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Evaluation of the effect of experimentally induced cartilage defect and intra-articular hyaluronan on synovial fluid biomarkers in intercarpal joints of horses

Evaluation of the effect of experimentally induced cartilage defect and intra-articular... Background: Inflammatory and degenerative activity inside the joint can be studied in vivo by analysis of synovial fluid biomarkers. In addition to pro ‑inflammatory mediators, several anabolic and anti‑inflammatory substances are produced during the disease process. They counteract the catabolic effects of the pro ‑inflammatory cytokines and thus diminish the cartilage damage. The response of synovial fluid biomarkers after intra‑articular hyaluronan injec‑ tion, alone or in combination with other substances, has been examined only in a few equine studies. The effects of hyaluronan on some pro‑inflammatory mediators, such as prostaglandin E , have been documented but especially the effects on synovial fluid anti‑inflammatory mediators are less studied. In animal models hyaluronan has been demonstrated to reduce pain via protecting nociceptive nerve endings and by blocking pain receptor channels. How‑ ever, the results obtained for pain‑relief of human osteoarthritis are contradictory. The aim of the study was to meas‑ ure the synovial fluid IL ‑1ra, PDGF‑BB, TGF‑β and TNF‑α concentrations before and after surgically induced cartilage defect, and following intra‑articular hyaluronan injection in horses. Eight Standardbred horses underwent bilateral arthroscopic surgeries of their intercarpal joints under general anaesthesia, and cartilage defect was created on the dorsal edge of the third carpal bone of one randomly selected intercarpal joint of each horse. Five days post‑surgery, one randomly selected intercarpal joint was injected intra‑articular with 3 mL HA (20 mg/mL). Results: Operation type had no significant effect on the synovial fluid IL ‑1ra, PDGF‑BB, TGF‑β and TNF‑α concentra‑ tions but compared with baseline, synovial fluid IL ‑1ra and TNF‑α concentrations increased. Intra‑articular hyaluronan had no significant effect on the biomarker concentrations but a trend of mild improvement in the clinical signs of intra‑articular inflammation was seen. Conclusions: Creation of the cartilage defect and sham‑ operation lead to an increase of synovial fluid IL ‑1ra and TNF‑α concentrations but changes in concentrations of anabolic growth factors TGF‑β and PDGF‑BB could not be documented 5 days after the arthroscopy. Intra‑articular hyaluronan was well tolerated. Further research is needed to document possible treatment effects of intra‑articular hyaluronan on the synovial fluid biomarkers of inflammation and cartilage metabolism. Keywords: Experimental study, Cartilage defect, Hyaluronan, Interleukin‑1 receptor antagonist, Non‑animal stabilized hyaluronic acid (NASHA), Platelet‑ derived growth factor BB, Synovitis, Transforming growth factor β , Tumour necrosis factor α *Correspondence: [email protected] Department of Equine and Small Animal Medicine, Faculty of Veterinary Medicine, University of Helsinki, P.O. Box 57, 00014 Helsinki, Finland Full list of author information is available at the end of the article © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Niemelä et al. Acta Vet Scand (2019) 61:24 Page 2 of 9 Background Methods Research on equine joint disease has been focused on The study protocol was approved by the National Ani - finding tools for early diagnosis and monitoring the treat - mal Experimental Board in Finland. Eight Standardbred ments and progression of the joint disease. Inflamma - horses (four mares, one stallion and three geldings) free tory and degenerative activity in the joint can be studied of lameness were used. Before recruiting the horses, in vivo by analysis of synovial fluid (SF) biomarkers, such combination of interventions was randomly picked for as pro-inflammatory cytokines in experimental setting each right intercarpal joint. As a result, each joint was [1–5], or in naturally occurring joint disease [6–8]. In selected for one of the following; cartilage defect (CD) addition to pro-inflammatory mediators, several anabolic with HA, CD without injection, sham-operation (SO) and anti-inflammatory mediators are produced during with HA or SO without injection. Interventions for each the inflammatory process. They counteract the catabolic of the contralateral intercarpal joints were determined by effects of the pro-inflammatory cytokines and diminish these randomly picked combinations; i.e. if CD with HA the cartilage damage. Moreover, the anti-inflammatory was selected for the right side, left side was for SO with- effect can produce temporal relief of the clinical symp - out injection. toms of the articular disease [9]. The median age of horses was 7  years and range Equine arthritis is commonly treated with intra-artic- 4–24  years. Examinations, grading of measured vari- ular (IA) corticosteroids and hyaluronan (HA) [10]. ables and surgical procedures were carried by the prin- However, the response of SF biomarkers after IA HA cipal veterinarian (TMN). Prior to inclusion, horses were injection, alone or in combination with other substances, subjected to a complete lameness examination. A stand- has been examined only in a few equine studies [3, 4, ardized American Association of Equine Practitioners’ 11]. In human medicine, clinical efficacy of HA has been (AAEP) scale of 0–5 [18] was used to grade lameness. widely studied [12]. In addition, the effect of IA HA on Effusion of the affected joint was recorded on a scale of some SF pro-inflammatory biomarkers, such as prosta - 0–4 (0 = no effusion, 1 = mild, 2 = moderate, 3 = severe glandin E , has been explored both in human and equine effusion, 4 = severe swelling of the joint region) [19]. studies [3, 4, 13–15]. However, research on the effect A flexion test of the affected and the contralateral limb of HA on anti-inflammatory mediators has been infre - was performed, and lameness was recorded on a scale quently conducted. Despite the observed effects of HA of 0–4 (0 = no increase, 1 = slight increase, 2 = moder- on pro-inflammatory mediators, results in pain-relief in ate increase, 3 = considerable increase compared with human osteoarthritis (OA) are contradictory [12]. In ani- the baseline lameness, 4 = non-weight-bearing lameness) mal models HA has been demonstrated to reduce pain [3]. Pain score for maximal flexion of the carpus was also via protecting nociceptive nerve endings [16] and by determined and recorded on a scale of 0–3 (0 = no pain blocking pain receptor channels [17]. on flexion, 1 = mild pain, i.e. the horse shows some reac- The aim of the study was to measure SF concentrations tion, such as moving the limb, 2 = moderate pain, i.e. the of the anti-inflammatory mediator interleukin-1 receptor horse retracts the limb repeatedly during the 1 min flex - antagonist (IL-1ra), the anabolic growth factors platelet- ion period, 3 = severe pain, i.e. the flexion test cannot be derived growth factor BB (PDGF-BB) and transforming properly performed). In addition, five radiographic views growth factor beta-1 (TGF-β ) and the pro-inflamma - (dorsopalmar, dorsolateral-palmaromedial, dorsomedial- tory cytokine tumour necrosis factor alpha (TNF-α) palmarolateral, flexed lateromedial and flexed dorsoprox - before and after surgically induced cartilage defect (CD) imal-dorsodistal) of the carpal joints were assessed. in healthy horses. Secondly, we wanted to explore if the Before the surgical procedure, 5  mL of the SF of both concentrations of the selected biomarkers changed fol- intercarpal joints of each horse were aspirated into a ster- lowing the IA HA (non-animal stabilized hyaluronic acid, ile 5 mL syringe for the biomarker measurements. The SF NASHA ) injection. The hypothesis was that both con - sample was immediately divided between a plain 4  mL centrations of anti-inflammatory cytokines and concen - tube on ice and into an etylenediaminetetraacetic acid tration of TNF-α will increase in SF after the induction of (EDTA) tube. White blood cell (WBC) count and total CD; and that in the HA-injected joints the pro-inflamma - protein (TP) concentration measurements were done tory TNF-α is decreased and the concentrations of anti- from the fresh sample in the EDTA tube. Within 1  h of inflammatory mediators are increased compared with collection, the plain sample was centrifuged at 4000 rpm the joints without the HA medication. for 10 min in 4 °C, aliquoted and stored at − 80 °C. The horses underwent bilateral arthroscopic surger - ies of their intercarpal joints under general anaesthesia. No pre-existing IA abnormalities were detected dur- ing the arthroscopy in any of the joints. Cartilage defect Durolane, Bioventus LLC, Durham, NC, USA. Niemelä et al. Acta Vet Scand (2019) 61:24 Page 3 of 9 was created on the dorsal edge of the third carpal bone Statistical methods of one randomly selected intercarpal joint of each horse. IL-1ra, PDGF-BB, TGF-β , TNF-α, WBC count and TP The lesion was generated using a 5.0 mm × 13 cm arthro- concentration were analysed with analysis of covariance scopic burr. After the procedure, the debris was left in models (ANCOVA). The study design had 3 time-points the joint, inducing synovitis and articular inflammation. and 2 different interventions (operation, treatment). The Synovial membrane and joint capsule (approximately two effects were analysed separately due to small sam - 3  mm × 3  mm) were harvested with a scalpel from the ple size. The change in biomarker and TP concentrations dorsal region of the joints for a study to be reported and WBC count from pre-operation to pre-treatment elsewhere. The sham-operated contralateral joints (SO) was examined in one analysis and the change from pre- served as controls and were similarly evaluated by arthro- treatment to end of follow-up in another analysis. In scopic examination, synovial membranes and joint cap- both analyses, the change in concentration was used as sules were harvested but the cartilage was left intact. The the response, operation type or treatment as the fixed arthroscopic portals were closed, forelimbs were band- effect and the corresponding baseline measurement as a aged, and horses were allowed to recover from the anaes- covariate. thesia and surgery. The horses were housed in stall boxes. As there were still some doubts about the normal- The status of each horse was monitored three times daily, ity of distributions after transformation, the changes in including comfort, lameness at walk, body temperature, biomarker and TP concentrations and WBC count were heart rate and respiratory rate. analysed also using the Wilcoxon signed rank test. The Five days post-surgery, the lameness examination was Wilcoxon test was conducted also for all 16 limbs (effect repeated, new SF samples from both intercarpal joints of operation regardless of type). A P-value < 0.05 was were harvested and one randomly selected intercarpal accepted as statistically significant for all tests. joint was injected IA with 3  mL HA (20  mg/mL). Nine days after the IA HA injection (i.e. 2 weeks after the sur- Results gical arthroscopic procedure) the third lameness evalua- Clinical outcomes tion was done, and the third SF sampling, and the second Five days after arthroscopy, all the horses showed signs synovial soft tissue sampling of both intercarpal joints of lameness and increased score in the flexion test of the were performed under general anesthesia, after which CD-limb and effusion of the affected joint. The mean horses were euthanized on the operating table. lameness score of the CD-limbs was 2.5, the mean flexion test score was 2.9 and the mean effusion score was 2.1, Laboratory analyses respectively. Two horses showed bilateral lameness, i.e. Synovial fluid samples obtained on day 0, day 5 and day had a mildly lame SO-limb. In the SO-group, the mean 14 were used to analyze for the concentration of IL-1ra, scores of the flexion test and effusion were 1 and 1.8, PDGF-BB, TGF-β and TNF-α. All markers were assayed respectively. No pain was elicited in the operated joint in 3 4 5 6 using commercial ELISA development kits , , , from maximal flexion in either of the groups (CD, SO). R&D Systems. Samples were analysed in triplicate. TGF- Following trends can be seen in Fig.  1. After IA HA β (see footnote 3) and PDGF-BB (see footnote 4) were injection, the mean lameness score of the CD group with determined using human antibodies. IL-1ra (see footnote IA HA (CD + HA) decreased more compared to the 5) and TNF-α (see footnote 6) were assayed with equine- group without HA-injection (CD − HA). Mild improve- specific antibodies. The standards provided for each ment in the flexion test score and more pronounced ELISA kit were used in preparing each standard curve improvement in the effusion score were observed in the according to the manufacturers’ instructions. Readings CD affected limbs of horses after IA HA (CD + HA) com- were performed at 450  nm. The inter-, and intra-assay pared to the limbs without HA injections (CD − HA). coefficient of variation was < 6% for each ELISA. Compared with CD groups, changes in the clinical scores and differences between the SO + HA and SO − HA groups were not consistent. Dyonics Arthroscopic Surgery Blade, Smith & Nephew, Andover, MA, USA. Biomarker outcomes In the ANCOVA models, no statistically significant dif - Human TGF-β1 DuoSet, DY240E, R&D Systems, Minneapolis, MN, USA. 4 ferences in the Type III tests of fixed effects were docu - Human PDGF-BB DuoSet, DY220, R&D Systems, Minneapolis, MN, USA. mented: no effects were documented by the type of Equine IL-1ra/IL1F3 DuoSet, DY1814, R&D Systems, Minneapolis, MN, operation (CD vs. SO) or treatment (HA vs. no HA) to USA. the concentrations measured in SF biomarkers. However, Equine TNF-alphaDuoSet, DY1814, R&D Systems, Minneapolis, MN, differences between the time points were revealed: the USA. Niemelä et al. Acta Vet Scand (2019) 61:24 Page 4 of 9 Fig. 1 Mean (± SEM) clinical outcome measures of treatment groups post operation (day 5) and post treatment (day 14). CD + HA cartilage defect joints with hyaluronan injection, CD − HA cartilage defect joints without hyaluronan injection, SO + HA sham‑ operated joints with hyaluronan injection, SO − HA sham‑ operated joints without hyaluronan injection Niemelä et al. Acta Vet Scand (2019) 61:24 Page 5 of 9 change from pre-operation to pre-treatment values were demonstrated, suggesting endogenous production of significant for IL-1ra (P = 0.0344 in the CD group and IL-1ra [2]. In the present study, IA HA had no appar- P = 0.0103 in the SO group), WBC count (P = 0.011 in the ent effect on SF IL-1ra concentration in the short-term. CD group and P = 0.002 in SO group) and TP (P = 0.0002 However, long-term effects of IA HA on SF IL-1ra war - in the CD group and P = 0.002 in the SO group). HA had rants further research. no significant effect on the biomarker concentrations PDGF is secreted in the early inflammatory phase pri - within the groups. marily by platelets, but also by macrophages, endothelial In the Wilcoxon signed rank tests similar results were cells and fibroblasts [36]. It is one of the earliest and the seen. The SF IL-1ra concentration was not significantly most sensible growth factors expressed after tissue injury different for the operation types (CD vs. SO) but regard - [37]. PDGF induces the synthesis of other growth fac- ing all limbs the SF IL-1ra concentration increased signif- tors [38], proliferation and differentiation of fibroblasts, icantly after arthroscopy (P = 0.0039). Also, the increase deposition of collagen and angiogenesis [39, 40]. There - in SF TNF-α concentration was significant (P = 0.0386) fore, it is an essential promoter of the healing process. In regarding all limbs. Induction of CD and SO both the present study, after sampling at the baseline on day caused a significant increase within both groups in WBC 0, the next sampling was performed on day 5, in a time count (P < 0.001 in both groups) and TP concentration point where PDGF-BB is supposed to play a major role in (P < 0.001 in both groups). the