Tumour necrosis factor alpha, interleukin 1 beta and interferon gamma have detrimental effects on equine tenocytes that cannot be rescued by IL-1RA or mesenchymal stromal cell-derived factors.

Tendon injuries occur commonly in both human and equine athletes, and poor tendon regeneration leads to functionally deficient scar tissue and an increased frequency of re-injury. Despite evidence suggesting inadequate resolution of inflammation leads to fibrotic healing, our understanding of the inflammatory pathways implicated in tendinopathy remains poorly understood, meaning successful targeted treatments are lacking. Here, we demonstrate IL-1ß, TNFα and IFN-γ work synergistically to induce greater detrimental consequences for equine tenocytes than when used individually. This includes altering tendon associated and matrix metalloproteinase gene expression and impairing the cells' ability to contract a 3-D collagen gel, a culture technique which more closely resembles the in vivo environment. Moreover, these adverse effects cannot be rescued by direct suppression of IL-1ß using IL-1RA or factors produced by BM-MSCs. Furthermore, we provide evidence that NF-κB, but not JNK, P38 MAPK or STAT 1, is translocated to the nucleus and able to bind to DNA in tenocytes following TNFα and IL-1ß stimulation, suggesting this signalling cascade may be responsible for the adverse downstream consequences of these inflammatory cytokines. We suggest a superior approach for treatment of tendinopathy may therefore be to target specific signalling pathways such as NF-κB.

Transdermal drug delivery in horses: An in vitro comparison of skin structure and permeation of two model drugs at various anatomical sites.

BACKGROUND: Oral and parenteral drug delivery in horses can be difficult. Equine-specific transdermal drug formulations offer improved ease of treatment; development of such formulations requires a deeper understanding of the structural and chemical tissue barrier of horse skin. HYPOTHESIS/OBJECTIVES: To compare the structural composition and barrier properties of equine skin. ANIMALS: Six warmblood horses (two males, four females) with no skin diseases. MATERIALS AND METHODS: Routine histological and microscopic analyses were carried out with image analysis for skin from six different anatomical locations. In vitro drug permeation was analysed using a standard Franz diffusion cell protocol coupled with reversed phase-high-performance liquid chromatography detailing flux, lag times and tissue partitioning ratios of two model drug compounds. RESULTS: Epidermal and dermal thicknesses varied between sites. The dermal and epidermal thicknesses of the croup were 1764 ± 115 µm and 36 ± 3.6 µm, respectively, and were significantly different (p < 0.05) from the inner thigh thicknesses which were 824 ± 35 µm and 49 ± 3.6 µm. Follicular density and size also varied. The highest flux for the model hydrophilic molecule (caffeine) was for the flank (3.22 ± 0.36 µg/cm2 /h), while that for the lipophilic molecule (ibuprofen) was for the inner thigh (0.12 ± 0.02 µg/cm2 /h). CONCLUSIONS AND CLINICAL RELEVANCE: Anatomical location differences in equine skin structure and small molecule permeability were demonstrated. These results can aid in the development of transdermal therapies for horses.

Effects of resveratrol and its analogues on the cell cycle of equine mesenchymal stem/stromal cells.

Resveratrol (RSV; trans-3,5,4'-trihydroxystilbene) strongly activates sirtuin 1, and it and its analogue V29 enhance the proliferation of mesenchymal stem/stromal cells (MSCs).Although culture medium containing 5-azacytydine and RSV inhibits senescence of adipose tissue-derived MSCs isolated from horses with metabolic syndrome, few studies have reported the effects of RSV on equine bone marrow-derived MSCs (eBMMSCs) isolated from horses without metabolic syndrome. The aim of this study was to investigate the effects of RSV and V29 on the cell cycle of eBMMSCs. Following treatment with 5 µM RSV or 10 µM V29, the cell proliferation capacity of eBMMSCs derived from seven horses was evaluated by EdU (5-ethynyl-2'-deoxyuridine) and Ki-67 antibody assays. Brightfield images of cells and immunofluorescent images of EdU, Ki-67, and DAPI staining were recorded by fluorescence microscopy, and the number of cells positive for each was quantified and compared by Friedman's test at P<0.05. The growth fraction of eBMMSCs was significantly increased by RSV and V29 as measured by the EdU assay (control 28.1% ± 13.8%, V29 31.8% ± 14.6%, RSV 32.0% ± 10.8%; mean ± SD; P<0.05) but not as measured by the Ki-67 antibody assay (control 27.0% ± 11.2%, V29 27.4% ± 10.8%, RSV 27.7% ± 6.8%). RSV and V29 promoted progression of the cell cycle of eBMMSCs into the S phase and may be useful for eBMMSC expansion.

