A Translational Model for Repeated Episodes of Joint Inflammation: Welfare, Clinical and Synovial Fluid Biomarker Assessment.

This study investigates repeated low-dose lipopolysaccharide (LPS) injections in equine joints as a model for recurrent joint inflammation and its impact on animal welfare. Joint inflammation was induced in eight horses by injecting 0.25 ng of LPS three times at two-week intervals. Welfare scores and clinical parameters were recorded at baseline and over 168 h post-injection. Serial synoviocentesis was performed for the analysis of a panel of synovial fluid biomarkers of inflammation and cartilage turnover. Clinical parameters and a final synoviocentesis were also performed eight weeks after the last sampling point to assess the recovery of normal joint homeostasis. Statistical methods were used to compare the magnitude of response to each of the 3 LPS inductions and to compare the baseline and final measurements. Each LPS injection produced consistent clinical and biomarker responses, with minimal changes in welfare scores. General matrix metalloproteinase (MMP) activity and joint circumference showed greater response to the second LPS induction, but response to the third was comparable to the first. Gylcosaminoglycans (GAG) levels showed a significantly decreased response with each induction, while collagen-cleavage neoepitope of type II collagen (C2C) and carboxypropetide of type II collagen epitope (CPII) showed quicker responses to the second and third inductions. All parameters were comparable to baseline values at the final timepoint. In conclusion, a consistent, reliable intra-articular inflammatory response can be achieved with repeated injections of 0.25 ng LPS, with minimal impact on animal welfare, suggesting potential as a refined translational model of recurrent joint inflammation.

Folate Receptor Expression by Human Monocyte-Derived Macrophage Subtypes and Effects of Corticosteroids.

OBJECTIVE: Folate receptor beta (FR-ß) has been used as a clinical marker and target in multiple inflammatory diseases, including osteoarthritis (OA) and rheumatoid arthritis (RA). However, the conditions under which FR-ß+ macrophages arise remain unclear and could be affected by corticosteroids. Therefore, we studied FR-ß expression in vitro in macrophage subtypes and determined their response to triamcinolone acetonide (TA), a clinically often-used corticosteroid. DESIGN: Human monocyte-derived macrophages were differentiated to the known M0, M1, or M2 macrophage phenotypes. The phenotype and FR-ß expression and plasticity of the macrophage subtypes were determined using flow cytometry, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and enzyme-linked immunosorbent assay (ELISA). RESULTS: FR-ß expression was low in granulocyte-macrophage colony-stimulating factor (GM-CSF)-generated (M1-like) macrophages and high in macrophage colony-stimulating factor (M-CSF)-generated (M0 and M2-like) macrophages. FR-ß expression remained high once the M0 or M2 macrophages were stimulated with pro-inflammatory stimuli (interferon-γ plus lipopolysaccharide) to induce M1-like macrophages. On the contrary, anti-inflammatory TA treatment skewed GM-CSF macrophage differentiation toward an M2 and FR-ß+ phenotype. CONCLUSIONS: As corticosteroids skewed monocytes toward an FR-ß-expressing, anti-inflammatory phenotype, even in an M1 priming GM-CSF environment, FR-ß has potential as a biomarker to monitor success of treatment with corticosteroids. Without corticosteroid treatment, M-CSF alone induces high FR-ß expression which remains high under pro-inflammatory conditions. This explains why pro-inflammatory FR-ß+ macrophages (exposed to M-CSF) are observed in arthritis patients and correlate with disease severity.

Treatment Effects of Intra-Articular Allogenic Mesenchymal Stem Cell Secretome in an Equine Model of Joint Inflammation.

