Palmar navicular bone fibrocartilage (NBF) and deep digital flexor tendon (DDFT) degeneration/pathologies are largely responsible for the clinical signs associated with navicular disease, a prevalent source of performance-limiting forelimb lameness and debilitation in equine athletes.1–4 These tissues heal mainly through fibrous tissue formation, resulting in chronic pain and an increased likelihood of re-injury.2–5 Further, unabated inflammation is often encountered after injury due in part to torn tendon fibers/tissue exposed into the intra-synovial and intra-bursal space.6,7 Identification of therapies with known efficacy to enhance intrabursal NBF and DDFT healing is an ongoing area of research. Intra-synovial or intra-bursal corticosteroid injections and systemic non-steroidal anti-inflammatory drugs (NSAIDs) for anti-inflammatory effects and pain relief, along with corrective farriery and rehabilitation, are mainstays for clinical management of horses afflicted with navicular pathologies.8–10
Methylprednisolone acetate (MPA) is a popular corticosteroid for distal interphalangeal joint and/or navicular bursa injections in horses diagnosed with navicular disease due to its potent anti-inflammatory properties;8,11 however, there is minimal understanding of the effects of corticosteroids on navicular tissues. In the context of osteoarthritis management, there is controversy regarding the degradative effects of MPA on the articular cartilage extracellular matrix (ECM).12,13 The dosage of MPA used and existing cartilage abnormalities have been shown to play a role in MPA-mediated chondrocyte ECM dysregulation and consequent glycosaminoglycan (GAG) catabolism.14–16 In vitro, articular chondrocytes exposed to interleukin-1β (IL-1β), the central inflammatory cytokine in osteoarthritis, exhibit a marked decrease in aggrecan gene expression; whereas MPA treatment when combined with IL-1β exposure restored the aggrecan gene expression to that of chondrocytes maintained under basal culture conditions.17 Although therapies for osteoarthritis are often extrapolated to navicular disease management, the equine navicular apparatus is an enthesis complex consisting of several tissue types such as the navicular bone and fibrocartilage, DDFT, distal phalanx, and peri-ligamentous structures enclosed within the joint/bursal capsule. Despite the widespread clinical use of MPA for navicular disease, investigations specifically evaluating the impact of MPA on navicular tissue or cell ECM biosynthesis are lacking. When DDFT cells maintained under non-inflammatory conditions were treated with 0.05 mg/mL and 0.5 mg/mL MPA, both concentrations of MPA downregulated the cartilage ECM gene expression, whereas only 0.5 mg/mL MPA downregulated the tendon-related ECM gene expression.18 To our knowledge, the effects of IL-1β and IL-1β combined with MPA treatments on DDFT or NBF cells have not been investigated.
The objective of this study was to examine the in vitro effects of MPA on equine forelimb DDFT and NBF cell ECM gene expression, matrix metalloproteinase-(MMP-)-3 and MMP-13 secretion, and glycosaminoglycan (GAG) secretion during non-adherent, aggregate culture and exposed to IL-1β induced inflammation. The secondary objective of this study was to determine if a differential response to IL-1β and MPA treatments is seen in DDFT and NBF cells. We hypothesized that the effects of IL-1β and IL-1β combined with MPA on DDFT and NBF cells will be equivalent.
Materials and Methods
Forelimb DDFT and NBF were harvested from 5 horses undergoing euthanasia for reasons unrelated to musculoskeletal disease that were donated to the university’s post-mortem research efforts. All study procedures were reviewed and approved by the university’s Institutional Animal Care and Use Committee. The horses included in this study were 2 mares and 3 geldings (3 Quarter horses, 1 Grade/mixed breed, and 1 Hanoverian) between 11 and 17 years of age. All horses had a good body condition score (BCS) > 6/9 and were free of forelimb lameness when trotted in hand on a smooth, hard surface. Horses were free of musculoskeletal abnormalities as determined through complete physical examination. The DDFT within the navicular bursa and NBF were determined to be normal based on gross assessment at the time of tissue harvest.