CD146 Delineates an Interfascicular Cell Sub-Population in Tendon That Is Recruited during Injury through Its Ligand Laminin-α4.

The interfascicular matrix (IFM) binds tendon fascicles and contains a population of morphologically distinct cells. However, the role of IFM-localised cell populations in tendon repair remains to be determined. The basement membrane protein laminin-α4 also localises to the IFM. Laminin-α4 is a ligand for several cell surface receptors, including CD146, a marker of pericyte and progenitor cells. We used a needle injury model in the rat Achilles tendon to test the hypothesis that the IFM is a niche for CD146+ cells that are mobilised in response to tendon damage. We also aimed to establish how expression patterns of circulating non-coding RNAs alter with tendon injury and identify potential RNA-based markers of tendon disease. The results demonstrate the formation of a focal lesion at the injury site, which increased in size and cellularity for up to 21 days post injury. In healthy tendon, CD146+ cells localised to the IFM, compared with injury, where CD146+ cells migrated towards the lesion at days 4 and 7, and populated the lesion 21 days post injury. This was accompanied by increased laminin-α4, suggesting that laminin-α4 facilitates CD146+ cell recruitment at injury sites. We also identified a panel of circulating microRNAs that are dysregulated with tendon injury. We propose that the IFM cell niche mediates the intrinsic response to injury, whereby an injury stimulus induces CD146+ cell migration. Further work is required to fully characterise CD146+ subpopulations within the IFM and establish their precise roles during tendon healing.

Development of a Cartilage Oligomeric Matrix Protein Neo-Epitope Assay for the Detection of Intra-Thecal Tendon Disease.

The diagnosis of tendon injury relies on clinical signs and diagnostic imaging but imaging is subjective and does not always correlate with clinical signs. A molecular marker would potentially offer a sensitive and specific diagnostic tool that could also provide objective assessment of healing for the comparison of different treatments. Cartilage Oligomeric Matrix Protein (COMP) has been used as a molecular marker for osteoarthritis in humans and horses but assays for the protein in tendon sheath synovial fluids have shown overlap between horses affected by tendinopathy and controls. We hypothesized that quantifying a COMP neoepitope would be more discriminatory of injury. COMP fragments were purified from synovial fluids of horses with intra-thecal tendon injuries and media from equine tendon explants, and mass spectrometry of a consistent and abundant fragment revealed a ~100 kDa COMP fragment with a new N-terminus at the 78th amino-acid (NH2-TPRVSVRP) located just outside the junctional region of the protein. A competitive inhibition ELISA based on a polyclonal antibody raised to this sequence yielded more than a 10-fold rise in the mean neoepitope levels for tendinopathy cases compared to controls (5.3 ± 1.3 µg/mL (n = 7) versus 58.8 ± 64.3 µg/mL (n = 13); p = 0.002). However, there was some cross-reactivity of the neoepitope polyclonal antiserum with intact COMP, which could be blocked by a peptide spanning the neoepitope. The modified assay demonstrated a lower concentration but a significant > 500-fold average rise with tendon injury (2.5 ± 2.2 ng/mL (n = 6) versus 1029.8 ± 2188.8 ng/ml (n = 14); p = 0.013). This neo-epitope assay therefore offers a potentially useful marker for clinical use.

Cryopreservation of canine cardiosphere-derived cells: Implications for clinical application.

