Recipient Organization
COLORADO STATE UNIVERSITY
(N/A)
FORT COLLINS,CO 80523
Performing Department
Clinical Science
Non Technical Summary
Orthopedic injuries represent one of the most common conditions treated by equine practitioners. Despite the high prevalence of orthopedic injuries in horses, there are no USDA or FDA approved therapeutics for the equine market. Nonetheless, multiple biological therapies for orthopedic injuries (mesenchymal stromal cells (MSC), autologous conditioned serum (ACS), platelet rich plasma (PRP), and autologous protein solution (APS)) are marketed to veterinarians, despite a lack of comparative activity data to guide equine practitioners in making informed decisions in selecting a most active compound. Therefore, the overall goal of this proposal is to use relevant bioassays to compare the anti-inflammatory and immunomodulatory disease-modifying activity of four commonly used therapies. The objectives are to 1) determine the macrophage and synoviocyte cytokine response to four orthobiologics, and 2) use RNA sequencing to identify unique potential disease-modifying pathways activated in macrophages after treatment with four biological therapies. We hypothesize that 1) inflammatory cytokine production by activated macrophages and synoviocytes will be suppressed following co-culture with biological products, with MSC conditioned medium (MSC-CM) inducing greatest immunomodulatory activity, and 2) that each treatment will activate unique and distinctive gene expression pathways in macrophages. Equine monocyte-derived macrophages and synoviocytes will be stimulated with cartilage degradation products, IL1-ß, or synovial fluid from horses with arthroscopically confirmed osteoarthritis, and treated with biological therapies (MSC-CM, ACS, PRP, APS) to compare unique cytokine response signatures. Following transient addition of orthobiologics, culture supernatants will be assessed by multiplex bead and ELISA immunoassays to characterize macrophage and synoviocyte response. RNA extracted from IL-1ß-stimulated macrophages transiently exposed to orthobiologics will be processed for RNA sequencing and analyzed to identify transcriptomic pathways activated by the four compounds. These studies are intended to fill a critical gap in our understanding of the relative immunomodulatory properties of regenerative therapies commonly used in equine practice to treat musculoskeletal disease.
Animal Health Component
30%
Research Effort Categories
Basic
70%
Applied
30%
Developmental
(N/A)
Goals / Objectives
The overall goal of this proposal is to use relevant bioassays to compare the anti-inflammatory and immunomodulatory disease-modifying activity of the four most popular marketed equine ortho-biologics, to ultimately provide clarity regarding relative potency and activity for modulating key disease-driving processes in orthopedic injuries.Uncontrolled inflammation is one of the key processes driving the progression of orthopedic injuries, and the development in some cases of chronic osteoarthritis.Therefore, we hypothesize that application of a standard panel of bioassays in a novelin vitroculture system to model joint inflammation can identify an ortho-biologic compound with the greatest disease-modifying activity, providing information needed to select the most active compounds for clinical application.To address this hypothesis, we propose the following two specific aims:Aim 1:Determine the macrophage and synovial cell cytokine response to four different ortho-biologics, using relevant cell culture assays.Equine monocyte-derived macrophages and synoviocytes from healthy donor horses (n=3) will be stimulated with either equine cartilage degradation products (CDP), IL1-ß, or synovial fluid obtained from horses with arthroscopically confirmed osteoarthritis. Macrophages and synoviocytes will be treated with biological therapies (MSC conditioned medium or MSC-CM, ACS, PRP, APS) obtained and processed from donor horses (n=3) to compare dose-responsiveness and unique cytokine response signatures. Following transient addition of ortho-biologics, culture supernatants will be assessed by multiplex bead assay (23 cytokines) and ELISA immunoassay (CRP, TGF-ß) to characterize the macrophage and synoviocyte response. Controls will include untreated macrophages and macrophages treated with an internal reference standard (recombinant IRAP).We hypothesize thatInflammatory cytokine production by activated macrophages and synoviocytes will be suppressed following co-culture with biological products, with MSC-CM inducing the greatest immunomodulatory activity.Aim 2: Use RNA sequencing to identify unique potential disease-modifying pathways activated in macrophages after treatment with four different ortho-biologic therapies.After transient exposure to standardized concentrations of ortho-biologics, RNA will be extracted from IL-1 ß stimulated macrophages of donor horses (n=3) and processed for RNA sequencing (Novogene). Data analysis will be performed using Partek and Ingenuity analysis platforms, to identify key transcriptomic pathways activated by the four compounds. Pathways will be compared between the four groups to identify unique gene expression signatures.Controls would include untreated and stimulated macrophages.We hypothesize that each of the biological treatments will activate unique and distinctive gene expression pathways in macrophages.