DDFT and NBF harvesting and cell isolation
Immediately after euthanasia with sodium pentobarbital (150 mg/kg IV), both forelimb digits were disarticulated at the metacarpophalangeal joint. The hooves were cleaned, hair was clipped, and the solar surfaces were pared with a hoof knife. The feet were rinsed with water to remove gross debris; the entire digits were thoroughly disinfected with 2% chlorohexidine scrub alternated with 70% isopropanol. Each digit was then disarticulated at the level of the distal interphalangeal joint (via incising the collateral ligaments at the level of the coronary band) to expose the proximal aspect of the navicular bone, without severing the DDFT. Using an aseptic technique, the navicular bone was dissected en-bloc from the foot by transecting the surrounding soft tissues. The fibrocartilage overlying the palmar surface of the navicular bone was sharply excised using a No. 10 scalpel blade and collected using an aseptic technique. The dorsal 2 mm thickness of DDFT tissue extending from distal to the T-ligament attachment up to the distal margin of the navicular bone was dissected.18,19 NBF and DDFT tissues were diced into 0.25 cm3 pieces and digested in 0.15% collagenase II (ThermoFisher Scientific) in Dulbecco’s modified Eagle’s medium (DMEM; Gemini Biomedicals) supplemented with 2% fetal bovine serum (ThermoFisher Scientific) in a humidified shaker incubator at 37 °C overnight. After digestion, the isolated cells were filtered through a 40-um filter (ThermoFisher Scientific). The cells were collected by centrifuging at 300 X g for 5 minutes at 37 °C. The supernatant was removed, and the cell pellet was resuspended in basal expansion medium (culture medium containing DMEM containing 4.5 g/L glucose and 300 ug/mL l-glutamine, supplemented with 10% fetal bovine serum, 100 U sodium penicillin/mL [Gemini Biomedicals], and 100 ug streptomycin sulfate/mL [Gemini Biomedicals]). Cell yield was determined by the use of a hemocytometer, and viability was estimated via trypan blue dye exclusion (Sigma).20 A cell viability of > 98% was ensured throughout.
DDFT and NBF cell culture
DDFT and NBF cells were seeded at 5,000 cells/cm2 in monolayer cultures in basal expansion medium and maintained at 37 °C, 95% air/5% CO2 in a humidified incubator.18,21 Non-adherent cells were removed on day 2 and the resulting colony-forming units trypsinized (0.05% Trypsin-EDTA, Gibco) on days 7–9. The cells were subsequently expanded in monolayers until 80% confluence was reached and passaged twice. Third passage DDFT and NBF cells were used in subsequent experiments as described below. DDFT and NBF cells were maintained as aggregate cultures to support their chondrogenic phenotype.18,21
Aggregate culture
Aggregate cultures were established from third passage DDFT and NBF cells by resuspending the cells in chondrogenic medium22 (OptiMEM medium [ThermoFisher Scientific] with supplemental 50 ng/mL ascorbic acid [Sigma], 100 U sodium penicillin/mL, 100 ug streptomycin sulfate/mL, 1% insulin-transferrin-selenium [Sigma], and 2% fetal bovine serum). Cell suspensions containing a total of 3 X 106 cells were aliquoted into each well of a 6-well Ultra Low Attachment, hydrogel-coated plate (Corning Inc.) and 0.5 X 106 cells into each well of a 24-well Ultra Low Attachment, hydrogel-coated plate (Corning Inc.). Cultures were maintained in a chondrogenic medium at 37 °C, 95% air/5% CO2 in a humidified incubator for 72 hours while the floating cells formed clearly visible cell pellets.22 Just before adding treatments, the media were collected and frozen at −80 °C for later analysis of total soluble GAG and MMP contents. Treatment groups consisted of chondrogenic medium alone (control), chondrogenic medium + 10 ng/mL interleukin-1β (IL-1β), chondrogenic medium + 10 ng/mL IL-1β + 0.05 mg/mL methylprednisolone acetate suspension (0.05 MPA; Depo-Medrol), or chondrogenic medium + 10 ng/mL IL-1β + 0.5 mg/mL MPA (0.5 MPA). Three replicates per treatment group per horse were established. After 24 hours of incubation with the aforementioned treatment groups, the media and cell pellets were collected separately, snap-frozen in liquid nitrogen, and stored at −80 °C for further analyses. The concentrations of IL-1β and MPA evaluated in this study were based on previous studies measuring corticosteroid-induced biosynthesis in equine chondrocytes, corticosteroid dose divided by the average estimated volume of navicular bursa/distal interphalangeal joint, and preliminary data collected in our laboratory.17,23–25
