Intraarticular treatment with integrin α10ß1-selected mesenchymal stem cells affects microRNA expression in experimental post-traumatic osteoarthritis in horses.

Osteoarthritis (OA) remains a major cause of lameness in horses, which leads to lost days of training and early retirement. Still, the underlying pathological processes are poorly understood. MicroRNAs (miRNAs) are small non-coding RNAs that serve as regulators of many biological processes including OA. Analysis of miRNA expression in diseased joint tissues such as cartilage and synovial membrane may help to elucidate OA pathology. Since integrin α10ß1-selected mesenchymal stem cell (integrin α10-MSC) have shown mitigating effect on equine OA we here investigated the effect of integrin α10-MSCs on miRNA expression. Cartilage and synovial membrane was harvested from the middle carpal joint of horses with experimentally induced, untreated OA, horses with experimentally induced OA treated with allogeneic adipose-derived MSCs selected for the marker integrin α10-MSCs, and from healthy control joints. miRNA expression in cartilage and synovial membrane was established by quantifying 70 pre-determined miRNAs by qPCR. Differential expression of the miRNAs was evaluated by comparing untreated OA and control, untreated OA and MSC-treated OA, and joints with high and low pathology score. A total of 60 miRNAs were successfully quantified in the cartilage samples and 55 miRNAs were quantified in the synovial membrane samples. In cartilage, miR-146a, miR-150 and miR-409 had significantly higher expression in untreated OA joints than in control joints. Expression of miR-125a-3p, miR-150, miR-200c, and miR-499-5p was significantly reduced in cartilage from MSC-treated OA joints compared to the untreated OA joints. Expression of miR-139-5p, miR-150, miR-182-5p, miR-200a, miR-378, miR-409-3p, and miR-7177b in articular cartilage reflected pathology score. Several of these miRNAs are known from research in human patients with OA and from murine OA models. Our study shows that these miRNAs are also differentially expressed in experimental equine OA, and that expression depends on OA severity. Moreover, MSC treatment, which resulted in less severe OA, also affected miRNA expression in cartilage.

Integrin α10ß1-Selected Mesenchymal Stem Cells Reduce Pain and Cartilage Degradation and Increase Immunomodulation in an Equine Osteoarthritis Model.

OBJECTIVE: Integrin α10ß1-selected mesenchymal stem cells (integrin α10-MSCs) have previously shown potential in treating cartilage damage and osteoarthritis (OA) in vitro and in animal models in vivo. The aim of this study was to further investigate disease-modifying effects of integrin α10-MSCs. DESIGN: OA was surgically induced in 17 horses. Eighteen days after surgery, horses received 2 × 107 integrin α10-MSCs intra-articularly or were left untreated. Lameness and response to carpal flexion was assessed weekly along with synovial fluid (SF) analysis. On day 52 after treatment, horses were euthanized, and carpi were evaluated by computed tomography (CT), MRI, histology, and for macroscopic pathology and integrin α10-MSCs were traced in the joint tissues. RESULTS: Lameness and response to carpal flexion significantly improved over time following integrin α10-MSC treatment. Treated horses had milder macroscopic cartilage pathology and lower cartilage histology scores than the untreated group. Prostaglandin E2 and interleukin-10 increased in the SF after integrin α10-MSC injection. Integrin α10-MSCs were found in SF from treated horses up to day 17 after treatment, and in the articular cartilage and subchondral bone from 5 of 8 treated horses after euthanasia at 52 days after treatment. The integrin α10-MSC injection did not cause joint flare. CONCLUSION: This study demonstrates that intra-articular (IA) injection of integrin α10-MSCs appears to be safe, alleviate pathological changes in the joint, and improve joint function in an equine post-traumatic osteoarthritis (PTOA) model. The results suggest that integrin α10-MSCs hold promise as a disease-modifying osteoarthritis drug (DMOAD).

Donor age effects on in vitro chondrogenic and osteogenic differentiation performance of equine bone marrow- and adipose tissue-derived mesenchymal stromal cells.

