The extracellular matrix (ECM) in horses is a complex network of proteins and polysaccharides that provides structural and biochemical support to surrounding cells. It is a dynamic entity involved in various physiological processes, including tissue repair, development, and homeostasis. In equine biology, the ECM is composed of components such as collagen, elastin, proteoglycans, and glycoproteins, which contribute to the integrity and functionality of tissues like cartilage, tendons, and skin. Alterations in the ECM can influence cellular behavior and are associated with conditions such as osteoarthritis and tendon injuries. This page aggregates peer-reviewed research studies and scholarly articles that explore the composition, function, and implications of the extracellular matrix in equine health and disease.
Semik-Gurgul E, Ocłoń E, Zubel-Łojek J, Pędziwiatr R, Pawlina-Tyszko K.Sarcoids are locally invasive skin tumors in equids, associated with bovine papillomavirus. Objective: Address potential applications of cannabidiol (CBD) in veterinary medicine. We evaluated the response of equine sarcoid cells to CBD in vitro, focusing on viability, invasiveness, and matrix remodeling. Methods: Three primary sarcoid cell lines. Methods: Cells were treated with CBD (20, 6.75, 2.25, 0.75 μM) and incubated for 6, 24, 48, 72 hours. Cell viability, cytotoxicity, and apoptosis were assessed using the ApoTox-Glo Assay. Based on these results, further analyses were performed for ...
Wójtowicz A, Sadowska A, Myszczyński K, Molcan T, Kaczmarek MM, Szóstek-Mioduchowska A.Fibrosis remains incompletely understood, particularly in terms of how immune mediators shape stromal programs. We used a spontaneous large‑animal model-endometrosis (equine endometrial fibrosis) to define how interleukin‑4 (IL‑4) reprograms fibroblasts from healthy and fibrotic endometrium. Primary fibroblasts were exposed to IL‑4 (10 ng/mL) for 48 or 96 h. At 48 h, bulk transcriptomes revealed 1307 differentially expressed genes (DEGs; 648 up, 659 down) and 1271 DEGs (645 up, 626 down) in fibroblasts derived from endometria without or with endometrosis, respectively. Enrichment an...
Amaral A, Cerveira-Pinto M, Santos C, Kordowitzki P, Skarzynski D, Ferreira-Dias G, Szóstek-Mioduchowska A.Transforming growth factor (TGF)-β1 is a pro-fibrotic cytokine that affects extracellular matrix (ECM) deposition and fibroblast activity. 17β-Estradiol (E), the dominant ovarian steroid during the follicular phase (FLP) of the estrous cycle, can also influence ECM remodeling and fibrosis, through prostaglandin (PG) synthesis. PGs have opposing roles in fibrosis, with PGE₂ showing anti-fibrotic effects and PGF₂α promoting fibrosis. Equine endometrosis, whose main pathological feature is fibrosis, is marked by chronic inflammation and ECM accumulation, and may involve mediators like TGF-...
Zanacchi A, Fruscione F, Grasselli S, Razzuoli E, Pastorino L, Di Lisa D.Developing advanced preclinical in vitro cancer models is essential for understanding tumor biology, improving drug discovery, and enhancing the clinical translation of new therapies. Papillomaviruses (PVs) cause diverse lesions in humans and animals; notably, bovine papillomavirus (BPV) is the main cause of equine sarcoid, the most common skin tumor in horses. Its pathogenesis involves viral infection, fibroblast transformation, and extracellular matrix (ECM) remodeling. These processes highlight the limitations of conventional two-dimensional (2D) culture systems, which fail to replicate the...
Verstraete MH, Norris JK, Loux SC, De Spiegelaere W, Carossino M, Daels P, Dini P.The placenta mediates critical interactions between mother and offspring that guide development and impact long-term health. Despite its importance, the cellular organization of the equine (Equus caballus) placenta remains poorly defined. The equine epitheliochorial placenta, characterized by the absence of trophoblast invasion and prominent endocrine activity, provides a unique comparative model for studying placental cellular composition and development. Methods: We applied single-cell RNA sequencing to chorioallantois tissue collected from first-trimester (n = 1) and term (n = 2) equine...
Milczek-Haduch D, Żmigrodzka M, Kiełbik P, Świderska B, Olędzki J, Witkowska-Piłaszewicz O.Extracellular vesicles (EVs) are promising biomarkers and mediators of intercellular communication, but their isolation from equine biofluids remains challenging. This study compared two isolation workflows-size-exclusion chromatography (SEC) and differential ultracentrifugation followed by SEC (UC + SEC)-to evaluate their efficiency, reproducibility, and the proteomic composition of EVs derived from equine serum and plasma. Blood from six healthy horses was processed to obtain platelet-free plasma and serum. EVs were isolated using SEC or UC + SEC and characterized by transmission ele...
