Objectives. Platelet-rich plasma (PRP) is being used increasingly often in the clinical setting to treat tendon-related pathologies. Yet the optimal PRP preparations to promote tendon healing in different patient populations are poorly defined. Here, we sought to determine whether increasing the concentration of platelet-derived proteins within a derivative of PRP, platelet lysate (PL), enhances tenocyte proliferation and migration in vitro, and whether the mitogenic properties of PL change with donor age. Methods. Concentrated PLs from both young (< 50 years) and aged (> 50 years) donors were prepared by exposing pooled PRP to a series of freeze-thaw cycles followed by dilution in plasma, and the levels of several platelet-derived proteins were measured using multiplex immunoassay technology. Human tenocytes were cultured with PLs to simulate a clinically relevant PRP treatment range, and cell growth and migration were assessed using DNA quantitation and gap closure assays, respectively. Results. Platelet-derived protein levels increased alongside higher PL concentrations, and PLs from both age groups improved tenocyte proliferation relative to control conditions. However, PLs from aged donors yielded a dose-response relationship in tenocyte behaviour, with higher PL concentrations resulting in increased tenocyte proliferation and migration. Conversely, no significant differences in tenocyte behaviour were detected when increasing the concentration of PLs from younger donors. Conclusion. Higher PL concentrations, when prepared from the PRP of aged but not young donors, were more effective than lower PL concentrations at promoting tenocyte proliferation and migration in vitro. Cite this article: D. R. Berger, C. J. Centeno, N. J. Steinmetz. Platelet lysates from aged donors promote human tenocyte proliferation and migration in a concentration-dependent manner. Bone Joint Res 2019;8:32–40. DOI: 10.1302/2046-3758.81.BJR-2018-0164.R1

Aims. Myokine developmental endothelial locus-1 (DEL-1) has been documented to alleviate inflammation and endoplasmic reticulum (ER) stress in various cell types. However, the effects of DEL-1 on inflammation, ER stress, and apoptosis in tenocytes remain unclear. Methods. Human primary tenocytes were cultured in palmitate (400 μM) and palmitate plus DEL-1 (0 to 2 μg/ml) conditions for 24 hours. The expression levels of ER stress markers and cleaved caspase 3, as well as phosphorylated 5' adenosine monophosphate-activated protein kinase (AMPK) and autophagy markers, were assessed by Western blotting. Autophagosome formation was measured by staining with monodansylcadaverine, and apoptosis was determined by cell viability assay and caspase 3 activity assay. Results. We found that treatment with DEL-1 suppressed palmitate-induced inflammation, ER stress, and apoptosis in human primary tenocytes. DEL-1 treatment augmented LC3 conversion and p62 degradation as well as AMPK phosphorylation. Moreover, small interfering RNA for AMPK or 3-methyladenine (3-MA), an autophagy inhibitor, abolished the suppressive effects of DEL-1 on inflammation, ER stress, and apoptosis in tenocytes. Similar to DEL-1, 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), an activator of AMPK, also attenuated palmitate-induced inflammation, ER stress, and apoptosis in tenocytes, which 3-MA reversed. Conclusion. These results revealed that DEL-1 suppresses inflammation and ER stress, thereby attenuating tenocyte apoptosis through AMPK/autophagy-mediated signalling. Thus, regular exercise or administration of DEL-1 may directly contribute to improving tendinitis exacerbated by obesity and insulin resistance. Cite this article: Bone Joint Res 2022;11(12):854–861

Peri-tendinous injection of local anaesthetic, both alone and in combination with corticosteroids, is commonly performed in the treatment of tendinopathies. Previous studies have shown that local anaesthetics and corticosteroids are chondrotoxic, but their effect on tenocytes remains unknown. We compared the effects of lidocaine and ropivacaine, alone or combined with dexamethasone, on the viability of cultured bovine tenocytes. Tenocytes were exposed to ten different conditions: 1) normal saline; 2) 1% lidocaine; 3) 2% lidocaine; 4) 0.2% ropivacaine; 5) 0.5% ropivacaine; 6) dexamethasone (dex); 7) 1% lidocaine+dex; 8) 2% lidocaine+dex; 9) 0.2% ropivacaine+dex; and 10) 0.5% ropivacaine+dex, for 30 minutes. After a 24-hour recovery period, the viability of the tenocytes was quantified using the CellTiter-Glo viability assay and fluorescence-activated cell sorting (FACS) for live/dead cell counts. A 30-minute exposure to lidocaine alone was significantly toxic to the tenocytes in a dose-dependent manner, but a 30-minute exposure to ropivacaine or dexamethasone alone was not significantly toxic. Dexamethasone potentiated ropivacaine tenocyte toxicity at higher doses of ropivacaine, but did not potentiate lidocaine tenocyte toxicity. As seen in other cell types, lidocaine has a dose-dependent toxicity to tenocytes but ropivacaine is not significantly toxic. Although dexamethasone alone is not toxic, its combination with 0.5% ropivacaine significantly increased its toxicity to tenocytes. These findings might be relevant to clinical practice and warrant further investigation

The purpose of this study was to evaluate the beneficial effects of r-Irisin (IR) on human primary tenocytes (hTCs) in vitro. Indeed, Irisin is secreted from muscles in response to exercise and mediates many beneficial effects on tissues and organs. Tissue samples (n=3) were analyzed by histology and immunohistochemistry for αVβ5 receptor. hTCs isolated, culture expanded were treated with: 1) RPMI medium as control; 2) IR at different concentrations; 3) IL-1β; 4) pre-treated with IL-1β for 24 h and then co-treated with IR; 5) pre-treated with IR for 24 h and then co-treated with IL-1β. We evaluated: cell metabolic activity (MTT); cell proliferation (trypan blue staining and PicoGreen); nitrite concentration (Griess). The analysis were performed in triplicate for each donor and each experiment was repeated at least three times. Data were expressed as mean ± S.D. One-way ANOVA analysis was used to compare the groups under exam. We found the presence of the αVβ5 receptor on hTCs plasma membrane supporting the potential interaction with irisin. Cell proliferation was significantly increased with IR at 5, 10 and 25 ng/mL. IR 25 ng/mL after IL1β pre-treatment was able to counteract the increase of nitrite production (p < 0.001) compared to the inflamed hTCs (p < 0.01; p < 0.0001), as well as IR at 10 and 25 ng/ml showed a protective role from oxidative damage. We observed a significant increase in cell metabolic viability in culture under IR at 5 and 25 ng/mL (p < 0.001; p < 0.05) in the pre-treated IR groups, whereas IR showed anti-inflammatory effects at the highest concentration of r-Irisin (p < 0.05). This is the first study reporting the capability of irisin to attenuate tendinopathy in vitro by acting on acute inflamed tenocytes. Our results confirmed and highlighted the potential cross-talk mechanism between muscle and tendon

Aims. The purpose of this study was to evaluate the in vitro effects of apocynin, an inhibitor of nicotinamide adenine dinucleotide phosphate oxidase (NOX) and a downregulator of intracellular reactive oxygen species (ROS), on high glucose-induced oxidative stress on tenocytes. Methods. Tenocytes from normal Sprague-Dawley rats were cultured in both control and high-glucose conditions. Apocynin was added at cell seeding, dividing the tenocytes into four groups: the control group; regular glucose with apocynin (RG apo+); high glucose with apocynin (HG apo+); and high glucose without apocynin (HG apo–). Reactive oxygen species production, cell proliferation, apoptosis and messenger RNA (mRNA) expression of NOX1 and 4, and interleukin-6 (IL-6) were determined in vitro. Results. Expression of NOX1, NOX4, and IL-6 mRNA in the HG groups was significantly higher compared with that in the RG groups, and NOX1, NOX4, and IL-6 mRNA expression in the HG apo+ group was significantly lower compared with that in the HG apo– group. Cell proliferation in the RG apo+ group was significantly higher than in the control group and was also significantly higher in the HG apo+ group than in the HG apo– group. Both the ROS accumulation and the amounts of apoptotic cells in the HG groups were greater than those in the RG groups and were significantly less in the HG apo+ group than in the HG apo– group. Conclusion. Apocynin reduced ROS production and cell death via NOX inhibition in high-glucose conditions. Apocynin is therefore a potential prodrug in the treatment of diabetic tendinopathy. Cite this article:Bone Joint Res 2020;9(1):23–28

Objectives. The aim of this study was to investigate the effect of hyperglycaemia on oxidative stress markers and inflammatory and matrix gene expression within tendons of normal and diabetic rats and to give insights into the processes involved in tendinopathy. Methods. Using tenocytes from normal Sprague-Dawley rats, cultured both in control and high glucose conditions, reactive oxygen species (ROS) production, cell proliferation, messenger RNA (mRNA) expression of NADPH oxidase (NOX) 1 and 4, interleukin-6 (IL-6), matrix metalloproteinase (MMP)-2, tissue inhibitors of matrix metalloproteinase (TIMP)-1 and -2 and type I and III collagens were determined after 48 and 72 hours in vitro. In an in vivo study, using diabetic rats and controls, NOX1 and 4 expressions in Achilles tendon were also determined. Results. In tenocyte cultures grown under high glucose conditions, gene expressions of NOX1, MMP-2, TIMP-1 and -2 after 48 and 72 hours, NOX4 after 48 hours and IL-6, type III collagen and TIMP-2 after 72 hours were significantly higher than those in control cultures grown under control glucose conditions. Type I collagen expression was significantly lower after 72 hours. ROS accumulation was significantly higher after 48 hours, and cell proliferation after 48 and 72 hours was significantly lower in high glucose than in control glucose conditions. In the diabetic rat model, NOX1 expression within the Achilles tendon was also significantly increased. Conclusion. This study suggests that high glucose conditions upregulate the expression of mRNA for NOX1 and IL-6 and the production of ROS. Moreover, high glucose conditions induce an abnormal tendon matrix expression pattern of type I collagen and a decrease in the proliferation of rat tenocytes. Cite this article: Y. Ueda, A. Inui, Y. Mifune, R. Sakata, T. Muto, Y. Harada, F. Takase, T. Kataoka, T. Kokubu, R. Kuroda. The effects of high glucose condition on rat tenocytes in vitro and rat Achilles tendon in vivo. Bone Joint Res 2018;7:362–372. DOI: 10.1302/2046-3758.75.BJR-2017-0126.R2

Objectives. Platelet-rich fibrin matrix (PRFM) has been proved to enhance tenocyte proliferation but has mixed results when used during rotator cuff repair. The optimal PRFM preparation protocol should be determined before clinical application. To screen the best PRFM to each individual’s tenocytes effectively, small-diameter culture wells should be used to increase variables. The gelling effect of PRFM will occur when small-diameter culture wells are used. A co-culture device should be designed to avoid this effect. Methods. Tenocytes harvested during rotator cuff repair and blood from a healthy volunteer were used. Tenocytes were seeded in 96-, 24-, 12-, and six-well plates and co-culture devices. Appropriate volumes of PRFM, according to the surface area of each culture well, were treated with tenocytes for seven days. The co-culture device was designed to avoid the gelling effect that occurred in the small-diameter culture well. Cell proliferation was analyzed by water soluble tetrazolium-1 (WST-1) bioassay. Results. The relative quantification (condition/control) of WST-1 assay on day seven revealed a significant decrease in tenocyte proliferation in small-diameter culture wells (96 and 24 wells) due to the gelling effect. PRFM in large-diameter culture wells (12 and six wells) and co-culture systems induced a significant increase in tenocyte proliferation compared with the control group. The gelling effect of PRFM was avoided by the co-culture device. Conclusion. When PRFM and tenocytes are cultured in small-diameter culture wells, the gelling effect will occur and make screening of personalized best-fit PRFM difficult. This effect can be avoided with the co-culture device. Cite this article: C-H. Chiu, P. Chen, W-L. Yeh, A. C-Y. Chen, Y-S. Chan, K-Y. Hsu, K-F. Lei. The gelling effect of platelet-rich fibrin matrix when exposed to human tenocytes from the rotator cuff in small-diameter culture wells and the design of a co-culture device to overcome this phenomenon. Bone Joint Res 2019;8:216–223. DOI: 10.1302/2046-3758.85.BJR-2018-0258.R1

During in vitro sub-culturing, tenocytes lose their phenotype which ultimately affects their functioning. As spindle-shaped fibroblasts, tenocytes have a unique thin elongated phenotype and they possess more spread-out shape through phenomena named dedifferentiation1. Given the link between cell shape and cell function, in this study, we first aimed to dedifferentiate tenocytes through in vitro sub-culturing in order to have a model system for dedifferentiation. For this, we isolated human flexor tendon cells from healthy female flexor digitorum longus and seeded at 5000 cells/cm. 2. cell density, passaged every two days for six passages. In order to assess cell phenotype, we fixed with 4% paraformaldehyde and stained with phalloidin and DAPI to visualize the actin cytoskeleton and DNA respectively. We noted that in each passage, cells lost their spindle-shaped phenotype and became more pancake-shaped. At passage 1 and 2, the main cell phenotype is spindle-shaped. However, as the cells are further passaged, the phenotype of the cell population becomes more heterogeneous and at passage 5 and 6, they already display a more spread-out shape. Based on these results, we further hypothesized that they can be re-differentiated through matrix-mediated mechano-transduction and regain their morphology and function. For this aim, we generated decellularized tendon from porcine Achilles tendon and setup a mechanical loading system where we can provide mechanical loadings at physiological levels. This system will provide a new approach on in vitro tenocyte culturing

Introduction. The hierarchical structure of tendon results in a complex mechanical strain environment, with tenocytes experiencing both tension and shear during loading. The mechanotransduction mechanisms involved in sensing these environments is currently unclear. To better understand the effects of shear and tension on cell behaviour, a fibre composite system able to recapitulate the physiological shear-tension ratio found in tendons, was used. Cell attachment within the composite was achieved by using either a collagen type I mimetic peptide, DGEA, or a fibronectin associated peptide, YRGDS, and the gene expression response analysed after loading. Materials and Methods. Fibre composites with 4 different shear-tension (S-T) ratios were made using both PEG-DGEA and PEG-YRGDS fibres. 4 composites were made for each S-T ratio, of which 2 were loaded and 2 used as non-strained controls. Bovine digital extensor tendon tenocytes were seeded within composites, with 3 biological repeats from different donors. Loaded samples were exposed to 5% cyclic strain (1Hz) for 24 hours maintained in an incubator. The gene expression of 14 matrix related genes were analysed after loading via RT-qPCR. Results. Tenocytes seeded on PEG-DGEA fibres were more mechano-sensitive than those seeded on PEG-YRGDS fibres; tenocytes in PEG-DGEA composites exhibited upregulation of COL-3, MMP-3 and IL-6, and downregulation of SCX with shear, while tenocytes in PEG-YRGDS composites downregulated TIMP-3 with shear. Discussion. The main integrin involved in DGEA binding is α2β1 while the integrins associated with YRGDS attachment include α5β1, αVβ3 and αIIbβ3. Consequently, the findings of this study emphasise the importance of integrins in the role of mechanotransduction, and suggest integrins involved in collagen type I binding induce functionally different responses in tenocytes to those not involved in collagen type I binding when sensing mechanical stimuli comprised of shear and tension. This information is critical in future studies investigating tenocyte behaviour and tissue engineering approaches, as physiological integrin binding may be key in maintaining normal tenocyte pathways

Introduction. The use of platelet-rich concentrate (PRC) to enhance the healing response in tendon repair is currently an area of considerable interest. Activated platelets release a cocktail of growth factors and ECM regulating molecules. Previous work suggests that tenocytes are activated by contact with these clot-derived molecules. Our studies on tenocytes and PRC aim to establish the direct molecular and functional effects of PRC on tenocytes and to support the clinical research on Achilles tendon repair taking place within our group. We hypothesise that applying PRC to human tenocytes in culture will increase proliferation rate and survival by activating relevant signalling pathways. Materials and Methods. Using a centrifugation method, PRC was extracted from fresh human whole blood. The PRC was immediately clotted and left in medium overnight to release biological factors (at least 95% of presynthesized growth factors are secreted in the first hour of activation). 1. Human tenocytes derived from explanted healthy hamstring were used for up to three passages. Cells were treated with varying concentrations of PRC-conditioned medium and assessed for viable cell number (Alamar Blue™ fluorescence) and proliferation (Ziva™ Ultrasensitive BrdU assay) after 72hrs. For western blotting, cells were treated with 10% PRC for 5 or 30 minutes. Antibodies to P-ERK and P-Akt detected the active protein state on the blot, followed by membrane stripping and re-probing with pan antibodies. Quantification was achieved by densitometry using Visionworks software v. 6.7.1. Results. PRC-conditioned medium affected tenocytes in a dose-dependent manner. Viable number of tenocytes was significantly increased by 10% PRC-conditioned medium compared to controls (One-way ANOVA, Tukey's post-hoc test P<0.001) after 72hrs. 10% PRC-conditioned medium also demonstrated time-dependence with viable tenocyte number significantly increasing between 24 and 72hrs (One-way ANOVA, Bonferroni's post-hoc test P<0.001). After 72hrs, tenocyte proliferation significantly increased in the presence of 5% and 10% PRC-conditioned media compared to controls (One-way ANOVA, Tukey's post-hoc test P<0.05 and P<0.001 respectively). ERK and Akt phosphorylation was strongly stimulated by treatment with 10% PRC-conditioned medium for 5 minutes compared to controls, and remained high after a 30 minute application time. Discussion and Conclusions. Factors released by activated PRC act upon human tendon cells to strongly increase viable cell number and proliferation, which would, in vivo, directly support the healing response, independent of any additional beneficial effects on vascular repair. Both ERK and Akt are pivotal kinases in signalling pathways that favour survival and proliferation. It is clear that both signalling pathways are immediately and strongly activated by PRC, suggesting a clear benefit via both stimulated cell cycle and cell survival in the environmentally compromised conditions of a healing ruptured tendon. This conclusion is strongly supported by previous work on platelet releasates and ERK signalling in other cell types

Introduction. Tendinopathies are debilitating and painful conditions. They are believed to result from repetitive overuse, which can create micro-damage that accumulates over time, and initiates a catabolic cell response. The aetiology of tendinopathy remains poorly understood, therefore the ideal treatment remains unclear. However, current data support the use of eccentric exercise as an effective treatment. In a previous study, we have shown that eccentric loading generates perturbations in the tendon at 10Hz, which is not present during other less effective loading regimes. Consequently, we hypothesis that 10Hz loading initiates an increased anabolic response in tenocytes, that can promote tendon repair. Materials and Methods. Human tenocytes from healthy hamstring tendons and tendinopathic Achilles tendons were derived by collagenase digest and outgrowth respectively. Tenocytes were seeded into 3D collagen gels. The gels were fixed in custom-made chambers and placed in an incubator for 24hrs whilst gene expression stabilised. After 24hrs, cyclic uniaxial strain at 1% ± 1% was applied to the cells, at either 1Hz (n=4) or 10Hz (n=4) using a Bose loading system. After 15 minutes of cyclic strain, the samples were maintained in chambers under 1% static strain for 24 hrs after which gene expression was characterised using RT-PCR. Results. In healthy cells, data showed an increase in expression of all analysed genes after loading (MMP1, MMP2, MMP13, COL1A1, COL3A1, COL5A1, ADAMTS5, IL6, IL8 and TIMP3). Furthermore, the increase in gene expression was larger in the higher frequency loading group, across all genes. Tendinopathic cells showed a more varied response, with upregulation of MMP1, MMP13, COL3A1, ADAMTS5, IL6 and IL8, and downregulation of COL1A1 and COL5A1. Once again, all changes were more pronounced in the higher frequency loading group. Discussion. These preliminary data suggest increased matrix turnover as a result of loading, particularly with high frequency loading in healthy tenocytes, whilst the profile of tendinopathic cells, may indicate an early healing response, where collagen type III is preferentially unregulated relative to types I and V. High frequency loading elicited a more pronounced cellular response, perhaps correlating with the improved repair seen with eccentric loading in vivo

Introduction and Aims: Large or recurrent rotator cuff tendon tears are difficult to treat effectively. Collagen bio-scaffolds have become available to reinforce a tendon repair or as an interpositional graft. This study compares the suitability of two collagen bio-scaffolds for autologous tenocyte implantation, and assesses the in vivo rotator cuff healing response with these grafts in a rabbit model. Method: Tenocytes were isolated from rabbit tendon, cultured and seeded onto the Restore patch (DePuy), or the Matricel (Verigen) collagen membrane. Serial scanning electron microscopy examined tenocyte integration with the bio-scaffold, and extra-cellular matrix synthesis over time. A rotator cuff tendon defect was created in 50 rabbits and repaired by either: a) direct suture to tuberosity; b) Matricel interposition graft; c) Matricel interposition with autologous tenocytes; d) Restore patch interposition graft; e) Restore patch interposition with autologous tenocytes. Gross and histological evaluation were performed at four weeks and eight weeks post-surgery. Results: Scanning electron microscopy of the Matricel membrane showed a rough surface characterised by a loose arrangement of collagen fibres capable of cell adhesion. SEM at one, three and five days after cell seeding, showed progressive integration of tenocytes into the three-dimensional membrane structure with extra-cellular matrix neosynthesis in the spaces between the native collagen fibres. SEM of the Restore patch showed a relatively smooth surface of highly compacted collagen fibres. Serial SEM after cell seeding showed relatively less tenocyte integration onto the membrane surface though tenocyte replication and matrix neo-synthesis was observed. All 50 rabbits regained normal gait at two weeks post-surgery. At sacrifice, no tendon ruptures had occurred at either time point in any of the five groups. At four weeks, the Matricel and Restore bio-scaffold membranes were partially absorbed, and a florid lymphocytic inflammatory response was evident surrounding the remaining membrane. By eight weeks, graft tissue had been resorbed further, the inflammatory response had decreased, and the regenerating tendon showed progressive remodelling. Autologous tenocyte implantation on both membranes improved the reparative tendon histological grade at eight weeks compared to membranes without cell implantation, and was equivalent to the direct repair group. Conclusion: Autologous tenocytes can be implanted onto both Matricel and Restore collagen bio-scaffolds. Though both Xeno grafts induce an anti-inflammatory response in vivo, membrane resorption subsequently occurs. The healing response of large rotator cuff defects treated with interpositional collagen grafts is improved with autologous tenocyte implantation in a rabbit model