BACKGROUND: Bone marrow (BM)- and adipose tissue (AT)-derived mesenchymal stromal cells (MSCs) have shown potential as cell-based therapies for cartilage and bone injuries and are used increasingly in human and veterinary practice to facilitate the treatment of orthopedic conditions. However, human and rodent studies have documented a sharp decline in chondrogenic and osteogenic differentiation potential with increasing donor age, which may be problematic for the important demographic of older orthopedic patients. The aim of this study was to identify the effect of donor age on the chondrogenic and osteogenic differentiation performance of equine BM- and AT-MSCs in vitro. BM- and AT-MSCs and dermal fibroblasts (biological negative control) were harvested from horses in five different age groups (n = 4, N = 60); newborn (0 days), yearling (15-17 months), adult (5-8 years), middle-aged (12-18 years), and geriatric (≥ 22 years). Chondrogenic differentiation performance was assessed quantitatively by measuring pellet size, matrix proteoglycan levels, and gene expression of articular cartilage biomarkers. Osteogenic differentiation performance was assessed quantitatively by measuring alkaline phosphatase activity, calcium deposition, and gene expression of bone biomarkers. RESULTS: Chondrogenic and osteogenic differentiation performance of equine BM- and AT-MSCs declined with increasing donor age. BM-MSCs had a higher chondrogenic differentiation performance. AT-MSCs showed minimal chondrogenic differentiation performance in all age groups. For osteogenesis, alkaline phosphatase activity was also higher in BM-MSCs, but BM-MSCs calcium deposition was affected by donor age earlier than AT-MSCs. Chondrogenic and osteogenic differentiation performance of BM-MSCs exhibited a decline as early as between the newborn and yearling samples. Steady state levels of mRNA encoding growth factors, chondrogenic, and osteogenic biomarkers were lower with increasing donor age in both MSC types. CONCLUSIONS: The data showed that chondrogenic and osteogenic differentiation performance of equine BM-MSCs declined already in yearlings, and that AT-MSCs showed minimal chondrogenic potential, but were affected later by donor age with regards to osteogenesis (calcium deposition). The results highlight the importance of donor age considerations and MSC selection for cell-based treatment of orthopedic injuries and will help inform clinicians on when to implement or potentially cryopreserve cells. Moreover, the study provides molecular targets affected by donor age.

Human integrin α10ß1-selected mesenchymal stem cells home to cartilage defects in the rabbit knee and assume a chondrocyte-like phenotype.

BACKGROUND: Mesenchymal stem cells (MSCs) have shown promising results in stimulating cartilage repair and in the treatment of osteoarthritis (OA). However, the fate of the MSCs after intra-articular injection and their role in cartilage regeneration is not clear. To address these questions, this study investigated (1) homing of labeled human adipose tissue derived integrin α10ß1-selected MSCs (integrin α10-MSCs) to a cartilage defect in a rabbit model and (2) the ability of the integrin α10-MSCs to differentiate to chondrocytes and to produce cartilage matrix molecules in vivo. DESIGN: Integrin α10-MSCs were labeled with superparamagnetic iron oxide nanoparticles (SPIONs) co-conjugated with Rhodamine B to allow visualization by both MRI and fluorescence microscopy. A cartilage defect was created in the articular cartilage of the intertrochlear groove of the femur of rabbits. Seven days post-surgery, labeled integrin α10-MSCs or vehicle were injected into the joint. Migration and distribution of the SPION-labeled integrin α10-MSCs was evaluated by high-field 9.4 T MRI up to 10 days after injection. Tissue sections from the repair tissue in the defects were examined by fluorescence microscopy. RESULTS: In vitro characterization of the labeled integrin α10-MSCs demonstrated maintained viability, proliferation rate and trilineage differentiation capacity compared to unlabeled MSCs. In vivo MRI analysis detected the labeled integrin α10-MSCs in the cartilage defects at all time points from 12 h after injection until day 10 with a peak concentration between day 1 and 4 after injection. The labeled MSCs were also detected lining the synovial membrane at the early time points. Fluorescence analysis confirmed the presence of the labeled integrin α10-MSCs in all layers of the cartilage repair tissue and showed co-localization between the labeled cells and the specific cartilage molecules aggrecan and collagen type II indicating in vivo differentiation of the MSCs to chondrocyte-like cells. No adverse effects of the α10-MSC treatment were detected during the study period. CONCLUSION: Our results demonstrated migration and homing of human integrin α10ß1-selected MSCs to cartilage defects in the rabbit knee after intra-articular administration as well as chondrogenic differentiation of the MSCs in the regenerated cartilage tissue.

Neutrophil Gelatinase-Associated Lipocalin in Synovial Fluid from Horses with and without Septic Arthritis.