RNA isolation and quantitative real-time polymerase chain reaction (rtPCR)
Total RNA was isolated using a previously described protocol.18,26,27 Briefly, the samples were homogenized in guanidinium thiocyanate phenol-chloroform solution reagent (TRIzol, Invitrogen) according to the manufacturer’s suggested protocol. The resultant pellet was purified using RNeasy silica columns that included on-column DNase digestion. The concentration of RNA was determined by measuring the absorbance at 260 nM (A260) and 320 nM (A320) in NanoDrop One/One® (Thermo Fisher Scientific). One microgram of RNA from each sample was reverse-transcribed (Superscript IV, Invitrogen) using oligo (dT) primers. Equine gene-specific primers (Appendix) for cardinal ECM markers (SOX9, ACAN, COL2A1, COL1A1, COL3A1, and COMP) were designed from published sequences in Genbank and using ClustalW multiple sequence alignment (available at http://www.ebi.ac.uk). Primer specificity was confirmed by cloning and sequencing the amplicons during optimization experiments, as previously described.18,21 PCR amplifications were catalyzed by Taq DNA polymerase (ABI QuantStudio 3™, Thermo Fisher Scientific) in the presence of SYBR green. Relative gene expression was quantified using the 2-ΔΔCT method,28 normalized to the expression of the reference gene, elongation factor-1α (EF1α; Appendix), and expressed as fold change from the respective DDFT or NBF cells maintained in chondrogenic medium alone (control).
Media MMP-3 and MMP-13 activity
MMP-3 and MMP-13 concentrations in the cell culture media were determined via equine-validated Quantikine ELISA (R&D Systems) by the manufacturer’s protocol.18,29 Briefly, the collected control, IL-1β, IL-1β + 0.05 MPA, or IL-1β + 0.5 MPA medium was combined with MMP-3 or MMP-13 conjugate. The samples were washed and incubated with a substrate solution, and optical density was measured with a microplate reader (Infinite M1000PRO; Tecan) set at 450 nm.
Total glycosaminoglycan (GAG) content
The pellets from 0.5 X 106 DDFT and NBF cells in control, IL-1β, IL-1β + 0.05 MPA, and IL-1β + 0.5 MPA treatment groups were individually digested in 250 uL of 0.5mg/mL papain (Worthington Biochemicals). The total GAG content of representative cell pellets and the culture media were determined via 1,9-dimethylmethylene blue binding (DMMB) assay.26,30 Two replicates from each treatment group were assayed to obtain the measurement. All sample values were compared against a standard curve of bovine cartilage chondroitin sulfate (Sigma Aldrich) values to estimate the total GAG content in the samples. All measurements (ug) were from 0.5 X 106 DDFT and NBF cells in 0.5 mL of the respective culture medium.
Statistical analysis
The normal distribution of data was assessed by Shapiro-Wilk’s test. The effects of the various treatments within each cell type were compared with 1-way ANOVA, and the treatment effects between cell types were compared with 2-way ANOVA, or the non-parametric equivalent, Kruskal-Wallis test on ranks. Post hoc comparisons for the detection of statistically significant differences between the control, IL-1β, IL-1β + 0.05 MPA, and IL-1β + 0.5 MPA treatment groups were conducted with the Holm-Sidak method. Differences were considered statistically significant at P ≤ .05 (Sigmastat 4; Systat Software Inc. and Graphpad Software).
Results
ECM mRNA expression
The mean and SD ECM mRNA of DDFT and NBF cell groups are depicted (Figure 1). IL-1β and IL-1β combined with MPA significantly downregulated SOX9 (P = .005), ACAN (P = .042), and COL1A1 (P = .048) mRNA in NBF cell groups compared with DDFT cell groups. Post hoc comparisons demonstrate that within DDFT cell groups, only ACAN mRNA was significantly downregulated with IL-1β (P = .008) and IL-1β combined with MPA (P = .006, P = .002) compared with respective control. Within NBF cell groups, IL-1β and IL-1β combined with MPA significantly downregulated SOX9, ACAN, COL2A1, COL1A1, COL3A1, and COMP mRNA compared with respective control. There were no significant differences among IL-1β alone, IL-1β+0.05, and IL-1β+0.5 MPA treatments in either DDFT or NBF cell groups.