Introduction: Tendon ruptures are increasingly common, repair can be difficult and healing poorly understood. Tissue engineering approaches often require expansion of cell numbers to populate a construct, and maintenance of cell phenotoype is essential for tissue regeneration. Methods: In this study we characterise the phenotype of human Achilles tenocytes and assess how this is affected by passaging. Tenocytes, isolated from tendon samples from 6 patients receiving surgery for rupture of the Achilles tendon, were passaged 8 times. Proliferation rates and cell morphology were recorded at passages 1, 4 and 8. Total collagen, the ratio of collagen types I and III and decorin were used as indicators of matrix formation and expression of the integrin ‘alpha’1 subunit as a marker of cell-matrix interactions. Results: With increasing passage number, cells became more rounded, were more widely spaced at confluence and confluent cell density declined from 18700 /cm2 to 16100 /cm2 (P=0.009). No change to total cell layer collagen was observed but the ratio of type III to type I collagen increased from 0.60 at passage 1 to 0.89 at passage 8 (P< 0.001). Decorin expression significantly decreased with passage number, from 22.9 ± 3.1 ng/ng DNA at passage 1, to 9.1 ± 1.8 ng/ngDNA at passage 8 (P< 0.001). Integrin expression did not change. Conclusion: We conclude that the phenotype of tenocytes in culture rapidly drifts with progressive passage

Introduction. We previously reported that disruption of TGFβ signaling in limb mesenchyme resulted in complete failure of tendon differentiation. Materials and Methods. To bypass this early function and study additional roles of TGFβ signaling in tendon development we disrupted TGFβ signaling in tenocytes after they assumed the tendon cell fate by using the tendon deletor ScxCre to target the floxed type2 TGFβ receptor. Results. Most mutant (Tgfbr2;ScxCre) pups appeared normal at birth but exhibited movement difficulties and splayed limbs by P3. ScxGFP signal revealed that tendon formation was not affected in CKO embryos. Nonetheless, three distinct tendon phenotypes were manifested later in development: (a) a single flexor tendon consistently snapped at late embryonic stage; whereas at post-natal stage, some tendons that appeared intact at birth were (b) eventually eliminated or (c) retained structural integrity with a substantial loss of the ScxGFP signal. Interestingly, the ScxGFP-negative cells also lost other tendon marker genes. Lineage tracing revealed that these cells were derived from Scx-expressing cells, suggesting a disruption of the tendon cell fate (dedifferentiation) but we found no evidence of transdifferentiation. Varying degrees of tendon degeneration were also seen in CKO pups, as indicated by disrupted collagen fibrils, septation of the tendon and altered epitenon. Another striking feature we identified in the Tgfbr2;ScxCre tendon phenotype was recruitment of new cells into the degenerating tendon. Finally, our data also indicates that the Tgfbr2f;ScxCre tendon phenotype is not due to a direct requirement for TGFβ signaling in tenocytes. Discussion. This analysis thus highlights an unexpected possibility for loss of differentiated characteristics in tenocytes as a key factor in a tendon degenerative process. We hypothesize that the tendon phenotypes may represent a disruption of cell-cell or cell-matrix interactions, and investigations are currently underway to test this hypothesis. Moreover, this is the first demonstration of active cell recruitment into a non-injured tendon that may be used to identify the origin and activation mechanisms for tendon stem/progenitor cells. Taken together, our findings reveal an essential and non-cell autonomous role of TGFβ signaling in maintenance of the tendon cell fate

Large and retracted rotator cuff tendon tears fail to repair, or re-tear following surgical intervention. This study attempted to develop novel tissue engineering approaches using tenocytes-seeded bioscaffolds for tendon reconstruction of massive rotator cuff tendon defect in rabbits. Porcine small intestine submucosa (Restore™) and type I/III collagen bioscaffold (ACI-MaixTM) were chosen as bioscaffold carriers for autologous tenocytes. Biological characterization of autologous tenocytes was conducted prior to the implantation. The tenocyte-seeded bioscaffolds were implanted as interposition grafts to reconstruct massive rotator cuff tendon defects in rabbits. In situ re-implantation of the autologous rotator cuff tendon, excised during defect creation served as a positive control. Histological outcomes were analysed and semi-quantitatively graded at four and eight weeks after surgery. The results demonstrate that at four weeks both tenocyte-seeded bioscaffolds display inflammatory reaction similar to bioscaffold-only cuff reconstruction and the histological grading were inferior to control repair. However, at eight weeks inflammatory reaction of both tenocyte-seeded bioscaffolds were dramatically reduced as compared to bioscaffold alone. In addition, bioscaf-folds seeded with tenocytes generated similar histological appearance to that of the positive control. The implantation of autologous tenocytes on collagen-based bioscaffold offers improved rotator cuff tendon healing and remodelling compared to the implantation of bioscaffold alone

Elastic energy-storing tendons including the equine superficial digital flexor tendon (SDFT) and human Achilles tendon significantly increase locomotor efficiency, but suffer high injury rates and experience increased core temperatures during exercise. Tenocytes are linked by gap junctions (GJ) comprised of connexin (Cx) proteins that mediate intercellular communication and are necessary for strain-induced collagen synthesis. The effects of hyperthermia on gap junction intercellular communication (GJIC) are unknown. We investigated the hypothesis that there is a down regulation in GJIC and Cx protein by tenocytes in response to hyperthermic episodes similar to those experienced in the SDFT of galloping horses. Monolayers of equine SDFT-derived cells were heated to 45°C for 10 minutes. GJIC, Cx43 and Cx32 protein expression and cell viability were measured by fluorescence recovery after photobleaching (FRAP) and immunofluorescent labelling respectively. There was a marked reduction in GJIC (p=< 0.0001) compared with controls (37oC) at 30min and 1h post-heating, with significant recovery by 2h (p=< 0.0001). The number of Cx43 plaques/cell also decreased significantly at 30mins (p=< 0.05), 1h (p=< 0.0001) and 2h (p=< 0.0001). There was however a 3-fold increase in the number of Cx32 plaques/cell at 1h (p=< 0.0001) that returned to normal by 2h. There was little change in cell viability up to 2h, however by 24h post-heating there was an 80% decrease in cell number indicating significant levels of cell death (p=< 0.0001). Reductions in GJIC following exercise-induced hyperthermia may inhibit tenocyte collagen synthesis. Connexin isotypes may differentially modulate tenocyte collagen synthesis, therefore the dissimilar alterations in Cx43 and Cx32 following heating could have functional importance. The return of GJIC a few hours post-heating might facilitate spread of apoptotic death signals, killing neighbouring cells which would have otherwise escaped death. Understanding the responses of GJ to increased temperature, and the effects of this on viability and collagen synthetic capacity is likely to increase our knowledge of how exercise-induced SDFT core degeneration accumulates

Autologous tendon cell injection (ATI) is a promising non-surgical treatment for tendinopathies and tendon tear that address its underlying pathology. The procedure involves harvesting autologous tendon tissue, the isolation of the tendon cells, expansion under quality assured GMP cell laboratory and the injection of the tendon cells via U/S into the degenerative tendon tissue. In clinical practice, the patella (PT) and palmaris longus (PL) tendons are common sites used for tendon tissue biopsy. The objective of this study is to compare the tendon cell quality, identity, purity, doubling time and yield of cells between PT and PL tendons for ATI.

Tendon tissue biopsies were harvested from PT via U/S using a 14-gauge needle or resected surgically from the PL tendon. The biopsies were transported to a GMP cell laboratory, where tendon cells were isolated, cultured and expanded for 4 to 6 weeks, and analysed for viability, cell doubling time, cellular characteristics including cell purity, potency and identity (PPI).

Tendon samples from 149 patients were analysed (63 PT). Average biopsy weight was 62mg for PT and 119mg for PI (p<0.001). Average cell doubling time (83.9 vs 82.7 hours), cellular yield (16.2 vs 15.2x106), viability (98.7 vs 99.0%) and passage number (3 vs 3) were not significantly different between tendons. Additionally, ddPCR analyses showed no differences of PPI including tendon cell markers of collagen type I, scleraxis and tenomodulin. No post-biopsy complications or contamination were reported for either group. Assessing tendon tissue from palmaris tendon is relatively easier.

Tendon tissue biopsy tissue for autologous tendon cell therapy can be obtained from either the PT or PL tendons. Tendon cells isolated from PT and PL were equal in growth characteristics and PPI. There are no differences in the quality of tendon cells isolated from the PT or PL.

The term macromolecular crowding is used to describe equilibria and kinetics of biochemical reactions and biological processes that occur via mutual volume exclusion of macromolecules in a highly crowded structureless medium. In vivo, the extracellular space is heavily crowded by a diverse range of macromolecules and thus, biological processes occur rapidly, whilst in vitro, in the absence of macromolecules, the same processes occur very slowly, if they are initiated at all (1-3). This talk will discuss the concept of macromolecular crowding, alone or in combination with other in vitro microenvironment modulators, in tendon engineering context.

Acknowledgements: This work has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme, grant agreement No. 866126. This publication has emanated from research supported by grants from Science Foundation Ireland (SFI) under grant number 19/FFP/6982.

Purpose. Platelet-Rich Plasma (PRP), an autologous derivative of whole blood that contains a supraphysiological concentration of platelets and growth factors. Most published studies have investigated the effect of PRP-conditioned media on cell cultures. We are not aware of any study that has investigated whole PRP with its cellular components on human tissue cultures. This study aims to investigate the effect of PRP on cell migration from human Achilles tendon explants, and the subsequent cellular proliferative effects in culture. Methods. This is an in-vitro study on tendon explants obtained from Achilles tendon rupture patients. The samples were collected in sterile DMEM F12 solution then carefully cut into approximately 1–3mm. 3. sections. Tendon explants were cultured in three media types: 1. 100% PRP; 2. 50% PRP; and 3. 50% fetal calf serum (FCS). 1 and 2 were made up using DMEM F12 media (standard culture medium). Explants and cells were incubated at 37°c in 5% CO. 2. for 48 hours. Results. Images of the explanted tissue were taken using a Nikon TE300 microscope with Retiga CCD camera and cells around each explant were counted. Kruskal-Wallis statistical test showed that 100%PRP and 50%PRP cultured explants have significantly higher number of cells (p ≤0.002 and 0.028 respectively) when compared with 50%FCS cultured explants. Ziva ultrasensitive proliferation assay revealed that 100%PRP significantly increased cell proliferation. In addition, PicoGreen assay showed that DNA content of 100% PRP cultured cells were significantly higher than the control. The concentration of TGF-b1, VEGF, PDGF-AB and IGF-1 growth factors were significantly higher in PRP comparing to 50% FCS medium. Conclusion. Our findings show that whole PRP strongly affect the behaviour of human tenocytes, indicating that PRP may have potential role as an orthobiological agent in ruptured tendon treatments

Interstitial supraspinatus tears can cause persistent subacromial impingement symptoms despite non operative treatment. Autologous tendon cell injection (ATI) is a non-surgical treatment for tendinopathies and tear. We report a randomised controlled study of ATI compared to corticosteroid injection (CS) as treatment for interstitial supraspinatus tears and tendinopathy.

Inclusion criteria were patients with symptom duration > 6 months, MRI confirmed intrasubstance supraspinatus tear, and prior treatment with physiotherapy and ≥ one CS or PRP injection. Participants were randomised to receive ATI to the interstitial tear or corticosteroid injection to the subacromial bursa in a 2:1 ratio, under ultrasound guidance. Assessments of pain (VAS) and function (ASES) were performed at baseline, and 1, 3, 6 and 12 months post treatment.

30 participants (19 randomised to ATI) with a mean age of 50.5 years (10 females) and a mean duration of symptoms of 23.5 months. Baseline VAS pain and ASES scores were comparable between groups. While mean VAS pain scores improved in both groups at 3 months after treatment, pain scores were superior with ATI at 6 months (p=0.01). Mean ASES scores in the ATI group were superior to the CS group at 3 months (p=0.026) and 6 months (p=0.012). Seven participants in the CS group withdrew prior to 12 months due to lack of improvement. At 12 months, mean VAS pain in the ATI group was 1.6 ± 1.3. The improvements in mean ASES scores in the ATI group at 6 and 12 months were greater than the MCID (12.0 points). At 12 months, 95% of ATI participants had an ASES score > the PASS (patient acceptable symptom state).

This is the first level one study using ATI to treat interstitial supraspinatus tear. ATI results in a significant reduction in pain and improvement in shoulder function.

Summary:

Hamstring tendons (HT) represent a widely used autograft for ACL reconstruction. Harvesting, processing and pretensioning procedures together with the time out of the joint could theoretically hamper tendon cells (TCs) viability. The authors hypothesize that HT cells are not impaired at the end of the surgical procedures and their tenogenic phenotype may be strongly improved by exposure to PEMF.

Aims. The effects of remnant preservation on the anterior cruciate ligament (ACL) and its relationship with the tendon graft remain unclear. We hypothesized that the co-culture of remnant cells and bone marrow stromal cells (BMSCs) decreases apoptosis and enhances the activity of the hamstring tendons and tenocytes, thus aiding ACL reconstruction. Methods. The ACL remnant, bone marrow, and hamstring tendons were surgically harvested from rabbits. The apoptosis rate, cell proliferation, and expression of types I and III collagen, transforming growth factor-β (TGF-β), vascular endothelial growth factor (VEGF), and tenogenic genes (scleraxis (SCX), tenascin C (TNC), and tenomodulin (TNMD)) of the hamstring tendons were compared between the co-culture medium (ACL remnant cells (ACLRCs) and BMSCs co-culture) and control medium (BMSCs-only culture). We also evaluated the apoptosis, cell proliferation, migration, and gene expression of hamstring tenocytes with exposure to co-culture and control media. Results. Compared to BMSCs-only culture medium, the co-culture medium showed substantially decreased early and late apoptosis rates, attenuation of intrinsic and extrinsic apoptotic pathways, and enhanced proliferation of the hamstring tendons and tenocytes. In addition, the expression of collagen synthesis, TGF-β, VEGF, and tenogenic genes in the hamstring tendons and tenocytes significantly increased in the co-culture medium compared to that in the control medium. Conclusion. In the presence of ACLRCs and BMSCs, the hamstring tendons and tenocytes significantly attenuated apoptosis and enhanced the expression of collagen synthesis, TGF-β, VEGF, and tenogenic genes. This in vitro study suggests that the ACLRCs mixed with BMSCs could aid regeneration of the hamstring tendon graft during ACL reconstruction. Cite this article: Bone Joint Res 2023;12(1):9–21

Tendinopathy is a tendon pathology often resulting from a failed healing response to tendon injury. Activated protein C (APC) is a natural anti-coagulant with anti-inflammatory and wound healing promoting functions, which are mainly mediated by its receptors, endothelial protein C receptor (EPCR) and protease activated receptors (PARs). This study aimed to determine whether APC stimulates tenocyte healing and if so, to assess the involvement of the receptors. Mouse-tail tenocytes were isolated from 3-week-old wild type (WT), PAR- 1 knockout (KO) and PAR-2 KO mice. The expression of EPCR, PAR-1 and −2 and the effect of APC on tenocytes tendon healing and the underlying mechanisms were investigated by Reverse transcription real time PCR, western blot, 3- (4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay, zymography, and scratch wound healing/ migration assay. When compared to WT cells, PAR-1 KO tenocytes showed increased cell proliferation (3.3-fold, p<0.0001), migration (2.7-fold, p<0.0001) and wound healing (3-fold, p<0.0001), whereas PAR-2 KO cells displayed decreased cell proliferation (0.6-fold, p<0.05) and no change in cell migration or wound healing. APC at 1 μg/ml stimulated WT and PAR-1 KO tenocyte proliferation (~1.3, respectively, p<0.05) and wound healing (~1.3-fold, respectively, p<0.05), and additionally promoted PAR1-KO cell migration (1.4-fold, p<0.0001). APC only increased the migration (2-fold, p<0.05) of PAR-2 KO tenocytes. The activation of AKT, extracellular signal-regulated kinase (ERK)-2, and glycogen synthase kinase (GSK)-β3, the intracellular molecules that are associated with cell survival/growth, and matrix metalloproteinase (MMP)-2 that is related to cell migration and wound healing, were increased in all three cell lines in response to APC treatment. These findings show that PAR-1 and PAR-2 act differentially in tenocyte proliferation/migration/wound healing. APC likely promotes tenocyte proliferation/ wound healing via PAR-2, not PAR-1

During aging, tendons demonstrate substantial disruptions in homeostasis, leading to impairments in structure-function. Impaired tendon function contributes to substantial declines quality of life during aging. Aged tendons are more likely to undergo spontaneous rupture, and the healing response following injury is impaired in aged tendons. Thus, there is a need to develop strategies to maintain tendon homeostasis and healing capacity through the lifespan. Tendon cell density sharply declines by ∼12 months of age in mice, and this low cell density is retained in geriatric tendons. Our data suggests that this decline in cellularity initiates a degenerative cascade due to insufficient production of the extracellular matrix (ECM) components needed to maintain tendon homeostasis. Thus, preventing this decline in tendon cellularity has great potential for maintaining tendon health. Single cell RNA sequencing analysis identifies two changes in the aged tendon cell environment. First, aged tendons primarily lose tenocytes that are associated with ECM biosynthesis functions. Second, the tenocytes that remain in aged tendons have disruptions in proteostasis and an increased pro-inflammatory phenotype, with these changes collectively termed ‘programmatic skewing'. To determine which of these changes drives homeostatic disruption, we developed a model of tenocyte depletion in young animals. This model decreases tendon cellularity to that of an aged tendon, including decreased biosynthetic tenocyte function, while age-related programmatic skewing is absent. Loss of biosynthetic tenocyte function in young tendons was sufficient to induce homeostatic disruption comparable to natural aging, including deficits in ECM organization, composition, and material quality, suggesting loss biosynthetic tenocytes as an initiator of tendon degeneration. In contrast, our data suggest that programmatic skewing underpins impaired healing in aged tendons. Indeed, despite similar declines in the tenocyte environment, middle-aged and young-depleted tendons mount a physiological healing response characterized by robust ECM synthesis and remodeling, while aged tendons heal with insufficient ECM

To address the current challenge of anterior cruciate ligament (ACL) reconstruction, this study is the first to fabricate a braided collagen rope (BCR) which mimics native hamstring for ACL reconstruction. The study aims to evaluate the biological and biomechanical properties of BCR both in vivo and vitro. Rabbit ACL reconstruction model using collagen rope and autograft (hamstring tendon) was conducted. The histological and biomechanical evaluations were conducted at 6-, 12-, 18, 26-week post-operation. In vitro study included cell morphology analysis, cell function evaluation and RNA sequencing of the tenocytes cultured on BCR. A cadaver study was also conducted to verify the feasibility of BCR for ACL reconstruction. BCR displays satisfactory mechanical strength similar to hamstring graft for ACL reconstruction in rabbit. Histological assessment showed BCR restore ACL morphology at 26 weeks similar to native ACL. The superior dynamic ligamentization in BCR over autograft group was evidenced by assessment of cell and collagen morphology and orientation. The in vitro study showed that the natural collagen fibres within BCR enables to signal the morphology adaptation and orientation of human tenocytes in bioreactor. BCR enables to enhance cell proliferation and tenogenic expression of tenocytes as compared to hydrolysed collagen. We performed an RNA-Sequencing (RNA-seq) experiment where RNA was extracted from tenocyte seeded with BCR. Analysis of enriched pathways of the up-regulated genes revealed that the most enriched pathways were the Hypoxia-inducible factor 1-alpha (HIF1A) regulated networks, implicating the possible mechanism BCR induced ACL regeneration. The subsequent cadaver study was conducted to proof the feasibility of BCR for ACL reconstruction. This study demonstrated the proof-of-concept of bio-textile braided collagen rope for ACL reconstruction, and the mechanism by which BCR induces natural collagen fibres that positively regulate morphology and function of tenocytes