vascular formation and proliferation of fibroblasts in ongoing repair [41]. PDGF-BB concentration increased Discussion in SF after induction of CD, although a significant dif - In the present study, the concentrations of the measured ference was not detected. The synthesis of PDGF-BB biomarkers in intact equine joints are mainly equivalent may have, however, occurred earlier in the course of compared with the results of a previous report [20]. To injury and inflammation. In contrast, PDGF-BB has not the best of our knowledge, IL-1ra, PDGF-BB, TGF-β been detected at all in the SF of osteoarthritic human measured in the present study have been evaluated sepa- knee joint [42]. In another study comparing OA joints to rately only in a few studies on SF of the equine joint [2, healthy controls, significant differences in the SF PDGF- 21–23]. A purely catabolic cytokine TNF-α has been BB concentrations were not detected [43]. These results studied in greater detail [5, 20, 21, 23–28]. Most of the are also suggestive for an early increase of SF PDGF- biomarkers have been documented in vitro in equine car- BB concentration and its role in the initial phase of the tilage as well as in the synovial membrane after a chal- pathogenesis of OA. SF PDGF concentration has been lenge, usually lipopolysaccharide (LPS) [20, 29–31]. documented to increase after IA platelet rich plasma- IL-1ra has been shown to increase after an acute intra- treatment [23]. In the present study, no changes in the articular fracture in humans. However, differences in PDGF-BB concentrations were documented 9  days after SF inflammatory cytokine concentrations between high IA HA treatment. and low-energy injuries have not been detected [32]. SF TGF-β has been studied in  vivo in normal joints Although not directly comparable with intra-articu- and joints with osteochondrosis in foals [22] and after lar fractures, in the present study no differences were LPS challenge in adult horses [21]. In the present study, detected between CD joints and SO joints. However, neither the creation of CD or IA HA injection caused the arthroscopy itself caused trauma to the synovial an increase in the SF TGF-β concentration. In contrast, soft tissues and may have caused the increase of IL-1ra TGF-β concentration decreased following the creation concentration. of CD, although no statistically significant difference Blocking the IL-1β receptor by IL-1ra has potentially between CD and SO joints was found (Fig. 2). This is dif - a wide positive effect on inhibiting deleterious events in ferent compared with the previous study on equine joints the joint. The binding of IL-1β to a receptor results in that reported increase of SF TGF-β concentration fol- activation of several transcription factors and expression lowing a challenge with LPS [21]. This may be explained of hundreds of genes leading to the synthesis of other by the more intense inflammation induced by LPS, com - cytokines, chemokines, adhesion molecules, inflamma - pared with inflammation induced by CD. tory mediators and enzymes [33]. Consequently, IL-1β Ríos et  al. [30] has shown an increase in TGF-β con- is has a significant effect on the metabolism of cells and centration after LPS challenge in cartilage inflammation the extracellular matrix [34]. A decrease in SF IL-1ra created in  vitro. The effect was suggested to result from has been documented in chronic stages of human OA a possible anti-inflammatory mechanism or by direct [35]. Delayed increase of SF IL-1ra (at day 35) follow- damage of LPS to the cartilage. A similar effect has been ing the IA administration of autologous conditioned seen after LPS challenge of synovial membrane explants serum in experimentally induced equine OA has been in vitro [31]. Our result is more consistent with the study Niemelä et al. Acta Vet Scand (2019) 61:24 Page 6 of 9 Fig. 2 SF biomarker concentrations (median, quartiles and minimum and maximum value) of treatment groups on different sampling days. CD + HA cartilage defect joints with hyaluronan injection, CD − HA cartilage defect joints without hyaluronan injection, SO + HA sham‑ operated joints with hyaluronan injection, SO − HA sham‑ operated joints without hyaluronan injection of human OA where SF TGF-β concentration was meas- TGF-β in the joint [51]. However, in the present study, ured both in healthy subjects and in patients with OA; no changes in the SF TGF-β concentrations were doc- in the latter, the concentration of SF TGF-β was low or umented after IA HA treatment, possibly implying that even undetectable [44]. Cell signaling pathways may be optimal SF TGF-β concentrations were already present intercepted by inflammatory cytokines, which may be a in the injected joints. possible reason for the reduced amount of TGF-β in the The results of studies on the synovial fluid TNF-α course of OA [45]. are somewhat contradictory. However, it seems to be TGF-β has an anabolic effect on cartilage; it has an quite a sensitive but not very specific marker of IA ability to induce chondrogenic differentiation of mes - insults. The results of this study are also suggestive of enchymal stem cells [46, 47] with rapid biosynthesis of that. TNF-α concentration increased in SF after induc- glycosaminoglycan and deposition of an extracellular tion of CD although significant differences were not matrix [47]. On the other hand, enhanced expression detected between the groups in the small population of TGF-β has been associated with developing osteo- of this experimental study. An increased concentration phytes [48] and hyperplasia of the synovium [49]. It has of SF TNF-α has been documented in horses in natu- been suggested that only a narrow range of bioactive rally occurring OA in carpi [27], and experimentally in TGF-β concentrations are beneficial to cartilage health amphotericin B- [5] and LPS-induced articular inflam - and any concentrations below or above this range may mation [21, 26]. In horses, even exercise alone leads to cause aberrant alterations in TGF-β pathways, resulting a significant increase in TNF-α levels for a short period in abnormal cartilage function [50]. As TGF-β is stored [25, 28]. However, an increase in SF TNF-α concentra- in the platelets, SF TGF-β concentration increases after tion as a result from serial arthrocentesis could not be IA administration of platelet rich plasma, in response to demonstrated [25]. In contrast to this, gas and liquid platelet activation [23]. HA has been suggested to play capsular distension during arthroscopy provoked an an important role in the mechanical activation of latent inflammatory response with increased concentration of Niemelä et al. Acta Vet Scand (2019) 61:24 Page 7 of 9 SF TNF-α [52]. In a clinical equine study, TNF-α was The number of horses in the experimental studies on found not to be a useful biomarker for different types equine joints has generally ranged from 6 to 13 [1, 3, 5, of joint lesions [53]. Similarly, in humans, increased 21, 26, 52]. Eight horses (16 joints) in the present study concentrations were not associated with any particular may have been too low to detect differences in concen - type of articular disease, such as rheumatoid arthritis, trations of SF biomarkers. Although horses with uniform although in OA patients, detectable concentrations of breed and sport discipline were selected, and all horses TNF-α were related with a long duration of the dis- were free of lameness and joint disease of the intercar- ease [24]. In the present study, SF TNF-α concentra- pal joint as verified by arthroscopy, differences in exercise tions were not affected by IA HA. Probably with more or training status and age may have caused some varia- horses, a bigger sample size and earlier and more fre- tion in the SF biomarker concentrations. Exercise may quent sampling significant results may have been dem - lead to increased biomarker concentrations in horses [7, onstrated in this study. 