The clinical application of cardiosphere-derived cells (CDCs) to treat cardiac disease has gained increasing interest over the past decade. Recent clinical trials confirm their regenerative capabilities, although much remains to be elucidated about their basic biology. To develop this new treatment modality, in a cost effective and standardized workflow, necessitates the creation of cryopreserved cell lines to facilitate access for cardiac patients requiring urgent therapy. Cryopreservation may however lead to alterations in cell behavior and potency. The aim of this study was to investigate the effect of cryopreservation on canine CDCs. CDCs and mesenchymal stem cells (MSCs) isolated from five dogs were characterized. CDCs demonstrated a population doubling time that was unchanged by cryopreservation (fresh vs. cryopreserved; 57.13 ± 5.27 h vs. 48.94 ± 9.55 h, P = 0.71). This was slower than for MSCs (30.46 h, P < 0.05). The ability to form clones, self-renew, and commit to multiple lineages was unaffected by cryopreservation. Cryopreserved CDCs formed larger cardiospheres compared to fresh cells (P < 0.0001). Fresh CDCs showed a high proportion of CD105+ (89.0% ± 4.98) and CD44+ (99.68% ± 0.13) cells with varying proportions of CD90+ (23.36% ± 9.78), CD34+ (7.18% ± 4.03) and c-Kit+ (13.17% ± 8.67) cells. CD45+ (0.015% ± 0.005) and CD29+ (2.92% ± 2.46) populations were negligible. Increasing passage number of fresh CDCs correlated with an increase in the proportion of CD34+ and a decrease in CD90+ cells (P = 0.003 and 0.03, respectively). Cryopreserved CDCs displayed increased CD34+ (P < 0.001) and decreased CD90+ cells (P = 0.042) when compared to fresh cells. Overall, our study shows that cryopreservation of canine CDCs is feasible without altering their stem characteristics, thereby facilitating their utilization for clinical trials. © 2017 International Society for Advancement of Cytometry.

Structural changes in cartilage and collagen studied by high temperature Raman spectroscopy.

Understanding the high temperature behavior of collagen and collagenous tissue is important for surgical procedures and biomaterials processing for the food, pharmaceutical, and cosmetics industries. One primary event for proteins is thermal denaturation that involves unfolding the polypeptide chains while maintaining the primary structure intact. Collagen in the extracellular matrix of cartilage and other connective tissue is a hierarchical material containing bundles of triple-helical fibers associated with water and proteoglycan components. Thermal analysis of dehydrated collagen indicates irreversible denaturation at high temperature between 135°C and 200°C, with another reversible event at ∼60-80°C for hydrated samples. We report high temperature Raman spectra for freeze-dried cartilage samples that show an increase in laser-excited fluorescence interpreted as conformational changes associated with denaturation above 140°C. Spectra for separated collagen and proteoglycan fractions extracted from cartilage indicate the changes are associated with collagen. The Raman data also show appearance of new features indicating peptide bond hydrolysis at high temperature implying that molecular H2 O is retained within the freeze-dried tissue. This is confirmed by thermogravimetric analysis that show 5-7 wt% H2 O remaining within freeze-dried cartilage that is released progressively upon heating up to 200°C. Spectra obtained after exposure to high temperature and re-hydration following recovery indicate that the capacity of the denatured collagen to re-absorb water is reduced. Our results are important for revealing the presence of bound H2 O within the collagen component of connective tissue even after freeze-drying and its role in denaturation that is accompanied by or perhaps preceded by breakdown of the primary polypeptide structure.

Exposure of a tendon extracellular matrix to synovial fluid triggers endogenous and engrafted cell death: A mechanism for failed healing of intrathecal tendon injuries.

AIM: The purpose of this study was to investigate the effect of normal synovial fluid (SF) on exposed endogenous tendon-derived cells (TDCs) and engrafted mesenchymal stem cells (MSCs) within the tendon extracellular matrix. METHODS: Explants from equine superficial digital flexor (extra-synovial) and deep digital flexor tendons (DDFTs) from the compressed, intra-synovial and the tensile, extra-synovial regions were cultured in allogeneic or autologous SF-media. Human hamstring explants were cultured in allogeneic SF. Explant viability was assessed by staining. Proliferation of equine monolayer MSCs and TDCs in SF-media and co-culture with DDFT explants was determined by alamarblue®. Non-viable Native Tendon matrices (NNTs) were re-populated with MSCs or TDCs and cultured in SF-media. Immunohistochemical staining of tendon sections for the apoptotic proteins caspase-3, -8, and -9 was performed. RESULTS: Contact with autologous or allogeneic SF resulted in rapid death of resident tenocytes in equine and human tendon. SF did not affect the viability of equine epitenon cells, or of MSCs and TDCs in the monolayer or indirect explant co-culture. MSCs and TDCs, engrafted into NNTs, died when cultured in SF. Caspase-3, -8, and -9 expression was the greatest in SDFT explants exposed to allogeneic SF. CONCLUSIONS: The efficacy of cells administered intra-synovially for tendon lesion repair is likely to be limited, since once incorporated into the matrix, cells become vlnerable to the adverse effects of SF. These observations could account for the poor success rate of intra-synovial tendon healing following damage to the epitenon and contact with SF, common with most soft tissue intra-synovial pathologies.