Yuan X, Yao X, Zeng Y, Wang J, Ren W, Wang T, Li X, Yang L, Yang X, Meng J.The effects of physical exercise on protein expression and metabolites in the plasma exosomes of horses are not yet clear. This study aims to elucidate the effects of exercise training on the expression of plasma extracellular vesicle proteins and metabolites in horses through multi-omics analysis, providing reference indicators for the training and performance evaluation of horses. Methods: Eight healthy 3-year-old horses were selected for the study, with four untrained horses as the control group and four trained horses as the training group. After conducting proteomic and metabolomic an...
To date, there is extensive scientific evidence affirming that physical exercise plays a fundamental role in both the prevention and treatment of various pathological conditions in humans as well as in animals. It is understood that the advantages of movement and exercise have a multifactorial origin and they depend on a category of bioactive molecules vehicolated by extracellular microvesicles known as exosomes. The exosomes act as potential delivery systems for messages within the organism. These findings have drawn significant attention, leading researchers to further investigate the role o...
Feng M, Hall TJ, MacHugh DE, Katz LM, Hill EW.Early skeletal muscle development is critical for young racehorses, yet research on the transcriptional changes during this period is limited. Additionally, the impact of age on the transcriptional response to exercise training in equine athletes is not well understood. A transcriptome-wide analysis of differential gene expression in skeletal muscle was performed for five untrained Thoroughbred horses sampled at rest at two years old (UR2) and three years old (UR3). A total of 136 differentially expressed genes (DEGs) were identified, with 95 increased and 41 decreased in expression. GO enrich...
Sadowska A, Molcan T, Słyszewska M, Skarzynski DJ, Ferreira-Dias G, Szóstek-Mioduchowska A.Equine endometrosis is a chronic degenerative condition of the endometrium. A hallmark feature of endometrosis is endometrial fibrosis accompanied by degenerative changes in the adjacent tissue structure. Tumor necrosis factor α (TNFα), a pleiotropic cytokine produced by various immune cells, plays a dual role in coordinating immune responses and regulating cell/tissue homeostasis, making it an important regulator of fibrotic-related disorders. However, the exact role of TNFα in the development of equine endometrial fibrosis remains to be discovered and explained. Therefore, the main aim of...
Partusch L, Rutland CS, Martens A, Du Cheyne C, De Spiegelaere W, Michler JK.Exuberant granulation tissue (EGT) is a second intention wound healing disorder. It commonly occurs in the distal limb of horses. EGT causes significant increase in the duration and cost of treatment, potentially leading to the decision not to pursue treatment and euthanize the patient. The underlying pathomechanisms of this fibroproliferative disorder remain unclear, particularly in terms of collagen composition and the association between myofibroblasts and blood vessels. This study investigated the collagen composition in naturally occurring EGT following trimming in 19 horses (EGT group). ...
DI Stefano DA, Coccoluto L, Panina-Bordignon P, Brambilla E, Ruffini F, Murtaj V, Orlando F, Colombo M, Frigerio C, DI Bona A, Recupero D, Morroni M....Bone grafting is a surgical procedure that involves the use of bone tissue or bone substitutes to repair damaged bone. In dentistry and maxillofacial surgery, bone graft substitutes from various sources are commonly used. Given their critical role in clinical outcomes, it is essential to thoroughly investigate the biological and mechanical properties of these materials. Methods: In this in vitro study, we evaluated the biological properties of two equine-derived bone graft substitutes in comparison to β-tricalcium phosphate. The materials included one equine-derived graft containing hydrolyze...
Salinas-Varas C, Espinosa G, Muñoz-Caro T, Conejeros I, Gärtner U, Fey K, Arnhold S, Taubert A, Hermosilla C.Neutrophil extracellular trap (NET) are thin and long web-like structures composed of DNA and antimicrobial proteins released by activated polymorphonuclear neutrophils (PMN) as part of the innate immune response. Adipose-derived stem cells (ADSCs) represent an accessible, abundant and minimal invasive source of mesenchymal stem cells (MSCs), with high regenerative potential, immunomodulatory and anti-inflammatory properties. Although recognized immunomodulatory properties of ADSCs, their interaction with PMN and their role on NET formation remains poorly characterized. The present study aimed...
Wójtowicz A, Sadowska A, Molcan T, Słyszewska M, Drzewiecka EM, Tobolski D, Ferreira-Dias G, Szóstek-Mioduchowska A.Mare endometrosis is a chronic degenerative condition of the endometrium, primarily characterized by fibrosis, involving interactions among fibroblasts, immune cells, and epithelial cells regulated by cytokines and growth factors. T helper (Th)1 and Th2 cells seem to play a pivotal role in fibrosis. However, their roles in equine endometrial fibrosis remain unknown. This study explores Th1 and Th2 cell distribution across different stages of endometrium histopathological Kenney and Doig categories; and evaluated their secretome effects on non-fibrotic endometrium derived fibroblast functional ...