28], which can be further enhanced in joints with com- In conclusion, after a mild increase in biomarker con- promised health [7, 25]. Moreover, also age has been centrations resulting from arthroscopy and induction of reported to influence SF biomarker concentrations or synovitis and CD, HA failed to produce any further effect gene expression so that they generally decrease with age on biomarkers. Minor improvement of clinical signs of [8, 29, 56]. IA inflammation was evident in the CD + HA group, The time from injury to collection of SF samples is when compared with scores of clinical signs of CD − HA an important issue as changes in biomarker concentra- group. However, in SO + HA and SO − HA groups tions can occur quickly, even within hours. On the other changes after IA HA/no injection were not that consist- hand, IA injection itself, especially repeatedly, causes ent. HA is reported to have an anti-inflammatory effect inflammatory reaction in the joint [57] and can cause [16] but IA HA injections have also been documented to increases in biomarker concentrations [56]. To minimize induce a transitional IA inflammatory reaction, either a this effect, it has been recommended there should be a flare with pronounced clinical signs of inflammation and period of even 14  days after the previous arthrocentesis pain [54] or an increase of WBC count in the SF [11]. before subsequent SF collection [28]. However, despite of To our knowledge, only a few studies have explored bio- these potentially interfering factors, repeated sampling markers in equine SF after IA HA [3, 11]. As HA is very and early time points have been generally used in equine frequently used in the IA treatment in horses, its mecha- SF biomarker studies. In studies using LPS induction nism of action warrants further research. for IA inflammation, first sampling point 8 h [19] or 1 h This study has its limitations. Firstly, the contralateral [21, 26] post injection were chosen which is reasonable limb of the horse served as a SO control. Concentra- considering very acute and strong inflammation caused tions of cartilage matrix products are elevated also in by LPS. On the other hand, in some experimental stud- the contralateral knee in patients with anterior cruciate ies, using surgical model [3, 4] comparable with model of ligament rupture, possibly as a consequence of an altered the present study or amphotericin B [5] for induction of loading [55]. Cytokines and degraded matrix products IA inflammation in horses, weekly samplings were cho - released from an operated joint may be transported to sen for exploring SF IL-1ra and TNF-α concentrations the contralateral joint by the circulation and initiate an (among other biomarkers), respectively. Finding an opti- inflammatory process. Therefore, the concentration of mal time point for SF aspiration is challenging, especially markers in the control joints may have resulted partly when several biomarkers are studied. In addition, avoid- by the transport from the CD operated joint. Measure- ing the effect of repeated aspiration and sham-opera - ment of serum concentrations of studied markers, as tion on biomarker concentrations complicates the issue. well as additional pro-inflammatory markers and mark - Although the effect of IA injection on biomarkers meas - ers of cartilage matrix metabolism, would have provided ured in this study is not known, except for TNF-α, 5 days additional information to test this hypothesis. However, after arthroscopy and 9  days after IA HA injection were this was outside the scope of this study. Moreover, the chosen to balance between the intervention time points. arthroscopy itself, as well as harvesting the synovial tis- sue samples, causes trauma to the synovial soft tissues. Conclusions These may have further affected the concentrations of the This study demonstrates that arthroscopy and both the measured markers in the SO joints. However, the effect creation of CD and SO, lead to an increase in SF IL-1ra was thought to be transient. To minimize the effect of and TNF-α concentrations but changes in concentrations arthroscopy and sampling of synovial soft tissue, sam- of anabolic growth factors TGF-β and PDGF-BB in SF pling of SF and IA HA injection were performed only were not documented 5 days after the arthroscopy. Intra- after 5 days. articular HA was well tolerated. However, changes in Niemelä et al. Acta Vet Scand (2019) 61:24 Page 8 of 9 3. Frisbie DD, Kawcak CE, McIlwraith CW, Werpy NM. Evaluation of concentrations of IL-1ra, PDGF-BB, TGF-β, and TNF-α polysulfated glycosaminoglycan or sodium hyaluronan administered were not seen after HA injection. The treatment effect of intra‑articularly for treatment of horses with experimentally induced IA HA on SF pro-inflammatory, anti-inflammatory and osteoarthritis. Am J Vet Res. 2009;70:203–9. 4. Frisbie DD, McIlwraith CW, Kawcak CE, Werpy NM. Evaluation of intra‑ anabolic biomarkers warrants further research. articular hyaluronan, sodium chondroitin sulfate and N‑acetyl‑ d ‑ glucosa‑ mine combination versus saline (0.9% NaCl) for osteoarthritis using an equine model. Vet J. 2013;197:824–9. Abbreviations 5. Ma TW, Li Y, Wang GY, Xin‑ran Li XR, Jiang RL, Song XP, et al. Changes in CD: cartilage defect; EDTA: etylenediaminetetraacetic acid; HA: hyaluronan; IA: synovial fluid biomarkers after experimental equine osteoarthritis. J Vet intra‑articular; IL ‑1ra: interleukin 1 receptor antagonist; LPS: lipopolysaccharide; Res. 2017;61(10):1515. NASHA: non‑animal stabilized hyaluronic acid; OA: osteoarthritis; PDGF‑BB: 6. Bertone A, Palmer JL, Jones J. Synovial fluid cytokines and eicosanoids as platelet‑ derived growth factor; PGE : prostaglandin E ; SF: synovial fluid; SO: 2 2 markers of joint disease in horses. Vet Surg. 2001;30:528–38. sham‑ operation; TGF‑β : transforming growth factor beta 1; TNF‑α: tumour 7. Frisbie DD, Al‑Sobayil F, Billinghurst RC, Kawcak CE, McIlwraith CW. necrosis factor alpha; WBC: white blood cell. Changes in synovial fluid serum biomarkers with exercise and early osteoarthritis in horses. Osteoarthr Cartilage. 2008;16:1197–204. Acknowledgements 8. Nicholson AM, Trumble TN, Merritt KA, Brown MP. Association of horse Financial support from the Finnish Foundation of Veterinary Research, the age, joint type, and osteochondral injury with serum and synovial fluid Finnish Veterinary Foundation, and the Erkki Rajakoski Foundation of the Finn‑ concentrations of type II collagen biomarkers in Thoroughbreds. Am J Vet ish Trotting Association is acknowledged. The authors thank Teppo Huttunen Res. 2010;71:741–9. for statistical assistance. 9. Wojdadiewicz LA, Poniatowski D, Szukiewicz D. The role of inflammatory and anti‑inflammatory cytokines in the pathogenesis of osteoarthritis. Authors’ contributions Mediators Inflamm. 2014. https ://doi.org/10.1155/2014/56145 9. TMN performed the clinical study and arthroscopy of all horses, evaluation 10. Goodrich LR, Nixon AJ. Medical treatment of osteoarthritis in the horse— of radiographs, and drafted the manuscript. CL and JUC contributed to the a review. Vet J. 2006;171:51–69. laboratory work, statistical analyses, interpretation of results and revision of 11. Niemelä TM, Tulamo R‑M, Aaltonen K, Sankari SM, Hielm‑Björkman AK. the manuscript. R‑MT participated in the design of the study, interpretation of Change in biomarkers in equine synovial fluid two weeks after intra‑ results and revision of the manuscript. All the authors read and approved the articular hyaluronan treatment: a randomized double‑blind clinical trial. final manuscript. BMC Vet Res. 2018;14:186. 12. Johansen M, Bahrt H, Altman RD, Bartels EM, Juhl CB, Bliddal H, et al. Funding Exploring reasons for the observed inconsistent trial reports on intra‑ The study was supported in part by a grant from the Finnish Foundation of articular injections with hyaluronic acid in the treatment of osteoarthritis: Veterinary Research, the Finnish Veterinary Foundation, and the Erkki Rajakoski meta‑regression analyses of randomized trials. Semin Arthritis Rheum. Foundation of the Finnish Trotting Association, which did not have any role in 2016;46:34–48. the design, collection, analysis, or interpretation of the data or in writing the 13. Punzi L, Sciavon F, Cavasin F, Ramonda R, Gambari PF, Todesco S. The manuscript. influence of intra‑articular hyaluronic acid on PGE and cAMP of synovial fluid. Clin Exp Rheumatol. 