Objective—To assess effects of zoledronic acid on biomarkers, radiographic scores, and gross articular cartilage changes in dogs with induced osteoarthritis.
Animals—21 purpose-bred hound-type dogs.
Procedures—The left stifle joint of each dog was examined arthroscopically to determine initial articular cartilage status, which was followed by cranial cruciate ligament (CrCL) transection to induce osteoarthritis. Dogs were assigned to 3 groups (control group, low dose [10 μg of zoledronic acid/kg], or high dose [25 μg of zoledronic acid/kg). Treatments were administered SC every 3 months for 1 year beginning the day after CrCL transection. Serum and synovial fluid samples and radiographs were obtained 0, 1, 3, 6, 9, and 12 months after transection. At 12 months, each joint was scored for cartilage defects. Serum and synovial fluid biomarkers of bone and cartilage turnover (bone-specific alkaline phosphatase, type I and II collagen, carboxy-propeptide of type II collagen, and chondroitin sulfate 846) were analyzed with ELISAs.
Results—The high-dose group had fewer total articular defects and lower severity scores in CrCL-transected stifle joints than did the control group. In addition, the high-dose group had significantly less change in collagenase cleavage of type I or II collagen in the synovial fluid at 1 and 3 months after CrCL transection than did the control group and also had greater changes in bone-specific alkaline phosphatase in synovial fluid at 3 months after CrCL transection than did the control group.
Conclusions and Clinical Relevance—Zoledronic acid had a chondroprotective effect in dogs with a transected CrCL.
Objective—To determine the response of cortical bone to a multicomponent and nanostructural polymeric matrix as a drug delivery system for enhancing bone healing.
Animals—20 healthy adult crossbred goats.
Procedures—A 3.5-mm-diameter unicortical defect was created in each tibia (day 0), and goats (4 goats/group) were treated as follows: not treated (control group), grafted with the matrix, grafted with antimicrobial (tigecycline and tobramycin)–impregnated matrix, grafted with recombinant human bone morphogenetic protein type 2 (rhBMP-2)–impregnated matrix, or grafted with antimicrobial- and rhBMP-2–impregnated matrix. Elution kinetics of antimicrobials was monitored through plasma concentrations. Bone response was assessed with radiographic scoring (days 1 and 30) and dual-energy x-ray absorptiometry (days 1, 14, and 30). Goats were euthanized on day 30, and histomorphologic analysis was performed. Categorical variables were analyzed with a generalized linear model, and continuous variables were analyzed with an ANOVA.
Results—Plasma antimicrobial concentrations indicated continued release throughout the study. Radiography and dual-energy x-ray absorptiometry did not reveal significant differences among treatments on day 30. Periosteal reactions were significantly greater surrounding bone defects grafted with rhBMP-2–impregnated matrix than those not treated or grafted with matrix or with antimicrobial-impregnated matrix; periosteal reactions were similar in bone defects grafted with rhBMP-2–impregnated matrix and antimicrobial- and rhBMP-2–impregnated matrix.
Conclusions and Clinical Relevance—The matrix served as an antimicrobial delivery system and stimulated bone proliferation when rhBMP-2 was present. Antimicrobial and rhBMP-2 can be used concurrently, but the presence of antimicrobials may affect the performance of rhBMP-2.
Objective—To determine the effects of interleukin (IL)-6 and IL-1β stimulation on expression of growth differentiation factor (GDF)-5 and Wnt signaling pathway genes in equine chondrocytes.
Sample—Macroscopically normal articular cartilage samples from 6 horses and osteochondral fragments (OCFs) from 3 horses.
Procedures—Chondrocyte pellets were prepared and cultured without stimulation or following stimulation with IL-6 or IL-1β for 1, 2, 12, and 48 hours; expression of GDF-5 was determined with a quantitative real-time PCR assay. Expression of genes in various signaling pathways was determined with microarrays for pellets stimulated for 1 and 2 hours. Immunohistochemical analysis was used to detect GDF-5, glycogen synthase kinase 3β (GSK-3β), and β-catenin proteins in macroscopically normal cartilage samples and OCFs.