Introduction and Objective. Achilles tendon defect is difficult problem for orthopedic surgeon, and therefore the development of new treatments is desirable. Platelet-rich fibrin (PRF), dense fibrin scaffold composed of a fibrin matrix containing many growth factors, is recently used as regenerative medicine preparation. However, few data are available on the usefulness of PRF on Achilles tendon healing after injury. The objective of this study is to examine whether PRF promotes the healing of Achilles tendon defect in vivo and evaluated the effects of PRF on tenocytes in vitro. Materials and Methods. PRF were prepared from rats according to international guidelines on the literature. To create rat model for Achilles tendon defect, a 4-mm portion of the right Achilles tendon was completely resected, and PRF was placed into the gap in PRF group before sewing the gap with nylon sutures. To assess the histological healing of Achilles tendon defect, Bonar score was calculated using HE, Alcian-blue, and Picosirius-red staining section. Basso, Beattie, Bresnahan (BBB) score was used for the evaluation of motor functional recovery. Biomechanical properties including failure tensile load, ultimate tensile stress, breaking elongation, and elastic modulus were measured. We examined the effects of PRF on tenocytes isolated from rat Achilles tendon in vitro. The number of viable cells were measured by MTS assay, and immunostaining of ki-67 was used for detection of proliferative cells. Migration of tenocytes was evaluated by wound closure assay. Protein or gene expression level of extracellular matrix protein, such as collagen, were evaluated by immunoblotting, immunofluorescence, or PCR. Phosphorylation level of AKT, FGF receptor, or SMAD3 was determined by western blotting. Inhibitory experiments were performed using MK-2206 (AKT inhibitor), FIIN-2 (FGFR inhibitor), SB-431542 (TGF-B receptor inhibitor), or SIS3 (SMAD3 inhibitor). All p values presented are two-sided and p values < 0.05 were considered statistically significant. Results. In rat Achilles tendon defects, Bonar score was significantly improved in PRF group compared to control group. Collagen deposition at the site of Achilles tendon defect was observed earlier in PRF group. Consistent with the histological findings, BBB score was significantly improved in PRF group. PRF also significantly improved the biomechanical properties of injured Achilles tendon. Furthermore, proliferating tenocytes, labelled by ki-67 were significantly increased in PRF group. These data suggested PRF prompted the healing of Achilles tendon defect. Thus, we further examined the effects of PRF on tenocytes in vitro. PRF significantly increased the number of viable cells, the proliferative cells labelled by ki-67, and migratory ability. Furthermore, PRF significantly increased the protein expression levels of collagen-I, collagen-III, α-SMA, and tenascin-C in tenocytes. Next, we examined the signalling pathway associated with PRF-induced proliferation of tenocytes. PRF increased the phosphorylation level and induced nuclear translocation of AKT, known as key regulator of cell survival. PRF also induced the phosphorylation of FGF receptor. Inhibition of AKT or FGF-receptor completely suppressed the positive effects of PRF on tenocytes. Furthermore, we found that inhibition of FGF receptor partially suppressed the phosphorylation of AKT by PRF. Thus, PRF induced the proliferation of tenocytes via FGFR/AKT axis. We further evaluated the signalling pathway associated with PRF-induced expression of extracellular matrix. PRF increased the phosphorylation levels of SMAD3 and induced nuclear translocation of SMAD3. Furthermore, inhibition of TGF-B receptor or SMAD3 suppressed increased expression level of extracellular matrix by PRF. Thus, PRF increased expression level of extracellular matrix protein via TGF-BR/SMAD3 axis. Conclusions. PRF promotes tendon healing of the Achilles tendon defect and recovery of exercise performance and biomechanical properties. PRF increases the proliferation ability or protein expression level of extracellular matrix protein in tenocytes via FGFR/AKT or TGF-βR/SMAD3 axis, respectively

Introduction and Objective. The rupture of the anterior cruciate ligament is a common sports injury and surgical reconstruction is often required to restore full function of the knee. Hamstring tendons are usually used as autografts. In addition to knee pain and stiffness, infections are feared complications after surgery. Incubation of the autograft in a vancomycin solution until implantation reduced the infection rate by about ten-fold. Recent studies showed no negative effect of vancomycin on the biomechanical properties of porcine tendons. A negative effect of high vancomycin concentrations on chondrocytes and osteoblast is reported, but the effect on tendon and tenocytes is not known. Materials and Methods. Rat Achilles tendons or isolated tenocytes were incubated with an increasing concentration of vancomycin (0 – 10 mg). Tendons were incubated for 0 – 40 minutes, while tenoyctes were incubated for 20 minutes followed by culturing for up to 7 days. Cell viability was assessed with PrestoBlue Assay and live/dead stain. The potential effect of vancomycin on the expression of tendon specific genes and extracellular matrix (ECM) genes was quantified. Possible structural changes of the tendon are analyzed. Results. Incubation of the tendons or tenocytes with 5 mg vancomycin for 20 minutes (clinical use) had no negative effects on the cell viability in the tendons or the isolated tenocytes, while incubation with the toxic control (ethanol) significantly reduced cell viability. Even twice the concentration and a longer incubation time had no negative effect on the cells in the tendons or the isolated cells. Vancyomycin did not affect the expression of Col1a1, Col3a1, and the tenocyte markers mohawk, scleraxis and tenomodulin. Conclusions. The results showed that clinical practice of wrapping the autograft in vancomycin did not impair the tenocyte viability. The expression of collagens and tenocyte markers was also not affected, neither in the incubated tendons nor in the isolated cells. This indicates that vancomycin had no effect on cell phenotype and the formation of the extracellular matrix, which, in addition to cell viability, is important for the performance of the autograft

Early identification of patients at risk for impaired tendon healing and corresponding novel therapeutic approaches are urgent medical needs. This study aimed to clarify the role of CD3+ T-cells during acute Achilles tendon (AT) healing. Blood and hematoma aspirate were taken from 26 patients during AT reconstruction, and additional blood samples were obtained during clinical follow-up at 6, 26 and 52 weeks after surgery. T-cell subsets were analyzed by flow cytometry using CD3, CD4, CD8, CD11a, CD57 and CD28 antibodies. Clinical follow-up included functional tests, MRI assessments, and subjective questionnaires. In vitro, the functional behavior of patient-derived tenocytes was investigated in co-cultures with autologous unpolarized CD4+ or CD8+ T-cells, or IFNy-polarized CD8+ or IL17-polarized CD4+ Tcells (n=5-6). This included alterations in gene expression (qPCR), MMP secretion (ELISA), migration rate (scratch wound healing assay) or contractility (collagen gels). Analysis revealed that elevated CD4+ T-cell levels and reduced CD8+ T-cell levels (increased CD4/CD8 ratio) in hematoma aspirate and pre-operative blood were associated with inferior clinical outcomes regarding pain and function at 26 and 52 weeks. Increased levels of CD8+ -memory T-cell subpopulations in blood 6 weeks after surgery were associated with less tendon elongation. In vitro, tenocytes showed increased MMP1/2/3 levels and collagen III/I ratio in co-culture with unpolarized and/or IL17-polarized CD4+ T-cells compared to unpolarized CD8+ T-cells. This coincided with increased IL17 receptor expression in tenocytes co-cultured with CD4+ T-cells. Exposure of tenocytes to IL17-polarized CD4+ T-cells decreased their migration rate and increased their matrix contractility, especially compared to IFNy-polarized CD8+ T-cells. The CD4+ /CD8+ T-cell ratio could serve as prognostic marker for early identification of patients with impaired AT healing potential. Local reduction of CD4+ T-cell levels or their IL17 secretion represent a potential therapeutic approach to improve AT healing and to prevent weakening of the tendon ECM

Tendon injuries present a major clinical challenge, as they necessitate surgical intervention and are prone to fibrotic progression. Despite advances in physical therapy and surgical technique, tendons fail to return to full native functioning, underlining the need for a biological therapeutic to improve tendon healing. Myofibroblasts are activated fibroblasts that participate in the proliferative and remodeling phases of wound healing, and while these matrix-producing cells are essential for proper healing, they are also linked to fibrotic initiation. A subset of tenocytes has been shown to give rise to the myofibroblast fate, and potentially contribute to fibrotic tendon healing. A viable anti-fibrotic therapy in other tissues has been reprogramming the fibroblast-myofibroblast differentiation route, avoiding a more pro-fibrotic myofibroblast phenotype. Thus, defining the molecular programs that underlie both physiological and pathological tendon healing is critical for the development of potential pharmacologic treatments. Towards that end, we have taken advantage of spatial transcriptomics, using the tenocyte marker Scleraxis as a tool, and have outlined three major spatiotemporally distinct tenocyte differentiation trajectories (synthetic, proliferative, and reactive) following acute tendon injury in mouse FDL. We have further outlined key transcriptional controls that may be manipulated to alter the differentiation process and influence the resulting myofibroblast phenotype, thereby promoting regenerative tendon healing

Depletion of Scleraxis-lineage (ScxLin) cells in adult tendon recapitulates age-related decrements in cell density, ECM organization and composition. However, depletion of ScxLin cells improves tendon healing, relative to age-matched wildtype mice, while aging impairs healing. Therefore, we examined whether ScxLin depletion and aging result in comparable shifts in the tendon cell environment and defined the intrinsic programmatic shifts that occur with natural aging, to define the key regulators of age-related healing deficits. ScxLin cells were depleted in 3M-old Scx-Cre+; Rosa-DTRF/+ mice via diphtheria toxin injections into the hindpaw. Rosa-DTRF/+ mice were used as wildtype (WT) controls. Tendons were harvested from 6M-old ScxLin depleted and WT mice, and 21-month-old (21M) C57Bl/6 mice (aged). FDL tendons (n=6) were harvested for single-cell RNAseq, pooled, collagenase digested, and sorted for single cell capture. Data was processed using Cell Ranger and then aligned to the annotated mouse genome (mm10). Filtering, unsupervised cell clustering, and differential gene expression (DEG) analysis were performed using Seurat. Following integration and sub-clustering of the tenocyte populations, five distinct subpopulations were observed. In both ScxLin depletion and aging, ‘ECM synthesizers’ and ‘ECM organizers’ populations were lost, consistent with disruptions in tissue homeostasis and altered ECM composition. However, in ScxLin depleted mice retention of a ‘specialized ECM remodeler’ population was observed, while aging tendon cells demonstrated inflammatory skewing with retention of a ‘pro-inflammatory tenocyte population’. In addition, enrichment of genes associated with protein misfolding clearance were observed in aged tenocytes. Finally, a similar inflammatory skewing was observed in aged tendon-resident macrophages, with this skewing not observed in ScxLin depleted tendons. These data suggest that loss of ‘ECM synthesizer’ populations underpins disruptions in tendon homeostasis. However, retention of ‘specialized remodelers’ promotes enhanced healing (ScxLin depletion), while inflammatory skewing may drive the impaired healing response in aged tendons

Cellular therapies play an important role in tendon tissue engineering with tenocytes being described as the most prominent cell population if available in large numbers. However, in vitro expansion of tenocytes in standard culture leads to phenotypic drift and cellular senescence. Recent work suggests that maintenance of tenogenic phenotype in vitro can be achieved by recapitulating different aspects of the native tendon microenvironment. One approach used to modulate the in vitro microenvironment and enhance extracellular matrix (ECM) deposition is macromolecular crowding (MMC). MMC is based on the addition of inert macromolecules to the culture media mimicking the dense extracellular matrix. In addition, as tendon has been described to be a relatively avascular and hypoxic tissue and low oxygen tension can stimulate collagen synthesis and cross-linking, we venture to assess the synergistic effect of MMC and low oxygen tension on human tenocyte phenotype maintenance by enhancing synthesis and deposition of tissue-specific ECM. Human tendons were kindly provided from University Hospital Galway, after obtaining appropriate licenses, ethical approvals and patient consent. Afterwards, tenocytes were extracted using the migration method. Experiments were conducted at passage three. Optimization of MMC conditions was assessed using 50 to 500 μg/ml carrageenan (Sigma Aldrich, UK). For variable oxygen tension cultures, tenocytes were incubated in a Coy Lab (USA) hypoxia chamber. ECM synthesis and deposition were assessed using SDS-PAGE (BioRad, UK) and immunocytochemistry (ABCAM, UK) analysis. Protein analysis for Scleraxis (ABCAM, UK) was performed using western blot. Gene analysis was conducted using a gene array (Roche, Ireland). Cell morphology was assessed using bright-field microscopy. All experiments were performed at least in triplicate. MINITAB (version 16, Minitab, Inc.) was used for statistical analysis. Two-sample t-test for pairwise comparisons and ANOVA for multiple comparisons were conducted. SDS-PAGE and immunocytochemistry analysis demonstrated that human tenocytes treated with the optimal MMC concentration at 2% oxygen tension showed increased synthesis and deposition of collagen type I, the major component of tendon ECM. Moreover, immunocytochemistry for the tendon-specific ECM proteins collagen type III, V, VI and fibronectin illustrated enhanced deposition when cells were treated with MMC at 2% oxygen tension. In addition, protein analysis revealed elevated dexpression of the tendon-specific protein Sclearaxis, while a detailed gene analysis revealed upregulation of tendon-related genes and downregulation of trans-differentiation markers again when cells cultured with MMC at 2% oxygen tension. Finally, low oxygen tension and MMC did not affect the metabolic activity, proliferation and viability of human tenocytes. Collectively, results suggest that the synergistic effect of MMC and low oxygen tension can accelerate the formation of ECM-rich substitutes, which stimulates tenogenic phenotype maintenance. Currently, the addition of substrate aligned topography together with MMC and hypoxia is being investigated in this multifactorial study for the development of an implantable device for tendon regeneration

Energy storing tendons such as the human Achilles and equine superficial digital flexor tendon (SDFT) are prone to age-related injury. Tendons have poor healing capacity and a lack of effective treatments can lead to ongoing pain, reduced function and re-injury. It is therefore important to identify the mechanisms underpinning age-related tendinous changes in order to develop more effective treatments. Our recent single cell sequencing data has shown that tendon cell populations have extensive heterogeneity and cells housed in the tendon interfascicular matrix (IFM) are preferentially affected by ageing. There is, however, a lack of established surface markers for cell populations in tendon, limiting the capacity to isolate distinct cell populations and study their contribution to age-related tendon degeneration. Here, we investigate the presence of the cell surface proteins MET proto-oncogene (MET), integrin subunit alpha 10 (ITGA10), fibroblast activation protein alpha (FAP) and platelet derived growth factor receptor alpha (PDGFRA) in the equine SDFT cell populations and their co-localisation with known markers. Using Western blot we validated the specificity of selected antibodies in equine tissue before performing immunohistochemistry to establish the location of the respective proteins in the SDFT. We subsequently used double labelling immunofluorescence with the established mural cell marker desmin (DES) to distinguish between tenocyte and mural cell populations. In situ, MET, ITGA10, and FAP presence was found in cells throughout the tendon whereas PDGFRA was present in cells within the IFM. Double labelling immunofluorescence with the mural cell marker DES showed lack of co-localisation between PDGFRA and DES suggesting PDGFRA is labelling an IFM cell population distinct from those associated with blood vessels. PDGFRA is a promising target for the specific cell sorting of IFM-localised tenocytes, enabling their isolation and subsequent characterisation. Acknowledgments: The authors acknowledge the Biotechnology and Biological Sciences Research Council (BB/W007282/1) for funding this work

Objectives. Intra-articular injections of local anaesthetics (LA), glucocorticoids (GC), or hyaluronic acid (HA) are used to treat osteoarthritis (OA). Contrast agents (CA) are needed to prove successful intra-articular injection or aspiration, or to visualize articular structures dynamically during fluoroscopy. Tranexamic acid (TA) is used to control haemostasis and prevent excessive intra-articular bleeding. Despite their common usage, little is known about the cytotoxicity of common drugs injected into joints. Thus, the aim of our study was to investigate the effects of LA, GC, HA, CA, and TA on the viability of primary human chondrocytes and tenocytes in vitro. Methods. Human chondrocytes and tenocytes were cultured in a medium with three different drug dilutions (1:2; 1:10; 1:100). The following drugs were used to investigate cytotoxicity: lidocaine hydrochloride 1%; bupivacaine 0.5%; triamcinolone acetonide; dexamethasone 21-palmitate; TA; iodine contrast media; HA; and distilled water. Normal saline served as a control. After an incubation period of 24 hours, cell numbers and morphology were assessed. Results. Using LA or GC, especially triamcinolone acetonide, a dilution of 1:100 resulted in only a moderate reduction of viability, while a dilution of 1:10 showed significantly fewer cell counts. TA and CA reduced viability significantly at a dilution of 1:2. Higher dilutions did not affect viability. Notably, HA showed no effects of cytotoxicity in all drug dilutions. Conclusion. The toxicity of common intra-articular injectable drugs, assessed by cell viability, is mainly dependent on the dilution of the drug being tested. LA are particularly toxic, whereas HA did not affect cell viability. Cite this article: P. Busse, C. Vater, M. Stiehler, J. Nowotny, P. Kasten, H. Bretschneider, S. B. Goodman, M. Gelinsky, S. Zwingenberger. Cytotoxicity of drugs injected into joints in orthopaedics. Bone Joint Res 2019;8:41–48. DOI: 10.1302/2046-3758.82.BJR-2018-0099.R1

Adherent cells are known to respond to physical characteristics of their surrounding microenvironment, adapting their cytoskeleton and initiating signaling cascades specific to the type of cue encountered. Scaffolds mimicking native biophysical cues have proven to differentiate stem cells towards tissue-specific lineages and to maintain the phenotype of somatic cells for longer periods of time in culture. Biomaterial-based tendon implants are designed to withstand high physiological loads but often lack the appropriate biochemical, biophysical and biological structure to drive tendon regeneration by populating cells. The objective of this study is to use tendon main component, collagen type I, to create scaffolds that reproduce tendon natural anisotropy and rigidity, in an effort to engineer functional tendon tissue with native organization and strength, able to maintain tenocyte phenotype and to differentiate stem cells towards the tenogenic lineage. Porcine collagen type I in solution was treated with one of the following cross-linkers: glutaraldehyde, genipin or 4-arm polyethylene glycol (4SP). The resulting mixture was poured on micro-grooved (2×2×2 um) or planar PDMS moulds and air-dried to obtain 5 mg/ml collagen films. Surface topography and elastic modulus were analyzed using SEM/AFM and rheometry, respectively. Human tendon cells were cultured on the micro-grooved/planar scaffolds for up to 10 days. Cell morphology, collagen III and tenascin C expression were analyzed by immunocytochemistry. Among the different cross-linkers used, only the treatment with 4SP resulted in scaffolds with a recognizable micro-grooved surface topography. Precise control over the micro-grooved topography and the rigidity of the scaffolds was achieved by cross-linking the collagen with varying concentrations of 4SP (0, 0.5, 1 and 1.5mM) at low pH and temperature. The elastic modulus of the scaffolds cross-linked with 4SP (0.5mM) matched the values previously reported to induce tenogenic differentiation in stem cells (50–90 kPa). Approximately eighty percent of the human tendon cells cultured on the micro-grooved collagen films aligned in the direction of the anisotropy for 10 days in culture, mimicking the alignment of tenocytes in the native tissue. Cell nuclei morphology, known to play a central role in the process of mechanotransduction, was significantly more elongated for the tenocytes cultured on the micro-grooved scaffolds after 4 days in culture for all the 4SP concentrations. Synthesis, deposition and alignment of collagen III and tenascin C, two important tenogenic markers, were up regulated selectively on the micro-grooved and rigid scaffolds after 10 days in culture, respectively. These results highlight the synergistic effect of matrix rigidity and cell alignment on tenogenic cell lineage commitment. Collectively, this study provides new insights into how collagen can be modulated to create scaffolds with precise imprinted topographies and controlled rigidities

Introduction and Objective. Chronic tendinopathy is a multifactorial disease and a common problem in both, athletes and the general population. Mechanical overload and in addition old age, adiposity, and metabolic disorders are among the risk factors for chronic tendinopathy but their role in the pathogenesis is not yet unequivocally clarified. Materials and Methods. Achilles tendons of young (10 weeks) and old (100 weeks) female rats bred for high (HCR) and low (LCR) intrinsic aerobic exercise capacity were investigated. Both Achilles tendons of 28 rats were included and groups were young HCR, young LCR, old HCR, and old LCR (n = 7 tendons per group/method). In this rat model, genetically determined aerobic exercise capacity is associated with a certain phenotype as LCR show higher body weight and metabolic dysfunctions in comparison to HCR. Quantitative real-time PCR (qPCR) was used to evaluate alterations in gene expression. For histological analysis, semi-automated image analysis and histological scoring were performed. Results. Age-related downregulation of tenocyte marker genes (Tenomodulin), genes related to matrix modelling and remodeling (Collagen type 1, Collagen type 3, Elastin, Biglycan, Fibronectin, Tenascin C), and Transforming growth factor beta 3 (Tgfb3) were detected in tendons from HCR and LCR. Furthermore, inflammatory marker Cyclooxygenase 2 (Cox2) was downregulated, while Microsomal prostaglandin E synthase 2 (Ptges2) was upregulated in tendons from old HCR and old LCR. No significant alteration was seen in Interleukin 6 (Il6), Interleukin 1 beta (Il1b), and Tumor necrosis factor alpha (Tnfa). Histological analysis revealed that Achilles tendons of old rats had fewer and more elongated tenocyte nuclei compared to young rats, indicating a reduced metabolic activity. Even though higher content of glycosaminoglycans as a sign of degeneration was found in tendons of old HCR and LCR, no further signs of tendinopathy were detectable in histological evaluation. Conclusions. Overall, aging seems to play a prominent role in molecular and structural alterations of Achilles tendon tissue, while low intrinsic exercise capacity did not cause any changes. Even though tendinopathy was not present in any of the groups, some of the shown age-related changes correspond to single characteristics of chronic tendon disease. This study gives an insight into tendon aging and its contribution to molecular and cellular changes in Achilles tendon tissue

