Accurate, reliable preclinical models are essential in the journey to understanding disease pathophysiology or the effects of a proposed therapeutic.1,2 Thus, preclinical testing is shifting away from monolayer cell culture in favor of novel modeling methodologies that recapitulate more of the driving biology of tissues of interest, especially the tissues’ 3-D structure, extracellular matrix components, and diverse cell types. This shift has already occurred in the study of cartilage, which increasingly relies on osteochondral explant culture.1–6 Multiple studies have validated reliable ways of measuring cartilage viability, metabolism, and function in both normal and diseased conditions ex vivo.6–8 This comes after comprehensive evaluations of cartilage explants in culture have characterized the structural and functional aspects of the tissue, such as chondrocyte metabolism and the mechanical properties of the complex cartilage matrix.1,3,6,9
Although the preclinical study of cartilage has resulted in the development of synthetic and biologic joint therapeutics, joint disease treatment remains challenging in human and veterinary medicine alike.10,11 Degradative joint conditions, such as traumatic and septic osteoarthritis, yield unpredictable and often poor prognoses that result in debilitation, loss of function, and, all too commonly in veterinary species, loss of life.12 Recently, it has been recognized that the synovium and its dysfunction play a significant role in driving early joint response to injury.13 Yet, a continuing lack of knowledge surrounding fundamental synovial biology drives an absence of therapeutics to support synovial function.14–18 Importantly, synovial biology is challenging to recapitulate in standard monolayer cell culture due to the lack of the fundamental chemical, biochemical, and mechanical signals delivered through its 3-D microenvironment. This is linked to the complexity of the structure of synovial tissue in vivo: it comprises an extensive villous lined intimal and subintimal layers of tissue, both of which contain fibroblast-like synovial cells as well as highly responsive, ciliated macrophagic synoviocytes. Although mammalian synovial explants have previously been used in sparse preclinical studies, there has been no comprehensive validation of the viability and functionality of these systems. Additionally, an accessible, straightforward method by which to maintain this delicate tissue fully submerged in an in vivo–like orientation has not yet been proposed. This lack of characterization of the baseline structure and function of synovial explants in culture has unfortunately resulted in heterogeneity in how they are assessed as well as a continued prevalence of monolayer synovium study in the literature. Thus, we aimed to describe and validate a short-term 3-D equine synovial explant culture system with the hypothesis that tethered 6-mm explants would maintain high viability, secretory abilities, and structural and metabolic soundness over 4 days of culture. Key to this protocol are our methods for synovial harvest, consistent perfusion in culture, and comprehensive tissue evaluation.
Methods
Preparation of the 3-D suspension system
Molecular-grade agar (Difco) was dissolved at a concentration of 5% weight to volume in PBS (Corning). The warm agar was dropped slowly onto the bottom of the sterile 12-well plates (Corning) at a volume of 2 mL/well using a serological pipette. After air drying (2 to 5 minutes), the plates were inverted and stored at 4 °C with a parafilm seal. Immediately prior to use, each agar “disc” was biopsy punched twice with a sterile 3.5-mm biopsy punch (Miltex), then removed carefully with a bent sterile needle and used in a separate dish to suspend the synovial explants villous-side down (Figure 1).
A—Explanted equine synovium from healthy adult horses was configured villous-side down in 2 mL of complete fetal bovine serum–containing medium and stabilized with a sterile agar ring containing two 3.5-mm holes. Prior to culture, macroscopic evaluation was performed by an investigator using a dissection microscope. B—Representative macroscopic villous presentation of day 4 explant at 10X magnification. C—Representative macroscopic villous presentation of day 4 explant at 25X magnification.