Results—Chondrocytes stimulated with IL-6 had significantly higher GDF-5 expression within 2 hours versus unstimulated chondrocytes. Microarray analysis of Wnt signaling pathway genes indicated expression of GSK-3β and coiled-coil domain containing 88C increased after 1 hour and expression of β-catenin decreased after 2 hours of IL-6 stimulation. Results of immunohistochemical detection of proteins were similar to microarray analysis results. Chondrocytes in macroscopically normal articular cartilage and OCFs had immunostaining for GDF-5.
Conclusion and Clinical Relevance—Results indicated IL-6 stimulation decreased chondrocyte expression of the canonical Wnt signaling pathway transactivator β-catenin, induced expression of inhibitors of the Wnt pathway, and increased expression of GDF-5. This suggested IL-6 may inhibit the Wnt signaling pathway with subsequent upregulation of GDF-5 expression. Anabolic extracellular matrix metabolism in OCFs may be attributable to GDF-5 expression. This information could be useful for development of cartilage repair methods.
Objective—To evaluate intra-articular autologous protein solution (APS) for the treatment of osteoarthritis in horses.
Animals—40 client-owned horses with naturally occuring osteoarthritis.
Procedures—APS was generated from a dual-device system that concentrated plasma and WBC proteins and enriched platelet growth factors. Horses were randomly assigned to receive an intra-articular injection of 5 mL of saline (0.9% NaCl) solution (n = 20) or APS (20), exercised on a treadmill, and evaluated on the basis of lameness grades, kinetic gait analysis, joint circumference, and range of motion for 14 days. Horses that received saline solution were administered APS at termination of the study, and clients scored horses for lameness and discomfort before, 12 weeks after, and 52 weeks after the APS injection.
Results—The APS group had significant improvements in lameness grade, asymmetry indices of vertical peak force, and range of joint motion by 14 days, compared with baseline or control group values. No adverse effects associated with APS treatment were evident. Clients assessed lameness and comfort as improved at 12 and 52 weeks. The APS had greater likelihood (OR, 4.3 to 30.0) of a therapeutic response in horses with a lameness score < 4, < 10% vertical force asymmetry, or absence of marked osteophyte formation, subchondral sclerosis, or joint space narrowing. Concentration of interleukin-1 receptor antagonist in APS was 5.8 times that in blood.
Conclusions and Clinical Relevance—Intra-articular administration of APS can be considered an effective treatment option for equine osteoarthritis, with the potential for disease-modifying effects.
Objective—To ultrasonographically quantify experimentally induced effusion of the distal interphalangeal (DIP) joint of horses and compare results with those obtained with palpation.
Sample—8 forelimbs from equine cadavers and forelimbs of 5 mares.
Procedures—Preliminary ex vivo experiments were performed to validate the methods. Then, the DIP joints of the forelimbs of standing horses were serially distended with saline (0.9% NaCl) solution (1, 4, and 10 mL) by injection through an intra-articular catheter. Two ultrasonographers measured distension of the dorsal recess of the DIP joint, and 2 surgeons, who were not aware of the volume injected, graded joint effusion by palpation.
Results—Intraobserver and interobserver repeatability was excellent for ultrasonographic measurements. Interobserver agreement for use of palpation to detect joint distension was moderate (κ = 0.45). There was an overall increase in the palpation distension grade with an increase in injected volume. Sensitivity for detection with palpation of larger volumes (4 and 10 mL) was high (92% and 100%, respectively). However, sensitivity was lower (57%) for detection with palpation of minimal distension (1 mL).
Conclusions and Clinical Relevance—Although palpation provided a reliable clinical assessment of DIP joint effusion for volumes of 4 to 10 mL, ultrasonographic measurements were easy to obtain, more accurate, and able to detect smaller amounts of distension. This may be clinically relevant for the assessment of effusion of the DIP joint that can arise in horses with early osteoarthritis or infectious arthritis with concomitant soft tissue swelling that precludes accurate assessment with palpation.
Objective—To validate use of stress MRI for evaluation of stifle joints of dogs with an intact or deficient cranial cruciate ligament (CrCL).
Sample—10 cadaveric stifle joints from 10 dogs.