Woodman MJ, Thomas MA, Colville MJ, Durgam S, McDonough SP, Reesink HL.To describe proteoglycan 4 (PRG4) expression, lubricin tissue distribution, and lubricin synovial fluid concentrations in healthy and injured equine flexor tendons and tenosynovial structures. Unassigned: Synovial fluid and tissues were collected over the span of 1 year. Proteoglycan 4 expression was quantified using quantitative real-time PCR, lubricin synovial fluid concentrations were measured using ELISAs, and lubricin tissue distribution was evaluated with immunohistochemistry. Histology and quantitative real-time PCR data for healthy samples were analyzed using paired t tests, repeated-m...
Muderspach ND, Daucke R, Nielsen MW, Scoggin KE, Fedorka CE, Miller LMJ, May MG, Troedsson MHT, Ferreira-Dias G, Agerholm JS, Schoof EM....Endometrial fibrosis leads to subfertility when periglandular fibrosis impairs endometrial gland function. Identifying distinct protein expressions in mares suffering from this condition is essential for understanding the pathogenesis and developing treatments to delay fibrotic progression. To gain a better understanding of protein dynamics underlying this condition, Data-Independent Acquisition Mass Spectrometry was utilized to comprehensively characterize and compare the equine endometrial and plasma proteome from mares with different severities of endometrial fibrosis. Mares in diestrus wer...
Sadowska A, Wójtowicz A, Molcan T, Drzewiecka EM, Kaczmarek MM, Słyszewska M, Ferreira-Dias G, Szóstek-Mioduchowska A.Equine endometrosis is a chronic degenerative condition with fibrosis being one of the most significant characteristics. A growing body of evidence indicates the critical role of interleukin (IL)-17 in fibrotic disorders. However, its exact role during equine endometrosis remains to be discovered and explained. The main aim of the current study was to establish the expression of IL-17A signaling components in equine endometria with and without endometrosis as well as the effects of IL-17A on the transcriptomic signature, cellular functional characteristics, expression of extracellular matrix (...
Méndez-Pérez L, Ibáñez BO, Rodríguez S, Sen Wong Y, Caamaño D, Navarrete FI, Cabezas J, Mançanares AC, Escudero C, Rodríguez-Álvarez L....Endometrial fibrosis in mares compromises fertility through aberrant extracellular matrix deposition and sustained myofibroblast activation. Conventional interventions fail to reverse these pathological alterations, necessitating innovative, mechanism-focused therapies. In this study, we pioneered the use of prostaglandin E2 (PGE2) preconditioning of equine endometrial-derived mesenchymal stem cells (ET-eMSCs) and their extracellular vesicles (EVs) to target fibrotic processes directly. ET-eMSCs were isolated from mare endometrial biopsies pretreated with PGE2 to enhance their anti-fibrotic se...
Sawicki S, Bugno-Poniewierska M, Żurowski J, Szmatoła T, Semik-Gurgul E, Bochenek M, Karnas E, Gurgul A. Mesenchymal stem cells (MSCs) are a promising tool in regenerative medicine due to their ability to secrete paracrine factors that modulate tissue repair. Extracellular vesicles (EVs) released by MSCs contain bioactive molecules (e.g., mRNAs, miRNAs, proteins) and play a key role in intercellular communication. This study compared the transcriptomic profiles (mRNA and miRNA) of equine MSCs derived from adipose tissue (AT-MSCs), bone marrow (BM-MSCs), and ovarian fibroblasts (as a differentiated control). Additionally, miRNAs present in EVs secreted by these cells were characterized using nex...
Durgam S.The mid-metacarpal SDFT, intrasynovial DDFT and suspensory ligament are frequently injured soft tissues in the equine athlete. The mid-metacarpal SDFT exhibits a hierarchical structure and encounters repetitive strains at its functional limit. The intrasynovial DDFT possesses fibrocartilage where opposing bony surfaces to facilitate resistance-free gliding and contains chondrocyte-like cells embedded in a proteoglycan-rich ECM. While suspensory ligament enthesis injuries are common, collagen and non-collagen ECM structure-function specializations at the bone-ligament interface are understudied...
Espinosa-López EM, Ortiz-Guisado B, Diez de Castro E, Durham A, Aguilera-Tejero E, Gómez-Baena G.Equine Metabolic Syndrome (EMS) is a multifactorial endocrine disorder characterized by obesity, insulin dysregulation (ID), and an increase in the risk of laminitis, a painful condition that can lead to euthanasia in severe cases. Diagnosing EMS is challenging and often relies on clinical history including obesity, difficulty in losing weight, and recurring episodes of laminitis. The gold standard for laboratory support of an EMS diagnosis is the identification of ID, with basal insulin being the simplest and most accessible method, especially in a field setting. However, various factors such...
