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Aging is hypothesized to be associated with changes in tendon matrix composition which may lead to alteration of tendon material properties and hence propensity to injury. Altered gene expression may offer insights into disease pathophysiology and thus open new perspectives toward designing pathophysiology‐driven therapeutics. Therefore, the current study aimed at identifying naturally occurring differences in tendon micro‐morphology and gene expression of newborn, young and old horses. Age‐related differences in the distribution pattern of tendon fibril thickness and in the expression of the tendon relevant genes collagen type 1 (Col1), Col3, Col5, tenascin‐C, decorin, tenomodulin, versican, scleraxis and cartilage oligomeric matrix protein were investigated. A qualitative and quantitative gene expression and collagen fibril diameter analysis was performed for the most frequently injured equine tendon, the superficial digital flexor tendon, in comparison with the deep digital flexor tendon. Most analyzed genes (Col1, Col3, Col5, tenascin‐C, tenomodulin, scleraxis) were expressed at a higher level in foals (age ≤ 6 months) than in horses of 2.75 years (age at which flexor tendons become mature in structure) and older, decorin expression increased with age. Decorin was previously reported to inhibit the lateral fusion of collagen fibrils, causing a thinner fibril diameter with increased decorin concentration. The results of this study suggested that reduction of tendon fibril diameters commonly seen in equine tendons with increasing age might be a natural age‐related phenomenon leading to greater fibril surface areas with increased fibrillar interaction and reduced sliding at the fascicular/fibrillar interface and hence a stiffer interfascicular/interfibrillar matrix. This may be a potential reason for the higher propensity to tendinopathies with increasing age., TEM pictures and histograms showing collagen fibril distribution changes for the SDFT and DDFT with age. Whereas a Gaussian‐like distribution of fibril diameters was observed in tendons of foals up to 6 months of age, a marked shift towards much thinner fibrils was seen in tendons of horses 2.75 years of age or older.

Tendinopathies are painful, disabling conditions that afflict 25% of the adult human population. Filling an unmet need for realistic large-animal models, we here present an ovine model of tendon injury for the comparative study of adult scarring repair and fetal regeneration. Complete regeneration of the fetal tendon within 28 days is demonstrated, while adult tendon defects remained macroscopically and histologically evident five months post-injury. In addition to a comprehensive histological assessment, proteome analyses of secretomes were performed. Confirming histological data, a specific and pronounced inflammation accompanied by activation of neutrophils in adult tendon defects was observed, corroborated by the significant up-regulation of pro-inflammatory factors, neutrophil attracting chemokines, the release of potentially tissue-damaging antimicrobial and extracellular matrix-degrading enzymes, and a response to oxidative stress. In contrast, secreted proteins of injured fetal tendons included proteins initiating the resolution of inflammation or promoting functional extracellular matrix production. These results demonstrate the power and relevance of our novel ovine fetal tendon regeneration model, which thus promises to accelerate research in the field. First insights from the model already support our molecular understanding of successful fetal tendon healing processes and may guide improved therapeutic strategies.

For research on tendon injury, many different animal models are utilized; however, the extent to which these species simulate the clinical condition and disease pathophysiology has not yet been critically evaluated. Considering the importance of inflammation in tendon disease, this study compared the cellular and molecular features of inflammation in tenocytes of humans and four common model species (mouse, rat, sheep, and horse). While mouse and rat tenocytes most closely equalled human tenocytes’ low proliferation capacity and the negligible effect of inflammation on proliferation, the wound closure speed of humans was best approximated by rats and horses. The overall gene expression of human tenocytes was most similar to mice under healthy, to horses under transient and to sheep under constant inflammatory conditions. Humans were best matched by mice and horses in their tendon marker and collagen expression, by horses in extracellular matrix remodelling genes, and by rats in inflammatory mediators. As no single animal model perfectly replicates the clinical condition and sufficiently emulates human tenocytes, fit-for-purpose selection of the model species for each specific research question and combination of data from multiple species will be essential to optimize translational predictive validity.

