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

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

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. 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

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.

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.

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

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