Koch DW.Our understanding of tendon homeostasis and repair following injury has made great strides through basic science, clinical veterinary medicine, and preclinical translational research. We have now gleaned a greater understanding of the cellular and matrix dynamics that are being orchestrated within the tendon, which provide therapeutic opportunities. This article will focus on how utilizing the horse and equine tissues have advanced our understanding of tendinopathy and the cellular and matrix dynamics at play while also identifying continued gaps in our knowledge where the horse as a veterinar...
Contentin R, Jehl C, Commenchail K, Legendre F, Galéra P, Cassé F, Demoor M.Osteoarthritis (OA) affects millions of people globally, causing irreversible cartilage damage, chronic inflammation, and progressive joint dysfunction. Similarly, horses can develop OA spontaneously or due to their athletic careers, influenced by mechanical and biochemical factors. Current treatments primarily focus on symptom relief without promoting cartilage regeneration. In line with the 3Rs principles (refine, reduce, replace), the development of OA models is essential for advancing new therapeutic approaches against OA. In response to this need, the present study aimed to develop an m...
Santiviparat S, Suthithanakom S, Bhanpattanakul S, Srisuwattanasagul S, Melde K, Stout TAE, Tharasanit T.Advances in endometrial tissue engineering have enabled the combination of modified scaffolding materials with modern cell culture technologies. Genipin and three-dimensional (3D) printing have advanced cell-tissue engineering by enabling the precise layering of cell-containing matrices while ensuring low cytotoxicity. This study aimed to advance equine endometrial tissue engineering by designing customized collagen scaffolds using 3D printing technology, while optimizing the genipin concentration to avoid toxicity. Genipin was tested at concentrations of 4, 2, 1, 0.5, 0.25, 0.125, and 0 mM on...
Marine L, Wilfried P, Joëlle P, Justine J, Axel D, Vinciane T, Benoît V, Laurent G, Luc G, Jean-François K, Nadine A.In situ injection of mesenchymal stem cells appears as a promising treatment of tendinopathies. Tendon-derived mesenchymal stem cells (TDSCs) are widely studied and show a lot of interesting characteristics for clinical use. The aim of this study is to confirm the tenogenic potential of cryopreserved TDSCs and to confirm their ability to produce type I and/or type III collagens fibers in culture. Tendon-derived mesenchymal stem cells are harvested from the tendon no later than 72 h post-mortem. Their tenogenic potential has been assessed by quantitative reverse transcriptase polymerase chain ...
Elena ZK, Reimer C, Herrmann D, Klein C.Equine endometrial fibrosis is a leading cause of subfertility in aging mares. Fibrosis is a reparative response involving excess deposition of extracellular matrix (ECM) and increasing tissue stiffness. Augmented rigidity itself can drive fibrosis, by stimulating transition from fibroblasts to myofibroblasts. Myofibroblasts release latent transforming growth factor beta 1 (TGF-β1) from the ECM, thereby activating this profibrotic cytokine. Tissue culture polystyrene (TCP) is commonly used for in vitro experiments. The endometrium, however, is considerably softer than TCP. This study critical...
Miranda MR, Montano C, Golino V, de Chiara M, Del Prete C, Pepe G, De Biase D, Ciaglia T, Bertamino A, Campiglia P, Sommella E, Vestuto V, Pasolini MP.Wounds are common in equine practice, and often lead to complications such as infections, delayed healing and hypertrophic scarring, which can be costly and difficult to manage. Developing affordable and effective treatments has become an increasingly important focus in veterinary research. Equine advanced-platelet-rich fibrin plus (A-PRF+) demonstrates regenerative properties comparable to its human counterpart, but cellular-level investigations exploring its molecular mechanisms remain limited. This study aimed to investigate the in vitro effects of equine A-PRF + on primary fibroblast ...
Pawlina-Tyszko K, Semik-Gurgul E, Podstawski P, Herc W, Witkowski M, Ropka-Molik K.Alterations in the genes involved in the creation of the extracellular matrix (ECM) were observed in our earlier transcriptome studies of sarcoids and their cell culture model. For a complete characterization of the underlying molecular pathways, it is imperative to comprehend the involvement of ECM modifications in the oncogenic transformation of sarcoid fibroblasts. Thus, the aim of this investigation was to describe the expression patterns of a set of genes that are essential for the rearrangements of the extracellular matrix, namely collagen genes, and elucidate possible mechanisms underly...