MMP culture media concentration
The mean and SD MMP-3 and MMP-13 culture media concentrations are depicted (Figure 2). Post hoc comparisons demonstrate that within DDFT cell groups, IL-1β (P = .015) and IL-1β combined with MPA (P = .024, P = .046) treatments significantly increased MMP-3 culture media concentrations. NBF cell culture media concentrations of MMP-3 were unchanged with IL-1β and IL-1β combined with MPA treatments.
MMP-13 concentrations in NBF cell culture media significantly increased compared with DDFT cell groups (P < .001). Post hoc comparisons showed that within DDFT and NBF cell groups, IL-1β (P < .001) and IL-1β + 0.05 MPA (P = .026 and P < .001, respectively) treatments significantly increased MMP-13 culture media concentrations compared with control. Additionally, within DDFT and NBF cell groups, IL-1β + 0.05 MPA (P = .013 and P < .001, respectively) and IL-1β + 0.5 MPA (P = .004 and P < .001, respectively) treatments significantly decreased MMP-13 compared with IL-1β treatment alone.
GAG content
IL-1β and IL-1β combined with MPA did not affect GAG content in DDFT or NBF cell pellets or their respective culture medium (Supplementary Figure 1).
Discussion
To our knowledge, this is the first report of IL-1β and IL-1β combined with MPA effects on DDFT and NBF cell ECM gene expression and MMP-3, -13 secretion. Contrary to our hypothesis, IL-1β and IL-1β combined with 0.05 or 0.5 mg/mL MPA treatments impacted NBF cells to a greater extent than DDFT cells. Specifically, IL-1β and IL-1β combined with MPA consistently downregulated ECM gene expression to a greater extent in NBF cells. Concerning MMP-3 and -13 bioactivity, IL-1β and IL-1β combined with MPA significantly increased culture medium MMP secretion in NBF cells compared with DDFT cells. There were no distinct dose-dependent effects of MPA when combined with IL-1β in either DDFT or NBF cells.
In our previous study conducted with DDFT cells and similar MPA concentrations without IL-1β, both MPA doses downregulated ECM genes.18 Consistent with these findings, the deleterious effects of both IL-1β alone and IL-1β combined with MPA on DDFT cell ECM gene expression in this study were also noted as progressive downregulation of ACAN and SOX9 gene expression with only ACAN being statistically significant. The large inter-horse variability noted in COL1A1, COL3A1, and COMP mRNA results of this study likely precluded identifying the impact of IL-1β alone and IL-1β combined with MPA on DDFT cells tendon ECM gene expression. In human rotator cuff tenocytes, IL-1β combined with dexamethasone (10−6 M) significantly decreased COL1A1 gene expression, whereas IL-1β alone increased COL1A1 gene expression.31 These discrepancies in equine and human intra-synovial tendon cells exposed to IL-1β and corticosteroids could be in part due to the differing IL-1β (10 ng/mL vs 1 ng/mL) and corticosteroid type and concentrations (approximately 0.1 and 1 μM MPA vs 1 μM dexamethasone) used. Considering the results of this study and existing evidence, further investigations that take into account the type and concentration of both inflammatory cytokine and corticosteroids are needed to elaborate on their impact on equine DDFT cells.
On the other hand, in NBF cells, all ECM genes assessed were significantly downregulated with all treatments investigated in this study, and overall, COL1A1, SOX9, and ACAN gene downregulations were significantly greater relative to DDFT cells. We are not aware of other studies assessing the effects of corticosteroids on NBF cells. Navicular bone fibrocartilage ECM is predominantly composed of fibrillar collagen types I and II as well as aggrecan, along with minor quantities of collagen III and other proteoglycans and glycoproteins such as COMP (with specific compositions varying with fibrocartilage to subchondral bone interface thickness).32 While collagen type II and aggrecan are present in the ECM of both articular cartilage and fibrocartilage, there is less collagen type I in articular cartilage compared with fibrocartilage.33 This study’s finding that MPA treatment did not mitigate the ECM gene expression downregulation seen with IL-1b in DDFT and NBF cells are consistent with equine articular chondrocyte in vitro studies.17,34,35 Specifically, 10 ng/mL IL-1β significantly decreased COL2A1 and ACAN gene expressions, and dexamethasone at 10−8, 10−7, and 10−6 M concentrations further decreased COL2A1 and ACAN mRNA.34 The significance of COL3A1 and COMP gene downregulations in NBF cells, both minor ECM constituents, will require tissue-level investigations and sequential IL-1β exposure and corticosteroid treatment.