1989;7:247–50. Availability of data and materials 14. Chang NS, Intrieri C, Mattison J, Armand G. Synthetic polysulfated hyalu‑ The datasets analysed during the current study are available from the cor‑ ronic acid is a potent inhibitor for tumour necrosis factor production. J responding author on reasonable request. Leucocyte Biol. 1994;55:778–84. 15. Cole BJ, Karas V, Hussey K, Pilz K, Fortier LA. Hyaluronic acid versus plate‑ Ethics approval and consent to participate let‑rich plasma: a prospective, double ‑blind randomized controlled trial The study protocol was approved by the National Animal Experimentation comparing clinical outcomes and effects on intra‑articular biology for the Board of Finland (ESAVI/1130/04.10.03/2011). treatment of knee osteoarthritis. Am J Sports Med. 2017;45:339–46. 16. Gomis A, Pawlak M, Balazs EA, Schmidt RF, Belmonte C. Eec ff ts of different Consent for publication molecular weight elastoviscous hyaluronan solutions on articular nocic‑ Not applicable. eptive afferents. Arthritis Rheum. 2004;50:314–26. 17. Caires R, Luis E, Taberner FJ, Fernandez‑Ballester G, Ferrer ‑Montiel A, Competing interests Balazs EA, et al. Hyaluronan modulates TRPV1 channel opening, reducing The authors declare that they have no competing interests. peripheral nociceptor activity and pain. Nat Commun. 2015;27:6. 18. Anonymous. Definition and Classification of Lameness. In: Guide for Vet ‑ Author details erinary Service and Judging of Equestrian Events. In: Lexington, American Department of Equine and Small Animal Medicine, Faculty of Veterinary Association of Equine Practitioners. 1991, pp. 19. Medicine, University of Helsinki, P.O. Box 57, 00014 Helsinki, Finland. Grupo de 19. de Grauw JC, van de Lest CHA, Brama PAJ, Rambags BPB, van Weeren PR. Investigación Terapia Regenerativa, Departamento de Salud Animal, Universi‑ In vivo effects of meloxicam on inflammatory mediators, MMP activity dad de Caldas, Calle 65 No26‑10, Manizales, Caldas, Colombia. and cartilage biomarkers in equine joints with acute synovitis. 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Evaluation of the effect of experimentally induced cartilage defect and intra-articular hyaluronan on synovial fluid biomarkers in intercarpal joints of horses

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Medicine & Public Health; Veterinary Medicine/Veterinary Science; Animal Anatomy / Morphology / Histology
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Abstract

Background: Inflammatory and degenerative activity inside the joint can be studied in vivo by analysis of synovial fluid biomarkers. In addition to pro ‑inflammatory mediators, several anabolic and anti‑inflammatory substances are produced during the disease process. They counteract the catabolic effects of the pro ‑inflammatory cytokines and thus diminish the cartilage damage. The response of synovial fluid biomarkers after intra‑articular hyaluronan injec‑ tion, alone or in combination with other substances, has been examined only in a few equine studies. The effects of hyaluronan on some pro‑inflammatory mediators, such as prostaglandin E , have been documented but especially the effects on synovial fluid anti‑inflammatory mediators are less studied. In animal models hyaluronan has been demonstrated to reduce pain via protecting nociceptive nerve endings and by blocking pain receptor channels. How‑ ever, the results obtained for pain‑relief of human osteoarthritis are contradictory. The aim of the study was to meas‑ ure the synovial fluid IL ‑1ra, PDGF‑BB, TGF‑β and TNF‑α concentrations before and after surgically induced cartilage defect, and following intra‑articular hyaluronan injection in horses. Eight Standardbred horses underwent bilateral arthroscopic surgeries of their intercarpal joints under general anaesthesia, and cartilage defect was created on the dorsal edge of the third carpal bone of one randomly selected intercarpal joint of each horse. Five days post‑surgery, one randomly selected intercarpal joint was injected intra‑articular with 3 mL HA (20 mg/mL). Results: Operation type had no significant effect on the synovial fluid IL ‑1ra, PDGF‑BB, TGF‑β and TNF‑α concentra‑ tions but compared with baseline, synovial fluid IL ‑1ra and TNF‑α concentrations increased. Intra‑articular hyaluronan had no significant effect on the biomarker concentrations but a trend of mild improvement in the clinical signs of intra‑articular inflammation was seen. Conclusions: Creation of the cartilage defect and sham‑ operation lead to an increase of synovial fluid IL ‑1ra and TNF‑α concentrations but changes in concentrations of anabolic growth factors TGF‑β and PDGF‑BB could not be documented 5 days after the arthroscopy. Intra‑articular hyaluronan was well tolerated. Further research is needed to document possible treatment effects of intra‑articular hyaluronan on the synovial fluid biomarkers of inflammation and cartilage metabolism. Keywords: Experimental study, Cartilage defect, Hyaluronan, Interleukin‑1 receptor antagonist, Non‑animal stabilized hyaluronic acid (NASHA), Platelet‑ derived growth factor BB, Synovitis, Transforming growth factor β , Tumour necrosis factor α *Correspondence: [email protected] Department of Equine and Small Animal Medicine, Faculty of Veterinary Medicine, University of Helsinki, P.O. Box 57, 00014 Helsinki, Finland Full list of author information is available at the end of the article © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Niemelä et al. Acta Vet Scand (2019) 61:24 Page 2 of 9 Background Methods Research on equine joint disease has been focused on The study protocol was approved by the National Ani - finding tools for early diagnosis and monitoring the treat - mal Experimental Board in Finland. Eight Standardbred ments and progression of the joint disease. Inflamma - horses (four mares, one stallion and three geldings) free tory and degenerative activity in the joint can be studied of lameness were used. Before recruiting the horses, in vivo by analysis of synovial fluid (SF) biomarkers, such combination of interventions was randomly picked for as pro-inflammatory cytokines in experimental setting each right intercarpal joint. As a result, each joint was [1–5], or in naturally occurring joint disease [6–8]. In selected for one of the following; cartilage defect (CD) addition to pro-inflammatory mediators, several anabolic with HA, CD without injection, sham-operation (SO) and anti-inflammatory mediators are produced during with HA or SO without injection. Interventions for each the inflammatory process. They counteract the catabolic of the contralateral intercarpal joints were determined by effects of the pro-inflammatory cytokines and diminish these randomly picked combinations; i.e. if CD with HA the cartilage damage. Moreover, the anti-inflammatory was selected for the right side, left side was for SO with- effect can produce temporal relief of the clinical symp - out injection. toms of the articular disease [9]. The median age of horses was 7  years and range Equine arthritis is commonly treated with intra-artic- 4–24  years. Examinations, grading of measured vari- ular (IA) corticosteroids and hyaluronan (HA) [10]. ables and surgical procedures were carried by the prin- However, the response of SF biomarkers after IA HA cipal veterinarian (TMN). Prior to inclusion, horses were injection, alone or in combination with other substances, subjected to a complete lameness examination. A stand- has been examined only in a few equine studies [3, 4, ardized American Association of Equine Practitioners’ 11]. In human medicine, clinical efficacy of HA has been (AAEP) scale of 0–5 [18] was used to grade lameness. widely studied [12]. In addition, the effect of IA HA on Effusion of the affected joint was recorded on a scale of some SF pro-inflammatory biomarkers, such as prosta - 0–4 (0 = no effusion, 1 = mild, 2 = moderate, 3 = severe glandin E , has been explored both in human and equine effusion, 4 = severe