Project Methods
Aim 1:Determine the macrophage and synovial cell cytokine response to four different orthobiologics, using relevant cell culture assays.Study overview.Three healthy skeletally mature gelded horses, aged 2 to 5 years, will be used for the study. All procedures are approved by the University's Institutional Animal Care and Use Committee (IACUC protocol #927) and will be performed in accordance with the NIH guide for the Care and Use of Laboratory Animals.Thispilot study will control for sex as a biological variable, as male mice develop PTOA more severely than female mice following destabilization of the medial meniscus, although this relationship has not been previously established in horses [36].Co-investigator on this proposal, Dr. Laurie Goodrich, an equine surgeon, has obtained funding from the Department of Defense to investigate the effect of localized gene therapy in post-traumatic osteoarthritis, which will fund the horses' purchase and per diem costs, thus making possible the studies proposed here within the allowed budget. Bone marrow aspirate and blood sampling for mesenchymal stromal cell expansion and processing of other orthobiological therapies (PRP, ACS, APS) will be performed after health certification and acclimation of horses and prior to initiation of DOD study procedures.Orthobiological therapy preparation.To isolate MSC, the sternum of donor horses (n=3) will be clipped and aseptically prepared. Bone marrow aspirate (5 mL) will be obtained from the sternum using jamshidi into a syringe containing 1 mL heparin (5,000 U/mL). Bone marrow aspirates will be centrifuged by ficoll (Ficoll-PaqueTMPlus; GE Healthcare BioSciences) density separation at 400g for 18 minutes to pellet red cells as previously described [37,38].Bone marrow derived MSC will be plated at 10,000 cells/cm2and expanded in culture (37?C, 5% CO2, 95% humidity) to 80% confluence for approximately seven days in complete growth medium (Dulbecco's Modified Eagle's Medium (DMEM) with 1000 mg/L glucose, 10% fetal bovine serum (FBS), penicillin (100 U/mL), streptomycin (100 µg/mL), 1M HEPES). Cells will be detached from flasks by trypsinization, then frozen at 5x106cells/mL in freeze media (90% FBS, 10% dimethyl sulfoxide [DMSO]) in liquid nitrogen vapor phase until further use. To generate MSC-CM for use in co-culture assays, cells will be thawed quickly in a 37?C water bath,recovered for 48 hours under standard incubation conditions (37°C with 5% CO2). MSC will be plated at 100,000 cells/well on 24-well plates for 24 hours and supernatants collected and frozen at -80°C for use in immunoassays.To prepare autologous protein solution (APS) blood will be drawn, processed according to manufacturers' instructions (Prostride®, Owl Manor, Warsaw, IN, USA 46581), and used fresh in co-culture assays. To prepare autologous conditioned serum (interleukin 1 receptor antagonist; IRAP), blood will be drawn and incubated according to manufacturers' instructions (IRAP II, Arthrex, Naples, FL, USA 34108), and frozen in 1 mL aliquots for later use in immunoassays. To prepare platelet rich plasma, blood will be drawn, processed according to manufacturers' instructions (Arthrex ACP Double Syringe System, Naples, FL, USA 34108), and used fresh in co-culture assays. Cartilage degredation product (CDP) will be prepared using ex vivo explants of equine cartilage; first treated with collagenase to digest cells, then sonicated to release proteins. All equipment necessary to prepare orthobiological therapies are available in the CSU Equine Veterinary Hospital.Monocyte-derived macrophage co-culture.To generate macrophages, equine peripheral blood mononuclear cells will be isolated from whole blood