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

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.

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.

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.

Introduction

Diabetes mellitus type 2 (DMT2) patients often develop Achilles tendon (AS) degeneration. The ZDF rat model is often used to study DMT2. Hence, this study investigated whether tenocytes isolated from diabetic and non diabetic ZDF rats respond differentially to normo- (NG) and hyperglycemic (HG) conditions in the presence of tumor necrosis (TNF)α.

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

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.

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.

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.

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.

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.

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.

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

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

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

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. 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

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.

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.