Procedures—A custom-made limb-holding device and a pulley system linked to a paw plate were used to apply axial compression across the stifle joint and induce cranial tibial translation with the joint in various degrees of flexion. By use of sagittal proton density–weighted MRI, CrCL-intact and deficient stifle joints were evaluated under conditions of loading stress simulating the tibial compression test or the cranial drawer test. Medial and lateral femorotibial subluxation following CrCL transection measured under a simulated tibial compression test and a cranial drawer test were compared.
Results—By use of tibial compression test MRI, the mean ± SD cranial tibial translations in the medial and lateral compartments were 9.6 ± 3.7 mm and 10 ± 4.1 mm, respectively. By use of cranial drawer test MRI, the mean ± SD cranial tibial translations in the medial and lateral compartments were 8.3 ± 3.3 mm and 9.5 ± 3.5 mm, respectively. No significant difference in femorotibial subluxation was found between stress MRI techniques. Femorotibial subluxation elicited by use of the cranial drawer test was greater in the lateral than in the medial compartment.
Conclusions and Clinical Relevance—Both stress techniques induced stifle joint subluxation following CrCL transection that was measurable by use of MRI, suggesting that both methods may be further evaluated for clinical use.
Objective—To assess effects of in vitro meloxicam exposure on metabolism in articular chondrocytes from dogs with naturally occurring osteoarthritis
Sample—Femoral head cartilage from 16 dogs undergoing total hip replacement
Procedures—Articular cartilage samples were obtained. Tissue sulfated glycosaminoglycan (SGAG), collagen, and DNA concentrations were measured. Collagen, SGAG, chondroitin sulfate 846, NO, prostaglandin E2 (PGE2), and matrix metalloproteinase (MMP)-2, MMP-3, MMP-9, and MMP-13 concentrations in culture medium were analyzed. Aggrecan, collagen II, MMP-2, MMP-3, MMP-9, MMP-13, ADAM metallopeptidase with thrombospondin type 1 motif (ADAMTS)-4, ADAMTS-5, tissue inhibitor of metalloproteinase (TIMP)-1, TIMP-2, TIMP-3, interleukin-1β, tumor necrosis factor-α, cyclooxygenase-1, cyclooxygenase-2, and nducible nitric oxide synthase gene expression were evaluated. Comparisons between tissues cultured without (control) and with meloxicam at concentrations of 0.3, 3.0, and 30.0 μg/mL for up to 30 days were performed by means of repeated-measures analysis.
Results—Meloxicam had no effect on chondrocyte SGAG, collagen, or DNA concentrations. Expression of ADAMTS-5 was significantly decreased in all groups on all days, compared with the day 0 value. On day 3, culture medium PGE2 concentrations were significantly lower in all meloxicam-treated groups, compared with values for controls, and values remained low. Culture medium MMP-3 concentrations were significantly lower on day 30 than on day 3 in all meloxicam-treated groups.
Conclusions and Clinical Relevance—Results suggested that in vitro meloxicam treatment of osteoarthritic canine cartilage for up to 30 days did not induce matrix degradation or stimulate MMP production. Meloxicam lowered PGE2 release from this tissue, and effects on tissue chondrocyte content and matrix composition were neutral.
Objective—To determine whether oxidative stress could be induced in canine chondrocytes in vitro.
Sample—Chondrocytes obtained from healthy adult mixed-breed dogs.
Procedures—Harvested chondrocytes were maintained at 37°C with 5% CO2 for 24 hours. To assess induction of oxidative stress, 2 stimuli were used: hydrogen peroxide and a combination of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α). To determine the effect of hydrogen peroxide, a set of chondrocyte-seeded plates was incubated with control medium alone or hydrogen peroxide (100, 200, or 300μM) for 24 hours. For inhibition of oxidative stress, cells were incubated for 24 hours with N-acetylcysteine (NAC; 10mM) before exposure to hydrogen peroxide. Another set of chondrocyte-seeded plates was incubated with control medium alone or with IL-1β (10 ng/mL) and TNF-α (1 ng/mL) for 24 hours. Supernatants were obtained for measurement of prostaglandin E2 production, and cell lysates were used for measurement of superoxide dismutase (SOD) activity and reduced-glutathione (GSH) concentration.