MMPs are critical for ECM turnover and play a significant role in tissue repair and remodeling.36 MMP-3 is a stromelysin-type MMP, and it plays a role in the breakdown of collagen type I, II, and III as well as proteoglycans.36 MMP-13 is a collagenase type MMP, and similarly breaks down collagen type I, II, and II as well as aggrecan within connective tissues.36 In equine articular chondrocytes, 10 ng/mL IL-1β significantly increased MMP-3 secretion, and with combined dexamethasone (10−6, 10−7, and 10−8 M) treatment the MMP-3 secretion was similar to that of the control group.31,34 Concurrently, Trahan et al. also demonstrated that 10 ng/mL IL-1β combined with MPA (10−4, 10−7, and 10−10 M) significantly decreased equine articular chondrocyte MMP-13 secretion compared IL-1β alone.14 Similar to articular chondrocyte studies, in both DDFT and NBF cells, MPA combined with IL-1β significantly decreased MMP-13 secretion when compared with respective cells treated with IL-1β alone. Additionally, MMP-13 secretions of DDFT and NBF cells with IL-1β and both MPA doses were similar to the respective control group cells. It is unclear as to why only the MMP-3 and -13 secretion patterns of DDFT and NBF cells after IL-1β and MPA treatments are similar to articular chondrocytes, whereas the ECM gene expression profiles of DDFT and NBF cells were not significantly different after IL-1β and IL-1β combined with MPA treatments. Follow-up investigations on DDFT and NBF cell tissue inhibitors of MMPs (TIMPs) bioactivities and ECM synthesis, as well as explant level assessments, may help clarify the disconnect in ECM gene expression and MMP secretion observed in this study.
The ECM of intrabursal fibrocartilage and opposing DDFT surfaces contain GAG with “rounded/chondrocyte-like” cells within lacunae and supports the resistance-free movement.5,37,38 Therefore, the GAG content in DDFT and NBF cell aggregates after IL-1β and IL-1β combined with MPA treatments were measured. In vitro equine articular cartilage explant studies have demonstrated that both IL-1β and IL-1β combined with MPA result in GAG release into culture media indicating GAG catabolism.14,39 In equine articular chondrocyte pellets, IL-1β alone did not affect GAG content whereas the addition of corticosteroids (0.5 and 2.0 mg/mL sodium hyaluronate or 0.06 and 0.6 mg/mL triamcinolone acetonide) significantly increased pellet GAG content.40 In this and our previous study,18 DDFT and NBF cell aggregates secreted minimal GAG during the 72-hour culture duration and consequent changes with IL-1β and MPA treatments were not detected. It must be noted that the total GAG contents in DDFT and NBF cell groups were not normalized for cell numbers via DNA content, and the results could have been impacted by differing cell proliferation rates with treatments. Follow-up experiments with normalization for DDFT and NBF cell numbers, longer in vitro culture duration, and assessment of GAG breakdown in DDFT and NBF tissue explants are needed to determine the impact of IL-1β and MPA on DDFT-NBF GAG catabolism.