Immunophenotypic characterization of ovine mesenchymal stem cells.

The clinical potential of multipotent mesenchymal stem cells (MSCs) has led to the essential development of analytical tools such as antibodies against membrane-bound proteins for the immunophenotypic characterization of human and rodent cells. Such tools are frequently lacking for emerging large animal models like the sheep that have greater relevance for the study of human musculoskeletal diseases. The present study identified a set of commercial nonspecies specific monoclonal antibodies for the immunophenotypic characterization of ovine MSCs. A protocol combining the less destructive proteolytic activity of accutase and EDTA was initially developed for the detachment of cells from plastic with minimum loss of cell surface antigens. A range of commercially available antibodies against human or rodent MSC antigens were then tested in single and multistain-based assays for their cross-reactivity to bone marrow derived ovine MSCs. Antibody clones cross-reactive to ovine CD73 (96.9% ± 5.9), CD90 (99.6% ± 0.3), CD105 (99.1 ± 1.5), CD271 (97.7 ± 2.0), and MHC1 (94.0% ± 7.2) antigens were identified using previously reported CD29, CD44, and CD166 as positive controls. Multistaining analysis indicated the colocalization of these antigens on MSCs. Furthermore, antibody clones identified to cross-react against white blood cell antigens exhibited either negative (CD117 (0.1% ± 0.1)) or low (MHCII (10.5% ± 16.0); CD31 (14.6% ± 4.2), and CD45 (39.4% ± 31.8)) cross-reactivity with ovine MSCs. The validation of these antibody clones to sheep MSC antigens is essential for studies utilizing this large animal model for stem cell-based therapies. © 2016 International Society for Advancement of Cytometry.

Modulation of mesenchymal stem cell genotype and phenotype by extracellular matrix proteins.

AIM: To investigate the effect of extracellular matrix (ECM) proteins on characteristics of mesenchymal stem cells (MSCs) and tendon-derived cells (TDCs). MATERIALS AND METHODS: MSCs and TDCs, cultured in a monolayer (2D) or hydrogels (3D), with or without ECM protein supplementation, and on a non-viable native tendon (NNT) matrix were assayed for adhesion, proliferation, gene expression, and integrin expression. RESULTS: MSCs exhibited a fibroblastic, spindle-shaped morphology on 2D matrices except in the presence of fibronectin. In 3D matrices, MSCs displayed a rounded phenotype except when cultured on NNTs where cells aligned along the collagen fibrils but, unlike TDCs, did not form inter-cellular cytoplasmic processes. MSC proliferation was significantly (p < 0.01) increased by collagen type I in 2D culture and fibronectin in 3D culture. TDC proliferation was unaffected by substrata. MSCs and TDCs differentially expressed α2 integrin. Adhesion to substrata was reduced by RGD-blocking peptide and ß1 integrin antibody. The presence of collagen I or fibronectin upregulated MSC expression of collagen type I and collagen type III, COMP, decorin, osteopontin, and fibronectin. CONCLUSIONS: The morphology, gene expression, and adhesion of both MSCs and TDCs are sensitive to the presence of specific ECM components. Interaction with the ECM is, therefore, likely to affect the mechanism of action of MSCs in vitro and may contribute to phenotypic modulation in vivo.

Proteomic analysis of tendon extracellular matrix reveals disease stage-specific fragmentation and differential cleavage of COMP (cartilage oligomeric matrix protein).

During inflammatory processes the extracellular matrix (ECM) is extensively remodeled, and many of the constituent components are released as proteolytically cleaved fragments. These degradative processes are better documented for inflammatory joint diseases than tendinopathy even though the pathogenesis has many similarities. The aims of this study were to investigate the proteomic composition of injured tendons during early and late disease stages to identify disease-specific cleavage patterns of the ECM protein cartilage oligomeric matrix protein (COMP). In addition to characterizing fragments released in naturally occurring disease, we hypothesized that stimulation of tendon explants with proinflammatory mediators in vitro would induce fragments of COMP analogous to natural disease. Therefore, normal tendon explants were stimulated with IL-1ß and prostaglandin E2, and their effects on the release of COMP and its cleavage patterns were characterized. Analyses of injured tendons identified an altered proteomic composition of the ECM at all stages post injury, showing protein fragments that were specific to disease stage. IL-1ß enhanced the proteolytic cleavage and release of COMP from tendon explants, whereas PGE2 had no catabolic effect. Of the cleavage fragments identified in early stage tendon disease, two fragments were generated by an IL-1-mediated mechanism. These fragments provide a platform for the development of neo-epitope assays specific to injury stage for tendon disease.