swelling of the joint region) [19]. studies [3, 4, 13–15]. However, research on the effect A flexion test of the affected and the contralateral limb of HA on anti-inflammatory mediators has been infre - was performed, and lameness was recorded on a scale quently conducted. Despite the observed effects of HA of 0–4 (0 = no increase, 1 = slight increase, 2 = moder- on pro-inflammatory mediators, results in pain-relief in ate increase, 3 = considerable increase compared with human osteoarthritis (OA) are contradictory [12]. In ani- the baseline lameness, 4 = non-weight-bearing lameness) mal models HA has been demonstrated to reduce pain [3]. Pain score for maximal flexion of the carpus was also via protecting nociceptive nerve endings [16] and by determined and recorded on a scale of 0–3 (0 = no pain blocking pain receptor channels [17]. on flexion, 1 = mild pain, i.e. the horse shows some reac- The aim of the study was to measure SF concentrations tion, such as moving the limb, 2 = moderate pain, i.e. the of the anti-inflammatory mediator interleukin-1 receptor horse retracts the limb repeatedly during the 1 min flex - antagonist (IL-1ra), the anabolic growth factors platelet- ion period, 3 = severe pain, i.e. the flexion test cannot be derived growth factor BB (PDGF-BB) and transforming properly performed). In addition, five radiographic views growth factor beta-1 (TGF-β ) and the pro-inflamma - (dorsopalmar, dorsolateral-palmaromedial, dorsomedial- tory cytokine tumour necrosis factor alpha (TNF-α) palmarolateral, flexed lateromedial and flexed dorsoprox - before and after surgically induced cartilage defect (CD) imal-dorsodistal) of the carpal joints were assessed. in healthy horses. Secondly, we wanted to explore if the Before the surgical procedure, 5  mL of the SF of both concentrations of the selected biomarkers changed fol- intercarpal joints of each horse were aspirated into a ster- lowing the IA HA (non-animal stabilized hyaluronic acid, ile 5 mL syringe for the biomarker measurements. The SF NASHA ) injection. The hypothesis was that both con - sample was immediately divided between a plain 4  mL centrations of anti-inflammatory cytokines and concen - tube on ice and into an etylenediaminetetraacetic acid tration of TNF-α will increase in SF after the induction of (EDTA) tube. White blood cell (WBC) count and total CD; and that in the HA-injected joints the pro-inflamma - protein (TP) concentration measurements were done tory TNF-α is decreased and the concentrations of anti- from the fresh sample in the EDTA tube. Within 1  h of inflammatory mediators are increased compared with collection, the plain sample was centrifuged at 4000 rpm the joints without the HA medication. for 10 min in 4 °C, aliquoted and stored at − 80 °C. The horses underwent bilateral arthroscopic surger - ies of their intercarpal joints under general anaesthesia. No pre-existing IA abnormalities were detected dur- ing the arthroscopy in any of the joints. Cartilage defect Durolane, Bioventus LLC, Durham, NC, USA. Niemelä et al. Acta Vet Scand (2019) 61:24 Page 3 of 9 was created on the dorsal edge of the third carpal bone Statistical methods of one randomly selected intercarpal joint of each horse. IL-1ra, PDGF-BB, TGF-β , TNF-α, WBC count and TP The lesion was generated using a 5.0 mm × 13 cm arthro- concentration were analysed with analysis of covariance scopic burr. After the procedure, the debris was left in models (ANCOVA). The study design had 3 time-points the joint, inducing synovitis and articular inflammation. and 2 different interventions (operation, treatment). The Synovial membrane and joint capsule (approximately two effects were analysed separately due to small sam - 3  mm × 3  mm) were harvested with a scalpel from the ple size. The change in biomarker and TP concentrations dorsal region of the joints for a study to be reported and WBC count from pre-operation to pre-treatment elsewhere. The sham-operated contralateral joints (SO) was examined in one analysis and the change from pre- served as controls and were similarly evaluated by arthro- treatment to end of follow-up in another analysis. In scopic examination, synovial membranes and joint cap- both analyses, the change in concentration was used as sules were harvested but the cartilage was left intact. The the response, operation type or treatment as the fixed arthroscopic portals were closed, forelimbs were band- effect and the corresponding baseline measurement as a aged, and horses were allowed to recover from the anaes- covariate. thesia and surgery. The horses were housed in stall boxes. As there were still some doubts about the normal- The status of each horse was monitored three times daily, ity of distributions after transformation, the changes in including comfort, lameness at walk, body temperature, biomarker and TP concentrations and WBC count were heart rate and respiratory rate. analysed also using the Wilcoxon signed rank test. The Five days post-surgery, the lameness examination was Wilcoxon test was conducted also for all 16 limbs (effect repeated, new SF samples from both intercarpal joints of operation regardless of type). A P-value < 0.05 was were harvested and one randomly selected intercarpal accepted as statistically significant for all tests. joint was injected IA with 3  mL HA (20  mg/mL). Nine days after the IA HA injection (i.e. 2 weeks after the sur- Results gical arthroscopic procedure) the third lameness evalua- Clinical outcomes tion was done, and the third SF sampling, and the second Five days after arthroscopy, all the horses showed signs synovial soft tissue sampling of both intercarpal joints of lameness and increased score in the flexion test of the were performed under general anesthesia, after which CD-limb and effusion of the affected joint. The mean horses were euthanized on the operating table. lameness score of the CD-limbs was 2.5, the mean flexion test score was 2.9 and the mean effusion score was 2.1, Laboratory analyses respectively. Two horses showed bilateral lameness, i.e. Synovial fluid samples obtained on day 0, day 5 and day had a mildly lame SO-limb. In the SO-group, the mean 14 were used to analyze for the concentration of IL-1ra, scores of the flexion test and effusion were 1 and 1.8, PDGF-BB, TGF-β and TNF-α. All markers were assayed respectively. No pain was elicited in the operated joint in 3 4 5 6 using commercial ELISA development kits , , , from maximal flexion in either of the groups (CD, SO). R&D Systems. Samples were analysed in triplicate. TGF- Following trends can be seen in Fig.  1. After IA HA β (see footnote 3) and PDGF-BB (see footnote 4) were injection, the mean lameness score of the CD group with determined using human antibodies. IL-1ra (see footnote IA HA (CD + HA) decreased more compared to the 5) and TNF-α (see footnote 6) were assayed with equine- group without HA-injection (CD − HA). Mild improve- specific antibodies. The standards provided for each ment in the flexion test score and more pronounced ELISA kit were used in preparing each standard curve improvement in the effusion score were observed in the according to the manufacturers’ instructions. Readings CD affected limbs of horses after IA HA (CD + HA) com- were performed at 450  nm. The inter-, and intra-assay pared to the limbs without HA injections (CD − HA). coefficient of variation was < 6% for each ELISA. Compared with CD groups, changes in the clinical scores and differences between the SO + HA and SO − HA groups were not consistent. Dyonics Arthroscopic Surgery Blade, Smith & Nephew, Andover, MA, USA. Biomarker outcomes In the ANCOVA models, no statistically significant dif - Human TGF-β1 DuoSet, DY240E, R&D Systems, Minneapolis, MN, USA. 4 ferences in the Type III tests of fixed effects were docu - Human PDGF-BB DuoSet, DY220, R&D Systems, Minneapolis, MN, USA. mented: no effects were documented by the type of Equine IL-1ra/IL1F3 DuoSet, DY1814, R&D Systems, Minneapolis, MN, operation (CD vs. SO) or treatment (HA vs. no HA) to USA. the concentrations measured in SF biomarkers. However, Equine TNF-alphaDuoSet, DY1814, R&D