of three horses by density gradient centrifugation (Ficoll-Paque TM plus, GE Healthcare Bio-Sciences) and cultured in macrophage medium (Dulbecco's Modified Eagle's Medium supplemented with 10% fetal bovine serum, non-essential amino acids, and penicillin/streptomycin antibiotics; SigmaAldrich) with human M-CSF (PeproTech, Rocky Hill, NJ USA 80553) at 25 ng/mL to stimulate differentiation into macrophages in three to five days, as previously described [39].Equine monocyte-derived macrophages and synoviocytes from healthy donor horses (n=3) will be stimulated with either equine cartilage degradation products (CDP), IL1-ß, or synovial fluid obtained from horses with arthroscopically confirmed osteoarthritis. Macrophages and synoviocytes will be treated with biological therapies (MSC conditioned medium or MSC-CM, ACS, PRP, APS) obtained and processed from donor horses (n=3) to compare dose-responsiveness and unique cytokine response signatures. Following transient addition of ortho-biologics (4 hours), macrophages and synoviocytes will be cultured an additional 24 hours. At that time, culture supernatants will be collected and assessed by multiplex bead assay (23 cytokines) and ELISA immunoassay (CRP, TGF-ß) to characterize the macrophage and synoviocyte response. Controls will include untreated macrophages and macrophages treated with an internal reference standard (recombinant IRAP).Determination of biomarkers of joint inflammation in synovial fluid.Competitive ELISA, previously validated for use in equine synovial fluid (Immunology Consultant Laboratories, Portland, Oregon, 97224) will be used to measure the concentration of C reactive protein (CRP) and TGFbin synovial fluid. Fluorescent bead-based multiplex assay (Milliplex MAP Equine Cytokine/Chemokine Magnetic Beads Multiplex Assay, Millipore Sigma, Burlington, MA, 01803) will be used to quantify the concentrations of 23 analytes (Eotaxin/CCL11, FGF-2. Fractalkine/CS3CL1, G-CSF, GM-CSF, GRO, IFN, IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8/CXCL8, IL-10, IL-12 (p70), IL-13, IL-17a, IL-18, IP-10, MCP-1, RANTES/CCL5 and TNFα) in cell culture supernatants.Aim 2: Use RNA sequencing to identify unique potential disease-modifying pathways activated in macrophages after treatment with four different orthobiologic therapies.RNA sequencing.Afterexposure to standardized concentrations of orthobiologics (24~ 48 hours ), RNA will be extracted from IL-1 ß in vitro stimulated monocyte derived macrophages of donor horses (n=3) and processed for RNA sequencing at external facility (Novogene). Controls would include untreated and stimulated macrophages. Macrophages will be derived from equine monocytes (n=3) as described in Aim 1.Data analysis.Raw data will be plotted and visually assessed for normality prior to statistical analysis. Data will be modeled individually using a linear mixed model (function lmer from the lme4(49) and lmerTest(50) packages) with donor as a random effect to account for differences in donor cell lines. For the cytokine secretion assays, orthobiologic treatment will be modeled as the sole fixed effect.Data analysis for RNA sequencing will be performed using Partek and GSEA, to identify key transcriptomic pathways activated by the four compounds. Pathways and differential expression patterns will be compared between the four groups to identify unique gene expression signatures.All statistical analyses will be performed using R for Mac (R version 4.0.0 "Arbor Day" and 3.6.0 "Planting of a Tree") [40]. Graphical analyses and graph generation will be performed using Prism software v8.4.1 (GraphPad Software Inc., La Jolla, CA). For all analyses, statistical significance will be assessed asp<0.05.