Cellular therapies play an important role in tendon tissue engineering and regenerative medicine with tenocytes being described as the most prominent cell population for these applications if available in large numbers. However, this is difficult to achieve, because in vitro expansion of tenocytes leads to phenotypic drift and loss of function. Recent work suggests that maintenance of tenogenic phenotype in vitro can be achieved by recapitulating different aspects of the native tendon microenvironment. One approach used to modulate in vitro microenvironment and enhance extracellular matrix (ECM) deposition is macromolecular crowding (MMC). MMC is based on the addition of inert macromolecules to the culture media to mimic the dense extracellular matrix and accelerate the production of ECM-rich substitutes. In addition, as tendon has been described to be a relatively avascular and hypoxic tissue and low oxygen tension can stimulate collagen synthesis and cross-linking through the activation of hypoxia-inducible factor 1-alpha (HIF1-α), we venture to assess the synergistic effect of MMC and low oxygen tension on human tenocyte phenotype maintenance by enhancing deposition of tissue-specific extracellular matrix. SDS-PAGE and immunocytochemistry analysis, demonstrated that human tenocytes treated with the optimal MMC concentration at 2% oxygen tension showed increased collagen type I synthesis and deposition after 7 days. Moreover, immunocytochemistry for collagen type III, type V, VI, elastin and fibronectin illustrated enhanced deposition when cells were treated with MMC at 2% oxygen tension. In addition, it was shown that low oxygen tension and MMC did not affect the spindle-shape morphology, metabolic activity, proliferation and viability of human tenocytes Collectively, these results suggest that the synergistic effect of optimal macromolecular crowding concentration and low oxygen tension (2%) can accelerate the formation of ECM-rich substitutes, which may stimulate tenogenic phenotype maintenance. Further gene and protein analysis for tendon specific markers should be performed to validate our promising results

A tendon is a fibrous connective tissue that acts to transmit tensile forces between muscles and bones. It mainly consists of soluble substance, collagen and small volume of elastic fibres, which are produced by tenoblasts and tenocytes. The Achilles tendon is the thickest tendon in the human body that subjects to some of the highest tensile force, thus disorders and ruptures commonly happen. As the insoluble fibrous components in Achilles tendons, the collagen fibrils and elastic fibres have unique spatial structure that plays important functional roles. Despite this, the understanding of relationship between them is still limited due to the lack of imaging evidence. Using confocal and second harmonic generation microscopy, this study aims to comprehensively investigate the spatial relationship of collagen, elastic fibres and tenocytes in hydrated tendons. Longitudinal sections of 50 µm thick and transverse sections of 20 µm thick were cryo-sectioned respectively from the mid-portion of ten rabbit Achilles tendons. Sections were stained with 0.03g/L Acridine Orange (AO) and 1mg/ml Sulforhodamine B (SRB) solution respectively for labelling the nucleus and elastic fibres. The Leica TCS SP2 multiphoton microscopy containing second harmonic generation microscopy can image collagen without labelling. The sections were scanned by the multiphoton microscopy, and images were processed and reconstructed into 3D images to study the spatial structure of collagen, elastic fibres and cells in Achilles tendons. A rabbit Achilles tendon consists of three sub-tendons named flexor digitorum superficialis tendon, medial gastrocnemius tendon and lateral gastrocnemius tendon. Loose connective tissue connects the three sub-tendons and ensures efficient sliding between sub-tendons. The 3D network shows that the mid-portion of Achilles tendons is composed of longitudinal collagen and elastic fibres, while spindle tenocytes rest along the collagen and elastic fibres. Tenocytes appear to have a closer microstructural relationship with the elastic fibres. In comparison with the collagen, tenocytes and elastic fibres only occupy a very small volume in the 3D network. The elastic fibres exist in both tendon proper and endotenons. The tendon sheath and loose connective tissue have a higher cell density, and the cells are large and round while compared with tenocytes. As a component of the extracellular matrix (ECM) in Achilles tendons that closely mediates with the tenocytes, the elastin may participate in the force transition and interaction between tenocytes and the ECM. The elastic fibres may also endow Achilles tendons with unique mechanical properties to stand for tensile force

Cellular therapies play an important role in tendon tissue engineering with tenocytes being described as the most prominent cell population if available in large numbers. In vitro expansion of tenocytes in standard culture leads to phenotypic drift and cellular senescence. Maintenance of tenogenic phenotype in vitro can be achieved by recapitulating different aspects of the tendon microenvironment. One approach used to modulate in vitro microenvironment and enhance extracellular matrix (ECM) deposition is macromolecular crowding (MMC). In addition, as tendon has been described to be a relatively avascular and hypoxic tissue and low oxygen tension can stimulate collagen synthesis and cross-linking through the activation of hypoxia-inducible factor 1-alpha (HIF1-α), we venture to assess the synergistic effect of MMC and low oxygen tension on human tenocyte phenotype maintenance. SDS-PAGE and immunocytochemistry analysis demonstrated that human tenocytes treated with MMC at 2 % oxygen tension showed increased synthesis and deposition of collagen type I. Moreover, immunocytochemistry for the tendon-specific ECM proteins collagen type III, V, VI and fibronectin illustrated enhanced deposition when cells were treated with MMC at 2 % oxygen tension. In addition, western blot analysis revealed increased expression of tendon-specific protein Scleraxis, while a detailed gene analysis illustrated upregulation of tendon-specific genes and downregulation of trans-differentiation genes again when cells cultured with MMC under hypoxic conditions. Collectively, results suggest that the synergistic effect of MMC and low oxygen tension can accelerate the formation of ECM-rich substitutes, which stimulates tenogenic phenotype maintenance

Tendon injuries are associated with the formation of inferior, disorganized scar tissue at the tendon bone insertion site and high failure rates. Two major processes are discussed being key players: the inflammatory reaction upon tear and the remodeling process of the tendon. In a previous study we demonstrated that the profile of MMPs and TIMPs, being key factors of tendon modeling and remodeling, is altered in tenocytes of rotator cuff tears from donors with higher age (>65 years) and degenerative status (high degree of muscle fatty infiltration)[1]. But do these cells also show different expression of inflammatory cytokines or react different upon cytokine stimulation? The aim of our project was to analyze the expression of inflammatory cytokines in human tenocyte-like cells (hTLCs) on mRNA-level and the responsiveness to cytokine stimulation regarding differences between varying donor characteristics such as age, sex and the degenerative status of the tendon. TLCs were isolated from SSP tendon biopsies from 16 male and 14 female donors undergoing arthroscopic or open shoulder surgery. Cells from each donor (passage 1 or 2) were seeded in a 6-well plate and RNA was isolated after 7 days of culture. Quantitative Real-Time PCR was performed to analyze the expression of IL-6, IL-1β, TNF-α, IL-10, IL-33, TGF-β1 and COX-2. Furthermore, hTLCs of 12 male donors were stimulated for 3 days with a combination of TNF-α and IFN-γ (10ng/ml). The effect of the cytokines was analyzed by flow cytometry regarding surface marker expression: ICAM (CD54), VCAM (CD106), and Major Histocompatibility Complex (MHC)-class I and MHC-class II. Statistics: Mann-Whitney-U-Test, Spearman´s-Rho-correlation, p≤0.05. Gene expression analysis revealed high levels of IL-6, TGF-β1 and COX-2 in hTLCs but low expression of TNF-α and IL-10. No differences in the expression of the inflammatory cytokines were found between low and high fatty infiltration or with respect to age. The stimulation of the hTLCs with TNF-α and IFN-γ increased the number of ICAM and VCAM positive cells up to 100% and 97±5%, respectively. MHC-class II was not expressed on unstimulated cells but 77±17% MHC-class II positive cells were present after stimulation. All unstimulated cells were positive for MHC-class I, but the MFI (Mean Fluorescent Intensity) increased after stimulation. No significant difference in the expression of surface markers was detected when comparing tenocytes of donors with low and high muscle fatty infiltration. In contrast to the significant changes in expression levels of MMPs and TIMPs in tenocytes of donors with different age and degenerative status[1], we could not detect any significant changes in the expression of inflammatory cytokines or in the responsiveness of these tenocytes upon cytokine stimulation. All tenocytes showed the potential to respond to inflammatory processes. This indicates that the response of the tenocytes to inflammatory stimuli seems to be independent of donor characteristics, whereas the tendon remodeling might depend on age and degenerative status of the donor

Introduction. Adult tendon injuries occur very frequently, but injured tendon heals very slowly and the mechanisms of the slow-healing response to injury are still largely unknown. Currently, the main barrier is our insufficient understanding of the mechanisms responsible for homeostasis, regeneration and repair of adult tendon. This gap in knowledge translates to a lack of experimental models. Therefore, using the combination of state-of-the-art genetic approaches, we have established novel cell biological tools to advance the understanding of tendon biology. Materials and Methods. Adult mouse tendon progenitor lines and Adult mouse tenocyte lines: Primary adult tenocytes were isolated from Achilles tendon in Scleraxis(fl/fl)/Scleraxis-GFP/p21(−/−) mice, then CD90.2- and subsequent Sca1-positive cells were sorted by Flow Cytometry. Then Scleraxis-null progenitor lines were generated by the treatment of those cells with adenovirus-Cre. Adult Scleraxis(+/+) and Scleraxis-null tenocyte lines were also generated from Scleraxis(fl/fl)/Scleraxis-GFP/p21(−/−) mice. To establish Scleraxis-Flag overexpressing tenocyte lines, Scleraxis and Flag-tag fusion-protein expression construct was generated and transfected into Scleraxis-null tenocytes (Scleraxis transgenic mouse strains were provided by Dr Ronen Schweitzer). Scleraxis antibody: DNA coding mouse Scleraxis residues were obtained by PCR, then the recombinant protein was expressed, immunized in rabbits, and an affinity-purified antibody was generated. Results. Established parental progenitor lines highly expressed Sca1 (98.9%), CD90.2 (97.3%), and CD44 (99.8%) and were almost negative for ScxGFP (2.3%). Interestingly, Scleraxis-null progenitors showed significantly increased clonogenicity. Furthermore, when stimulated toward mesenchymal lineages, Scleraxis-null progenitors enhanced differentiation into chondrocytes. Our Scleraxis antibody reacted with lysates from cells expressing Scleraxis-Flag fusion proteins (∼30 kDa), whereas it did not react with Scleraxis-null cells by Western analysis. Immunofluorescence analysis of adult mouse Achilles tendons further confirmed intense Scleraxis protein expression in wild-type tenocytes, whereas considerably decreased expression of Scleraxis was evident in Cre-treated Scleraxis(fl/fl) tenocytes. Discussion. These novel tools will be the promising resources to get an insight into molecular framework for Scleraxis in adult tendons. It is anticipated that the establishment of experimental models using these resources will fill major gaps in the current knowledge of adult tendon biology and will facilitate development of novel strategies to treat adult tendon injury

Rotator cuff repair is performed to treat shoulder pain and disability. Failure of the tendon repair site is common; one strategy to improve healing is to enforce a period of post-operative immobilisation. Immobilisation may have unintended effects on tendon healing. Tenocytes under uniaxial strain form more organised collagen and up regulate expression of proliferative genes. Vitamin C (ascorbic acid), an anti-oxidant that is a co-factor for collagen synthesis, has also been reported to enhance collagen deposition and organisation. The purpose of this study was to compare human tenocyte cultures exposed to uniaxial cyclical strain with or without slow-release ascorbic acid (ascorbyl-2 phosphate) to determine their individual and combined effects on tissue remodelling and expression of tissue repair genes. Rotator cuff tissues were collected from degenerative supraspinatus tears from eight patients. Tenocytes were incorporated into 3D type I collagen culture matrices. Cultures were divided into four groups: 1) ascorbic acid (0.6mMol/L) + strain (1%–20% uniaxial cyclic strain at 0.1 Hz), 2) ascorbic acid unstrained, 3) strain + vehicle 4) unstrained + vehicle. Samples were fixed in paraffin, stained with picrosirius red and analysed with circular polarising light. A second set of cultures were divided into three groups: 1) 0.5mM ascorbic acid, 2) 1mM ascorbic acid, 3) vehicle cultured for 24, 72, 120 and 168 hours. Cell-free collagen matrix was used as a control. Tenocyte proliferation was assessed using the water soluble tetrazolium-1 (WST1) assay and f tissue repair gene expression (TGFB1, COL1A1, FN1, COLIII, IGF2, MMP1, and MMP13), were analysed by qPCR. The data were analysed using a Split model ANOVA with contrast and bonferroni correction and a one-way ANOVAs and Tukey's test (p<0.05 was significant). Our results indicated that unstrained cultures with or without exposure to slow release ascorbic acid exhibited greater picrosirius red birifringency and an increase in collagen fiber deposition in a longitudinal orientation compared to strained tenocytes. We found that slow release ascorbic acid promoted significant dose and culture-time dependent increases in tenocyte proliferation (p<0.05) but no obvious enhancement in collagen deposition was evident over cultures without ascorbic acid supplementation. Based on these data, applying strain to tenocytes may result in less organised formation of collagen fibers, suggestive of fibrotic tissue, rather than tendon remodelling. This may indicate that a short period of immobilisation post-rotator cuff repair is beneficial for the healing of tendons. Exposure to slow release ascorbic acid enhanced tenocyte proliferation, suggesting that supplementation with Vitamin C may improve tendon repair post-injury or repair. Future studies will assess levels of tissue repair-associated proteins as well as comparing traumatic and degenerative rotator cuff tears to healthy uninjured rotator cuff tissue

Tendons mainly consist of collagen in order to withstand high tensile forces. Compared to other, high turnover tissues, cellularity and vascularity in tendons are low. Thus, the natural healing process of tendons takes long and can be problematic. In case of injury to the enthesis, the special transition from tendon over cartilage to bone is replaced by a fibrous scar tissue, which remains an unsolved problem in rotator cuff repair. To improve tendon healing, many different approaches have been described using scaffolds, stem cells, cytokines, blood products, gene therapy and others. Despite promising in vitro and in vivo results, translation to patient care is challenging. In clinics however, tendon auto- or allografts remain still first choice to augment tendon healing if needed. Therefore, it is important to understand natural tendon properties and natural tendon healing first. Like in other tissues, senescence of tenocytes seems to play an important role for tendon degeneration which is interestingly not age depended. Our in vivo healing studies have shown improved and accelerated healing by adding collagen type I, which is now used in clinics, for example for augmentation of rotator cuff repair. Certain cytokines, cells and scaffolds may further improve tendon healing but are not yet used routinely, mainly due to missing clinical data, regulatory issues and costs. In conclusion, the correct diagnosis and correct first line treatment of tendon injuries are important to avoid the necessity to biologically augment tendon healing. However, strategies to improve and accelerate tendon healing are still desirable. New treatment opportunities may arise with further advances in tendon engineering in the future

Cell-based tissue engineering strategies for tendon repair have limited clinical applicability due to delayed extracellular matrix (ECM) deposition and subsequent prolonged culture periods, which lead to tenogenic phenotypic drift. Deposition of ECM in vitro can be enhanced by macromolecular crowding (MMC), a biophysical phenomenon that governs the intra- and extra-cellular milieu of multicellular organisms, which has been described to accelerate ECM deposition in human tenocytes. A variety of cell sources have been studied for tendon repair including tenocytes, dermal fibroblasts (DFs) and mesenchymal stem cells (MSCs) and various biophysical, biochemical and biological tools have been used to mimic tendon microenvironment. Therefore, we propose to assess the combined effect of MMC and mechanical loading on different cell sources to determine their suitability for the in vitro fabrication of tendon-like tissue. The uniaxial strain induced differential cell orientation based on the differentiation state of the cells: tenocytes and DFs, both permanently differentiated cells exhibited alignment perpendicular to the direction of the load, similarly to what is seen in native tendon environment. Immunocytochemistry showed that, when MMC is used, the DFs and MSCs showed increased deposition of collagen type I, one of the main components in tendon ECM. It is also seen that the ECM deposited follows the alignment of the cell cytoskeleton. However, for tenocytes, deposition of collagen type I is only seen when MMC is used in combination with mechanical loading, indicating that mechanical loading led to increased synthesis of collagen I, suggesting maintenance of the tenogenic phenotype. Other collagen types relevant to native tendon composition were also analysed, including types III, V and VI, and their deposition was also shown to be modulated by the use of MMC and mechanical loading. This appears to recreate the events of tendon tissue formation during development, where these collagen types are involved in regulation of collagen I fibrillogenesis and fibril diameter. Preliminary data also indicates that, under mechanical loading and MMC, expression of tenogenic genes is upregulated whilst chondrogenic and osteogenic markers are downregulated. This indicates the suitability of the combination of MMC and mechanical stimulation for modulating tenogenic phenotype of various cell sources and fabricating tendon-like tissue

Worldwide, tendon disorders are one of the main causes of disability that decrease the quality of life of individuals and represent a substantial economic burden on society. Currently, the main therapies used for tendon injuries are not able to restore tendon functionality, and due to tendons' hypovascular and hypocellular nature, they present a reduced healing capacity, which also limits the success of the available therapies. In order to discover new therapies, extracellular vesicles (EVs), key players in cell-cell communication, have been widely explored for tissue engineering and regenerative medicine applications. Thus, the aim of this study is to assess the role of EVs derived from platelets in stem cell tenogenic commitment using a bioengineered tendon in vitro model for potential use as tendon therapeutic agents. Biomimetic platelet-derived EVs were produced by freeze-thaw cycles of platelets and isolation at different centrifugation speed. To recreate the architecture of tendons, a 3D system consisting of electrospun anisotropic nanofiber scaffolds coated with collagen encapsulating human adipose stem cells (hASCs) and different types of platelet-derived EVs, were produced. Then, the influence of the tendon-mimetic constructs and the distinct EVs populations in the hASCs tenogenic differentiation were assessed over culture time. We observed that the hASCs on the nanofibrous tendon scaffolds, show high cytoskeleton anisotropic organization that is characteristic of tenocytes. Moreover, acting as biological cues, platelet-derived EVs boosted hASCs tenogenic commitment, supported by the increased gene expression of tendon-related markers (SCX and TNMD). Additionally, EVs enhanced the deposition of tendon like extracellular matrix (ECM), as evidenced by the increased gene expression of ECM-related markers such as COL1, COL3, DCN, TNC, and MMP-3, which are fundamental for ECM synthesis and degradation balance. Moreover, EVs induced lower collagen matrix contraction on hASCs, which has been related with lower myofibroblast differentiation. Overall, the results revealed that EVs are capable of modulating stem cells' behavior boosting their tenogenic commitment, through the increased expression of healthy tendon cell markers, potentiating ECM deposition and decreasing cell contractility. Therefore, platelet EVs are a promising biochemical tool, worthy to be further explored, as paracrine signaling that might potentiate tendon repair and regeneration

Introduction. Tendons are critical to mobility, and are susceptible to degeneration through injury and ageing. Type I collagen is the most abundant protein in vertebrates; it is the main structural protein of the extracellular matrix in numerous musculoskeletal tissues, including tendons. Type I collagen predominantly is a heterotrimer, which consists of two alpha-1 chains and one alpha-2 chain (α1). 2. (α2) encoded by the COL1A1 and COL1A2 genes, respectively. However, type I collagen can form homotrimers (α1). 3. which are protease-resistant, and are associated with age-related musculoskeletal diseases, fibrotic and connective tissue pathologies. Transforming growth factor beta (TGFβ) enhances collagen (I) gene expression, is involved in tendon mechanobiology and repair processes, while its effect on homotrimer formation is unknown. Our aim is to investigate the relative expressions of collagen (I) α1 and α2 polypeptide chains in tenocytes (tendon fibroblasts) stimulated with TGFβ. Materials and Methods. Included RT-qPCR to measure the relative expression of COL1A1 and COL1A2 genes. [. 14. C]-proline metabolic labelling was used to measure the expression of the collagen (I) α1 and α2 polypeptide chains. These techniques were performed in equine superficial digital flexor tendon (SDFT) tenocytes (n=3) and murine tail tendon tenocytes (n=3) with different concentrations of TGFβ (0.01 ng/ml-100 ng/ml). Results. There was an increase in both COL1A1 and COL1A2 gene expression when stimulated with TGFβ in both cell types. In equine tenocytes the gene expression ratio of COL1A1:COL1A2 increased from 1.73 ± 0.75 to 7.87 ± 2.9 (p=0.003) when stimulated with 100 ng/ml of TGFβ3. TGFβ upregulated collagen (I) protein in both cell types. In equine tenocytes (n=3) when stimulated with 100 ng/ml of TGFβ3, the α1:α2 protein chain ratio increased from 1.93 ± 0.54 to 3.02 ± 0.32 (p=0.059) in comparison with serum-starved cells, which alongside the changes in gene expression, may be indicative of collagen (I) homotrimer production. Discussion. There were biosynthetic alterations in collagen production, and putative collagen (I) homotrimer when equine tenocytes were stimulated with 100 ng/ml TGFβ3. Future work will focus isolating different collagens by repeated differential salt precipitation. The level of TGFβ receptors and Smad signaling molecules will be also analysed using RT-qPCR and western blotting