Neutrophil gelatinase-associated lipocalin (NGAL) has been suggested to be a highly sensitive and specific marker of joint infection in humans. The aim of the study was to investigate NGAL concentrations in synovial fluid (SF) from horses with septic synovitis, horses without septic synovitis, and horses with uncertain status. NGAL was measured in 177 admission samples obtained from 152 horses. From a subset of horses (n = 35), additional samples obtained sequentially over the course of treatment were available. Concentrations of NGAL were significantly higher in septic synovitis (n = 47 samples) than in samples classified as non-septic (n = 103) or samples with uncertain status (n = 27), with median NGAL concentrations in the three groups being 1236, 16.8, and 266.4 µg/L, respectively. NGAL discriminated nearly perfectly between septic and non-septic (area under the receiver operating characteristic curve 0.98, 95% confidence interval 0.95-1.00). The optimal cut-off value for maximal sensitivity (87.2%) and specificity (75.0%) to discriminate septic samples from those with uncertain status was 444.6 µg/L, with an area under the receiver operating characteristic curve of 0.85 (95% confidence interval 0.74-0.93). Concentrations declined over time in horses undergoing treatment. NGAL is a novel biomarker that seems to have great potential for identifying septic synovitis and for monitoring the response to treatment of synovial infection in horses.

[Stem cell therapy in knee osteoarthritis].

Stem cell therapy is a promising new treatment option for a range of conditions including osteoarthritis (OA). Recent clinical studies in patients with knee OA have shown that stem cell therapy may improve pain scores and joint mobility for up to 12-24 months. However, the studies vary in study design and quality making comparisons difficult, and there is a lack of controls. The mode-of-action of stem cells in the joint is still largely unknown. A large number of clinical studies are underway and will increase our understanding of the efficacy and function of stem cells in OA.

Mass spectrometric analysis of the in vitro secretome from equine bone marrow-derived mesenchymal stromal cells to assess the effect of chondrogenic differentiation on response to interleukin-1ß treatment.

BACKGROUND: Similar to humans, the horse is a long-lived, athletic species. The use of mesenchymal stromal cells (MSCs) is a relatively new frontier, but has been used with promising results in treating joint diseases, e.g., osteoarthritis. It is believed that MSCs exert their main therapeutic effects through secreted trophic biomolecules. Therefore, it has been increasingly important to characterize the MSC secretome. It has been shown that the effect of the MSCs is strongly influenced by the environment in the host compartment, and it is a crucial issue when considering MSC therapy. The aim of this study was to investigate differences in the in vitro secreted protein profile between naïve and chondrogenic differentiating bone marrow-derived (BM)-MSCs when exposed to an inflammatory environment. METHODS: Equine BM-MSCs were divided into a naïve group and a chondrogenic group. Cells were treated with normal expansion media or chondrogenic media. Cells were treated with IL-1ß for a period of 5 days (stimulation), followed by 5 days without IL-1ß (recovery). Media were collected after 48 h and 10 days. The secretomes were digested and analyzed by nanoLC-MS/MS to unravel the orchestration of proteins. RESULTS: The inflammatory proteins IL6, CXCL1, CXCL6, CCL7, SEMA7A, SAA, and haptoglobin were identified in the secretome after 48 h from all cells stimulated with IL-1ß. CXCL8, OSM, TGF-ß1, the angiogenic proteins VCAM1, ICAM1, VEGFA, and VEGFC, the proteases MMP1 and MMP3, and the protease inhibitor TIMP3 were among the proteins only identified in the secretome after 48 h from cells cultured in normal expansion media. After 10-day incubation, the proteins CXCL1, CXCL6, and CCL7 were still identified in the secretome from BM-MSCs stimulated with IL-1ß, but the essential inducer of inflammation, IL6, was only identified in the secretome from cells cultured in normal expansion media. CONCLUSION: The findings in this study indicate that naïve BM-MSCs have a more extensive inflammatory response at 48 h to stimulation with IL-1ß compared to BM-MSCs undergoing chondrogenic differentiation. This extensive inflammatory response decreased after 5 days without IL-1ß (day 10), but a difference in composition of the secretome between naïve and chondrogenic BM-MSCs was still evident.

Identification and Quantification of Transient Receptor Potential Vanilloid 1 (TRPV1) in Equine Articular Tissue.

