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

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.