Introduction. Tendon is prone to degeneration through ageing and injury and current therapies are largely ineffective. The recent identification of a cell population within tendon with stem cell-like characteristics holds potential for regeneration of tendon. The local stem cell environment (niche) is important for stem cell maintenance and function. This study aims to characterize extracellular matrix (ECM) components of the stem cell niche in equine tendon, which is prone to age-related degeneration and rupture. Materials and Methods. Putative tendon stem cells (TSCs) were isolated from equine superficial digital flexor tendon by low-density plating and differential adhesion to fibronectin. Cells were analysed by flow cytometry using antibodies to mesenchymal stem cell markers, as well as qRT-PCR for stem cell and tenogenic markers. The multipotency of cells was assessed using tri-lineage differentiation assays. ECM components of the tenocyte and TSC niche were analysed using radio-isotope labelling, immunohistochemistry and histology. Results. Putative TSCs were able to form colonies, and both tenocytes and TSCs expressed CD90, CD105 and CD73 as determined by flow cytometry. However, TSCs did not exhibit increased expression of stem cell marker genes when compared with tenocytes. TSCs and tenocytes both displayed osteogenic and chondrogenic differentiation, however not adipogenic differentiation. Tenocytes and TSCs labelled with 14C-labelled amino acids both displayed similar labelling profiles. Histological analysis of tendon tissue highlighted the varied structure and composition of tendon, with tenascin C expression confined to the interfascicular matrix. Discussion. TSCs do not highly express stem cell markers when compared with tenocytes, indicating that these cells may not be true stem cells. In addition the similar labelling profiles of the two cell types indicates that a stem cell population has not been differentially isolated, however the tri-lineage differentiation assays suggest the cells may possess some stem cell-like properties. It is possible that the equine tendon cell population consists of a heterogeneous mixture of cells at different stages of differentiation

Background and objectives: Tenocytes change their structure, composition and mechanical properties to adapt to mechanical loading. Voltage gated and mecha-nosensitive ion channels may play a key role in human tenocytes to regulate some or all initial responses to mechanical stimulation. To date, there has been no direct investigation of ion channel expression by human tenocytes. Methods: Human tenocytes were cultured from patellar tendon samples harvested from five patients undergoing routine total knee replacement surgery (mean age: 66 years; range 63-73 years). RT-PCR, Western Blotting and whole cell electrophysiological studies were performed to investigate the expression of different classes of ion channels within tenocytes. Results: Human tenocytes express mRNA and protein encoding voltage operated calcium channel (VOCCs) sub-units (Ca alpha 1A, Ca alpha 1C, Ca alpha 1D, Ca alpha2 delta1) and the mechanosensitive tandem pore domain potassium channel (2PK+) TREK-1. They exhibit whole cell currents consistent with the functional expression of these channels. In addition, other ionic currents were detected within these tenocytes consistent with the expression of voltage gated potassium channels, voltage gated sodium channels, and other outwardly rectifying leak currents. Discussion and conclusions: Human tendon cells show increased levels of intracellular calcium when stress is applied to them. One of the mechanisms by which this occurs is by the influx of extracellular calcium into the cell via ion channels. VOCCs and TREK channels have been implicated in mechanotransduction signalling pathways in numerous connective tissue cell types. This study suggests that these mechanisms may be present in human tenocytes. In addition, human tenocytes may express other channel currents. Ion channels may represent potential targets for the pharmacological management of chronic tendinopathies

The energy-storing human Achilles tendon and equine superficial digital flexor tendon (SDFT) show no adaptation to exercise unlike muscle and bone, and are prone to injury. Injury involves microdamage accumulation until there is sufficient weakening for rupture to occur during normal athletic activity. Anatomically opposing positional tendons, such as the common digital extensor tendon (CDET) in the horse rarely suffer exercise–induced injury. Tenocytes maintain the extra-cellular matrix, but in energy-storing tendons they appear unable to adequately repair microdamage as it occurs. Tenocytes have been classified subjectively into 3 subtypes on the basis of histological nuclear morphology. Long, thin type 1 cells are thought to be less synthetically active than cigar-shaped type 2 cells, but their exact morphology and relative proportions in different tendon sites and ages has not been clearly defined. We hypothesised that tenocytes are separable into morphologically distinct subtypes, reflecting differences in age and functional requirements within and between specific tendons. Samples were taken from tensional and compressed regions of the SDFT and CDET of 5 neonates, 5 foals (1–6 m), 5 young adults (2–6 y) and 5 old horses (18–33 y) Cell nuclei were counted and measured in digital images from histological sections by computerised image analysis. Total tenocyte densities and proportions of the 3 subtypes were calculated for each age group, as were nuclear length:width ratios. Length:width ratio distributions for all horses were evaluated using a normality test followed by a paired t-test. There was a significantly higher total cellularity in the SDFT than the CDET, with a higher proportion of type 1 tenocytes in the CDET. With age, total cellularity decreased in all tendon sites and an increase in the proportion of type 1 tenocytes was observed in tensional regions. Foal and neonatal tendons contained significantly higher proportions of type 2 tenocytes than older tendons. The morphology of the two main subtypes in all age groups was significantly different; type 1 tenocytes had a higher nuclear length:width ratio (mean ± SD = 9.6 ± 2.5) than type 2 (mean ± SD =4.7 ±1.1) (p< 0.001). We were able to objectively separate tenocytes into 3 distinct subtypes based on nuclear length:width ratio measurements. There were significant differences in proportions of subtypes with tendon site and age. The positional tendon had significantly lower cellularity and a higher proportion of type 1 tenocytes; these cells may be less functionally active but sufficient to maintain the matrix in a tendon which is not subjected to high levels of strain. The SDFT continues to grow up to 2 years of age and is subjected to high strains, explaining the need for relatively higher proportions of type 2 cells. There is however an age-related increase in type 1 cells in both tendons which may explain an inability of the adult energy-storing tendon to adapt to exercise and to repair microdamage. Understanding the stimulus for age-related changes in tenocyte subtype proportions in tendons with different functions may help us understand the pathogenesis of exercise-induced tendon injury and to develop more appropriate training regimens

Objectives. Tranexamic acid (TXA) is an anti-fibrinolytic medication commonly used to reduce perioperative bleeding. Increasingly, topical administration as an intra-articular injection or perioperative wash is being administered during surgery. Adult soft tissues have a poor regenerative capacity and therefore damage to these tissues can be harmful to the patient. This study investigated the effects of TXA on human periarticular tissues and primary cell cultures using clinically relevant concentrations. Methods. Tendon, synovium, and cartilage obtained from routine orthopaedic surgeries were used for ex vivo and in vitro studies using various concentrations of TXA. The in vitro effect of TXA on primary cultured tenocytes, fibroblast-like synoviocytes, and chondrocytes was investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assays, fluorescent microscopy, and multi-protein apoptotic arrays for cell death. Results. There was a significant (p < 0.01) increase in cell death within all tissue explants treated with 100 mg/ml TXA. MTT assays revealed a significant (p < 0.05) decrease in cell viability in all tissues following treatment with 50 mg/ml or 100 mg/ml of TXA within four hours. There was a significant (p < 0.05) increase in cell apoptosis after one hour of exposure to TXA (100 mg/ml) in all tissues. Conclusion. The current study demonstrates that TXA caused significant periarticular tissue toxicity ex vivo and in vitro at commonly used clinical concentrations. Cite this article: M. McLean, K. McCall, I. D. M. Smith, M. Blyth, S. M. Kitson, L. A. N. Crowe, W. J. Leach, B. P. Rooney, S. J. Spencer, M. Mullen, J. L. Campton, I. B. McInnes, M. Akbar, N. L. Millar. Tranexamic acid toxicity in human periarticular tissues. Bone Joint Res 2019;8:11–18. DOI: 10.1302/2046-3758.81.BJR-2018-0181.R1

Alarmins- also referred to as damage associated molecular patterns (DAMPS)- are endogenous molecules mobilized in response to tissue damage known to activate the innate immune system and regulate tissue repair and remodelling. The molecular mechanisms that regulate inflammatory and remodelling pathways in tendinopathy are largely unknown therefore identifying early immune effectors is essential to understanding the pathology. S100A8 and S100A9 are low molecular weight calcium binding proteins primarily released by activated phagocytes in an inflammatory setting and also secreted as a heterodimeric complex that exhibits cytokine like functions. Based on our previous investigations we sought evidence of S100A8/A9 expression in human tendinopathy and thereafter, to explore mechanisms whereby S100 proteins may regulate inflammatory mediators and matrix regulation in human tenocytes. Torn supraspinatus tendon (established pathology) and matched intact subscapularis tendon (representing ‘early pathology’) biopsies were collected from patients undergoing arthroscopic shoulder surgery. Control samples of subscapularis tendon were collected from patients undergoing arthroscopic stabilisation surgery. S100A8/A9 expression was analysed at transcript and protein level using quantitative RT-PCR and immunohistochemistry, respectively. Primary human tenocytes were cultured from hamstring tendon tissue obtained during hamstring tendon ACL reconstruction. The in vitro effect of recombinant human S100 A8/A9 on primary human tenocytes was measured using quantitative RT-PCR and ELISA. Immunohistochemistry of tendinopathic tissues demonstrated the presence of S100 A8/A9 in diseased tissues compared to control tissue. In addition, early pathological diseased tissue indicated greater S100A9 expression compared with established diseased pathology. These findings were reflected by data obtained at transcript level from diseased tissues. Recombinant human S100A8, A9 and A8/A9 complex led to significant increase in expression of inflammatory mediators, including IL-6 in vitro. Further analysis via quantitative RT-PCR demonstrated recombinant S100A8, A9 and A8/A9 complex treatment on tenocytes, in vitro, had no direct effect on the expression of genes involved in matrix remodelling. The presence of S100A8 and S100A9 in early tendinopathic lesions suggests expression is upregulated in response to cellular damage. S100A8 and S100A9 are endogenous ligands of Toll-like receptors (TLRs) and receptor for advanced glycation end products (RAGE). These receptors have known regulatory effects on immune mediated cytokine production. We propose S100A8 and S100A9 as active alarmins in the early stages of tendinopathy and thus targeting of its downstream signalling may offer novel therapeutic approaches in the management of human tendon disorders

Injury to the core region of energy-storing tendons is a frequent occurrence in both human and equine athletes, the incidence of which increases with age. Such energy-storing tendons include the human Achilles tendon (AT) and the equine superficial digital flexor tendon (SDFT). By definition, energy-storing tendons experience high strains during high-speed athletic activity. In contrast, anatomically opposing tendons (“positional” tendons), such as the common digital extensor tendon (CDET) in the horse and extensor digitorum longus tendon in man act only to transmit muscular force and rarely suffer exercise–induced injury. Functional adaptation of muscle and bone in response to exercise is well – documented, but there has been no convincing evidence to suggest that the energy-storing tendons in adults have the ability to adapt to exercise. We hypothesised that adaptive increases in tenocyte cellularity would occur in the energy-storing and positional tendons of young horses subjected to three specific exercise regimens. Samples were taken from midmeta-carpal regions of the SDFT (periphery and core) and CDET of young Thoroughbred horses from the following groups. Group 1: 6 horses exercised on a high-speed treadmill for 18 months from 21.3 months of age (SD 1.1) with 6 age-matched controls that underwent walking exercise only (long-term); Group 2: 6 horses exercised on a high-speed treadmill for 18 weeks from 19.4 months of age (SD 0.6) with 6 age-matched controls that underwent walking exercise only (short-term) and Group 3: 6 horses trained on pasture in New Zealand for 18 months beginning at 7–10 days of age, with 6 age-matched controls kept at pasture with no additional enforced exercise (Global Equine Research Alliance). Tenocyte nuclei were counted and measured in digital images from histological sections stained with haematoxylin and eosin, by computerised image analysis. Tenocyte densities (per mm2) for exercised and control groups for each study were evaluated using paired t-tests. Tenocyte density was significantly higher in the CDET of exercised horses in Group 3 (mean ± SD =260.4 ± 23.4) compared with the non – exercised controls (mean ± SD =226.9 ± 23.8) (p < 0.01). There was no such difference in the SDFT (core or periphery). There was also no significant exercise-related difference in tenocyte density in either the SDFT (core or periphery) or CDET for Groups 1 or 2. No previous data is available on the effect of exercise on tenocyte populations in equine tendons. The lack of other adaptive changes in previous studies of mature equine tendons had raised the question as to whether immature tendons would be more able to adapt to mechanical stimuli. In this study we were able to show that beginning training of horses shortly after birth (Group 3) stimulated an adaptive response by tenocytes in the positional CDET but not the SDFT. The inability of energy-storing tendons to show functional adaptation to exercise in immature or mature animals may explain the high incidence of strain-induced injury. Understanding the pathway by which exercise-related increases in tenocyte densities occur in immature positional but not energy-storing tendons may increase our understanding of the pathogenesis of strain-induced tendon injury

The objective was to seek evidence of hypoxia in early human tendinopathy and thereafter, to explore mechanisms whereby tissue hypoxia may regulate apoptosis, inflammatory mediators and matrix regulation in human tenocytes. Fifteen torn supraspinatus tendon (established pathology) and matched intact subscapularis tendon (representing ‘early pathology’) biopsies were collected from patients undergoing arthroscopic shoulder surgery. Control samples of subscapularis tendon were collected from 10 patients undergoing arthroscopic stabilisation surgery. Markers of hypoxia were quantified by immunohistochemical methods. Human tendon-derived primary cells were derived from hamstring tendon tissue obtained during hamstring tendon ACL reconstruction. The impact of hypoxia upon tenocyte biology ex vivo was measured using quantitative RT-PCR, multiplex cytokine assays, apoptotic proteomic profiling, immunohistochemistry and annexin V FACS staining. Increased expression of HIF 1a, Bcl-2 and clusterin (hypoxic and apoptotic markers) was detected in subscapularis tendon samples compared to both matched torn samples and non matched control samples (p<0.01). Hypoxic tenocytes exhibited increased production of proinflammatory cytokines (p<0.001), altered matrix regulation (p<0.01) with increased production of Collagen type III operating through a MAPK dependent pathway. Finally, hypoxia increased expression of several mediators of apoptosis and thereby promoted tenocyte apoptosis. Hypoxia promotes expression of proinflammatory cytokines, key apoptotic mediators and drives matrix component synthesis towards a collagen type III profile by human tenocytes. We propose hypoxic cell injury as a critical pathophysiological mechanism in early tendinopathy offering novel therapeutic opportunities in the management of tendon disorders

Introduction. The two-dimensional (2D) monolayer culture paradigm has limited translational potential to physiological systems; chondrocytes and tenocytes in monolayer lose expression of hallmarks of differentiated status (dedifferentiation). Qualitative assessment of three-dimensional (3D) cultures in musculoskeletal biology relative to native tissues has been limited. An understanding of prevailing gene regulatory networks is required to define whether 3D culture systems faithfully restitute the native tissue phenotype (redifferentiation). Using a systems biology approach to explore the gene networks associated with de- and re-differentiation may define targetable regulators associated with phenotypic plasticity of adult musculoskeletal cells. Materials and Methods. Global transcriptomic and proteomic profiling of matrix-depleted chondrocytes and tenocytes from the rat was performed for each of three conditions (native tissue, monolayer at passage three, or tissue-appropriate 3D cultures). Differential analysis of mRNA and protein abundance, gene ontology annotation, pathway topology impact analysis, and derivation of common mechanistic networks was undertaken to define consensus expression profiles, signalling pathways, and upstream regulators for de- and re-differentiation in each cell type. Results. Principal component analysis demonstrated a convergence of gene expression profiles in monolayer, including the expression of musculoskeletal progenitor markers scleraxis (Scx) and Mohawk (Mkx). Three-dimensional culture systems failed to demonstrate parity with native tissue and incited the expression of Il-6 and Ptgs2 (COX2). The CCN-family member Ctgf (CCN2), and the marker of skeletal differentiation Grem1 (gremlin 1), were consistently differentially abundant in de- and re-differentiation at both the mRNA and protein level. Pathway topology impact analysis defined PI-3K/Akt as the common signalling pathway in de- and re-differentiation. Discussion. Historically, the terms de- and re-differentiation have been used with no mechanistic definition. Additionally, there is no standardised phenotype for 3D cultures to benchmark novel progress in bioengineering. Consensus upstream regulators yielded a unified mechanistic network for chondrocyte and tenocyte phenotypes in three conditions. The PI-3K/Akt signalling pathway has been implicated in a range of physiological activities including dedifferentiation, proliferation, matrix synthesis, and cell survival. Pathway analysis suggests that the PI-3K/Akt signalling pathway may contribute to the de- and re-differentiation phenotypes for both chondrocytes and tenocytes and represents a rational target for further network-level analysis

All cells exist within a 3D microenvironment where they are exposed to a multitude of mechanobiological cues, from nano-level cell/matrix interactions, to tissue-level mechanical strain. These cues are fundamental to maintaining tissue homeostasis, but when disrupted during disease, can promote pathological outcomes and impair healing. This is particularly true in tendons; 3D load bearing connective tissue structures composed of a complex arrangement of matrix proteins, organised in a highly aligned manner and maintained by tendon cells (tenocytes). When diseased or injured (termed tendinopathy), the tendon begins a journey of poor healing, characterised by mechanically inferior disorganised scar tissue which ultimately results in compromised or total loss of function. In both health and disease, the mechanobiological stimuli experienced by tenocytes will directly affect their behaviour, yet this is a poorly studied area of research. We have used decellularised tendon slices to mimic the structure of healthy tendon, and induced degradation to mimic tendinopathic tendon. We have re-seeded these slices with tenocytes or immune cells and are building a greater picture of the role that the structure and stiffness of the matrix has on cell behaviour in health and disease

Mesenchymal stem cells (MSCs), characterised by their self-renewal and multidifferentiation potential, are a favoured cell population for future tissue engineering applications. Differentiation of MSCs towards a specific lineage has been extensively studied, mainly through the use of growth factors or conditioned media. However, growth factor supplementation is a mono-domain approach and considering the number of permutations, it is unlikely to find the optimal cocktail. Although PRPs are used extensively, its use is controversial, and standardization is impossible. Conditions media have various limitations, including how much, when and how effective it is at the time that it would be aspirated. Thus, co-culture systems are at forefront of scientific research and technological innovation. Co-culture system gives access to the complete cell secretome and offers the advantages of autologous therapy. However, several weeks of co-culture are necessary to observe stem cell differentiation. We hypothesize that, by using macromolecular crowding, which has been shown to recapitulate the dense in vivo microenvironment of the extracellular area and enhance matrix deposition in vitro with its excluded volume effect, it will accelerate stem cell differentiation towards tenogenic lineage. Further, we will assess if tendon specific extracellular matrix deposited by tenocytes is sufficient for stem cell differentiation without the necessity of cell contact between tenocytes and stem cells

Tranexamic acid (TXA) is an anti-fibrinolytic medication commonly used to reduce peri-operative bleeding. Increasingly, topical administration as an intra-articular injection or peri-operative wash is being administered at concentrations between 10–100mg/ml. This study investigated effects of TXA on human periarticular tissues and primary cell cultures using clinically relevant concentrations. Tendon, synovium and cartilage obtained from routine orthopaedic surgeries were used ex vivo or cultured for in vitro studies using various concentrations of TXA. They were stained with 5-chloromethylfluorescein diacetate and propidium iodide and imaged using confocal microscopy to identify the proportion of live and dead cells. The in vitro effect of TXA on primary cultured tenocytes, synovial like fibroblast (FLS) cells and chondrocytes was investigated using cell viability assays (MTT), fluorescent microscopy and multi-protein apoptotic arrays for cell death. There was significant (p<0.01) increase in cell death in all tissue treated with 100mg/ml TXA, ex vivo. MTT assays revealed significant (p<0.05) decrease in cell viability following treatment with 50 or 100mg/ml of TXA within 4 hours of all cell types cultured in vitro. Additionally, there was significant (p<0.05) increase in cell apoptosis detected by fluorescent microscopy within 1 hour of exposure to TXA. Furthermore, multi-protein apoptotic arrays detected increased apoptotic proteins within 1 hour of TXA treatment in tenocytes and FLS cells. Our study provides evidence of TXA cytotoxicity to human peri-articular tissues ex vivo and in vitro at concentrations and durations of treatment routinely used in clinical environments. Clinicians should therefore show caution when considering use of topical TXA administration

Collagen materials are extensively used in regenerative medicine. However, they still present limitations such as a mono-domain composition and poor mechanical properties. On the other hand, tissue grafts overcome most of these limitations. In addition, the potential of tissue grafts in musculoskeletal tissue engineering has not been fully investigated. Herein, we ventured to assess the potential of a decellularised porcine peritoneum for musculoskeletal applications by comparing its characteristics with a commercial collagen scaffold employed in tendon. Results indicated that the porcine peritoneum had higher mechanical properties and a lower crosslinking ratio (p < 0.01). Furthermore, it presented a lower resistance to collagenase digestion, which suggests a faster remodelling in vivo of the tissue graft. Immunohistochemistry analysis showed a preserved and multicomponent structure in the porcine peritoneum contrary to the collagen matrix, confirming the multifunctional nature of the tissue graft. Regarding the cell-response assessment, tenocytes and ADSCs were able to grow on both materials, however, proliferation was enhanced by the porcine peritoneum (p<0.01). Immune response by THP-1 showed an acute inflammatory response by macrophages to the collagen matrix, contrary to that observed in the porcine peritoneum which triggered a mild reaction. The in-progress in vivo study in a rabbit tendon model will elucidate the potential of porcine peritoneum for tendon repair applications. The present study shows how the multifunctionality of the porcine peritoneum provides higher cytocompatibility than a mono-domain collagen matrix with human tenocytes and ADSC. Besides, its lower immune response in vitro suggests better remodelling after implantation