Meniscal injuries are the most frequently encountered soft tissue injuries in the equine stifle joint. Due to the inherent limited repair potential of meniscal tissue, meniscal injuries do not only affect the meniscus itself but also lead to impaired joint homeostasis and secondary osteoarthritis. The presented study compares 3D coculture constructs of primary equine mesenchymal stem cells (MSC) and meniscus cells (MC) seeded on three different scaffolds-a cell-laden collagen type I hydrogel (Col I gel), a tissue-derived small intestinal matrix scaffold (SIS-muc) and a combination thereof-for their qualification to be applied for meniscus tissue engineering. To investigate cell attachment of primary MC and MSC on SIS-muc matrix SEM pictures were performed. For molecular analysis, lyophilized samples of coculture constructs with different cell ratios (100% MC, 100% MSC, and 50% MC and 50% MSC, 20% MC, and 80% MSC) were digested and analyzed for DNA and GAG content. Active matrix remodeling of 3D coculture models was indicated by matrix metalloproteinases detection. For comparison of tissue-engineered constructs with the histologic architecture of natural equine menisci, paired lateral and medial menisci of 15 horses representing different age groups were examined. A meniscus phenotype with promising similarity to native meniscus tissue in its GAG/DNA expression in addition to Col I, Col II, and Aggrecan production was achieved using a scaffold composed of Col I gel on SIS-muc combined with a coculture of MC and MSC. The results encourage further development of this scaffold-cell combination for meniscus tissue engineering.

In this work, we describe a microfluidic three-dimensional (3D) chondrocyte culture mimicking in vivo articular chondrocyte morphology, cell distribution, metabolism, and gene expression. This has been accomplished by establishing a physiologic nutrient diffusion gradient across the simulated matrix, while geometric design constraints of the microchambers drive native-like cellular behavior. Primary equine chondrocytes remained viable for the extended culture time of 3 weeks and maintained the low metabolic activity and high Sox9, aggrecan, and Col2 expression typical of articular chondrocytes. Our microfluidic 3D chondrocyte microtissues were further exposed to inflammatory cytokines to establish an animal-free, in vitro osteoarthritis model. Results of our study indicate that our microtissue model emulates the basic characteristics of native cartilage and responds to biochemical injury, thus providing a new foundation for exploration of osteoarthritis pathophysiology in both human and veterinary patients. Keywords: Organ-on-a-Chip, Cartilage, Primary cells, Tissue-on-a-Chip

The purpose of the current study was to establish an in vitro model for osteoarthritis (OA) by co-culture of osteochondral and synovial membrane explants. Osteochondral explants were cultured alone (control-1) or in co-culture with synovial membrane explants (control-2) in standard culture medium or with interleukin-1β (IL1β) and tumor necrosis factor (TNFα) added to the culture medium (OA-model-1 = osteochondral explant; OA-model-2 = osteochondroal-synovial explant). In addition, in OA-model groups a 2-mm partial-thickness defect was created in the centre of the cartilage explant. Changes in the expression of extracellular matrix (ECM) genes (collagen type-1 (Col1), Col2, Col10 and aggrecan) as well as presence and quantity of inflammatory marker genes (IL6, matrix metalloproteinase-1 (MMP1), MMP3, MMP13, a disintegrin and metalloproteinase with-thrombospondin-motif-5 (ADAMTS5) were analysed by immunohistochemistry, qPCR and ELISA. To monitor the activity of classically-activated pro-inflammatory (M1) versus alternatively-activated anti-inflammatory/repair (M2) synovial macrophages, the nitric oxide/urea ratio in the supernatant of osteochondral-synovial explant co-cultures was determined. In both OA-model groups immunohistochemistry and qPCR showed a significantly increased expression of MMPs and IL6 compared to their respective control group. ELISA results confirmed a statistically significant increase in MMP1and MMP3 production over the culturing period. In the osteochondral-synovial explant co-culture OA-model the nitric oxide/urea ratio was increased compared to the control group, indicating a shift toward M1 synovial macrophages. In summary, chemical damage (TNFα, IL1β) in combination with a partial-thickness cartilage defect elicits an inflammatory response similar to naturally occurring OA in osteochondral explants with and without osteochondral-synovial explant co-cultures and OA-model-2 showing a closer approximation of OA due to the additional shift of synovial macrophages toward the pro-inflammatory M1 phenotype.