Background: Allogenic mesenchymal stem cell (MSC) secretome is a novel intra-articular therapeutic that has shown promise in in vitro and small animal models and warrants further investigation. Objectives: To investigate if intra-articular allogenic MSC-secretome has anti-inflammatory effects using an equine model of joint inflammation. Study Design: Randomized positively and negatively controlled experimental study. Method: In phase 1, joint inflammation was induced bilaterally in radiocarpal joints of eight horses by injecting 0.25 ng lipopolysaccharide (LPS). After 2 h, the secretome of INFy and TNFα stimulated allogeneic equine MSCs was injected in one randomly assigned joint, while the contralateral joint was injected with medium (negative control). Clinical parameters (composite welfare scores, joint effusion, joint circumference) were recorded, and synovial fluid samples were analyzed for biomarkers (total protein, WBCC; eicosanoid mediators, CCL2; TNFα; MMP; GAGs; C2C; CPII) at fixed post-injection hours (PIH 0, 8, 24, 72, and 168 h). The effects of time and treatment on clinical and synovial fluid parameters and the presence of time-treatment interactions were evaluated. For phase 2, allogeneic MSC-secretome vs. allogeneic equine MSCs (positive control) was tested using a similar methodology. Results: In phase 1, the joint circumference was significantly (p < 0.05) lower in the MSC-secretome treated group compared to the medium control group at PIH 24, and significantly higher peak synovial GAG values were noted at PIH 24 (p < 0.001). In phase 2, no significant differences were noted between the treatment effects of MSC-secretome and MSCs. Main Limitations: This study is a controlled experimental study and therefore cannot fully reflect natural joint disease. In phase 2, two therapeutics are directly compared and there is no negative control. Conclusions: In this model of joint inflammation, intra-articular MSC-secretome injection had some clinical anti-inflammatory effects. An effect on cartilage metabolism, evident as a rise in GAG levels was also noted, although it is unclear whether this could be considered a beneficial or detrimental effect. When directly comparing MSC-secretome to MSCs in this model results were comparable, indicating that MSC-secretome could be a viable off-the-shelf alternative to MSC treatment.

Treatment effects of intra-articular triamcinolone acetonide in an equine model of recurrent joint inflammation.

BACKGROUND: Intra-articular triamcinolone acetonide is a widely used treatment for joint inflammation despite limited scientific evidence of its efficacy. OBJECTIVES: To investigate if intra-articular triamcinolone acetonide has sustained anti-inflammatory effects using an equine model of repeated joint inflammation. STUDY DESIGN: Randomised controlled experimental study. METHOD: For three consecutive cycles 2 weeks apart, inflammation was induced in both middle carpal joints of eight horses by injecting 0.25 ng lipopolysaccharide (LPS). After the first LPS injection only, treatment with 12 mg triamcinolone acetonide (TA) followed in one randomly assigned joint, while the contralateral joint was treated with sterile saline (control). Clinical parameters (composite welfare scores, joint effusion, joint circumference) were recorded and synovial fluid samples were analysed for various biomarkers (total protein, WBCC; PGE2 ; CCL2; TNFα; MMP; GAGs; C2C; CPII) at fixed timepoints (post injection hours 0, 8, 24, 72 and 168). The effects of time and treatment on clinical and synovial fluid parameters and the presence of time-treatment interactions were tested using a linear mixed model for repeated measures with horse as a random effect, and time and treatment as fixed effects. RESULTS: The TA treated joints showed significantly higher peak synovial GAG concentrations (Difference in means 283.1875 µg/mL, 95% CI 179.8, 386.6, P < 0.000), and PGE2 levels (Difference in means 77.8025 pg/mL, 95% CI 21.2, 134.4, P < 0.007) after the first inflammation induction. Significantly lower TP levels were seen with TA treatment after the second induction (Difference in means -7.5 g/L, 95% CI -14.8, -0.20, P < 0.04) . Significantly lower WBCC levels were noted with TA treatment after the first (Difference in means -23.7125 × 109  cells/L, 95% CI -46.7, -0.7, P < 0.04) and second (Difference in means -35.95 × 109  cells/L, 95% CI -59.0, -12.9, P < 0.002) inflammation inductions. Significantly lower general MMP activity was also seen with TA treatment after the second inflammation inductions (Difference in means -51.65 RFU/s, 95% CI -92.4, -10.9, P < 0.01). MAIN LIMITATIONS: This experimental study cannot fully reflect natural joint disease. CONCLUSIONS: In this model, intra-articular TA seems to have some anti-inflammatory activity (demonstrated by reductions in TP, WBCC and general MMP activity) up to 2 weeks post treatment but not at 4 weeks. This anti-inflammatory effect appeared to outlast a shorter-lived, potentially detrimental effect illustrated by increased synovial GAG and PGE2 levels after the first induction.

Sustained Intra-Articular Release and Biocompatibility of Tacrolimus (FK506) Loaded Monospheres Composed of [PDLA-PEG1000]-b-[PLLA] Multi-Block Copolymers in Healthy Horse Joints.