This in vitro study has several limitations. Firstly, the DDFT and NBF tissues were deemed normal at the time of tissue harvest based on gross evaluations alone, and the presence of histologic pathologies may have been overlooked. The low (n = 5) number of horses and large inter-horse variability within DDFT cell groups may have precluded the detection of small differences between groups. While these results provide foundational data, it is important to recognize that tissue degeneration is a complex process. This experiment used grossly normal DDFT and NBF tissues under IL-1β inflammatory conditions alone. Although IL-1β is an important inflammatory cytokine implicated in osteoarthritis,41,42 the role of IL-1β or other inflammatory mediators is less defined in tendonitis and bursitis.43–45 Further, higher IL-1β and MPA concentrations, and concurrent IL-1β and MPA treatments evaluated in this study do not reflect in vivo clinical scenarios. In this respect, investigating a range of clinically relevant IL-1β and MPA concentrations, as well as evaluating DDFT and NBF cell responses to sequential MPA treatment after an inflammatory challenge will be more informative for clinical interpretation. Lastly, these study results are restricted to DDFT and NBF cell responses evaluated separately. Experiments with navicular tissue explants and cell/explant co-cultures that consider crosstalk effects or the complex interactions between cells/tissues and biomechanical loading present in vivo are warranted.
In-depth characterization of the specific pathologies involved in horses with navicular disease is possible due to the increasing use of diagnostic magnetic resonance imaging (MRI). Horses with primary DDFT lesions and navicular bursitis have been reported to respond better to navicular bursa corticosteroid injections than do horses with concurrent pathologies involving the navicular bone fibrocartilage, cartilage surfaces, and the collateral ligaments of the distal interphalangeal joint.8 We observed minimal differences between IL-1β + 0.05 MPA and IL-1β + 0.5 MPA treatments in both DDFT and NBF cells. Despite the high doses of MPA tested in this study, neither dose was found sufficient to overcome the negative effects of IL-1β inflammation. Results of this study demonstrate that MPA treatment (concentrations in the range of 10−5 M) downregulated tendon ECM and cartilage ECM gene expression while upregulating MMP-13 cell culture media concentrations of equine NBF cells maintained under inflammatory conditions to a greater extent than DDFT cells. While MPA injections continue to hold value in clinical practice for inflammatory bursitis management, identifying the timing and suitable dosage of steroid used as well as what specific pathologies are appropriate for such treatment will help guide the judicious use of MPA to most effectively harness its potent anti-inflammatory effects.8,25,46 At present, therapies are largely focused on improving or modifying clinical symptoms, and assessing existing and potential novel therapies for their efficacy to limit or resolve ongoing local or intrasynovial inflammation both as a primary cause and secondarily due to tissue injury or degeneration are necessary to enhance navicular tissue healing. Accepting the in vitro nature, these results serve as a first step for future work to determine intrabursal corticosteroid regimens that limits or resolves inflammation as well as takes into consideration NBF and DDFT cell ECM biosynthesis in horses with navicular disease.
Supplementary Materials
Supplementary materials are posted online at the journal website: avmajournals.avma.org.
Acknowledgments
Funding was obtained from the Ohio State University Equine Research Fund by the Ohio State Racing Commission. This publication was supported in part, by the National Center for Advancing Translational Sciences of the National Institutes of Health under Grant No. KL2TR002734. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
The authors have no conflicts of interest to disclose.
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Appendix
Real time polymerase chain reaction (rtPCR) primer sequences.
Gene | Accession number | Sequence | Amplicon (bp) | |
---|---|---|---|---|
SOX9 | XM_023452130 | S | 5' GAA CGC ACA TCA AGA CGG AG | 304 |
A | 5' CTG GTG GTC TGT GTA GTC GT | |||
ACAN | XM_023650277.1 | S | 5' GAC GCC GAG AGC AGG TGT | 202 |
A | 5' AAG AAG TTG TCG GGC TGG TT | |||
COL2A1 | NM_001081764.1 | S | 5' AGC AGG AAT TTG GTG TGG AC | 223 |
A | 5' TCT GCC CAG TTC AGG TCT CT | |||
COL1A1 | NC_009154 | S | 5' GAA AAC ATC CCA GCC AAG AA | 231 |
A | 5' GAT TGC CAG TCT CCT CAT CC | |||
COL3A1 | AW261123 | S | 5' AGG GGA CCT GGT TAC TGC TT | 215 |
A | 5' TCT CTG GGT TGG GAC AGT CT | |||
COMP | NM_001081856 | S | 5' TCA TGT GGA AGC AGA TGG AG | 223 |
A | 5' TAG GAA CCA GCG GTA GGA TG | |||
EF1α | NM_001081781.1 | S | 5' CCC GGA CAC AGA GAC TTC AT | 328 |
A | 5' AGC ATG TTG TCA CCA TTC CA |