Item does not contain fulltext Human bone marrow-derived mesenchymal stem cells (BMSCs) are clinically promising to repair damaged articular cartilage. This study investigated TWIST1, an important transcriptional regulator in mesenchymal lineages, in BMSC chondrogenesis. We hypothesized that downregulation of TWIST1 expression is required for in vitro chondrogenic differentiation. Indeed, significant downregulation of TWIST1 was observed in murine skeletal progenitor cells during limb development (N = 3 embryos), and during chondrogenic differentiation of culture-expanded human articular chondrocytes (N = 3 donors) and isolated adult human BMSCs (N = 7 donors), consistent with an inhibitory effect of TWIST1 expression on chondrogenic differentiation. Silencing of TWIST1 expression in BMSCs by siRNA, however, did not improve chondrogenic differentiation potential. Interestingly, additional investigation revealed that downregulation of TWIST1 in chondrogenic BMSCs is preceded by an initial upregulation. Similar upregulation is observed in non-chondrogenic BMSCs (N = 5 donors); however, non-chondrogenic cells fail to downregulate TWIST1 expression thereafter, preventing their chondrogenic differentiation. This study describes for the first time endogenous TWIST1 expression during in vitro chondrogenic differentiation of human BMSCs, demonstrating dynamic regulation of TWIST1 expression whereby upregulation and then downregulation of TWIST1 expression are required for chondrogenic differentiation of BMSCs. Elucidation of the molecular regulation of, and by, TWIST1 will provide targets for optimization of BMSC chondrogenic differentiation culture.

Meniscal pathologies are among the most common injuries of the femorotibial joint in both human and equine patients. Pathological forces and ensuing injuries of the cranial horn of the equine medial meniscus are considered analogous to those observed in the human posterior medial horn. Biomechanical properties of human menisci are site- and depth- specific. However, the influence of equine meniscus topography and composition on its biomechanical properties is yet unknown. A better understanding of equine meniscus composition and biomechanics could advance not only veterinary therapies for meniscus degeneration or injuries, but also further substantiate the horse as suitable translational animal model for (human) meniscus tissue engineering. Therefore, the aim of this study was to investigate the composition and structure of the equine knee meniscus in a site- and age-specific manner and their relationship with potential site-specific biomechanical properties. The meniscus architecture was investigated histologically. Biomechanical testing included evaluation of the shore hardness (SH), stiffness and energy loss of the menisci. The SH was found to be subjected to both age and site-specific changes, with an overall higher SH of the tibial meniscus surface and increase in SH with age. Stiffness and energy loss showed neither site nor age related significant differences. The macroscopic and histologic similarities between equine and human menisci described in this study, support continued research in this field., PLoS ONE, 13 (3), ISSN:1932-6203

Osteoarthritis (OA), a degenerative joint disease characterized by progressive cartilage degeneration, is one of the leading causes of disability worldwide owing to the limited regenerative capacity of adult articular cartilage. Currently, there are no disease-modifying pharmacological or surgical therapies for OA. Fetal mammals, in contrast to adults, are capable of regenerating injured cartilage in the first two trimesters of gestation. A deeper understanding of the properties intrinsic to the response of fetal tissue to injury would allow us to modulate the way in which adult tissue responds to injury. In this study, we employed secretome proteomics to compare fetal and adult protein regulation in response to cartilage injury using an ovine cartilage defect model. The most relevant events comprised proteins associated with the immune response and inflammation, proteins specific for cartilage tissue and cartilage development, and proteins involved in cell growth and proliferation. Alarmins S100A8, S100A9 and S100A12 and coiled-coil domain containing 88A (CCDC88A), which are associated with inflammatory processes, were found to be significantly upregulated following injury in adult, but not in fetal animals. By contrast, cartilage-specific proteins like proteoglycan 4 were upregulated in response to injury only in fetal sheep postinjury. Our results demonstrate the power and relevance of the ovine fetal cartilage regeneration model presented here for the first time. The identification of previously unrecognized modulatory proteins that plausibly affect the healing process holds great promise for potential therapeutic interventions., Summary: Secretome proteomics identifies differential regulation of inflammation modulators during fetal and adult articular cartilage defect healing, offering novel strategies for therapy.

Sheep are one of the most frequently used large animal models in stem cell research. However, minimal invasive or noninvasive sources of mesenchymal stem cells (MSCs) in sheep are scarce. In the light of the principles of the 3Rs (reduce, refine, replace), it would therefore be desirable to identify a minimally invasive or noninvasive ovine MSC source. In humans, the chorionic villi of the placenta, which can be noninvasively harvested as part of the afterbirth, have been identified as a rich source of MSCs. Therefore, in the present study, ovine placenta cotyledons, which have similar function and structure to human chorionic villi, were tested for their potential use as a noninvasive source of ovine MSCs. Through mincing of the placental cotyledons, collagenase digestion, and Ficoll density gradient centrifugation, combined with plastic adherence selection, MSCs were successfully isolated. Their morphological, immunophenotypical, and cellular growth characteristics, as well as their proliferation, differentiation, and migration potential, were evaluated and compared to the currently best-researched MSC source, bone marrow-derived stem cells. Ovine cotyledons were shown to be a reliable, abundant source for the noninvasive, pain- and risk-free harvest of MSCs. The collection procedure does not interfere with partum or the initial bonding phase between ewes and lambs and is therefore exempt from ethical debate. Ovine placenta cotyledon-derived MSCs exhibit multipotential characteristics and can be cryopreserved for later use.