There is an increasing interest in controlled release systems for local therapy in the treatment of human and equine joint diseases, aiming for optimal intra-articular concentrations with no systemic side effects. In this study, the intra-articular tolerability and suitability for local and sustained release of tacrolimus (FK506) from monospheres composed of [PDLA-PEG1000]-b-PLLA multiblock copolymers were investigated. Unloaded and tacrolimus-loaded (18.4 mg tacrolimus/joint) monospheres were injected into the joints of six healthy horses, with saline and hyaluronic acid (HA) in the contralateral joints as controls. Blood and synovial fluid were analysed for the tacrolimus concentration and biomarkers for inflammation and cartilage metabolism. After an initial burst release, sustained intra-articular tacrolimus concentrations (>20 ng/mL) were observed during the 42 days follow-up. Whole-blood tacrolimus levels were below the detectable level (<0.5 ng/mL). A transient inflammatory reaction was observed for all substances, evidenced by increases of the synovial fluid white blood cell count and total protein. Prostaglandin and glycosaminoglycan release were increased in joints injected with unloaded monospheres, which was mitigated by tacrolimus. Both tacrolimus-loaded monospheres and HA transiently increased the concentration of collagen II cleavage products (C2C). A histologic evaluation of the joints at the endpoint showed no pathological changes in any of the conditions. Together, these results indicate the good biocompatibility of intra-articular applied tacrolimus-loaded monospheres combined with prolonged local drug release while minimising the risk of systemic side effects. Further evaluation in a clinical setting is needed to determine if tacrolimus-loaded monospheres can be beneficial in the treatment of inflammatory joint diseases in humans and animals.

Early Signs of Bone and Cartilage Changes Induced by Treadmill Exercise in Rats.

This study aims to investigate the earliest alterations of bone and cartilage tissues as a result of different exercise protocols in the knee joint of Wistar rats. We hypothesize that pretraining to a continuous intense running protocol would protect the animals from cartilage degeneration. Three groups of animals were used: (i) an adaptive (pretraining) running group that ran for 8 weeks with gradually increasing velocity and time of running followed by a constant running program (6 weeks of 1.12 km/hour running per day); (ii) a non-adaptive running (constant running) group that initially rested for 8 weeks followed by 6 weeks of constant running; and (iii) a non-running (control) group. At weeks 8, 14, and 20 bone and cartilage were analyzed. Both running groups developed mild symptoms of cartilage irregularities, such as chondrocyte hypertrophy and cell clustering in different cartilage zones, in particular after the adaptive running protocol. As a result of physical training in the adaptive running exercise a dynamic response of bone was detected at week 8, where bone growth was enhanced. Conversely, the thickness of epiphyseal trabecular and subchondral bone (at week 14) was reduced due to the constant running in the period between 8 and 14 weeks. Finally, the intermediate differences between the two running groups disappeared after both groups had a resting period (from 14 to 20 weeks). The adaptive running group showed an increase in aggrecan gene expression and reduction of MMP2 expression after the initial 8 weeks running. Thus, the running exercise models in this study showed mild bone and cartilage/chondrocyte alterations that can be considered as early-stage osteoarthritis. The pretraining adaptive protocol before constant intense running did not protect from mild cartilage degeneration. © 2017 The Authors. JBMR Plus is published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

No Effects of Hyperosmolar Culture Medium on Tissue Regeneration by Human Degenerated Nucleus Pulposus Cells Despite Upregulation Extracellular Matrix Genes.

STUDY DESIGN: An in vitro study using human degenerated nucleus pulposus cells. OBJECTIVE: To determine the effect of osmolality and different osmolytes on the regeneration by human nucleus pulposus cells through gene expression and extracellular matrix production. SUMMARY OF BACKGROUND DATA: Intervertebral disc (IVD) degeneration is a major problem in developed countries. Regeneration of the IVD can prevent pain and costs due to diminished work absence and health care, and improve quality of life. The osmotic value of a disc decreases during degeneration due to loss of proteoglycans and might increase degeneration. It is known that gene expression of matrix genes of nucleus pulposus (NP) cells increases when cultured in hyperosmotic medium. Thus, increasing the osmolality of the disc might be beneficial for disc regeneration. METHODS: In the current study, isolated degenerated human NP cells were used in regeneration culture with medium of different osmolalities, adjusted with different osmolytes. NaCl, urea and sucrose. The cells were cultured for 28 days and expression of matrix genes and production of glycosaminoglycans and collagen II were measured. RESULTS: Gene expression for both collagen II and aggrecan increased with increasing osmolality using NaCl or sucrose, but not urea. Protein production however, was not affected by increasing osmolality and was decreased when using urea and sucrose. Expression of genes for Col1A1, MMP13, and MMP14 decreased with increasing osmolality, whereas expression of LOXL2 and LOXL3 increased. Transient expression of TonEBP was found 6 hours after the start of culture, but not at later time points. CONCLUSION: Although expression of matrix genes is upregulated, hyperosmolality does not enhance matrix production by nucleus pulposus cells. Raising osmolality can potentially increase matrix production, but in itself is not sufficient to accomplish regeneration in the current in vitro culture system. LEVEL OF EVIDENCE: N /A.