Wójtowicz A, Sadowska A, Piotrowska-Tomala K, Szóstek-Mioduchowska A.The role of AREG in the development of fibrosis in the progression of endometrosis in mare remains unknown. We aimed to determine the effects of AREG on fibroblast functional characteristics as well as the expression of extracellular matrix (ECM)-associated genes in fibroblast derived from non-fibrotic and fibrotic equine endometria. Our findings suggest that the mechanisms associated with ECM remodeling regulated by AREG in non-fibrotic fibroblasts may be dysregulated in the progression of fibrosis in endometrosis.
Klymiuk MC, Speer J, Marco I, Elashry MI, Heimann M, Wenisch S, Arnhold S.Osteoarthritis (OA) is a common and incurable disease in humans and animals. To gain a better understanding of the pathogenesis and identify potential treatments, miRNAs will be extracted and analysed from extracellular vesicles (EVs) of equine adipose derived mesenchymal stem cells (AdMSCs). Methods: For this purpose we cultivated and pretreated AdMSCs under different conditions: interleukin 1β, shock wave, chondrogenic differentiation, chondrogenic differentiation under hypoxia, or after senescence. After treatment, EVs were harvested from the cell culture supernatants. Next-generation sequ...
Frisbie DD, Oxford JT, Southwood L, Trotter GW, Rodkey WG, Steadman JR, Goodnight JL, McIlwraith CW.The current study investigated healing of large full-thickness articular cartilage defects during the first 8 weeks with and without penetration of the subchondral bone using microfracture in an established equine model of cartilage healing. Chondral defects in the weightbearing portion of the medial femoral condyle were made bilaterally; one defect in each horse was microfractured whereas the contralateral leg served as the control. The expression of cartilage extracellular matrix components (Types I and II collagen and aggrecan) was evaluated using histologic techniques, reverse transcriptio...
Caliari SR, Harley BA.Current surgical and tissue engineering approaches for treating tendon injuries have shown limited success, suggesting the need for new biomaterial strategies. Here we describe the development of an anisotropic collagen-glycosaminoglycan (CG) scaffold and use of growth factor supplementation strategies to create a 3D platform for tendon tissue engineering. We fabricated cylindrical CG scaffolds with aligned tracks of ellipsoidal pores that mimic the native physiology of tendon by incorporating a directional solidification step into a conventional lyophilization strategy. By modifying the freez...
Worster AA, Brower-Toland BD, Fortier LA, Bent SJ, Williams J, Nixon AJ.This study evaluated chondrogenesis of mesenchymal progenitor stem cells (MSCs) cultured initially under pre-confluent monolayer conditions exposed to transforming growth factor-beta1 (TGF-beta1), and subsequently in three-dimensional cultures containing insulin-like growth factor I (IGF-I). Bone marrow aspirates and chondrocytes were obtained from horses and cultured in monolayer with 0 or 5 ng of TGF-beta 1 per ml of medium for 6 days. TGF-beta 1 treated and untreated cultures were distributed to three-dimensional fibrin disks containing 0 or 100 ng of IGF-I per ml of medium to establish fou...
Kisiday JD, Kopesky PW, Evans CH, Grodzinsky AJ, McIlwraith CW, Frisbie DD.Bone marrow mesenchymal stem cells (BM-MSCs) and adipose-derived progenitor cells (ADPCs) are potential alternatives to autologous chondrocytes for cartilage resurfacing strategies. In this study, the chondrogenic potentials of these cell types were compared by quantifying neo-tissue synthesis and assaying gene expression and accumulation of extracellular matrix (ECM) components of cartilage. Adult equine progenitor cells encapsulated in agarose or self-assembling peptide hydrogels were cultured in the presence or absence of TGFbeta1 for 3 weeks. In BM-MSCs-seeded hydrogels, TGFbeta1 stimulate...
Thorpe CT, Peffers MJ, Simpson D, Halliwell E, Screen HR, Clegg PD.Tendon is a simple aligned fibre composite, consisting of collagen-rich fascicles surrounded by a softer interfascicular matrix (IFM). The composition and interactions between these material phases are fundamental in ensuring tissue mechanics meet functional requirements. However the IFM is poorly defined, therefore tendon structure-function relationships are incompletely understood. We hypothesised that the IFM has a more complex proteome, with faster turnover than the fascicular matrix (FM). Using laser-capture microdissection and mass spectrometry, we demonstrate that the IFM contains more ...
Dudhia J, Scott CM, Draper ER, Heinegård D, Pitsillides AA, Smith RK.Age-associated and degenerative loss of functional integrity in soft tissues develops from effects of cumulative and subtle changes in their extracellular matrix (ECM). The highly ordered tendon ECM provides the tissue with its tensile strength during loading. As age and exercise collide in the high incidence of tendinopathies, we hypothesized that aged tendons fail due to cumulative damage resulting from a combination of diminished matrix repair and fragmentation of ECM proteins induced by prolonged cyclical loading, and that this is an active cell-mediated process. We developed an equine ten...