Objective—To describe the anatomic and histologic features of the collateral ligaments (CLs) of the metacarpophalangeal (MCP) and metatarsophalangeal (MTP) joints in cadaveric limbs obtained from nonlame horses and to compare the histologic findings with the ultrasonographic appearance of the CLs. Sample—Medial and lateral CLs of the MCP and MTP joints of 28 limbs (16 forelimbs and 12 hind limbs) from 9 adult nonlame horses euthanized for reasons unrelated to the study. Procedures—26 limbs of 8 horses were examined by ultrasonography immediately after euthanasia. Postmortem gross and histologic examinations were performed for all 28 limbs. Histologic and ultrasonographic images were graded and compared. Results—Ultrasonographically, the mean ± SD depth and width of the superficial CL were 5.1 ± 0.7 mm and 20.5 ± 1.7 mm, respectively. On histologic examination, only 125 of 319 (39%) specimens obtained from 56 medial and lateral CLs appeared normal. Histopathologic findings varied from mild changes in cellular density and collagen fiber orientation to severe fibrocartilaginous metaplasia. The degree of CL lesion severity increased distally, and the lateral CL was affected more frequently than was the medial CL. Ultrasonographically detectable abnormalities were not correlated with the histologic findings. Conclusions and Clinical Relevance—In horses, histologic abnormalities within the CLs of the MCP and MTP joints may be an adaptive response to joint hyperextension and compression and might predispose horses to desmopathy and ligament failure in the event of trauma. Ultrasonography did not detect morphologic changes of the CL matrix. For an accurate diagnosis of subclinical lesions, more sensitive imaging techniques (eg, MRI) should be considered.

Summary A comminuted fracture of the central tarsal bone, fragmentation of the fourth tarsal bone and associated joint instability was diagnosed in a 14-year-old Sport Horse mare who presented for investigation of acute nonweightbearing hindlimb lameness. The mare responded well to conservative management and, unlike previously reported cases of similar injuries, returned to a similar level of athletic activity.

Summary Objectives: To review the outcome of equine shoulder osteochondrosis (OC) with surgical or conservative treatment. Methods: Retrospective study of 32 horses, less than two years of age, with scapulohumeral joint (SHJ) OC. The lesion severity was graded based on measurements taken from lateromedial radiographs. Follo–up information was obtained from racing records or telephone conversations with owners. Successful outcome was defined as the ability of the horse to perform its intended use. Results: Sixteen of 32 horses were affected bilaterally (48 joints). Eleven of 16 horses with bilateral OC underwent arthroscopic surgery; five had bilateral arthroscopy, six had unilateral arthroscopy of the most severely affected joint. Eight of 16 horses with unilateral OC underwent arthroscopy. The overall outcome was ′poor′. Only 15.4% (4/26) of potential race horses started a race, whereas 67% (4/6) non-racehorses were ′sound′ for the intended use. Statistical analysis evaluating the effect of breed on outcome showed a statistically significant difference. There were no significant interactions between outcome and gender, affected limb, unilateral versus bilateral involvement, treatment or severity of the radiographic lesion. However, radiographic lesion severity on the humerus and glenoid showed significant positive correlation. Clinical significance: The overall poor prognosis for shoulder OC in young horses appears to be, in part, dependent on breed and intended use. There was not any difference in outcome between surgically and conservatively treated horses.