Systems, Minneapolis, MN, differences between the time points were revealed: the USA. Niemelä et al. Acta Vet Scand (2019) 61:24 Page 4 of 9 Fig. 1 Mean (± SEM) clinical outcome measures of treatment groups post operation (day 5) and post treatment (day 14). CD + HA cartilage defect joints with hyaluronan injection, CD − HA cartilage defect joints without hyaluronan injection, SO + HA sham‑ operated joints with hyaluronan injection, SO − HA sham‑ operated joints without hyaluronan injection Niemelä et al. Acta Vet Scand (2019) 61:24 Page 5 of 9 change from pre-operation to pre-treatment values were demonstrated, suggesting endogenous production of significant for IL-1ra (P = 0.0344 in the CD group and IL-1ra [2]. In the present study, IA HA had no appar- P = 0.0103 in the SO group), WBC count (P = 0.011 in the ent effect on SF IL-1ra concentration in the short-term. CD group and P = 0.002 in SO group) and TP (P = 0.0002 However, long-term effects of IA HA on SF IL-1ra war - in the CD group and P = 0.002 in the SO group). HA had rants further research. no significant effect on the biomarker concentrations PDGF is secreted in the early inflammatory phase pri - within the groups. marily by platelets, but also by macrophages, endothelial In the Wilcoxon signed rank tests similar results were cells and fibroblasts [36]. It is one of the earliest and the seen. The SF IL-1ra concentration was not significantly most sensible growth factors expressed after tissue injury different for the operation types (CD vs. SO) but regard - [37]. PDGF induces the synthesis of other growth fac- ing all limbs the SF IL-1ra concentration increased signif- tors [38], proliferation and differentiation of fibroblasts, icantly after arthroscopy (P = 0.0039). Also, the increase deposition of collagen and angiogenesis [39, 40]. There - in SF TNF-α concentration was significant (P = 0.0386) fore, it is an essential promoter of the healing process. In regarding all limbs. Induction of CD and SO both the present study, after sampling at the baseline on day caused a significant increase within both groups in WBC 0, the next sampling was performed on day 5, in a time count (P < 0.001 in both groups) and TP concentration point where PDGF-BB is supposed to play a major role in (P < 0.001 in both groups). the vascular formation and proliferation of fibroblasts in ongoing repair [41]. PDGF-BB concentration increased Discussion in SF after induction of CD, although a significant dif - In the present study, the concentrations of the measured ference was not detected. The synthesis of PDGF-BB biomarkers in intact equine joints are mainly equivalent may have, however, occurred earlier in the course of compared with the results of a previous report [20]. To injury and inflammation. In contrast, PDGF-BB has not the best of our knowledge, IL-1ra, PDGF-BB, TGF-β been detected at all in the SF of osteoarthritic human measured in the present study have been evaluated sepa- knee joint [42]. In another study comparing OA joints to rately only in a few studies on SF of the equine joint [2, healthy controls, significant differences in the SF PDGF- 21–23]. A purely catabolic cytokine TNF-α has been BB concentrations were not detected [43]. These results studied in greater detail [5, 20, 21, 23–28]. Most of the are also suggestive for an early increase of SF PDGF- biomarkers have been documented in vitro in equine car- BB concentration and its role in the initial phase of the tilage as well as in the synovial membrane after a chal- pathogenesis of OA. SF PDGF concentration has been lenge, usually lipopolysaccharide (LPS) [20, 29–31]. documented to increase after IA platelet rich plasma- IL-1ra has been shown to increase after an acute intra- treatment [23]. In the present study, no changes in the articular fracture in humans. However, differences in PDGF-BB concentrations were documented 9  days after SF inflammatory cytokine concentrations between high IA HA treatment. and low-energy injuries have not been detected [32]. SF TGF-β has been studied in  vivo in normal joints Although not directly comparable with intra-articu- and joints with osteochondrosis in foals [22] and after lar fractures, in the present study no differences were LPS challenge in adult horses [21]. In the present study, detected between CD joints and SO joints. However, neither the creation of CD or IA HA injection caused the arthroscopy itself caused trauma to the synovial an increase in the SF TGF-β concentration. In contrast, soft tissues and may have caused the increase of IL-1ra TGF-β concentration decreased following the creation concentration. of CD, although no statistically significant difference Blocking the IL-1β receptor by IL-1ra has potentially between CD and SO joints was found (Fig. 2). This is dif - a wide positive effect on inhibiting deleterious events in ferent compared with the previous study on equine joints the joint. The binding of IL-1β to a receptor results in that reported increase of SF TGF-β concentration fol- activation of several transcription factors and expression lowing a challenge with LPS [21]. This may be explained of hundreds of genes leading to the synthesis of other by the more intense inflammation induced by LPS, com - cytokines, chemokines, adhesion molecules, inflamma - pared with inflammation induced by CD. tory mediators and enzymes [33]. Consequently, IL-1β Ríos et  al. [30] has shown an increase in TGF-β con- is has a significant effect on the metabolism of cells and centration after LPS challenge in cartilage inflammation the extracellular matrix [34]. A decrease in SF IL-1ra created in  vitro. The effect was suggested to result from has been documented in chronic stages of human OA a possible anti-inflammatory mechanism or by direct [35]. Delayed increase of SF IL-1ra (at day 35) follow- damage of LPS to the cartilage. A similar effect has been ing the IA administration of autologous conditioned seen after LPS challenge of synovial membrane explants serum in experimentally induced equine OA has been in vitro [31]. Our result is more consistent with the study Niemelä et al. Acta Vet Scand (2019) 61:24 Page 6 of 9 Fig. 2 SF biomarker concentrations (median, quartiles and minimum and maximum value) of treatment groups on different sampling days. CD + HA cartilage defect joints with hyaluronan injection, CD − HA cartilage defect joints without hyaluronan injection, SO + HA sham‑ operated joints with hyaluronan injection, SO − HA sham‑ operated joints without hyaluronan injection of human OA where SF TGF-β concentration was meas- TGF-β in the joint [51]. However, in the present study, ured both in healthy subjects and in patients with OA; no changes in the SF TGF-β concentrations were doc- in the latter, the concentration of SF TGF-β was low or umented after IA HA treatment, possibly implying that even undetectable [44]. Cell signaling pathways may be optimal SF TGF-β concentrations were already present intercepted by inflammatory cytokines, which may be a in the injected joints. possible reason for the reduced amount of TGF-β in the The results of studies on the synovial fluid TNF-α course of OA [45]. are somewhat contradictory. However, it seems to be TGF-β has an anabolic effect on cartilage; it has an quite a sensitive but not very specific marker of IA ability to induce chondrogenic differentiation of mes - insults. The results of this study are also suggestive of enchymal stem cells [46, 47] with rapid biosynthesis of that. TNF-α concentration increased in SF after induc- glycosaminoglycan and deposition of an extracellular tion of CD although significant differences were not matrix [47]. On the other hand, enhanced expression detected between the groups in the small population of TGF-β has been associated with developing osteo- of this experimental study. An increased concentration phytes [48] and hyperplasia of the synovium [49]. It has of SF TNF-α has been documented in horses in natu- been suggested that only a narrow range of bioactive rally occurring OA in carpi [27], and experimentally in TGF-β concentrations are beneficial to cartilage health