Surgical repair of rotator cuff tears have high failure rates (20–70%), often due to a lack of biological healing. Augmenting repairs with extracellular matrix-based scaffolds is a common option for surgeons, although to date, no commercially available product has proven to be effective. In this study, a novel collagen scaffold was assessed for its efficacy in augmenting rotator cuff repair. The collagen scaffold was assessed in vitro for cytocompatability and retention of tenocyte phenotype using alamarBLUE assays, confocal imaging and real-time PCR. Immunogenicity was assessed in vitro by the activation of pre-macrophage cells. In vivo, using a modified rat rotator cuff defect model, supraspinatus tendon repairs were carried out in 46 animals. Overlay augmentation with the collagen scaffold was compared to unaugmented repairs. At 6- and 12-weeks post-op the repairs were tested biomechanically to evaluate repair strength, and histologically for quality of healing. The collagen scaffold supported human tenocyte growth in vitro, with cells appearing morphologically tenocytic and expressing higher tendon gene markers compared to plastic controls. No immunogenic responses were provoked compared to suture material control. In vivo, augmentation with the scaffold improved the histological scores at 12 weeks (8.37/15 vs. 6.43/15, p=0.0317). However, no significant difference was detected on mechanical testing. While the collagen scaffold improved the quality of healing of the tendon, a meaningful increase in biomechanical strength was not achieved. This is likely due to its inability to affect the bone-tendon junction. Future materials/orthobiologics must target both the repaired tendon and the regenerating bone-tendon junction

Cell-based tissue engineering strategies for tendon repair have limited clinical applicability due to delayed extracellular matrix (ECM) deposition and subsequent prolonged culture periods, which lead to tenogenic phenotypic drift. Deposition of ECM in vitrocan be enhanced by macromolecular crowding (MMC), a biophysical phenomenon that governs the intra- and extra-cellular milieu of multicellular organisms. 2. , which has been described to accelerate ECM deposition in human tenocytes. 1. A variety of cell sources have been studied for tendon repair including tenocytes, dermal fibroblasts and mesenchymal stem cells (MSCs). 3. and various biophysical, biochemical and biological tools have been used to mimic tendon microenvironment and induce phenotype maintenance in long term cultures or differentiation. 4. Therefore, we propose to assess the combined effect of macromolecular crowding and mechanical loading on different cell sources to determine their suitability for the in vitro fabrication of tendon-like tissue. Human dermal fibroblasts, tenocytes and bone marrow mesenchymal stem cells were cultured for 3 days with 100 µg/ml of carrageenan (MMC) under static and dynamic culture conditions. Cyclic uniaxial strain was applied using a MechanoCulture FX (CellScale) at 1 Hz and 10% strain for 12 hours a day. Cell morphology and alignment were evaluated by fluorescein isothiocyanate (FITC) labelled phalloidin and 4’,6-diamidino-2-phenylindole (DAPI) staining. Extracellular matrix composition was evaluated by immunocytochemistry. Cell phenotype maintenance/differentiation (tenogenic, chondrogenic and osteogenic lineages) were assessed by gene and protein analysis. After 12 hours of exposure to the uniaxial load, permanently differentiated cells are strictly aligned in the direction perpendicular to the load while the MSCs do not show preferential alignment. ECM deposition (e.g. collagens type I, III, V, VI) is increased in the presence of MMC and this effect is maintained under mechanical loading. ECM deposited under mechanical loading is also aligned in the direction perpendicular to the load. Tenogenic, osteogenic and chondrogenic markers are being tested to assess cell phenotype. Mechanical loading and macromolecular crowding can induce cell and ECM alignment and increased ECM deposition without affecting cell metabolic activity or viability. Cell and ECM alignment alongside ECM composition and tenogenic marker expression suggest this approach might be suitable to maintain or differentiate towards tenogenic lineage

Introduction: Tendinopathy entails pain and degenerative tissue proliferation such as tenocyte transformation and increased numbers of sensory nerves and microvessels. Pain and tissue proliferation are suggested to be modulated via nerve transmitters, including substance P (SP) and glutamate, both detected in tendinopathy. Substance P and glutamate are known to activate glutamate receptors in a variety of pain conditions and additionally to be implicated in cell transformation. However, the presence of different glutamate receptors, eg. ionotropic (NMDA) and metabotropic (mGlu), and whether they are up- or downregulated in tendinopathy is still unknown. In this study we assessed the. presence,. the tissue density and. the co-existence of different glutamate receptors together with glutamate in tendinopathic biopsies and controls. Methods: All procedures were conducted with local ethical committee approval and patient consent. Human patellar tendon biopsies of tendinopathic patients (n=10) and controls (n=8) were single- and double-stained immunohistochemically for glutamate, glutamate receptors NMDAR1, mGluR1, mGluR5 and mGluR6,7, the nerve marker PGP9.5 and SP and assessed subjectively and semi-quantitatively with image analysis. Images were taken using an epifluorescence microscope with camera and were subjectively assessed by two independent observers blinded with regard to the identity of the slides. Tenocyte density and morphologic characteristics were assessed. Non-parametric Mann-Whitney U-tests for independent samples were used, and the level for significance was set at p< 0.05. Results: Of the glutamate receptors tested all except mGluR1 was identified in the tendons, however only NMDAR1 was found significantly altered between both groups. The chronic painful tendons exhibited a significant elevation of NMDAR1 (9-fold) and also of glutamate (10-fold). This up-regulation of NMDAR1 and glutamate was found to be co-localized on sensory nerve fibers, blood vessels as well as on transformed tenocytes. None of the controls exhibited neuronal co-existence of glutamate with NMDAR1. Conclusions: This study establishes for the first time that patients with tendinopathy exhibit an elevation of peripheral glutamate receptor NMDAR1, morphologically co-localized with increased glutamate expression. The up-regulated NMDAR1/glutamate system may represent hyper-excitability of the cells – leading to cell proliferative effects observed as angiogenesis, tenocyte transformation, and nerve sprouting. Moreover, the neuronal co-existence of glutamate and NMDAR1 observed in painful tendinosis, but not seen in any of the controls, strongly suggests a role in pain signalling. Future studies will focuse on interventional approaches to investigate if modulation of NMDAR1 pathways can ameliorate the symptoms of tendinopathic patients

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

Summary Statement. We have shown that integrin mRNA expression is regulated by the application of mechanical load. This indicates that mechanical loading may modify cell sensitivity to perceive further load through increased interaction with the ECM. Introduction. Tendinopathies are a range of diseases characterised by pain and insidious degeneration. Although poorly understood, onset is often associated with physical activity. We have previously investigated the regulation by mechanical strain of metalloproteinase gene expression in human tenocyte in a 3D collagen matrix. Integrins are important in cellular interaction with the ECM and are reported to mediate mechanotransduction in various non-tendon tissues. We have reported that TGFbeta activation is a key player in the regulation of metalloproteinases in response to mechanical load, which may be mediated by integrins. This project aims to investigate the effect of cyclic loading and TGFbeta stimulation on integrin expression by human tenocytes, in collagen and fibrin matrices. Methods. Human tenocytes were seeded at 1.5×106 cells/ml into collagen (rat tail type I, 1mg/ml) or fibrin (fibrinogen 6mg/ml, Thrombin 0.2u/ml) gels and stretched using a sinusoidal waveform of 0–5% at 1Hz using the Flexcell FX4000T(trade mark) system. Cultures were treated with or without 1ng/ml TGFbeta1 and load for 0–48 hours. Taqman Low density Array was used to asses a range of integrin, including ITGA1-6, ITGA10 and ITGA11 as well as ITGB1-5 (n=3). Results. In collagen cultures all integrins assayed were detectable (Ct < 35). ITGB1 was increased 2 fold with 48 hours of cyclic strain (p=0.006). ITGA6 and ITGA10 were decreased 1.4 and 2 fold with TGFbeta treatment after 24 hours (p=0.019, p=0.006). ITGA3 and ITGB3 were significantly decreased 7.6 and 8.3 fold with TGFbeta treatment after 48 hours (p=0.012, p=0.023). ITGA5 and ITGB1 showed similar responses with strain and TGFbeta, i.e. an increased trend. However, the other integrins showed a dissimilar response to strain and TGFbeta. Here we compare these responses to those in fibrin under the same conditions. Discussion. We have shown that integrin mRNA expression is regulated by the application of mechanical load. This indicates that mechanical loading may modify cell sensitivity to perceive further load through increased interaction with the ECM. Any differences in the cellular response to load in collagen and fibrin cultures, indicates that cellular interaction with the ECM is an important factor in the detection of load. Due to the differential regulation of some of the integrins with strain and TGFbeta, it appears that TGFbeta may not be responsible for the regulation of all integrins with strain. However this remains unconfirmed and may be explained by a temporal difference. Further analysis of how integrins are regulated in response to mechanical load and how this expression is translated to the protein level is required

Anterior cruciate ligament injury is the most common and economically costly sport injuries, frequently requiring expensive surgery and rehabilitation. Post-operative knee septic arthritis represents a serious complication with an incidence rate between 0.14% and 1.7%. A common practice to avoid septic arthritis is the “vancomycin wrap”, consisting in the soaking of the graft for 10–15 minutes within a sterile gauze swab previously saturated with 5 mg/mL vancomycin. Even though several studies have been conducted to investigate vancomycin toxicity on different musculoskeletal tissues or cells, little is known about the effect of such antimicrobial on tendon-derived cells. The aim of this study was to determine the in vitro toxicity of different concentrations of vancomycin at different time points on human primary tenocytes (hTCs). hTCs were isolated from hamstring grafts of patients undergoing anterior cruciate ligament reconstruction. After expansion, cells were treated with different concentrations of vancomycin (2.5, 5, 10, 25, 50 and 100 mg/mL) for 10, 15, 30 and 60 minutes. In vitro toxicity was evaluated measuring: metabolic activity through the reduction of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT Assay); cytotoxicity (Live/Dead assay); and cell apoptosis (Annexin V apoptosis kit). The metabolic activity of hTCs was affected by vancomycin treatment starting from 10 mg/mL at all time points (p < 0.05) and dropped down at 100 mg/mL at all time points (0.05 < p < 0.001). Cells viability resulted to be unaffected only by 2.5 mg/mL vancomycin at all time points. Vancomycin resulted to be cytotoxic starting from 10 mg/mL after 15 minutes of treatment and at all higher concentrations under study at all time points. Cells died when treated with vancomycin concentrations higher than 5 mg/mL but not through apoptosis, as confirmed by negative staining for Annexin V. In our experimental conditions, vancomycin resulted to be toxic on hTCs at concentrations higher than 5 mg/mL. The use of this antibiotic on tendons to prevent infections could be useful and safe for resident cells if used at a concentration of 2.5 mg/mL up to 1 hour of treatment

Summary Statement. Tendon micromechanics were investigated using 2 methods. When collagen deformation was measured directly, higher levels of inter-fibre sliding were observed than when tenocyte nuclei were tracked. This suggests that under high strain tenocytes become unattached from the collagen fibres. Introduction. Fibre extension and inter-fibre sliding have both been reported during tendon extension, but fibre sliding is believed to be the predominant mechanism in normal healthy tendon function. Fatigue damage is known to result in structural changes and reduced mechanical properties, but its influence on micromechanics is unknown. This work aimed:. 1. To investigate the effect of fatigue loading on bovine digital extensor fascicle micromechanics, comparing fibre extension and fibre sliding, hypothesising that the relative importance of these may change due to fatigue damage. 2. To compare two techniques for characterising micromechanics: bleaching of a grid to directly measure collagen deformation, and using the cells as fiducial markers of fibre movement. Methods. The tensional regions of healthy digital extensor tendons were removed within 24 hours of slaughter and frozen. Tendons were defrosted, hydrated and fascicles dissected and loaded into custom-designed chambers allowing the mechanical loading of fully hydrated tendon fascicles. Fascicles were loaded for 0, 300 or 900 cycles under creep conditions at a frequency of 1Hz and to a maximum applied stress of 25% of the mean UTS of the fascicles. Fascicles were stained using either Acridine Orange to stain the cell nuclei or DTAF solution to stain the collagen. After DTAF staining, a grid consisting of 4 squares of side 50 μm was photo-bleached using the FRAP system on a Leica TCS SP2 confocal scanning microscope. To investigate micromechanics, fascicles were secured in a uniaxial rig and strained in 2% increments to 10% total strain at a rate of 1%s. −1. Imaging was carried out at each increment and local strains calculated from grid deformation or nuclei movement. Results. No significant changes in micromechanics were observed with increasing numbers of creep cycles, as measured with either technique. This was despite quite significant matrix damage being observed particularly after 900 cycles. When using the grid deformation measure of strains, a continual increase in fibre sliding was seen above 4% applied strain, correlating with the levelling off of intra-fibre strains. This same move towards dominant fibre sliding was not observed with techniques using the nuclei as fiducial markers. Using the nuclei as markers consistently reported significantly lower levels of fibre sliding than those measured from grid deformation at strains of 6% and above, under all creep conditions. Discussion/Conclusion. The apparent absence of any effect of creep on the measured microstructural deformation may be a result of the localised nature of the measurement techniques. At sites where matrix structure broke down both the tracking of nuclei and the photo-bleaching of the grid proved problematic and it is these regions where the greatest degree of deformation would perhaps be expected, with remaining areas of the tissue stress-deprived. The smaller levels of fibre shear reported when measured through nuclei tracking suggests that the tenocytes may not be well adhered to the fibres and may be protected from some of the matrix deformation in response to loading

Summary. Silver nanoparticles improve the tensile property of the repaired Achilles tendon by modulating the synthesis and deposition of collagen. This makes silver nanoparticles a potential drug for tendon healing process with less undesirable side effect. Introduction. Tendon injury is a common injury that usually takes a long time to fully recover and often lead to problems of joint stiffness and re-rupture due to tissue adhesions and scarring on the repaired tendon respectively. Recently, it has been proven that silver nanoparticles (AgNPs) are capable of regenerating skin tissue with minimal scarring and comparable tensile property to normal skin. Hence, it is hypothesised that AgNPs could also improve the healing in tendon injury as both tissues are predominating with fibroblasts. The objective of this study is to look at the in vitro response of primary tenocytes to AgNPs and to investigate the mechanical and histological outcome in vivo. Methods and Materials. Primary tenocytes were harvested from 4 weeks old Sprague Dawley rat. 1.5×10. 4. cells per cm. 2. were seeded in triplicate for BrdU incorporation assay and Sirius red/ fast green staining to study the proliferation and collagen synthesis respectively. In vivo rat Achilles tendon injury model was used to investigate the effect of AgNPs to tendon regeneration. Briefly, the Achilles tendon was transected at 0.5cm from its insertion. The wound was either treated with 1mM AgNPs every 5 days or left untreated as the control. Skin incision was done without transecting the tendon in the sham group. The tendons were harvested on day 42 post operation. Tensile test and immunohistological staining on 7μm cryosections were performed to assess the mechanical property and biological events in healing respectively. SHG imaging was used to determine the collagen fibre orientation and abundance. Results. In vitro BrdU incorporation and Sirius red fast green assay suggested that AgNPs promoted the proliferation and collagen synthesis of tenocytes between 1 to 20μM and 10 to 20μM respectively. Tensile test on in vivo tissue showed that AgNPs-treated samples had significantly better tensile modulus compared to the untreated ones (p<0.05). SHG imaging suggested a better collagen alignment and density in AgNPs-treated samples. Immunohistochemistry demonstrated that AgNPs suppressed tumor necrosis factor (TNF α) whilst promoted fibromodulin (Fmod) and proliferating cell nucleus antigen (PCNA) expression. Discussion. Collagen is the major component that contributes to the tensile strength of a tendon. Its thickness, abundance and alignment directly affect the strength. In this study, it is found that AgNPs stimulate cell proliferation both in vitro and in vivo which is believed to be the reason of the increase in collagen synthesis. Fmod is an important proteoglycan responsible for collagen fibrillogenesis and TNF α is related to ECM degradation which directly affects collagen integrity. Stimulation of Fmod and alleviation of TNF α therefore promote collagen maturity and integrity which attributes to the improvement in the tensile property of the regenerated tissue. Furthermore, inflammation is known to relate to fibrosis and scarring in healing of many types of tissue. It is therefore postulated that the anti-inflammatory effect of AgNPs is one of the major reasons for this phenomenal healing of tendon. To conclude, this study demonstrates a positive effect of AgNPs to the early events of tendon healing which is important for accelerating the whole healing process and shortening of rehabilitation time

Cell-based therapies require removal of cells from their optimal in vivotissue context and propagation in vitroto attain suitable number. However, bereft of their optimal tissue niche, cells lose their phenotype and with it their function and therapeutic potential. Biophysical signals, such as surface topography and substrate stiffness, and biochemical signals, such as collagen I, have been shown to maintain permanently differentiated cell phenotype and to precisely regulate stem cell lineage commitment (1, 2). Herein, we developed and characterised substrates of variable rigidity and constant nanotopographical features to offer control over cellular functions during ex vivoexpansion. PDMS substrates with varying ratios of monomer to curing agent (0:1, 1:1, 5:1) were fabricated based on established protocols. Grooved substrates were created using a silinated wafer with groove dimensions of 2µm × 2µm × 2µm; planar control groups were created using flat glass. The aforementioned PDMS solutions were poured onto the wafer/glass, cured at 200 ºC and treated with oxygen plasma. Substrates were then investigated with/without collagen I coating. (0.1, 0.5, and 1 mg/ml). Atomic force microscopy (AFM) and optical profilometry were used to assess the topographical features of the substrates. Dynamic mechanical analysis (DMA) was used to determine the mechanical properties of the substrates. The simultaneous effect of surface topography / substrate rigidity on cell phenotype and function was assessed using human permanently differentiated cells (dermal fibroblasts, tenocytes) and stem cells (human bone marrow stem cells) and various morphometric and gene / protein assays. PDMS substrates of varying stiffness (1000 kPa, 130 kPa, 50 kPa) can be made by varying the Sylgard ratio, while maintaining topographical features. Human adult dermal fibroblasts, tenocytes, and tenocytes attach, align, elongate and deposit aligned extracellular matrix on the grooved PDMS substrate surface of all 3 stiffnesses. Preliminary in vitrodata indicate that surface topography and substrate stiffness play crucial role in maintaining cell phenotype and the prevention of phenotypic drift in vitro

Tendon is a bradytrophic and hypovascular tissue, hence, healing remains a major challenge. The molecular key events involved in successful repair have to be unravelled to develop novel strategies that reduce the risk of unfavourable outcomes such as non-healing, adhesion formation, and scarring. This review will consider the diverse pathophysiological features of tendon-derived cells that lead to failed healing, including misrouted differentiation (e.g. de- or transdifferentiation) and premature cell senescence, as well as the loss of functional progenitors. Many of these features can be attributed to disturbed cell-extracellular matrix (ECM) or unbalanced soluble mediators involving not only resident tendon cells, but also the cross-talk with immigrating immune cell populations. Unrestrained post-traumatic inflammation could hinder successful healing. Pro-angiogenic mediators trigger hypervascularization and lead to persistence of an immature repair tissue, which does not provide sufficient mechano-competence. Tendon repair tissue needs to achieve an ECM composition, structure, strength, and stiffness that resembles the undamaged highly hierarchically ordered tendon ECM. Adequate mechano-sensation and -transduction by tendon cells orchestrate ECM synthesis, stabilization by cross-linking, and remodelling as a prerequisite for the adaptation to the increased mechanical challenges during healing. Lastly, this review will discuss, from the cell biological point of view, possible optimization strategies for augmenting Achilles tendon (AT) healing outcomes, including adapted mechanostimulation and novel approaches by restraining neoangiogenesis, modifying stem cell niche parameters, tissue engineering, the modulation of the inflammatory cells, and the application of stimulatory factors.

Cite this article: Bone Joint Res 2022;11(8):561–574.