Low density lipoprotein receptor-related protein 1 (LRP1)-mediated endocytic clearance of a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4): functional differences of non-catalytic domains of ADAMTS-4 and ADAMTS-5 in LRP1 binding.

Degradation of the cartilage proteoglycan aggrecan is an early event in the development of osteoarthritis, and a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4) and ADAMTS-5 are considered to be the major aggrecan-degrading enzymes. We have recently found that ADAMTS-5 is rapidly endocytosed via low density lipoprotein receptor-related protein 1 (LRP1) and degraded by chondrocytes. Here we report that this regulatory mechanism also applies to ADAMTS-4, although its rate of endocytosis is slower than that of ADAMTS-5. Domain deletion mutagenesis of ADAMTS-4 identified that the cysteine-rich and spacer domains are responsible for binding to LRP1, whereas the thrombospondin 1 and spacer domains are responsible in ADAMTS-5. The estimated t½ value of ADAMTS-4 endocytosis was about 220 min, whereas that of ADAMTS-5 was 100 min. The difference in half-lives between the two enzymes is explained by the 13-fold lower affinity of ADAMTS-4 for LRP1 compared with that of ADAMTS-5. Studies using soluble ligand binding clusters of LRP1 showed that ADAMTS-4 binds to clusters II and IV with similar KD,app values of 98 and 73 nm, respectively, whereas ADAMTS-5 binds to cluster II, III, and IV with KD,app values of 3.5, 41, and 9 nm, respectively. Thus, ADAMTS-5 competitively inhibits ADAMTS-4 endocytosis but not vice versa. This study highlights that the affinity between a ligand and LRP1 dictates the rate of internalization and suggests that LRP1 is a major traffic controller of the two aggrecanases, especially under inflammatory conditions, where the protein levels of ADAMTS-4 increase, but those of ADAMTS-5 do not.

CRISPR/Cas9 gene editing in induced pluripotent stem cells to investigate the feline hypertrophic cardiomyopathy causing MYBPC3/R820W mutation.

Hypertrophic cardiomyopathy (HCM) is the most common heart disease in domestic cats, often leading to congestive heart failure and death, with current treatment strategies unable to reverse or prevent progression of the disease. The underlying pathological processes driving HCM remain unclear, which hinders novel drug discovery. The aim of this study was to generate a cellular model of the feline HCM-causing MYBPC3 mutation R820W. Using CRISPR/Cas9 gene editing we introduced the R820W mutation into a human induced pluripotent stem cell (iPSC) line. We differentiated both homozygous mutant clones and isogenic control clones to cardiomyocytes (iPSC-CMs). Protein quantification indicated that haploinsufficiency is not the disease mechanism of the mutation. Homozygous mutant iPSC-CMs had a larger cell area than isogenic controls, with the sarcomere structure and incorporation of cMyBP-C appearing similar between mutant and control iPSC-CMs. Contraction kinetic analysis indicated that homozygous iPSC-CMs have impaired relaxation and are hypocontractile compared to isogenic control iPSC-CMs. In summary, we demonstrate successful generation of an iPSC model of a feline MYBPC3 mutation, with the cellular model recapitulating aspects of HCM including cellular hypertrophy and impaired relaxation kinetics. We anticipate that further study of this model will lead to improved understanding of the disease-causing molecular mechanism, ultimately leading to novel drug discovery.

Equine Embryonic Stem Cell-Derived Tenocytes are Insensitive to a Combination of Inflammatory Cytokines and Have Distinct Molecular Responses Compared to Primary Tenocytes.