Objective: Articular chondrocytes originate from a distinct cohort of progenitor cells located in the so-called interzone in embryonic developing joints. We conducted this study 1) to determine if it is possible to identify the intermediate and outer interzone layers histologically (using cresyl violet) without adjunct in situ hybridization or immunohistochemistry; 2) to establish whether sufficient amounts of RNA can be harvested from each interzone layer of individual embryos to allow gene expression analysis; 3) to develop measurements that can provide an estimation of the RNA yield prior to costly amplification steps. Methods: Cells from the outer (OI) and intermediate (II) interzone of the nascent femorotibial joint and the epiphyseal cartilage (EC) of femur and tibia of murine embryos of 13.5 and 15.5 days gestation were harvested using laser capture microdissection (LCM). Subsequently, microarray analysis was performed to confirm appropriate layer selection. The surface area harvested and the grey value (gv) of photomicrographs taken during LCM was measured and the corresponding relative optical density (ROD) was calculated and degree and significance of the correlation with RNA yield were determined. Results: Cells from the OI, II and EC could be identified histologically using cresyl violet staining and were successfully harvested with LCM yielding sufficient amounts of RNA for linear amplification and microarray analysis. The RNA yield correlated significantly with the tissue surface area harvested, the mean gv and the corresponding ROD. Conclusions: This study provides a technique for selective laser capture microdissection and subsequent microarray analysis of murine interzone cells of the intermediate and outer layer and presents a method to estimate the RNA yield by measuring the tissue area harvested and calculating the ROD. We recommend to harvest a minimum of 1×106 μm2 of 13.5 days murine embryos and 3×106 μm2 of 15.5 days murine embryos to obtain approximately 10 ng total RNA that can be used for linear T7-based amplification and subsequent microarray analysis.

Objective—To evaluate the use of hydrothermal ablation of articular cartilage for arthrodesis in horses through investigation of the effects of joint lavage with physiologic saline (0.9% NaCl) solution (80°C) for various treatment times on chondrocyte viability in the articular cartilage of the metacarpophalangeal and metatarsophalangeal joints of cadaveric horse limbs. Sample Population—7 pairs of metacarpophalangeal and 8 pairs of metatarsophalangeal joints from 8 Thoroughbreds. Procedure—The horses were euthanatized for reasons unrelated to musculoskeletal disease. On a random basis, 1 joint of each pair underwent intra-articular lavage for 5, 10, or 15 minutes with heated saline solution (80°C); the other joint underwent sham treatment of similar duration with saline solution at 22°C (control). Cartilage samples from the distal articular surface of metacarpus III (or metatarsus III), the proximal surface of the proximal phalanx, and the lateral and medial proximal sesamoid bones were assessed for chondrocyte viability via confocal microscopy and viability staining following enzymatic digestion. Results—Compared with the control joints, findings of both viability assays indicated that the percentage of sites containing viable chondrocytes in heat-treated joints was decreased. Treatment hazard ratios of 0.048 (confocal microscopy) and 0.2 (digestion assay) were estimated. Histologically, periarticular soft tissues had minimal detrimental effects after heat treatment. Conclusions and Clinical Relevance—Ex vivo intraarticular lavage with saline solution at 80°C resulted in the death of almost all articular chondrocytes in the joint. This technique may be a satisfactory method for extensive cartilage ablation when performing arthrodesis by minimally invasive techniques. (Am J Vet Res 2005;66:36–42)

Objective—To determine whether triamcinolone acetonide diffuses from the distal interphalangeal joint (DIPJ) to the navicular bursa, diffusion is direct or systemic, and addition of sodium hyaluronan has an effect on diffusion in horses. Animals—11 adult horses without forelimb lameness. Procedures—1 randomly chosen forelimb DIPJ of each horse received an injection of 10 mg of triamcinolone acetonide plus 20 mg of sodium hyaluronan (group 1), and the contralateral forelimb DIPJ received an injection of 10 mg of triamcinolone acetonide plus 2 mL of lactated Ringer's solution (group 2). Synovial fluid samples were taken from both forelimb navicular bursae and 1 hind limb navicular bursa (systemic control group) at 6 hours. Triamcinolone acetonide concentrations in synovial fluid were quantified by use of high-performance liquid chromatography plus tandem mass spectrometry. Data were logarithmically transformed, and contrast analysis was performed on the 3 groups. Results—Triamcinolone acetonide was detected in navicular bursal samples in all groups. Groups 1 and 2 had significantly greater concentrations of triamcinolone acetonide than the systemic control group. There was no significant difference between groups 1 and 2. Conclusions and Clinical Relevance—Triamcinolone acetonide diffused directly from the DIPJ into the navicular bursa in clinically normal horses, and diffusion was not affected by addition of hyaluronan. Injection into the DIPJ with triamcinolone acetonide or a triamcinolone acetonide–hyaluronan combination can potentially be used for treatment of navicular syndrome, but further studies are needed to determine whether triamcinolone acetonide diffuses similarly in horses with navicular syndrome.