Dahlgren LA, van der Meulen MC, Bertram JE, Starrak GS, Nixon AJ.Flexor tendinitis is a common and debilitating injury of elite and recreational athletes. Healing may be improved through intratendinous injection of insulin-like growth factor-I (IGF-I), which has been shown in vitro to stimulate mitogenesis and enhance tendon matrix production. This study investigated the effects of intratendinous injection of IGF-I on tendon healing in an equine model of flexor tendinitis. Collagenase-induced lesions were created in the tensile region of theflexor digitorum superficialis tendon of both forelimbs of eight horses. Treated tendons were injected with 2 microg r...
Peffers MJ, Thorpe CT, Collins JA, Eong R, Wei TK, Screen HR, Clegg PD.Energy storing tendons, such as the human Achilles and equine superficial digital flexor tendon (SDFT), are highly prone to injury, the incidence of which increases with aging. The cellular and molecular mechanisms that result in increased injury in aged tendons are not well established but are thought to result in altered matrix turnover. However, little attempt has been made to fully characterize the tendon proteome nor determine how the abundance of specific tendon proteins changes with aging and/or injury. The aim of this study was, therefore, to assess the protein profile of normal SDFTs ...
Mansfield J, Yu J, Attenburrow D, Moger J, Tirlapur U, Urban J, Cui Z, Winlove P.A combination of two-photon fluorescence (TPF), second harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) imaging has been used to investigate the elastin fibre network in healthy equine articular cartilage from the metacarpophalangeal joint. The elastin fibres were identified using their intrinsic two-photon fluorescence and immuno-staining was used to confirm the identity of these fibres. SHG was used to reveal the collagen matrix and the collagen fibre orientations were determined from their SHG polarization sensitivity, while CARS was used to clearly delineate the c...
Peffers M, Liu X, Clegg P.Age is an important factor in the development of osteoarthritis. Microarray studies provide insight into cartilage aging but do not reveal the full transcriptomic phenotype of chondrocytes such as small noncoding RNAs, pseudogenes, and microRNAs. RNA-Seq is a powerful technique for the interrogation of large numbers of transcripts including nonprotein coding RNAs. The aim of the study was to characterise molecular mechanisms associated with age-related changes in gene signatures. Methods: RNA for gene expression analysis using RNA-Seq and real-time PCR analysis was isolated from macroscopicall...
Klymiuk MC, Balz N, Elashry MI, Heimann M, Wenisch S, Arnhold S.Mesenchymal stem cells are used for different therapeutic approaches, e.g. for osteoarthritis, lesions of the tendon as well as for bone defects. Current research on the mechanism of stem cells on the repair of damaged tissue suggest an important role of a cell-to-cell communication through secreted extracellular vesicles, mainly represented by exosomes. To enhance the scarce knowledge on the functional role of exosomes we compared as a first step different techniques to isolate and identify exosomes from the supernatant of equine adipose derived mesenchymal stem cells for further characteriza...
Visser J, Levett PA, te Moller NC, Besems J, Boere KW, van Rijen MH, de Grauw JC, Dhert WJ, van Weeren PR, Malda J.Decellularized tissues have proven to be versatile matrices for the engineering of tissues and organs. These matrices usually consist of collagens, matrix-specific proteins, and a set of largely undefined growth factors and signaling molecules. Although several decellularized tissues have found their way to clinical applications, their use in the engineering of cartilage tissue has only been explored to a limited extent. We set out to generate hydrogels from several tissue-derived matrices, as hydrogels are the current preferred cell carriers for cartilage repair. Equine cartilage, meniscus, a...
Williams IF, McCullagh KG, Silver IA.During tissue response to injury the glycoproteins fibronectin and Type III collagen are synthesized in increased amounts. We have studied the distribution of these molecules in the healing tendon at various times after injury by comparison with that of the major constituent of normal tendon, Type I collagen. Immunofluorescent localization demonstrated the presence of fibronectin throughout the tendon within one week after injury. Staining was found in the matrix, both around capillaries and around fibroblast-like cells. Fibronectin was still apparent in the healing tendon at one month after i...
Caliari SR, Harley BA.Biomolecular environments encountered in vivo are complex and dynamic, with combinations of biomolecules presented in both freely diffusible (liquid-phase) and sequestered (bound to the extracellular matrix) states. Strategies for integrating multiple biomolecular signals into a biomimetic scaffold provide a platform to simultaneously control multiple cell activities, such as motility, proliferation, phenotype, and regenerative potential. Here we describe an investigation elucidating the influence of the dose and mode of presentation (soluble, sequestered) of five biomolecules (stromal cell-de...