Joint pain and osteoarthritis (OA) are some of the most common causes of lameness in horses, and most of the available treatments focus on symptomatic relief without a disease-modifying effect. TRPV1 is a potential target for treating joint diseases, including OA, and the present study aims to investigate if the TRPV1 receptor is present in equine articular tissue and determine whether the number of receptors is upregulated in joint inflammation. Metacarpo/metatarsophalangeal (MCP/MTP) joints from 15 horses euthanised for reasons unrelated to this study were included. Based on synovial fluid analysis, macroscopic evaluation, and magnetic resonance imaging (MRI), joints were divided into two groups: healthy joints and joints with pathology. ELISA analysis was performed on synovial tissue harvested from all joints. TPRV1 was found in all joints. The mean concentration of TRPV1 compared to total protein in healthy joints (8.4 × 10-7 ng/mL) and joints with pathology (12.9 × 10-7 ng/mL) differed significantly (p = 0.01, t-test with Welch correction). Quantitative real-time reverse transcriptase PCR analysis was performed on RNA isolates from synovial tissue from all joints. TRPV1 mRNA expression ratio normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in healthy joints (0.16 (SD: 0.19)) and joints with pathology (0.24 (SD: 0.14)) did not differ significantly (p = 0.43, t-test with Welch correction). mRNA expression of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-) was very low for both groups. In conclusion, TRPV1 was detected both on mRNA and the protein level, with a higher expression of TRPV1 in samples from joints with pathology. Future studies will determine the clinical potential of equine TRPV1 as a target in the management of joint pain and inflammation.

Mapping of equine mesenchymal stromal cell surface proteomes for identification of specific markers using proteomics and gene expression analysis: an in vitro cross-sectional study.

BACKGROUND: Stem cells have great potential for tissue regeneration, but before stem cell populations can be used in the clinic, it is crucial that the stem cells have been definitely characterized by a set of specific markers. Although there have been attempts to identify a set of immunophenotypic markers to characterize equine mesenchymal stromal cells (MSCs), immunophenotyping of equine MSCs is still challenging due to the limited availability of suitable antibodies of high quality and consistent performance across different laboratories. The aim of this study was to evaluate a strategy for mapping the equine MSCs surface proteome by use of biotin-enrichment and mass spectrometry (MS) analysis and mine the proteome for potential equine MSCs surface markers belonging to the cluster of differentiation protein group. Gene expression analysis was used for verification. METHODS: Equine MSCs derived from bone marrow (BM) (n = 3) and adipose tissue (AT) (n = 3) were expanded to P3 and either used for (1) cell differentiation into mesodermal lineages (chondrogenic and osteogenic), (2) enrichment of the MSCs surface proteins by biotinylation followed by in-gel digest of the isolated proteins and nanoLC-MS/MS analysis to unravel the enriched cell surface proteome, and (3) RNA isolation and quantitative real-time reverse transcriptase PCR analysis of the CD29, CD44, CD90, CD105, CD166, CD34, CD45, and CD79a gene expression. RESULTS: A total of 1239 proteins at 1% FDR were identified by MS analysis of the enriched MSCs surface protein samples. Of these proteins, 939 were identified in all six biological samples. The identified proteins included 19 proteins appointed to the cluster of differentiation classification system as potential cell surface targets. The protein and gene expression pattern was positive for the commonly used positive MSCs markers CD29, CD44, CD90, CD105, and CD166, and lacked the negative MSCs markers CD34, CD45, and CD79a. CONCLUSIONS: The findings of this study show that enrichment of the MSCs surface proteome by biotinylation followed by MS analysis is a valuable alternative to immunophenotyping of surface markers, when suitable antibodies are not available. Further, they support gene expression analysis as a valuable control analysis to verify the data.

FOXO1, PXK, PYCARD and SAMD9L are differentially expressed by fibroblast-like cells in equine synovial membrane compared to joint capsule.

BACKGROUND: The synovial membrane lines the luminal side of the joint capsule in synovial joints. It maintains joint homeostasis and plays a crucial role in equine joint pathology. When trauma or inflammation is induced in a joint, the synovial membrane influences progression of joint damage. Equine synovial membrane research is hampered by a lack of markers of fibroblast-like synoviocytes (FLS) to distinguish FLS from other fibroblast-like cells in musculoskeletal connective tissues. The aim of this study is to identify potential FLS markers of the equine synovial membrane using microarray to compare between gene expression in equine synovial membrane and the joint capsule in metacarpophalangeal joints. RESULTS: Microarray analysis of tissues from 6 horses resulted in 1167 up-regulated genes in synovial membrane compared with joint capsule. Pathway analysis resulted in 241 candidate genes. Of these, 15 genes were selected for further confirmation as genes potentially expressed by fibroblast-like synoviocytes. Four genes: FOXO1, PXK, PYCARD and SAMD9L were confirmed in 9 horses by qPCR as differentially expressed in synovial membrane compared to joint capsule. CONCLUSIONS: In conclusion, FOXO1, PXK, PYCARD and SAMD9L were confirmed as differentially expressed in synovial membrane compared to joint capsule. These four genes are potential markers of fibroblast-like synoviocytes of the synovial membrane. As these genes are overexpressed in synovial membrane compared to joint capsule, these genes could shed light on synovial membrane physiology and its role in joint disease.