amphotericin B- [5] and LPS-induced articular inflam - and any concentrations below or above this range may mation [21, 26]. In horses, even exercise alone leads to cause aberrant alterations in TGF-β pathways, resulting a significant increase in TNF-α levels for a short period in abnormal cartilage function [50]. As TGF-β is stored [25, 28]. However, an increase in SF TNF-α concentra- in the platelets, SF TGF-β concentration increases after tion as a result from serial arthrocentesis could not be IA administration of platelet rich plasma, in response to demonstrated [25]. In contrast to this, gas and liquid platelet activation [23]. HA has been suggested to play capsular distension during arthroscopy provoked an an important role in the mechanical activation of latent inflammatory response with increased concentration of Niemelä et al. Acta Vet Scand (2019) 61:24 Page 7 of 9 SF TNF-α [52]. In a clinical equine study, TNF-α was The number of horses in the experimental studies on found not to be a useful biomarker for different types equine joints has generally ranged from 6 to 13 [1, 3, 5, of joint lesions [53]. Similarly, in humans, increased 21, 26, 52]. Eight horses (16 joints) in the present study concentrations were not associated with any particular may have been too low to detect differences in concen - type of articular disease, such as rheumatoid arthritis, trations of SF biomarkers. Although horses with uniform although in OA patients, detectable concentrations of breed and sport discipline were selected, and all horses TNF-α were related with a long duration of the dis- were free of lameness and joint disease of the intercar- ease [24]. In the present study, SF TNF-α concentra- pal joint as verified by arthroscopy, differences in exercise tions were not affected by IA HA. Probably with more or training status and age may have caused some varia- horses, a bigger sample size and earlier and more fre- tion in the SF biomarker concentrations. Exercise may quent sampling significant results may have been dem - lead to increased biomarker concentrations in horses [7, onstrated in this study. 28], which can be further enhanced in joints with com- In conclusion, after a mild increase in biomarker con- promised health [7, 25]. Moreover, also age has been centrations resulting from arthroscopy and induction of reported to influence SF biomarker concentrations or synovitis and CD, HA failed to produce any further effect gene expression so that they generally decrease with age on biomarkers. Minor improvement of clinical signs of [8, 29, 56]. IA inflammation was evident in the CD + HA group, The time from injury to collection of SF samples is when compared with scores of clinical signs of CD − HA an important issue as changes in biomarker concentra- group. However, in SO + HA and SO − HA groups tions can occur quickly, even within hours. On the other changes after IA HA/no injection were not that consist- hand, IA injection itself, especially repeatedly, causes ent. HA is reported to have an anti-inflammatory effect inflammatory reaction in the joint [57] and can cause [16] but IA HA injections have also been documented to increases in biomarker concentrations [56]. To minimize induce a transitional IA inflammatory reaction, either a this effect, it has been recommended there should be a flare with pronounced clinical signs of inflammation and period of even 14  days after the previous arthrocentesis pain [54] or an increase of WBC count in the SF [11]. before subsequent SF collection [28]. However, despite of To our knowledge, only a few studies have explored bio- these potentially interfering factors, repeated sampling markers in equine SF after IA HA [3, 11]. As HA is very and early time points have been generally used in equine frequently used in the IA treatment in horses, its mecha- SF biomarker studies. In studies using LPS induction nism of action warrants further research. for IA inflammation, first sampling point 8 h [19] or 1 h This study has its limitations. Firstly, the contralateral [21, 26] post injection were chosen which is reasonable limb of the horse served as a SO control. Concentra- considering very acute and strong inflammation caused tions of cartilage matrix products are elevated also in by LPS. On the other hand, in some experimental stud- the contralateral knee in patients with anterior cruciate ies, using surgical model [3, 4] comparable with model of ligament rupture, possibly as a consequence of an altered the present study or amphotericin B [5] for induction of loading [55]. Cytokines and degraded matrix products IA inflammation in horses, weekly samplings were cho - released from an operated joint may be transported to sen for exploring SF IL-1ra and TNF-α concentrations the contralateral joint by the circulation and initiate an (among other biomarkers), respectively. Finding an opti- inflammatory process. Therefore, the concentration of mal time point for SF aspiration is challenging, especially markers in the control joints may have resulted partly when several biomarkers are studied. In addition, avoid- by the transport from the CD operated joint. Measure- ing the effect of repeated aspiration and sham-opera - ment of serum concentrations of studied markers, as tion on biomarker concentrations complicates the issue. well as additional pro-inflammatory markers and mark - Although the effect of IA injection on biomarkers meas - ers of cartilage matrix metabolism, would have provided ured in this study is not known, except for TNF-α, 5 days additional information to test this hypothesis. However, after arthroscopy and 9  days after IA HA injection were this was outside the scope of this study. Moreover, the chosen to balance between the intervention time points. arthroscopy itself, as well as harvesting the synovial tis- sue samples, causes trauma to the synovial soft tissues. Conclusions These may have further affected the concentrations of the This study demonstrates that arthroscopy and both the measured markers in the SO joints. However, the effect creation of CD and SO, lead to an increase in SF IL-1ra was thought to be transient. To minimize the effect of and TNF-α concentrations but changes in concentrations arthroscopy and sampling of synovial soft tissue, sam- of anabolic growth factors TGF-β and PDGF-BB in SF pling of SF and IA HA injection were performed only were not documented 5 days after the arthroscopy. Intra- after 5 days. articular HA was well tolerated. However, changes in Niemelä et al. Acta Vet Scand (2019) 61:24 Page 8 of 9 3. Frisbie DD, Kawcak CE, McIlwraith CW, Werpy NM. Evaluation of concentrations of IL-1ra, PDGF-BB, TGF-β, and TNF-α polysulfated glycosaminoglycan or sodium hyaluronan administered were not seen after HA injection. The treatment effect of intra‑articularly for treatment of horses with experimentally induced IA HA on SF pro-inflammatory, anti-inflammatory and osteoarthritis. Am J Vet Res. 2009;70:203–9. 4. Frisbie DD, McIlwraith CW, Kawcak CE, Werpy NM. Evaluation of intra‑ anabolic biomarkers warrants further research. articular hyaluronan, sodium chondroitin sulfate and N‑acetyl‑ d ‑ glucosa‑ mine combination versus saline (0.9% NaCl) for osteoarthritis using an equine model. Vet J. 2013;197:824–9. Abbreviations 5. Ma TW, Li Y, Wang GY, Xin‑ran Li XR, Jiang RL, Song XP, et al. Changes in CD: cartilage defect; EDTA: etylenediaminetetraacetic acid; HA: hyaluronan; IA: synovial fluid biomarkers after experimental equine osteoarthritis. J Vet intra‑articular; IL ‑1ra: interleukin 1 receptor antagonist; LPS: lipopolysaccharide; Res. 2017;61(10):1515. NASHA: non‑animal stabilized hyaluronic acid; OA: osteoarthritis; PDGF‑BB: 6. Bertone A, Palmer JL, Jones J. Synovial fluid cytokines and eicosanoids as platelet‑ derived growth factor; PGE : prostaglandin E ; SF: synovial fluid; SO: 2 2 markers of joint disease in horses. Vet Surg. 2001;30:528–38. sham‑ operation; TGF‑β : transforming growth factor beta 1; TNF‑α: tumour 7. 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Published: May 30, 2019

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