were slightly lower but of the same order of magnitude as tendon fibers (Failure stress: 33.7±19.2 MPa; Failure strain: 21.0±9.1 %; Young Modulus: 257±101 MPa [2]). The bundles made of pure PLLA had a failure stress of 13.2±0.8 MPa; failure strain of 84.7±9.4%; Young Modulus of 78.6±7.5 MPa. The bundles made of PLLA/Coll 50/50 had: failure stress of 10.5±1.5 MPa; failure strain of 21.4±2.7%, Young Modulus of 65.7±9.8 MPa. The most promising composition was the PLLA/Coll 75/25, with a failure stress of 14.0±0.7 MPa; failure strain of 40.3±2.2 %, Young Modulus of 98.6±12 MPa.

We also tested bundles mechanical properties after aging samples in phosphate buffer at 37 °C for 48 hours, 7 and 14 days. After ageing, stress and strain values were progressively lower, while the toughness increased, compared to the dry samples.

The promising results found in this work for the electrospun PLLA-collagen blends confirm their potential use for tendon tissue regeneration. This is a starting point for developing multiscale scaffolds mimicking the structure of tendon tissue, which can potentially be used in human regenerative medicine both as bioresorbable prosthesis, or inserted in a bioreactor for in vitro production of tendon tissue.

Intra-synovial tendon injuries affect compressed tendon within a synovial environment (eg Rotator cuff tears of the shoulder) and frequently demonstrate ‘failed healing'. Current therapeutic methods for tendon tears (intra-synovial corticosteroid medication and surgical debridement) offer poor outcomes and new strategies for enhancing repair are needed. We have therefore evaluated two different approaches involving the use of mesenchymal stem cells and scaffolds. Bone marrow- and synovial-derived stem cells were capable of adhering to cut surfaces of tendon in vitro and modulating the release of extracellular matrix into the media. However, when administered in vivo into the digital flexor tendon sheath in naturally-occurring deep digital flexor tendon tears in horses and in an experimental model in sheep, neither cell type was capable of healing the tendon defect. Superparamagnetic iron oxide particle labelling of the implanted cells imaged using MRI and histologically revealed that cells only engraft into the synovium. In contrast a non-cellularised bilayered electrospun and woven polydioxanone scaffold, when used in the same experimental sheep model via a modified open approach and sutured over the created defect resulted in no local or systemic signs of excessive inflammation 3 months after implantation. All the tendon lesions healed with only a mild local inflammatory reaction and minimal-to-mild adhesion formation. Significant proliferative fibroblast infiltration was observed within and immediately adjacent to the implanted scaffold. The cellular infiltrate was accompanied by an extensive network of new blood vessel formation within the new tissue. In conclusion, the use of a scaffold to cover the defect appears to be a more successful strategy to repair intra-synovial tendon defects than intra-synovially injected mesenchymal stem cells. It remains to be tested whether the combination of the two techniques might offer an even better healing response

Frictional resistance to tendon gliding is minimised by surrounding loose areolar tissues. During periods of prolonged immobilisation, for example post tendon-repair, adhesions can form between the two adjacent tissues, thereby limiting function. Whilst agents applied during surgery are recognised to succeed in adhesion prevention, they have also been reported to provide some reduction in friction during in vitro tendon-bony pulley investigations. This study investigated the effectiveness of common anti-adhesion agents in lubricating the tendon-surrounding tissue contact by comparison with a control study. By using a validated apparatus and with reference to the Stribeck curve, it was determined that the natural in vivo contact is likely to be lubricated by a film of synovial-like fluid. Application of all anti-adhesives generated a similarly efficient lubricating system, and hence administration of these agents should be encouraged to all regions of the tendon disrupted during surgery. Minimising frictional resistance to gliding will reduce the likelihood of tendon ‘gapping’ - and subsequently failure - at the repair site.

The fibrocartilaginous enthesis displays a complex interface between two mechanically dissimilar tissues, namely tendon and bone. This graded transition zone consists of parallel collagen type I fibres arising from the tendon and inserting into bone across zones of fibrocartilage with aligned collagen type I and collagen type II fibres and mineralised fibrocartilage. Due the high stress concentrations arising at the interface, entheses are prone to traumatic and chronic overuse injuries such as rotator cuff and anterior cruciate ligament (ACL) tears. Treatment strategies range from surgical reattachment for complete tears and conservative treatments (physiotherapy, anti-inflammatory drugs) in chronic inflammatory conditions. Generally, the native tissue architecture is not re-established and mechanically inferior scar tissue is formed. Current interfacial tissue engineering approaches pose scaffold-associated drawbacks and limitations, such as foreign body response. Using a thermo-responsive electrospun scaffold that provides architectural signals similar to native tissues and can be removed prior to implantation, we aim to develop an ECM-rich, cell-based implant for tendon-enthesis regeneration. Alcian blue staining revealed highest sGAG deposition in cell (human adipose derived stem cells) sheets grown on random electrospun fibres and lowest sGAG deposition in collagen type I sponges. Cells did not show an equal distribution throughout the collagen type II scaffolds but tended to form localised aggregates. Thermo-responsive electrospun fibres with random and aligned fibre orientation provided an adequate three-dimensional environment for chondrogenic differentiation of multilayer hADSC-sheets shown by high ECM-production, especially high sGAG deposition. Chondrogenic cell sheets showed increased expression of SOX9, COL2A1, COL1A1, COMP and ACAN after 7 days of chondrogenic induction when compared to pellet culture. Anisotropic fibres enabled the generation of aligned chondrogenic cell sheets, shown by cell and collagen fibre alignment. Thermoresponsive electrospun fibres showed high chondro-inductivity due to their three-dimensionality and therefore pose a promising tool for the generation of scaffold-free multilayer constructs for tendon-enthesis repair within short culture periods. Aligned chondrogenic cell sheets mimic the zonal orientation of the native enthesis as the fibrocartilaginous zone exhibits high collagen alignment.