Fortier LA, Nixon AJ, Williams J, Cable CS.To isolate mesenchymal stem cells from adult horses and determine specific monolayer culture conditions required to enhance biochemically and phenotypically defined chondrocytic differentiation. Methods: 2 adult horse bone marrow donors without skeletal or hematologic abnormalities. Methods: Bone marrow was aspirated from the sternebra, and mesenchymal stem cells were isolated by centrifugation and cultured in monolayers. Subcultures were established in 24-well plates on day 13. Culture medium was harvested every 2 days, and culture of 12 of the 24 wells was terminated on day 6 and of the rema...
Richardson DW, Dodge GR.To assess the effects of glucocorticoids on the expression of multiple matrix-related genes in normal and cytokine-treated cultured equine articular chondrocytes in a phenotypically correct suspension culture. Methods: Articular cartilage harvested from the joints of 15 foals, 7 yearling horses, and 16 adult horses. Methods: Glucocorticoids (dexamethasone, prednisolone, triamcinolone) at 10(-10) to 10(-4) M. Methods: Equine articular chondrocytes maintained in suspension cultures were treated with glucocorticoids with and without human recombinant interleukin 1-beta (IL1-beta) and tumor necros...
Crovace A, Lacitignola L, Rossi G, Francioso E.The aim of this study was to compare treatment with cultured bone marrow stromal cells (cBMSCs), bone marrow Mononucleated Cells (BMMNCs), and placebo to repair collagenase-induced tendinitis in horses. In six adult Standardbred horses, 4000 IU of collagenase were injected in the superficial digital flexor tendon (SDFT). Three weeks after collagenase treatment, an average of either 5.5 x 10(6) cBMSCs or 1.2 x 10(8) BMMNCs, fibrin glue, and saline solution was injected intralesionally in random order. In cBMSC- and BMMNCS-treated tendons, a high expression of cartilage oligomeric matrix protein...
Birch HL, Bailey AJ, Goodship AE.Injuries to the superficial digital flexor tendon are common in horses required to gallop and jump at speed. Partial rupture of this tendon usually occurs in the central core of the midmetacarpal region and may be preceded by localised degenerative changes. Post mortem examination of apparently normal equine flexor tendons has revealed an abnormal macroscopic appearance in the central core, characterised by a reddish discolouration. We have previously shown that there is also physical damage to the collagen fibres. In the present study we tested the hypothesis that the abnormal appearance is a...
Clutterbuck AL, Mobasheri A, Shakibaei M, Allaway D, Harris P.Osteoarthritis (OA) is a degenerative and inflammatory disease of synovial joints that is characterized by the loss of articular cartilage, for which there is increasing interest in natural remedies. Curcumin (diferuloylmethane) is the main polyphenol in the spice turmeric, derived from rhizomes of the plant Curcuma longa. Curcumin has potent chemopreventive properties and has been shown to inhibit nuclear factor kappaB-mediated inflammatory signaling in many cell types, including chondrocytes. In this study, normal articular cartilage was harvested from metacarpophalangeal and metatarsophalan...
Szóstek-Mioduchowska AZ, Lukasik K, Skarzynski DJ, Okuda K.Transforming growth factor (TGF)-β1 not only regulates cell growth, development, and tissue remodeling, but it also participates in the pathogenesis of tissue fibrosis. In the equine endometrium, the concentration of TGF-β1 is correlated with endometrosis (equine endometrial fibrosis). In other tissues, TGF-β1 induces differentiation of many cell types into myofibroblasts. These cells are characterized by α-smooth muscle actin (α-SMA) expression and an ability to deposit excessive amounts of extracellular matrix (ECM) components. The aim of the study was to determine whether TGF-β1 plays...
Raabe O, Reich C, Wenisch S, Hild A, Burg-Roderfeld M, Siebert HC, Arnhold S.Adipose-derived stromal cells (ADSCs) are multipotent cells which, in the presence of appropriate stimuli, can differentiate into various lineages such as the osteogenic, adipogenic and chondrogenic. In this study, we investigated the effect of transforming growth factor beta 1 (TGF-β1) in comparison to hydrolyzed fish collagen in terms of the chondrogenic differentiation potential of ADSCs. ADSCs were isolated from subcutaneous fat of horses by liposuction. Chondrogenesis was investigated using a pellet culture system. The differentiation medium was either supplemented with TGF-β1 (5 ng/ml)...
Godinho MSC, Thorpe CT, Greenwald SE, Screen HRC.Tendon is composed of fascicles bound together by the interfascicular matrix (IFM). Energy storing tendons are more elastic and extensible than positional tendons; behaviour provided by specialisation of the IFM to enable repeated interfascicular sliding and recoil. With ageing, the IFM becomes stiffer and less fatigue resistant, potentially explaining why older tendons become more injury-prone. Recent data indicates enrichment of elastin within the IFM, but this has yet to be quantified. We hypothesised that elastin is more prevalent in energy storing than positional tendons, and is mainly lo...