Tissue fibrosis following tendon injury is a major clinical problem due to the increased risk of re-injury and limited treatment options; however, its mechanism remains unclear. Evidence suggests that insufficient resolution of inflammation contributes to fibrotic healing by disrupting tenocyte activity, with the NF-κB pathway being identified as a potential mediator. Equine embryonic stem cell (ESC) derived tenocytes may offer a potential cell-based therapy to improve tendon regeneration, but how they respond to an inflammatory environment is largely unknown. Our findings reveal for the first time that, unlike adult tenocytes, ESC-tenocytes are unaffected by IFN-γ, TNFα, and IL-1ß stimulation; producing minimal changes to tendon-associated gene expression and generating 3-D collagen gel constructs indistinguishable from unstimulated controls. Inflammatory pathway analysis found these inflammatory cytokines failed to activate NF-κB in the ESC-tenocytes. However, NF-κB could be activated to induce changes in gene expression following stimulation with NF-κB pharmaceutical activators. Transcriptomic analysis revealed differences between cytokine and NF-κB signalling components between adult and ESC-tenocytes, which may contribute to the mechanism by which ESC-tenocytes escape inflammatory stimuli. Further investigation of these molecular mechanisms will help guide novel therapies to reduce fibrosis and encourage superior tendon healing.

A SIRT1-independent mechanism mediates protection against steroid-induced senescence by resveralogues in equine tenocytes.

Tendinopathy is a common age-related disease which causes significant morbidity for both human athletes and performance horses. In the latter, the superficial digital flexor tendon is an excellent model for human tendinopathies because it is a functional homologue of the human Achilles tendon and a primary site of injuries with strong similarities to the human disease. Corticosteroids have been previously used clinically to treat tendinopathic inflammation, but they upregulate the p53-p21 axis with concomitant reductions in cell proliferation and collagen synthesis in human tenocytes. This phenotype is consistent with the induction of cellular senescence in vitro and in vivo and probably represents an important clinical barrier to their effective use. Because of the many differences in senescence mechanisms between species, this study aimed to evaluate these mechanisms after corticosteroid treatment in equine tenocytes. Exposure to clinically reflective levels of dexamethasone for 48 hours drove equine tenocytes into steroid induced senescence (SIS). This was characterised by permanent growth arrest and upregulation of p53, the cyclin dependent kinase inhibitors p21waf and p16ink4a as well as the matrix degrading enzymes MMP1, MMP2 and MMP13. SIS also induced a distinctive equine senescence associated secretory phenotype (eSASP) characterised by enhanced secretion of IL-8 and MCP-1. Preincubation with resveratrol or the potent SIRT1 activator SRT1720 prevented SIS in equine tenocytes, while treatment with the non-SIRT1 activating resveratrol analogue V29 was equally protective against SIS, consistent with a novel, as yet uncharacterised SIRT1-indendent mechanism which has relevance for the development of future preventative and therapeutic strategies.

Influence of Rho/ROCK inhibitor Y-27632 on proliferation of equine mesenchymal stromal cells.

Mesenchymal stromal cells (MSC) isolated form bone marrow and adipose tissue are the most common cells used for cell therapy of orthopedic diseases. MSC derived from different tissues show differences in terms of their proliferation, differentiation potential and viability in prolonged cell culture. This suggests that there may be subtle differences in intracellular signaling pathways that modulate these cellular characteristics. The Rho/ROCK signaling pathway is essential for many cellular functions. Targeting of this pathway by the ROCK inhibitor Y-27632 has been shown to be beneficial for cell viability and proliferation of different cell types. The aim of this study was to investigate the effects of Rho/ROCK inhibition on equine MSC proliferation using bone marrow-derived MSC (BMSC) and adipose-derived MSC (ASC). Primary ASC and BMSC were stimulated with or without 10 ng/mL TGF-ß3 or 10 µM Y-27632, as well as both in combination. Etoposide at 10 µM was used as a positive control for inhibition of cell proliferation. After 48 h of stimulation, cell morphology, proliferation activity and gene expression of cell senescence markers p53 and p21 were assessed. ASC showed a trend for higher basal proliferation than BMSC, which was sustained following stimulation with TGF-ß3. This included a higher proliferation with TGF-ß3 stimulation compared to Y-27632 stimulation (p < 0.01), but not significantly different to the no treatment control when used in combination. Expression of p21 and p53 was not altered by stimulation with TGF-ß3 and/or Y-27632 in either cell type. In summary, the Rho/ROCK inhibitor Y-27632 had no effect on proliferation activity and did not induce cell senescence in equine ASC and BMSC.

Technical note: Cartilage imaging with sub-cellular resolution using a laboratory-based phase-contrast x-ray microscope.