Adherent cells are known to respond to physical characteristics of their surrounding microenvironment, adapting their cytoskeleton and initiating signaling cascades specific to the type of cue encountered. Scaffolds mimicking native biophysical cues have proven to differentiate stem cells towards tissue-specific lineages and to maintain the phenotype of somatic cells for longer periods of culture time. Although the characteristic anisotropy of tendon tissue is commonly replicated in scaffolds, relevant physical cues such as tendon rigidity or mechanical loading are often neglected. The objective of this study is to use tendons' main extracellular matrix component, collagen type I, to create scaffolds with an anisotropic surface topography and controlled rigidity, in an effort to engineer functional tendon tissue equivalents, with native organization and strength. Porcine collagen type I in solution was treated with one of the following cross-linkers: glutaraldehyde, genipin or 4-arm polyethylene glycol (4SP). The resulting mixture was poured on micro-grooved (2×2×2 μm) or planar polydimethylsiloxane (PDMS) molds and dried in a laminar flow hood to obtain 5 mg/ml collagen films. Surface topography and elastic modulus of the final scaffolds were analyzed using SEM/AFM and rheometry, respectively. Human tendon cells were isolated from adult tendon tissue and cultured on micro-grooved/planar scaffolds for 4, 7 and 10 days. Cell morphology, collagen III and tenascin C expression were analyzed by immunocytochemistry. Among the different cross-linkers used, only the treatment with 4SP resulted in scaffolds with a recognizable micro-grooved surface topography. Precise control over the micro-grooved topography and the rigidity of the scaffolds was achieved by cross-linking the collagen with varying concentrations of 4SP at low pH and temperature. The elastic modulus of the scaffolds cross-linked with the highest concentration of 4SP matched the physiological values reported in developing tendons (∼15 kPa). Around eighty percent of the human tendon cells cultured on the cross-linked collagen films aligned in the direction of the anisotropy for 10 days in culture. At 4 days, tenoyctes cultured on micro-grooved substrates presented a significant higher nuclei aspect ratio than tenocytes cultured on planar substrates for all the 4SP concentrations. Synthesis, deposition and alignment of collagen III and tenascin C, two important tenogenic markers, were up regulated selectively in the rigid micro-grooved scaffolds after 7 days in culture. These results highlight the synergistic effect of matrix rigidity and cell alignment on tenogenic cell lineage commitment. Collectively, this study provides new insights into how collagen can be modulated to create scaffolds with precise imprinted topographies and controlled rigidities. Gene expression analysis and a replicate study with hBMSCs will be carried out to support the first results and to further identify the optimal biophysical conditions for tenogenic cell lineage commitment. This potentially leads to the design of smart implants that not only restore immediate tendon functionality but also provide microscopic cues that drive cellular synthesis of organized tissue-specific matrix

Summary Statement. This study describes the design and preliminary in vitro testing of a novel patch for the repair of rotator cuff tendon tears. The laminated design incorporates woven and electrospun components. The woven element provides the patch with excellent mechanical strength and the electrospun layer improves cell attachment and promotes cell orientation and diferentiation. Introduction. Aligned nanofibrous electrospun scaffolds have been previously proposed as ideal scaffolds for tendon repair, replicating the anisotropy of tendon and providing a biomimetic design to encourage tissue regeneration (Hakimi et al., 2012). However, such scaffolds are still limited in terms of mechanical properties. This paper presents the design of a novel patch for rotator cuff repair in which the electrospun scaffold is supported by a woven component. Patients & Methods. Aligned polydioxanone (PDO) electrospun scaffolds were produced using a single nozzle electrospinning set-up with a rotating collector. The woven component was created by weaving PDO monofilaments with a manual loom. The woven and non-woven constituents were bound by a non-destructive method which preserves the surface morphology of the electrospun material. For each type of scaffold, a minimum of 3 specimens were tested to failure in tension using Zwick machine at rate of 0.3 mm/min until failure. For in vitro work, human-derived tendon cells were extracted from rotator cuff tendon tissue obtained during surgical repair, with appropriate ethical approval. Cells were cultured on the scaffolds for at least 14 days. Results. The contribution of the woven component to the tensile strength of the assembled patch is about 20 times more when compared to the electrospun scaffold. In vitro work shows that human tenocytes grown on the nanofibrous non-woven electrospun component align in the direction of the fibre orientation. The appearance of the woven component is shown. Discussion/Conclusion. While the woven component provides most of the mechanical strength, the aligned electrospun fibres enable cell orientation along the axis of the patch. These cells display a similar morphology to tenocytes in native tendons. With the combination of biomimetic features and good mechanical properties, this novel PDO patch is an excellent candidate material to support tendon repair

Human mesenchymal stem cells (MSCs) are multipotent stem cells with the ability to differentiate into mesoderm-type cells such as osteoblasts, chondroblast, tenocytes etc. They can be retrieved by different sources, but the number of cells obtained suggested the adipose tissue as a primary harvest site of MSCs. Cells can be harvested using the Coleman procedure, obtaining stromal vascular fraction (SVF), enriched with MSCs, after collagenase digestion. The availability of SVF storage has been envisioned for multiple treatments of the degenerated tissue. Indeed, the use of SVF has been introduced into clinical trials for tissue regeneration for orthopaedic patients. Difficulties of a selective delivery of SVF locally have been previously discussed. Thus, the use of biological scaffolds in order to better localize SVF in the tissue site has been studied. The methodological evolution for the use of SVF in the best possible biological conditions is a milestone for good clinical results

Aims

To test the hypothesis that reseeded anterior cruciate ligament (ACL)-derived cells have a better ability to survive and integrate into tendon extracellular matrix (ECM) and accelerate the ligamentization process, compared to adipose-derived mesenchymal stem cells (ADMSCs).

Current cell-based tissue engineering strategies have limited clinical applicability due to the need for large cell numbers and prolonged culture periods that lead to phenotypic drift. In vitro microenvironmental modulators have been proposed to mimic the native tendon. Standard in vitro culture conditions result in delayed extracellular matrix (ECM) deposition, impairing the development of scaffold-free approaches. ECM deposition can be enhanced by macromolecular crowding (MMC), a biophysical phenomenon that governs the milieu of multicellular organisms. We assessed a multifactorial biophysical approach, using MMC and mechanical loading, on different cell sources to determine their suitability for in vitro fabrication of tendon-like tissue. Human dermal fibroblasts (DFs), tenocytes (TCs) and bone marrow mesenchymal stem cells (BMSCs) were cultured with MMC under static and uniaxial strain culture conditions. TCs and DFs exhibited alignment perpendicular to the load, whilst BMSCs did not show preferential alignment. When MMC was used, DFs and BMSCs showed increased deposition of collagen I, the main component in tendon ECM. DFs presented ECM composition similar to TCs with collagen types III, V and VI present. Gene expression analysis revealed upregulation of tenogenic markers by TCs and DFs, such as scleraxis and thrombospondin-4, under both loading and MMC. The combined use of MMC and mechanical stimulation is suitable for TCs phenotype maintenance and can modulate the phenotype of DFs and BMSCs differentially. This study provides insight into response of different cell sources to biophysical cues and contributes to further development of cell therapies for tendon repair and regeneration

Aims

A revision for periprosthetic joint infection (PJI) in total hip arthroplasty (THA) has a major effect on the patient’s quality of life, including walking capacity. The objective of this case control study was to investigate the histological and ultrastructural changes to the gluteus medius tendon (GMED) in patients revised due to a PJI, and to compare it with revision THAs without infection performed using the same lateral approach.

R Appleyard, Murray Maxwell Biomechanics Lab, Royal North Shore Hospital, Sydney. The fundamental mechanisms that underlie tendon breakdown are ill understood. There is an emerging hypothesis that altered mechanical strain modulates the metabolism and/or phenotype of tenocytes, disrupting the balance of matrix synthesis and degradation, and that rupture then occurs through an abnormal tendon matrix. The critically regulated genes have not yet been determined. We have developed sheep model in sheep where both stress-deprived and over-stressed areas can be examined in the one tendon, to evaluate the pathological and molecular changes over time. We have also used ‘wild type’ and genetically modified mice to determine the role of specific enzymes and proteoglycans in tendon degeneration. Stress-deprived and over-stressed regions showed classical changes of increased cellularity and vascularity, rounded tenocytes and interfascicular matrix infiltration. These structural changes resolved for up to one year after injury. Resolution was more rapid in over-stressed regions. Irrespective of the initiating stress, proteoglycan staining and chondroid metaplasia increased in tendon with time. There were distinct molecular and temporal differences between regions, which are reviewed here. While tendon degeneration has traditionally been regarded as a single field of change, our studies show that at a molecular level, the injured tendon may be regarded as a number of distinct regions—overloaded and underloaded, adjacent to bone or adjacent to muscle. Each region manifests distinct molecular changes, driven by relevant gene expression. While collagen metabolism in pathological tendon has received much attention, accumulation of proteoglycan is also consistently induced by altered mechanical loading. We suggest that ADAMTS enzymes, which cleave aggrecan, versican and small proteoglycans, may play a significant role in tendon homeostasis and pathology. Regulating proteoglycan turnover may represent a novel target for treating tendon degeneration. We have initiated studies using mesenchymal stem cells (MSC), not to directly augment healing but to modify the molecular pathology in tendon resulting from altered loading. Preliminary data indicates that injection of MSC into an acute tendon defect significantly abrogates the increase in expression of aggrecan and collagen degrading metalloproteinases in the adjacent over-stressed tendon. This may decrease the resultant degeneration. The effects of MSC in treating tendon degeneration are reviewed here, as are the possible benefits of radiofrequency microtenotomy

Tenocytes from several mammal species have been shown to be prone to phenotypic drift at early sub-culture passages. In the present study we compared allogenic and xenogenic serum supplementation suitability as a supplement for the in vitro expansion of equine tenocytes (eTCs), in combination with the presence or absence of crowding conditions. eTCs were isolated from superficial digital flexor tendon and expanded in normal growth medium containing DMEM, 10% appropriate serum, 1% penicillin/streptomycin solution. Isolation was performed by migration method in growth medium containing the selected serum. Silver staining, densitometry, zymography, immunofluorescence, metabolic activity, proliferation, viability and morphology were performed after 3, 5 and 7 days in culture with a seeding density of 10,000 cells/cm2. Treatment conditions were equine serum (ES) or foetal bovine serum (FBS), with or without 75 μg/mL of crowding agent carrageenan (CR). Viability and metabolic activity of eTCs were affected by FBS. eTCs in ES reached higher cell density than in FBS in day 7, especially with CR. Morphology of eTCs was maintained under different sera. Silver staining on pepsin digested cell layers shows that collagen type I deposition rate is remarkably enhanced in the presence of CR in all conditions. Immunofluorescence showed increased expression for collagen I, III, V and VI in both sera in the presence of CR. Deposition of all collagen types but type VI was increased by ES supplementation. We conclude that ES in combination with CR can represent a reliable choice for the ex vivo expansion of eTCs

Rotator cuff tears are common in middle-aged and elderly patients. Despite advances in the surgical repair of rotator cuff tears, the rates of recurrent tear remain high. This may be due to the complexity of the tendons of the rotator cuff, which contributes to an inherently hostile healing environment. During the past 20 years, there has been an increased interest in the use of biologics to complement the healing environment in the shoulder, in order to improve rotator cuff healing and reduce the rate of recurrent tears. The aim of this review is to provide a summary of the current evidence for the use of forms of biological augmentation when repairing rotator cuff tears.

Cite this article: Bone Joint J 2024;106-B(9):978–985.

As we grow older, the risk of tendon degeneration and injuries increases, which can result in pain, disability, healthcare cost, and lost productivity. Even after surgical repair the results are often unsatisfactory. The cellular reasons for the differences in the healing potential, however, are not well studied. To get a deeper insight into the biological characteristics of tenocyte-like cells from different patient groups we established a biobank with material from over 150 human donors. The patients/donors suffered from rotator cuff tears and were operated to restore the function. A proportion of the isolated cells showed stem cell-like characteristics and was able to differentiate into the osteoblastic, chondrogenic and adipogenic linage. Investigating the differentiation potential of the cells with regard to donor characteristics, we were able to demonstrate that age, sex but also the “degeneration” has an impact of the cellular potential. A possibility to stimulate the cellular activity is the application of growth factors, as already clinically used for stimulation of bone healing. Therefore, the responsiveness of the cells to the growth factors Bone Morphogenetic protein-2/7 (BMP-2/7) was analysed in vitro. Independent of the donor characteristics, the cells responded to the BMP-stimulation by increased proliferation and collagen-1 synthesis. However, cells isolated from donors with high fatty infiltration of the muscle or older females were less responsive. Looking into the intracellular signalling pathway, the data showed that the BMP-signal is mainly mediated by the canonical-pathway with samd8 playing a major role. This basic research gives first information regarding the differences in tenocytes biology with respect to the donor and is important for the understanding of tendon regeneration and the future development of new treatment strategies

The December 2022 Foot & Ankle Roundup³⁶⁰ looks at: Evans calcaneal osteotomy and multiplanar correction in flat foot deformity; Inflammatory biomarkers in tibialis posterior tendon dysfunction; Takedown of ankle fusions and conversion to total ankle arthroplasty; Surgical incision closure with three different materials; Absorbable sutures are not inferior to nonabsorbable sutures for tendo Achilles repair; Zadek’s osteotomy is a reliable technique for treating Haglund’s syndrome; How to best assess patient limitations after acute Achilles tendon injury; Advances in the management of infected nonunion of the foot and ankle.

Aims

Mechanical stimulation is a key factor in the development and healing of tendon-bone insertion. Treadmill training is an important rehabilitation treatment. This study aims to investigate the benefits of treadmill training initiated on postoperative day 7 for tendon-bone insertion healing.

Aims

Rotator cuff (RC) tears are common musculoskeletal injuries which often require surgical intervention. Noninvasive pulsed electromagnetic field (PEMF) devices have been approved for treatment of long-bone fracture nonunions and as an adjunct to lumbar and cervical spine fusion surgery. This study aimed to assess the effect of continuous PEMF on postoperative RC healing in a rat RC repair model.

Introduction. Primary cilia are organelles found singularly on almost every cell in the body, including tenocytes. Tendon is a hierarchical, composite structure, and previous work from our group has suggested that the cell populations in the inter-fascicular matrix (IFM) may be different from those within the fascicle matrix (FM). This study investigated how stress deprivation influenced the primary cilia of both cell types, and the mechanics of the IFM and the FM. Materials and Methods. Rat tail tendons were dissected and then either tested immediately (fresh), or maintained in media for 1 week, either stress deprived or at 4% static strain. Fascicles and IFM were then either, fixed and imaged to determine cilia length (n = 80–160 cilia per group from across 3 rats), or mechanically tested to determine the static and viscoelastic properties of both the fascicles and the IFM (n = 6–8 per group). Results. Cilia length in the IFM and FM of fresh samples were not significantly different. After 1 week of stress deprivation, the cilia had significantly increased in length in both the IFM and FM, however the increase in length in the IFM was significantly greater than that in the FM. Cilia in tissue maintained at 4% static strain were significantly shorter than those in stress deprived tissue, however they remained longer than those in fresh tissue. The tensile strength of the fascicles was not affected by stress deprivation or static strain conditions. However, the viscoelastic properties of the stress deprived fascicles were significantly reduced. By contrast, the tensile strength of the IFM was significantly reduced in the stress deprived samples, indicative of greater degradation in this region. Discussion. This is the first time differences in the cilia have been observed between tendon regions. Their different response to stress deprivation provides further evidence that these populations of cells respond differently to changes in mechanical stimulation. Cilia length increased more in the region where there was more mechanical degradation, suggesting that cilia are responding to their local mechanical environment

Achilles tendinopathy is a common cause of disability. Despite the economic and social relevance of the problem, the causes and mechanisms of Achilles tendinopathy remain unclear. Tendon vascularity, gastrocnemius-soleus dysfunction, age, gender, body weight and height, pes cavus, and lateral ankle instability are considered common intrinsic factors. The essence of Achilles tendinopathy is a failed healing response, with haphazard proliferation of tenocytes, some evidence of degeneration in tendon cells and disruption of collagen fibres, and subsequent increase in non-collagenous matrix. Tendinopathic tendons have an increased rate of matrix remodelling, leading to a mechanically less stable tendon which is more susceptible to damage. The diagnosis of Achilles tendinopathy is mainly based on a careful history and detailed clinical examination. The latter remains the best diagnostic tool. Over the past few years, various new therapeutic options have been proposed for the management of Achilles tendinopathy. Despite the morbidity associated with Achilles tendinopathy, many of the therapeutic options described and in common use are far from scientifically based. New minimally invasive techniques of stripping of neovessels from the Kager's triangle of the tendo Achillis have been described, and seem to allow faster recovery and accelerated return to sports, rather than open surgery. A genetic component has been implicated in tendinopathies of the Achilles tendon, but these studies are still at their infancy

Purpose of the study: Platelet rich fibrin (PRF) favours proliferation of tenocytes and synthesis of extracellular matrix. The purpose of this study was to demonstrate the technical feasibility of adding a PRF envelope during arthroscopic rotator cuff repair to favour short-term vascularisation of the tendon-trochiter zone vascularization. Material and method: Twenty patients aged over 55 years with a posterosuperior rotator cuff tear were included in this prospective randomized controlled study. The double strand technique was used for all patients. Patients were selected at random for insertion of a PRF envelope between the tendon and the trochiter. There were thus two groups of ten patients. The SSV, SST, VAS and Constant scores were noted. Vascularization was assessed with Power Doppler ultrasound at 6 weeks and 3 months by an independent operator unaware of the study group. Results: There were no complications during or after the operations. Postoperatively, all patients increased their SSV, SST, VAS and Constant scores significantly. Vascularization of the tendon-trochiter zone, as assessed by Power Doppler, was significantly higher in the PRF group at 6 weeks. It was unchanged in the two groups at 3 months. Discussion: Arthroscopic rotator cuff repair with adjunction of a PRF envelope is technically feasible and increases vascularizaton of the tendon-trochiter zone at 6 weeks. Conclusion: PRF can improve the tendon healing rate for rotator cuff tears

Introduction: We studied the extracellular matrix (ECM) of 19 ruptured human Achilles tendons, comparing the tissue composition of specimens taken from area close to the rupture with specimens harvested from an apparently healthy area in the same tendon. The hypothesis was that the metabolism of these molecules is altered in patients with Achilles tendon rupture. Materials and Methods: We compared the gene expression and the protein localization of the main ECM molecules (collagen type I, decorin and versican) including enzymes involved in their metabolism as matrix metallo-proteases (MMP2 and 9) and tissue inhibitory of metal-loproteinase (TIMP 1 and 2) using a Real Time PCR, zymography and FACE analysis. Results: The gene expression of proteoglycans core protein, collagen type I, MMPs and TIMPs is more represented in the area close to the tendon rupture (p< 0.05). The expression of MMPs was confirmed by zymography analysis, showing a marked increase of gelatinolytic activity in area close to the tendon rupture (p< 0.05). The chemical composition of tendon changes showing that in the healthy area the carbohydrate content is higher than the ruptured area (p< 0.05). Discussion/Conclusions: In the ruptured area, the tenocytes tried to restore the normal proteoglycan pattern increasing the core protein synthesis but without the normal glycosaminoglycan production. Our data support the hypothesis that, in human tendons, the tissue in the area of rupture undergoes marked rearrangement at molecular levels based on the MMP2 activity, and support the role of MMPs in the tendon pathology

Purpose: Ultrastructural analysis of PT graft for ACL single bundle reconstruction. Materials and methods: Arthroscopical biopsies for new meniscal lesions at 6-12-24mm-5-10 ys. All cases with IKDC normal/nearly normal and KT2000 excellent/good. Samples prepared with Karnowsky fixing and urani-lacetate solution. Fibril diameter and transversal area measured by LEICA QUIN in 5 cuts randomly selected for each sample. Results: 6 months biopsy showed severe P.T. modifications, with a decrease of larger fibrils, substituted with smaller one with plenty of extra cellular matrix. Oxitalan fibers, macrophagic cells and tenocytes were observed. At 12 months compact fascicles of small fibrils (50–60 nm) divided the larger one, similar to a normal tendon. At 24 months graft modifications were increased with wide compact fasciclesvariously oriented. At 5 and 10 years the modifications were similar to those observed at 2 years, with the graft not completely transformed in native ACL structure. Discussion: The results showed that PT graft used for ACL single bundle reconstruction certainly undergoes a neoligamentization process up to two years. At longer follow-up the foresaw complete remodelling in a normal ACL was not observed. Heterogeneous fibrils presence suggests incomplete ligamentization or its impossible complete realization in single bundle ACL reconstructions

Aims

Proliferation, migration, and differentiation of anterior cruciate ligament (ACL) remnant and surrounding cells are fundamental processes for ACL reconstruction; however, the interaction between ACL remnant and surrounding cells is unclear. We hypothesized that ACL remnant cells preserve the capability to regulate the surrounding cells’ activity, collagen gene expression, and tenogenic differentiation. Moreover, extracorporeal shock wave (ESW) would not only promote activity of ACL remnant cells, but also enhance their paracrine regulation of surrounding cells.