Sustained intra-articular release of celecoxib in an equine repeated LPS synovitis model.

Synovial inflammation is an important characteristic of arthritic disorders like osteoarthritis and rheumatoid arthritis. Orally administered non-steroidal anti-inflammatory drugs (NSAIDs) such as celecoxib are among the most widely prescribed drugs to manage these debilitating diseases. Intra-articular delivery in biodegradable in situ forming hydrogels overcomes adverse systemic effects and prolongs drug retention in the joint. In this study two formulations of celecoxib (40 mg/g and 120 mg/g) in a propyl-capped PCLA-PEG-PCLA triblock copolymer were sequentially evaluated in a multiple LPS challenge equine synovitis model. Intra-articular release and systemic exposure to celecoxib and local changes at joint level were evaluated longitudinally. A single intra-articular injection of the high dose (HCLB)-gel or low dose (LCLB)-gel showed a sustained and controlled intra-articular release in both inflamed and healthy joints together with very low systemic exposure. Synovitis and lameness were moderate respectively very mild in this model due to the low concentration LPS (0.25 ng/joint). Both celecoxib formulations had a mild, transient effect on inflammatory and structural synovial fluid biomarkers but these returned to baseline within one week of administration. The HCLB-gel showed a significant inhibition in peak white blood cell concentration at 8 h after LPS induction. Elevated levels of celecoxib were observed in the joint for up to 30 days but no overall anti-inflammatory effects could be observed, which was thought to be due to the moderate synovitis. As there were no long-term adverse effects, sustained intra-articular release of celecoxib from in situ forming hydrogels should be evaluated further for its effects on longer-term relief of inflammatory joint pain in humans and animals.

Intradiscal application of a PCLA-PEG-PCLA hydrogel loaded with celecoxib for the treatment of back pain in canines: What's in it for humans?

Chronic low back pain is a common clinical problem in both the human and canine population. Current pharmaceutical treatment often consists of oral anti-inflammatory drugs to alleviate pain. Novel treatments for degenerative disc disease focus on local application of sustained released drug formulations. The aim of this study was to determine safety and feasibility of intradiscal application of a poly(ε-caprolactone-co-lactide)-b-poly(ethylene glycol)-bpoly(ε-caprolactone-co-lactide) PCLA-PEG-PCLA hydrogel releasing celecoxib, a COX-2 inhibitor. Biocompatibility was evaluated after subcutaneous injection in mice, and safety of intradiscal injection of the hydrogel was evaluated in experimental dogs with early spontaneous intervertebral disc (IVD) degeneration. COX-2 expression was increased in IVD samples surgically obtained from canine patients, indicating a role of COX-2 in clinical IVD disease. Ten client-owned dogs with chronic low back pain related to IVD degeneration received an intradiscal injection with the celecoxib-loaded hydrogel. None of the dogs showed adverse reactions after intradiscal injection. The hydrogel did not influence magnetic resonance imaging signal at long-term follow-up. Clinical improvement was achieved by reduction of back pain in 9 of 10 dogs, as was shown by clinical examination and owner questionnaires. In 3 of 10 dogs, back pain recurred after 3 months. This study showed the safety and effectiveness of intradiscal injections in vivo with a thermoresponsive PCLA-PEG-PCLA hydrogel loaded with celecoxib. In this set-up, the dog can be used as a model for the development of novel treatment modalities in both canine and human patients with chronic low back pain.

Intradiscal application of rhBMP-7 does not induce regeneration in a canine model of spontaneous intervertebral disc degeneration.