Results—Chondrocytes responded to the oxidative stressor hydrogen peroxide with a decrease in SOD activity and GSH concentration. Exposure to the antioxidant NAC caused an increase in SOD activity in hydrogen peroxide–stressed chondrocytes to a degree comparable with that in chondrocytes not exposed to hydrogen peroxide. Similarly, NAC exposure induced significant increases in GSH concentration. Activation with IL-1β and TNF-α also led to a decrease in SOD activity and increase in prostaglandin E2 production.
Conclusions and Clinical Relevance—Canine chondrocytes responded to the oxidative stress caused by exposure to hydrogen peroxide and cytokines. Exposure to oxidative stress inducers could result in perturbation of chondrocyte and cartilage homeostasis and could contribute to the pathophysiology of osteoarthritis. Use of antioxidants, on the other hand, may be helpful in the treatment of arthritic dogs.
Objective—To determine the efficiency of a novel point-of-care gravitational marrow separator and bone marrow aspiration needle for concentrated bone marrow production and bone marrow-derived mesenchymal stem cell (MSC) separation and assess the effect of repeated bone marrow collections in horses.
Animals—8 healthy adult horses.
Procedures—Bone marrow aspiration was performed twice (1 month apart) from sternebral bodies with a standard or prototype multidirectional needle. Concentrated bone marrow was obtained by gravitational marrow separation and evaluated for WBC and platelet counts, automated and cytomorphologic cell differential counts, MSCs, and cell viability.
Results—Concentrated bone marrow samples obtained with the marrow separator had 5- to 19-fold bone marrow-derived MSC, WBC, and platelet counts, compared with original bone marrow samples. Use of a multidirectional needle increased the frequency of obtaining MSC-richer concentrated bone marrow. Repeating bone marrow aspiration at 1 month yielded greater MSC numbers but slightly lower cell viability after processing.
Conclusions and Clinical Relevance—The gravitational bone marrow separator and multidirectional needle were used to effectively harvest bone marrow and improve the quality of concentrated bone marrow. Comparable, or even greater, numbers of bone marrow-derived MSCs were collected by repeated bone marrow aspiration after a 1-month interval from the same aspiration sites. Use of the marrow separator and multidirectional bone marrow aspiration needle can facilitate a 1-step, point-of-care, nonlaboratory method to obtain concentrated bone marrow as a mixture of bone marrow-derived MSCs and growth factors from platelets and plasma.
Objective—To compare the effects of autologous equine serum (AES) and autologous conditioned serum (ACS) on equine articular chondrocyte metabolism when stimulated with recombinant human (rh) interleukin (IL)-1β.
Sample—Articular cartilage and nonconditioned and conditioned serum from 6 young adult horses.
Procedures—Cartilage samples were digested, and chondrocytes were isolated and formed into pellets. Chondrocyte pellets were treated with each of the following: 10% AES, 10% AES and rhIL-1β, 20% AES and rhIL-1β, 10% ACS and rhIL-1β, and 20% ACS and rhIL-1β, and various effects of these treatments were measured.
Results—Recombinant human IL-1β treatment led to a decrease in chondrocyte glycosaminoglycan synthesis and collagen II mRNA expression and an increase in medium matrix metalloproteinase-3 activity and cyclooxygenase-2 mRNA expression. When results of ACS and rhIL-1β treatment were compared with those of AES and rhIL-1β treatment, no difference was evident in glycosaminoglycan release, total glycosaminoglycan concentration, total DNA content, or matrix metalloproteinase-3 activity. A significant increase was found in chondrocyte glycosaminoglycan synthesis with 20% AES and rhIL-1β versus 10% ACS and rhIL-1β. The medium from ACS and rhIL-1β treatment had a higher concentration of IL-1β receptor antagonist, compared with medium from AES and rhIL-1β treatment. Treatment with 20% ACS and rhIL-1β resulted in a higher medium insulin-like growth factor-I concentration than did treatment with 10% AES and rhIL-1β. No difference in mRNA expression was found between ACS and rhIL-1β treatment and AES and rhIL-1β treatment.
Conclusions and Clinical Relevance—Minimal beneficial effects of ACS treatment on proteoglycan matrix metabolism in equine chonrocytes were evident, compared with the effects of AES treatment.