Summary. Our results prove that Demineralised Cortical Bone (DCB) can be used as biological tendon graft substitute, combined with correct surgical technique and the use of suture bone anchor early mobilisation can be achieved. Introduction. Surgical repair of tendon injuries aims to restore length, mechanical strength and function. In severe injuries with loss of tendon substance a tendon graft or a substitute is usually used to restore functional length. This is usually associated with donor site morbidity, host tissue reactions and lack of remodelling of the synthetic substitutes which may result in suboptimal outcome. In this study we hypothesise that DCB present in biological tendon environment with early mobilisation and appropriate tension will result in remodelling of the DCB into ligament tissue rather that ossification of the DCB at traditional expected. Our preparatory cadaveric study (abstract submitted to CORS 2013) showed that the repair model used in this animal study has sufficient mechanical strength needed for this animal study. Methods. 6 mature female sheep undergone surgical resection of the distal 1 cm of the right patellar tendon and osteotomy of patellar tendon attachment at the tibial tuberosity under general anaesthesia. Repair was done using DCB with 2 suture bone anchor. Animals were allowed immediate mobilisation after surgery and were sacrificed at 12 weeks. The force passing through the operated and non-operated legs was assessed preoperatively and at week 3, week 6, week 9 and week 12 bay walking the animals over a force plate. Radiographs were taken immediately after euthanasia, the Patella-Tendon-tibia constructs were retrieved and pQCT scan was done. Histological analysis included tenocytes and chondrocytes cell counts, semi-quantitative scoring of the neo-enthesis and polarised microscopy. Result. In this study, none of the retrieved specimens showed any evidence of ossification of the DCB as proved by the pQCT analysis. One animal failed to show satisfactory progress after week 3, X-rays showed patella alta, on specimen retrieval no damage to the DCB was found, sutures and stitches were intact and no evidence of anchor pullout was found. Force plate analysis of the other 5 animals showed satisfactory progression over time with 44% functional weight bearing at week 3 progressing to 79% at week 12. There was full range of movement of the stifle joint after 12 weeks. Histological analysis proved formation of neo-enthesis with evidence of cellulisation, vascularisation and remodelling of the collagen leading to ligamentisation of the DCB. Discussion. Surgical reconstruction of damaged tendons is technically challenging, patellar tendon injuries presents even more challenging situation as it involves weight bearing joint. It is generally accepted that a period of immobilisation with passive range of movement exercises and protected weight bearing for up to 6 weeks post operatively is usually advised. Some surgeons use offloading metal wire to protect the repair for 6 weeks involving second surgical procedure to remove the wire. Demineralised bone is usually used in orthopaedics to utilise its osteogenic properties as bone graft substitute and to enhance osteogenesis in load bearing situations. In our study we explored a potential new use of the demineralised bone as tendon graft substitute, it acts as collagen scaffold allowing host cells to remodel its fibres into ligament like structure

Summary Statement. A novel biomimetic polydioxanone tendon patch with woven and electrospun components is biocompatible, recapitulates native tendon architecture and creates a tissue-healing microenvironment directed by a subpopulation of regenerative macrophages. The woven component provides tensile strength while the tendon heals. Introduction. There is great interest in the use of biomimetic devices to augment tendon repairs. Ideally, implants improve healing without causing adverse local or systemic reactions. Biocompatibility remains a critical issue prior to implantation into humans, as some implants elicit a foreign body response (FBR) involving inflammation, poor wound healing and even fistulae formation. Additionally, the effect on articular cartilage locally or systemically with placement of a juxta-articular implant has not been examined. The purpose of this study is to test the in vivo biocompatibility of a novel hybrid woven and electrospun polydioxanone patch in a rat tendon transection model. Patients and Methods. Sixty Lewis rats were divided into 4 groups in which the infraspinatus was surgically transected 3 mm from its insertion. Tendons were repaired with a woven and electrospun polydioxanone patch (PDOe) and 5-0 Prolene sutures. Vicryl and Silk patches or a simple Prolene suture repair served as comparators. Animals were sacrificed at 1, 2, 4, 6 and 12 weeks to examine the biocompatibility of the implants. Immunohistochemistry was used to examine macrophage subpopulations and hematoxylin and eosin staining was used to assess foreign-body giant cells and both analyzed with a one-way ANOVA with significance set at p<.05. Articular cartilage was scrutinised with semi-quantitative analysis. Hind paw inflammatory indices were used to determine the systemic effects and biomechanical testing the tensile strength of the materials over time. Results. The PDOe patch remained grossly quiescent at all time-points. There was a severe inflammatory reaction to Vicryl at one and 2-week time-points with gross exudate. Silk patches were associated with larger fibrous capsules at each time point. There were no adverse systemic effects and articular cartilage remained normal with no differences between materials to controls. Immunohistochemistry showed a significantly higher ratio of regenerative to inflammatory macrophages for the PDOe patch compared to other constructs at each time-point and similar to controls. Silk and Vicryl patches had a greater than 10-fold increase in foreign-body giant cells compared to the PDOe patch and controls (p<.05) suggesting incorporation rather than rejection and walling off of the biomaterial. Tensile strength of the PDOe patch increased in the first 2 weeks to greater than 90 N and gradually declined to a mean of 22 N at 12 weeks. Discussion/Conclusion. The novel PDOe patch appears to be biocompatible and illicit very little FBR in this rat tendon injury model. Importantly, there was no joint reaction to the biomaterial which has not been addressed previously. We believe the electrospun component of the patch recapitulates native tendon architecture creating a tissue healing microenvironment directed by a regenerative macrophage subpopulation. These results corroborate earlier in vitro work that showed incorporation of tenocytes within the electrospun scaffold. The woven component of the scaffold provides tensile strength as the tendon heals and begins to degrade after healing is underway making it less likely to elicit a FBR. Based on these and earlier in vitro data we believe this implant shows excellent biocompatibility and is ready to proceed to human trials

Introduction: Tendon tissue engineering entails the generation of a highly ordered collagen matrix with several organization scales that confer the tendon its mechanical functionality. Endogenous production of proteoglycans account for the typical microscopic organization in bundles of the tendon extracellular matrix, as they prevent lateral fusion of collagen fibril by binding the shaft of the fibres and promoting tip to tip fusion. The approach developed in this study is to rely on this molecular endogenous production and to induce a supramolecular uniaxial alignment of collagen fibres bundles with the help of specially designed scaffolds under continuous fluid shear stress. Methods: Microchannel chitosan scaffolds were produced by casting 2% chitosan gel on a mould equipped with stainless steel needles array that was imaged by optical coherence tomography with a resolution at ~10microns. From OCT measurements, regularly spaced microchannels with clearly delimited boundaries are obtained inside a microporous core of chitosan. By varying the number and the diameter of needles (from 250 μm (microns)to 500 μm (microns)) different types of microstructure have been produced. Microchannels scaffolds were seeded with primary tenocytes explanted from pig tendons and cultured in static culture, as nonstimulated group, and in a perfusion bioreactor. Results: There was a general increase in the channels occupation ratio for the group stimulated by perfusion, and inversely proportional to the microchannel diameter. Tenocytes were able to proliferate and to produce collagen extracellular matrix from the inner surface of the microchannel up to the whole channel volume. Conclusion: The proposed microstructure was appropriate for tendon engineering and its channel structure is adequate for direct OCT monitoring

Embryonic chick tenocytes cultured in fixed-length three-dimensional fibrin gels synthesise a taught collagen fibril-rich extracellular matrix that closely resembles embryonic tendon (. Kapacee et al., . Matrix Biology. , . 27. : . 371. –375, . 2008. ). Importantly, the cells replace fibrin with parallel arrays of collagen fibrils that are able to resist pulling forces. Regenerative medicine strategies for musculoskeletal applications require the development of tissue with mechanical strength comparable to that of native tissues. How the cells generate tension is not understood and the mechanical properties and the cellular behaviour of this culture system have not been described. This project aimed to describe the morphology of cells in the tendon-like constructs and to quantitate the mechanical properties of the constructs. Multiphoton imaging (MPI) can image deep into objects with reduced phototoxicity, allowing live-cell applications. MPI of the tendon constructs revealed that cells under tension were aligned longitudinally with the matrix. However, when tension was released the cells became rounded. The results suggested that embryonic tendon cells align along lines of force. Mechanical testing of newly-formed tendon constructs (T0), then at weekly intervals to six weeks (T7 to T42) was performed using an INSTRON. ®. failure-testing machine. An initial increase in ultimate tensile strength (UTS) was seen from T0 to T7 (1.023±0.031N to 1.150±1.150N, p=0.006), followed by a gradual decline at T35 to 0.350±0.043N, after which there was no further decrease. The UTS of the constructs was comparable with embryonic day 14 chick tendon. The initial increase in strength between T0 and T7 was cell dependent; constructs immersed in Triton-X 100 to remove cells were weaker than cellular constructs (1.277±0.096 versus 0.508±0.099, p< 0.001). Stress-strain plots demonstrated toe, heel, linear and failure regions that are classically observed in tendon. The results show that embryonic tendon cells synthesise an extracellular matrix of collagen fibrils that are tensioned by the cells, and that the tendon constructs have mechanical strength comparable to in vivo generated tissue. The research is generously supported by grants from The Wellcome Trust

Summary. In an in vitro tendon cell model, the tendon-specific gene expression up-regulation induced by PEMF negatively correlates with field intensity; moreover repeated lower-intensity PEMF treatments (1.5 mT) provokes a higher release of anti-inflammatory cytokines respect to the single treatment. Introduction. Tendon disorders represent a diagnostic and therapeutic challenge for physicians. Traditional treatments are characterised by a long recovery time and a high occurrence of injury relapses. Despite the growing clinical interest in pulsed electromagnetic fields (PEMFs) few studies on their effect on tendons and ligaments have been conducted. Tendon resident cells (TCs) are a mixed population, made up mostly by tenocytes and tendon stem/progenitor cells, which are responsible of the tissue homeostasis. Since studies on the effect of PEMFs on this cell population are conflicting, we evaluated the possible relation between PEMFs dosage and TCs’ response. In particular, we compared the in vitro effect of low and high PEMFs on TCs (PEMF-1.5 mT; PEMF-3 mT); moreover we assessed the results of repeated treatments (R-PEMF-1.5mT). Methods. TCs were isolated from the waste portion of semitendinosus and gracilis tendons of 7 healthy donors undergoing ACL reconstruction; at P4 they were exposed to different PEMF treatments (intensity: 1.5mT or 3mT; duration: 8 or 12 hours; periodicity: single or 3 treatments with an interval of 48h). Viability and DNA content were assessed by MTT and CyQuant, respectively, immediately at the end of the treatment (0d) and two days after (2d). Moreover, in order to accurately detected live and dead cells after the different treatments, Live&Dead staining was also assessed. At the same time points the expression of SCX, COL1A1 and VEGF were evaluated with RT-Real Time PCR, as well as the release of the cytokines TGFβ, IL6, IL10, IL1β, and TNFα by ELISA. Results. All the treatments applied for 12h increased TCs viability respect to untreated cells. However, respect to single PEMF-1.5mT, R-PEMF-1.5mT slightly decreased the TCs viability 2 days after 8 (−15%) and 12 hours (−9%) of exposure, whereas PEMF-3mT showed similar viability values. Nevertheless, the number of dead cells detected with Live&Dead assay was very low in all samples. All the tested PEMF treatments were able to relevantly enhance cell proliferation, with the exception of 12h R-PEMF-1.5mT, that reduces DNA content 2 day after treatment (−33%). All the treatments induced a significant increase of IL6, IL10 and TGFβ release respect to untreated cells (p<0.05), especially R-PEMF-1.5mT that showed higher values in comparison to the single PEMF-1.5mT treatment (p<.001). On the other hand pro-inflammatory cytokines (IL-1β and TNFα) production were not relevantly affected by any treatment. PEMF-3mT reduced the expression of tendon specific markers (SCX, COL1A1), whereas PEMF-1.5mT, above all as a single exposure, induced their up-regulation as well as the VEGF one, in comparison to untreated cells. Discussion/Conclusion. All PEMF treatments did not induced any cytotoxic events. Overall, a low intensity treatment, both single or repeated, allows to obtain a better in vitro TCs response in terms of anti-inflammatory cytokines release and tissue specific gene expression in comparison to higher electromagnetic field intensity (3 mT). In conclusion, these results suggest that PEMFs intensity negatively correlate with TCs in vitro response, whereas a repetition of low intensity treatment could positively influence tendon recovery. Further analyses on different models are needed to confirm these observations

Introduction: The pathogenesis of chronic tendinopathy is unclear. A role for increased apoptosis of tenocytes has been suggested. Nitric oxide is thought to be a mediator of apoptosis and nitric oxide synthase (NOS) isoforms have been shown to be up regulated in rotator cuff tendons as a result of chronic overuse. We found, the same up regulation of NOS in the Achilles tendon in non-insertional Achilles tendinopathy in a previous study. The purpose of this study was to investigate whether apoptotic cells were present in these tissues with raised endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) levels. Methods: Consent was obtained preoperatively from all patients and the research and ethics committee granted ethical approval. Samples were obtained from the Achilles Tendons of patients with non-insertional Achilles tendinopathy who had failed conservative treatment for at least six months and were undergoing a surgical procedure. Several biopsies were taken of the visibly abnormal tendon tissue. Control samples were taken from macroscopically normal tendon correlating with areas of normal tissue on MRI. Standard immunohistochemical techniques were used to identify the expression of eNOS and iNOS. Apoptotic cells were identified using terminal deoxynucleotidyl transferase-mediated dUTP neck end labelling (TUNEL reaction) with TdT-FragEL and the demonstration of Caspase-3 activation. Results: Significant differences were found between the diseased tendon and the controls for all of the parameters measured. The mean Caspase-3 cell count for diseased tendon was 51.9 compared to 28.3 for the controls (p=0.000001). The mean TUNEL cell count for diseased tendon was 24.1 compared to 14.8 (p=0.00014). iNOS densitometry revealed a mean of 26.1 for the diseased tissue verses 15.0 for the controls (p=0.000009) and the values for eNOS were 48.3 and 23.7 respectively (p=0.015). Conclusions: Apoptosis clearly plays a role in the development of non-insertional Achilles tendinopathy and appears to be related to the presence of raised eNOS and iNOS levels. It is possible that, by blocking the apoptotic pathway, the tendinopathic process could be halted. This may lead to the development of treatments strategies for early Achilles tendinopathy

Introduction: New biotechnologies create opportunities for gene therapy to promote rotator cuff healing. We have previously demonstrated that genetically engineered mesenchymal stem cells (MSCs) over expressing BMP-2 and SMAD8 signaling molecule differentiate to tenocytes in vitro and in vivo. Therefore, we hypothesized that rotator cuff defect could be regenerated using genetically engineered MSCs. Method: Nonviral methods were utilized to establish genetically engineered MSCs that co-express BMP-2 and the Smad8 signaling molecule. A previously validated animal model was utilized to examine rotator cuff healing. A 2mm x2mm full thickness defect was created in the infraspinatus tendon of 8 nude rats. A collagen-I biomembrane (TissueMend) containing 3 x 10. 6. engineered cells was sewn into the defect. An identical control procedure was repeated on the contralateral side with biomembrane containing non-engineered MSCs. Results: 4 weeks post implantation the area of implantation was isolated and analyzed by light microscopy and histochemical staining. Analysis of the engineered implants revealed the formation of dense connective tissue with parallel-organized fibers and spindle shaped cells, unlike the control samples. Proton Double Quantum Filtered Magnetic Resonance Imaging technique of the rotator cuff tendons demonstrated an increased presence of organized collagen fibers within the engineered rotator cuff tissue when compared with either native rotator cuff or those treated with non-engineered MSCs. Conclusion: This is the first report showing rotator cuff tendon repair using genetically engineered MSCs. Moreover these findings may have considerable importance for tendon healing and may indicate a clinical gene therapy platform to augment surgical repair

Introduction: The pathogenesis of chronic tendinopathy is unclear. The role of the increased apoptosis of tenocytes has been suggested by high intratendinous levels of glutamate being demonstrated in patients with tendinosis. Nitric oxide is a known mediator of apoptosis and nitric oxide synthase (NOS) isoforms have been shown to be upregulated in rotator cuff tendons as a result of chronic overuse. We found, the same upregulation of NOS in the Achilles tendon in non-insertional Achilles tendinopathy in a previous study. The purpose of this study was to investigate whether apoptotic cells were present in these tissues with raised eNOS and iNOS levels. Methods: Samples were obtained from the Achilles Tendons of patients with in non-insertional Achilles tendinopathy who had failed conservative treatment for at least six months and were undergoing a surgical procedure. Consent was obtained preoperatively from all patients and ethical approval was granted by the research ethics committee. Several biopsies were taken of the visibly abnormal tendon tissue. Control samples were taken from macroscopically normal tendon correlating with areas of normal tissue on MRI. Standard immunohistochemical techniques were used to identify the expression of endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS). Apoptotic cells were identified using terminal deoxynucleotidyl transferase-mediated dUTP neck end labelling (TUNEL reaction) with TdT-FragEL and the demonstration of Caspase-3 activation. A power calculation was performed which showed that 14 patients in each group would be required to show a 50% difference between the two groups using a level of significance of 5%. Results: Significant differences were found between the diseased tendon and the controls for all of the parameters measured. The mean Caspase-3 cell count for diseased tendon was 51.9 compared to 28.3 for the controls (p=0.000001). The mean TUNEL cell count for diseased tendon was 24.1 compared to 14.8 (p=0.00014). iNOS densitometry revealed a mean of 26.1 for the diseased tissue verses 15.0 for the controls (p=0.000009) and the values for eNOS were 48.3 and 23.7 respectively (p=0.015). Conclusions: Apoptosis clearly plays a role in the development of non-insertional Achilles tendinopathy and appears to be related to the presence of raised eNOS and iNOS levels. It is possible that, by blocking the apoptotic pathway, the tendinopathic process could be halted. This may lead to the development of treatments strategies for early Achilles tendinopathy

Introduction: Rotator cuff tears are a common injury which affects both the young athlete and the sedentary elderly alike. This condition is commonly treated with glucocorticoid injections as part of initial management. The effects, however, of these injections on the histology of collagen and the metabolism of tendon fibroblasts are still controversial. Materials and methods: In this study, samples from 19 patients with rotator cuff tears were taken during definitive surgery to manage these tears. There was a history of glucocorticoid injections in all of the patients. The samples were examined in terms of histopathology using light microscopy, in situ hybridization to detect the presence of glucocorticoid receptor mRNA and TUNEL assay to determine the incidence of apoptosis. Results: Light microscopy of hematoxylin-eosin stained samples from the study group showed marked cellularity although there were no signs of inflammation. The nuclei were noted to be rounded and a significant number showed pyknosis. Angiogenesis was also noted in the sections, consistent with previous finding of angio-fibroblastic hyperplasia as a characteristic of tendinosis. Collagen structure was noted to be abnormal, with longitudinal clefts and focal areas of marked disorganization of fibers. In situ hybridization showed a strong signal for glucocorticoid receptor mRNA in all of the samples. TUNEL assay also showed a strong signal for apoptosis of the tendon fibroblasts in the study group as compared to the control group which showed almost no signal. Conclusion: Our results suggest that although an overall picture of hypercellularity is seen in cases of tendinosis and tendon tears, a high percentage of these cells are undergoing apoptosis. This may reflect a natural high rate of turnover of cells during the process of repair or may be due to exogenous factors. Glucocorticoids almost certainly affect metabolism of tendon fibroblasts and subsequently collagen structure as seen by the abundant expression of the receptor mRNA. However, a causal relationship between glucocorticoids and apoptosis of tenocytes is yet to be established

Introduction: The anterior cruciate ligament (ACL) is the most frequently damaged ligament in the knee joint. The patella tendon autograft is the current replacement of choice, however autografts are not always available and grafting often leads to donor site morbidity. Allogeneic implants may cause an adverse immunological reaction [. 1. ] The aim of this study was to develop an acellular tendon scaffold with the mechanical and biochemical properties of tissue which could be rapidly recellularised for use in tissue engineering of the anterior cruciate ligament. Materials and Methods: Porcine patella tendons were dissected less than 24 hours after slaughter and washed in PBS. The tendons were decellularised using 0.1% (w/ v) SDS for 24 hours. Decellularisation was assessed by haematoxylin and eosin staining and light microscopy. The glycosaminoglycan and hydroxyproline (measure of collagen) content of the scaffold were also assessed quantitatively following decellularisation. Following decellularisation the scaffolds were subject to various levels of ultrasonication in order to modify the acellular scaffold prior to reseeding in an attempt to achieve recellularisation of the scaffold. Denaturation of the collagen within the scaffold following ultrasonication was assessed using the ƒÑ-chymotrypsin assay. Decellularised and ultrasonicated scaffolds were subject to uniaxial tensile loading to failure in a Howden tensile testing machine. The sonicated scaffolds were reseeded with human tenocytes (1x105 cells.cm2) and cultured in 5% CO2 in air at 37°C for three weeks. One scaffold was removed every seven days and either fixed in 10% neutral buffered formalin prior to dehydration and H& E staining or was stained with Live/Dead stain (Molecular Probes) and observed using confocal microscopy. Results: Porcine patella tendons were successfully decellularised using 0.1% (w/v) SDS. Following decellularisation there was no change in the biochemical composition of the scaffold. Ultrasonication of the scaffold at 360W was shown to open up spaces between collagen bundles without damaging the collagen matrix and this was confirmed with the ƒÑ-chymotrypsin assay. Following decellularisation and ultrasonication there was no change in the ultimate force (N) needed to break the tendon scaffold. When cells were seeded onto the sonicated scaffold, the cells were shown to penetrate to the centre of the scaffold within just 3 weeks of culture. Following staining with Live/Dead stain it was shown that after three weeks in static culture approximately 50% of the cells in the centre of the scaffold were viable. In comparison the cells cultured on the acellular non-sonicated scaffold remained on the surface of the scaffold and did not penetrate the matrix during this culture period. Conclusion: An acellular scaffold with excellent biochemical and mechanical properties has been developed which can be recellularised in an important first step towards tissue engineering of the anterior cruciate ligament. Future work will investigate culture of the reseeded scaffold under appropriate physical stimulation with a view to maintaining tissue homeostasis and increasing cell viability

Objectives

Re-rupture is common after primary flexor tendon repair. Characterization of the biological changes in the ruptured tendon stumps would be helpful, not only to understand the biological responses to the failed tendon repair, but also to investigate if the tendon stumps could be used as a recycling biomaterial for tendon regeneration in the secondary grafting surgery.

Objectives

The long head of the biceps (LHB) is often resected in shoulder surgery and could therefore serve as a cell source for tissue engineering approaches in the shoulder. However, whether it represents a suitable cell source for regenerative approaches, both in the inflamed and non-inflamed states, remains unclear. In the present study, inflamed and native human LHBs were comparatively characterized for features of regeneration.

Aims

Femoroacetabular impingement (FAI) is a potential cause of hip osteoarthritis (OA). The purpose of this study was to investigate the expression profile of matrix metalloproteinases (MMPs) in the labral tissue with FAI pathology.