BACKGROUND: Microscopic imaging of cartilage is a key tool for the study and development of treatments for osteoarthritis. When cellular and sub-cellular resolution is required, histology remains the gold standard approach, albeit limited by the lack of volumetric information as well as by processing artifacts. Cartilage imaging with the sub-cellular resolution has only been demonstrated in the synchrotron environment. PURPOSE: To provide a proof-of-concept demonstration of the capability of a laboratory-based x-ray phase-contrast microscope to resolve sub-cellular features in a cartilage sample. METHODS: This work is based on a laboratory-based x-ray microscope using intensity-modulation masks. The structured nature of the beam, resulting from the mask apertures, allows the retrieval of three contrast channels, namely, transmission, refraction and dark-field, with resolution depending only on the mask aperture width. An ex vivo equine cartilage sample was imaged with the x-ray microscope and results were validated with synchrotron tomography and histology. RESULTS: Individual chondrocytes, that is, cells responsible for cartilage formation, could be detected with the laboratory-based microscope. The complementarity of the three retrieved contrast channels allowed the detection of sub-cellular features in the chondrocytes. CONCLUSIONS: We provide the first proof-of-concept of imaging cartilage tissue with sub-cellular resolution using a laboratory-based x-ray microscope.

Exploration of Mediators Associated with Myocardial Remodelling in Feline Hypertrophic Cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) affects both humans and cats and exhibits considerable interspecies similarities that are exemplified by underlying pathological processes and clinical presentation to the extent that developments in the human field may have direct relevance to the feline disease. Characteristic changes on histological examination include cardiomyocyte hypertrophy and interstitial and replacement fibrosis. Clinically, HCM is characterised by significant diastolic dysfunction due to a reduction in ventricular compliance and relaxation associated with extracellular matrix (ECM) remodelling and the development of ventricular hypertrophy. Studies in rodent models and human HCM patients have identified key protein mediators implicated in these pathological changes, including lumican, lysyl oxidase and TGF-ß isoforms. We therefore sought to quantify and describe the cellular location of these mediators in the left ventricular myocardium of cats with HCM and investigate their relationship with the quantity and structural composition of the ECM. We identified increased myocardial content of lumican, LOX and TGF-ß2 mainly attributed to their increased expression within cardiomyocytes in HCM cats compared to control cats. Furthermore, we found strong correlations between the expressions of these mediators that is compatible with their role as important components of cellular pathways promoting remodelling of the left ventricular myocardium. Fibrosis and hypertrophy are important pathological changes in feline HCM, and a greater understanding of the mechanisms driving this pathology may facilitate the identification of potential therapies.

The tendon interfascicular basement membrane provides a vascular niche for CD146+ cell subpopulations.

Introduction: The interfascicular matrix (IFM; also known as the endotenon) is critical to the mechanical adaptations and response to load in energy-storing tendons, such as the human Achilles and equine superficial digital flexor tendon (SDFT). We hypothesized that the IFM is a tendon progenitor cell niche housing an exclusive cell subpopulation. Methods: Immunolabelling of equine superficial digital flexor tendon was used to identify the interfascicular matrix niche, localising expression patterns of CD31 (endothelial cells), Desmin (smooth muscle cells and pericytes), CD146 (interfascicular matrix cells) and LAMA4 (interfascicular matrix basement membrane marker). Magnetic-activated cell sorting was employed to isolate and compare in vitro properties of CD146+ and CD146- subpopulations. Results: Labelling for CD146 using standard histological and 3D imaging of large intact 3D segments revealed an exclusive interfascicular cell subpopulation that resides in proximity to a basal lamina which forms extensive, interconnected vascular networks. Isolated CD146+ cells exhibited limited mineralisation (osteogenesis) and lipid production (adipogenesis). Discussion: This study demonstrates that the interfascicular matrix is a unique tendon cell niche, containing a vascular-rich network of basement membrane, CD31+ endothelial cells, Desmin+ mural cells, and CD146+ cell populations that are likely essential to tendon structure and/or function. Contrary to our hypothesis, interfascicular CD146+ subpopulations did not exhibit stem cell-like phenotypes. Instead, our results indicate CD146 as a pan-vascular marker within the tendon interfascicular matrix. Together with previous work demonstrating that endogenous tendon CD146+ cells migrate to sites of injury, our data suggest that their mobilisation to promote intrinsic repair involves changes in their relationships with local interfascicular matrix vascular and basement membrane constituents.