Youngstrom DW, Barrett JG, Jose RR, Kaplan DL.Natural extracellular matrix provides a number of distinct advantages for engineering replacement orthopedic tissue due to its intrinsic functional properties. The goal of this study was to optimize a biologically derived scaffold for tendon tissue engineering using equine flexor digitorum superficialis tendons. We investigated changes in scaffold composition and ultrastructure in response to several mechanical, detergent and enzymatic decellularization protocols using microscopic techniques and a panel of biochemical assays to evaluate total protein, collagen, glycosaminoglycan, and deoxyribo...
Hortensius RA, Harley BA.The design of biomaterials for regenerative medicine can require biomolecular cues such as growth factors to induce a desired cell activity. Signal molecules are often incorporated into the biomaterial in either freely-diffusible or covalently-bound forms. However, biomolecular environments in vivo are often complex and dynamic. Notably, glycosaminoglycans (GAGs), linear polysaccharides found in the extracellular matrix, are involved in transient sequestration of growth factors via charge interactions. Biomaterials mimicking this phenomenon may offer the potential to amplify local biomolecular...
Burk J, Plenge A, Brehm W, Heller S, Pfeiffer B, Kasper C.Tendon and ligament pathologies are still a therapeutic challenge, due to the difficulty in restoring the complex extracellular matrix architecture and biomechanical strength. While progress is being made in cell-based therapies and tissue engineering approaches, comprehensive understanding of the fate of progenitor cells in tendon healing is still lacking. The aim of this study was to investigate the effect of decellularized tendon matrix and moderate cyclic stretching as natural stimuli which could potentially direct tenogenic fate. Equine adipose-derived mesenchymal stromal cells (MSC) were...
Stewart AA, Byron CR, Pondenis H, Stewart MC.To determine whether fibroblast growth factor-2 (FGF-2) treatment of equine mesenchymal stem cells (MSCs) during monolayer expansion enhances subsequent chondrogenesis in a 3-dimensional culture system. Methods: 6 healthy horses, 6 months to 5 years of age. Methods: Bone marrow-derived MSCs were obtained from 6 horses. First-passage MSCs were seeded as monolayers at 10,000 cells/cm(2) and in medium containing 0, 1, 10, or 100 ng of FGF-2/mL. After 6 days, MSCs were transferred to pellet cultures (200,000 cells/pellet) and maintained in chondrogenic medium. Pellets were collected after 15 days....
Thorpe CT, Karunaseelan KJ, Ng Chieng Hin J, Riley GP, Birch HL, Clegg PD, Screen HR.Although the predominant function of all tendons is to transfer force from muscle to bone and position the limbs, some tendons additionally function as energy stores, reducing the energetic cost of locomotion. To maximise energy storage and return, energy-storing tendons need to be more extensible and elastic than tendons with a purely positional function. These properties are conferred in part by a specialisation of a specific compartment of the tendon, the interfascicular matrix, which enables sliding and recoil between adjacent fascicles. However, the composition of the interfascicular matr...
Cadby JA, Buehler E, Godbout C, van Weeren PR, Snedeker JG.The role of intrinsic and extrinsic healing in injured tendons is still debated. In this study, we characterized cell plasticity, proliferative capacity, and migration characteristics as proxy measures of healing potential in cells derived from the peritenon (extrinsic healing) and compared these to cells from the tendon core (intrinsic healing). Both cell populations were extracted from horse superficial digital flexor tendon and characterized for tenogenic and matrix remodeling markers as well as for rates of migration and replication. Furthermore, colony-forming unit assays, multipotency as...
Clutterbuck AL, Smith JR, Allaway D, Harris P, Liddell S, Mobasheri A.This study employed a targeted high-throughput proteomic approach to identify the major proteins present in the secretome of articular cartilage. Explants from equine metacarpophalangeal joints were incubated alone or with interleukin-1beta (IL-1β, 10ng/ml), with or without carprofen, a non-steroidal anti-inflammatory drug, for six days. After tryptic digestion of culture medium supernatants, resulting peptides were separated by HPLC and detected in a Bruker amaZon ion trap instrument. The five most abundant peptides in each MS scan were fragmented and the fragmentation patterns compared to m...
Böer U, Lohrenz A, Klingenberg M, Pich A, Haverich A, Wilhelmi M.Decellularized equine carotid arteries (dEAC) may represent a reasonable alternative to alloplastic materials in vascular replacement therapy. Acellularity of the matrix is standardly evaluated by DNA quantification what however may not record sufficiently the degree of matrix immunogenicity. Thus, our aim was to analyze dEAC with a low DNA content for residual cellular proteins. A detergent-based decellularization protocol including endonuclease treatment resulted in dEAC with 0.6 ± 0.15 ng DNA/mg dry weight representing 0.33 ± 0.14% of native tissue DNA content. In contrast, when matrices ...