Tendon injuries occur frequently in athletes and the general population, with inferior healing leading to deposition of fibrotic scar tissue. New treatments are essential to limit fibrosis and enable tendon regeneration post-injury. In this study, we tested the hypothesis that rapamycin improves tendon repair and limits fibrosis by inhibiting the mTOR pathway. The left hindlimb of female adult Wistar rats was injured by needle puncture and animals were either given daily injections of rapamycin (2mg/kg) or vehicle. Animals were euthanized 1 week or 3 weeks post-injury (n=6/group). Left and right Achilles tendons were harvested, with the right limbs acting as controls. Tendon sections were stained with haematoxylin & eosin, and scored by 2 blinded scorers, assessing alterations in cellularity, cell morphology, vascularity, extracellular matrix (ECM) organization and peritendinous fibrosis. Immunohistochemistry was performed for the tendon pan-vascular marker CD146 and the autophagy marker LC3. Injury resulted in significantly altered ECM organization, cell morphology and cellularity in both rapamycin and vehicle-treated groups, but no alterations in vascularity compared to uninjured tendons. Rapamycin had a limited effect on tendon repair, with a significant reduction in peritendinous fibrosis 3 weeks after injury (p=0.028) but no change in cell morphology, cellularity or ECM organization compared to vehicle treated tendons at either 1 week or 3 weeks post injury. CD146 labelling was increased at the site of injury, but there was no apparent difference in CD146 or LC3 labelling in rapamycin and vehicle treated tendons. The decrease in peritendinous fibrosis post-injury observed in rapamycin treated tendons indicates rapamycin as a potential therapy for tendon adhesions. However, the lack of improvement of other morphological parameters in response to rapamycin treatment indicates that rapamycin is not an effective therapy for injuries to the tendon core. Acknowledgements: This study was funded by Versus Arthritis (22607)

Tendons are characterised by an inferior healing capacity when compared to other tissues, ultimately resulting in the formation of a pathologically altered extracellular matrix structure. Although our understanding of the underlying causes for the development and progression of tendinopathies remains incomplete, mounting evidence indicates a coordinated interplay between tendon-resident cells and the ECM is critical. Our recent results demonstrate that the matricellular protein SPARC (Secreted protein acidic and rich in cysteine) is essential for regulating tendon tissue homeostasis and maturation by modulating the tissue mechanical properties and aiding in collagen fibrillogenesis [1,2]. Consequently, we speculate that SPARC may also be relevant for tendon healing. In a rat patellar tendon window defect model, we investigated whether the administration of recombinant SPARC protein can modulate tendon healing. Besides the increased mRNA expression of collagen type 1 and the downregulation of collagen type 3, a robust increase in the expression of pro-regenerative fibroblast markers in the repair tissue after a single treatment with rSPARC protein was observed. Additionally, pro-fibrotic markers were significantly decreased by the administration of rSPARC. Determination of structural characteristics was also assessed, indicating that the ECM structure can be improved by the application of rSPARC protein. Therefore, we believe that SPARC plays an important role for tendon healing and the application of recombinant SPARC to tendon defects has great potential to improve functional tendon repair

Poor tendon repair is an unsolved issue in clinical practice, due to complex tendon structure. Tendon stem/progenitor cells (TSPCs) play key roles in homeostasis, regeneration, and inflammation regulation in acute tendon injuries, and rely on TGF-β signaling for recruitment into degenerative tendons. In this study, we aimed to develop an in vitro model for tenogenesis adopting a dynamic culture of a fibrin 3D scaffold, bioengineered with human TSPCs collected from both healthy and tendinopathic surgery explants (Review Board prot./SCCE n.151, 29 October 2020). 3D culture was maintained for 21 days under perfusion provided by a custom-made bioreactor, in a medium supplemented with hTGF-β1 at 20 ng/mL. The data collected suggested that the 3D in vitro model well supported survival of both pathological and healthy cells, and that hTGF-β signaling, coupled to a dynamic environment, promoted differentiation events. However, pathological hTSPCs showed a different expression pattern of tendon-related genes throughout the culture and an impaired balance of pro-inflammatory and anti-inflammatory cytokines, compared to healthy hTSPCs, as indicated by qRT-PCT and immunofluorescence analyses. Additionally, the expression of both tenogenic and cytokine genes in hTSPCs was influenced by hTGF-β1, indicating that the environment assembled was suitable for studying tendon stem cells differentiation. The study offers insights into the use of 3D cultures of hTSPCs as an in vitro model for investigating their behavior during tenogenic events and opens perspectives for following the potential impact on resident stem cells during regeneration and healing events