INTRODUCTION: Strategies for biological repair and regeneration of the intervertebral disc (IVD) by cell and tissue engineering are promising, but few have made it into a clinical setting. Recombinant human bone morphogenetic protein 7 (rhBMP-7) has been shown to stimulate matrix production by IVD cells in vitro and in vivo in animal models of induced IVD degeneration. The aim of this study was to determine the most effective dose of an intradiscal injection of rhBMP-7 in a spontaneous canine IVD degeneration model for translation into clinical application for patients with low back pain. METHODS: Canine nucleus pulposus cells (NPCs) were cultured with rhBMP-7 to assess the anabolic effect of rhBMP-7 in vitro, and samples were evaluated for glycosaminoglycan (GAG) and DNA content, histology, and matrix-related gene expression. Three different dosages of rhBMP-7 (2.5 µg, 25 µg, and 250 µg) were injected in vivo into early degenerated IVDs of canines, which were followed up for six months by magnetic resonance imaging (T2-weighted images, T1rho and T2 maps). Post-mortem, the effects of rhBMP-7 were determined by radiography, computed tomography, and macroscopy, and by histological, biochemical (GAG, DNA, and collagen), and biomolecular analyses of IVD tissue. RESULTS: In vitro, rhBMP-7 stimulated matrix production of canine NPCs as GAG deposition was enhanced, DNA content was maintained, and gene expression levels of ACAN and COL2A1 were significantly upregulated. Despite the wide dose range of rhBMP-7 (2.5 to 250 µg) administered in vivo, no regenerative effects were observed at the IVD level. Instead, extensive extradiscal bone formation was noticed after intradiscal injection of 25 µg and 250 µg of rhBMP-7. CONCLUSIONS: An intradiscal bolus injection of 2.5 µg, 25 µg, and 250 µg rhBMP-7 showed no regenerative effects in a spontaneous canine IVD degeneration model. In contrast, intradiscal injection of 250 µg rhBMP-7, and to a lesser extent 25 µg rhBMP-7, resulted in extensive extradiscal bone formation, indicating that a bolus injection of rhBMP-7 alone cannot be used for treatment of IVD degeneration in human or canine patients.

Biocompatibility and intradiscal application of a thermoreversible celecoxib-loaded poly-N-isopropylacrylamide MgFe-layered double hydroxide hydrogel in a canine model.

INTRODUCTION: Chronic low back pain due to intervertebral disc (IVD) degeneration is associated with increased levels of inflammatory mediators. Current medical treatment consists of oral anti-inflammatory drugs to alleviate pain. In this study, the efficacy and safety of a novel thermoreversible poly-N-isopropylacrylamide MgFe-layered double hydroxide (pNIPAAM MgFe-LDH) hydrogel was evaluated for intradiscal controlled delivery of the selective cyclooxygenase (COX) 2 inhibitor and anti-inflammatory drug celecoxib (CXB). METHODS: Degradation, release behavior, and the ability of a CXB-loaded pNIPAAM MgFe-LDH hydrogel to suppress prostaglandin E2 (PGE2) levels in a controlled manner in the presence of a proinflammatory stimulus (TNF-α) were evaluated in vitro. Biocompatibility was evaluated histologically after subcutaneous injection in mice. Safety of intradiscal application of the loaded and unloaded hydrogels was studied in a canine model of spontaneous mild IVD degeneration by histological, biomolecular, and biochemical evaluation. After the hydrogel was shown to be biocompatible and safe, an in vivo dose-response study was performed in order to determine safety and efficacy of the pNIPAAM MgFe-LDH hydrogel for intradiscal controlled delivery of CXB. RESULTS: CXB release correlated to hydrogel degradation in vitro. Furthermore, controlled release from CXB-loaded hydrogels was demonstrated to suppress PGE2 levels in the presence of TNF-α. The hydrogel was shown to exhibit a good biocompatibility upon subcutaneous injection in mice. Upon intradiscal injection in a canine model, the hydrogel exhibited excellent biocompatibility based on histological evaluation of the treated IVDs. Gene expression and biochemical analyses supported the finding that no substantial negative effects of the hydrogel were observed. Safety of application was further confirmed by the absence of clinical symptoms, IVD herniation or progression of degeneration. Controlled release of CXB resulted in a nonsignificant maximal inhibition (approximately 35 %) of PGE2 levels in the mildly degenerated canine IVDs. CONCLUSIONS: In conclusion, this study showed biocompatibility and safe intradiscal application of an MgFe LDH-pNIPAAM hydrogel. Controlled release of CXB resulted in only limited inhibition of PGE2 in this model with mild IVD degeneration, and further studies should concentrate on application of controlled release from this type of hydrogel in animal models with more severe IVD degeneration.