Citation: American Journal of Veterinary Research 2025; 10.2460/ajvr.24.11.0357
Explant cultures
Middle carpal and radiocarpal joints synovium from a randomly chosen front limb of healthy adult horses donated to the University of Georgia Veterinary School for reasons unrelated to this study was harvested 30 to 60 minutes posteuthanasia with 20 mg/kg sodium pentobarbital, IV. Horses were chosen based on a lack of history of known orthopedic disease, no signs of lameness or swelling as evaluated by an equine surgeon, and no macroscopic evidence of arthritis on cartilage surfaces when the joints were opened. Tissue extraction was performed aseptically by an equine surgeon using the sterile surgical technique. The synovium was taken en bloc after removal of the skin, SC tissue, and extensor carpi radialis tendon. After rinsing the synovium with PBS, the tissue block from each horse underwent a 24-hour equilibration in 150 mL of synoviocyte culture medium comprised of 84% Dulbecco modified Eagle medium (Gibco), 10% fetal bovine serum (Gibco), 4% penicillin-streptomycin (Thermo Fisher Scientific), and 2% L-glutamine (Gibco).
After equilibration, the tissue was rinsed again and held vertically with moderate tension using towel clamps affixed to the outer fibrous capsular layer of tissue and onto a sterile, flexible biopsy cup lid (Corning). The fixture was kept still with the use of the lid as a fixed point to ensure that the synovium was not stretched excessively during the harvesting process to avoid potential cell loss or warping of the tissue. Surgical scissors and a scalpel were then used to trim deep to the synovium, removing adipose and fibrous tissue until homogenous depth was achieved across the tissue piece (approx 12 mm). Care was taken to minimize touching the synovial surface with instruments, and the tissue was kept moist with repeated rinses of sterile PBS. At least 24 homogenous whole-thickness explants were extracted using a 6-mm biopsy punch (Corning) while the tissue was secured fibrous-side down using 22- or 25-gauge needles bent at 90°. To ensure consistency, tissue cleaning and sampling was performed by the same investigator each time. All techniques were performed sterilely within a biosafety cabinet.
Immediately after extraction (time = 0 days), 4 explants were fixed in 10% neutral-buffered formalin for histology, and 4 were frozen and kept at −80 °C for mitochondrial enzyme analysis. Two further explants were set aside for viability quantitation, and cell-free supernatant was stored at −80 °C to ascertain baseline cytokine levels in media originating from fetal bovine serum. Fine-tipped dissecting forceps were used to pull the fibrous portion of each explant through the center of an agar disk, and the construct was subsequently lowered into warmed synoviocyte medium (2 mL/well; 12-well, nontreated tissue culture plate; Corning). Plates were maintained in incubators at 37 °C with 5% CO2. On day 2 of culture, the full volume of medium from each well was harvested and spun down at 1,957 X g for 10 minutes to remove cellular debris, then the debris-free medium was vortexed and aliquoted for storage at −80 °C. Meanwhile, explants were washed twice with 2 to 3 mL sterile PBS for a maximum of 5 minutes before new, warmed medium was replaced into wells for the final 2 days of culture. After a total of 4 days of culture, media were harvested in an identical manner, and explants were again harvested for histology (n = 4/horse), mitochondrial analysis (n = 4/horse), and LIVE/DEAD (ABCAM, Cambrige, UK) staining (n = 2/horse), and an additional 4 explants were dried and weighed. Dry weights were determined from explants placed on weighed coverslips in an oven at 200 °C overnight and then cooled for at least 24 hours but were found to be very similar and thus not used for normalization within subsequent analyses.
Viability staining
Preliminary work suggested that the percentage of deviation from the mean viability ascertained via manual cell counting was minimized once at least 1,200 cells were counted from the villous surfaces of each explant. Thus, between 2 and 3 representative images of explant villi containing at least 1,200 clearly distinguishable total cells were manually counted for calcein acetoxymethyl (AM; green, 490/525 nm excitation/emission; metabolically active and intact cells; Invitrogen) or ethidium homodimer-1 (red, 528/617 nm ex/em; dead cells with compromised membranes; Invitrogen) staining after incubation with both probes. Stains were applied for 20 minutes in cold Hanks balanced salt solution at a total volume of 3 mL/explant and at concentrations of 5 µM calcein AM and 1 µM ethidium homodimer-1 before explants were moved to 2 mL of Hanks balanced salt solution alone for imaging. Counting was performed over a maximum of 1 h/explant as evident efflux of calcein AM was noted after that point. Fluorescent imaging was performed immediately after explants were created (day 0) or after 96 hours in culture (4 days). The percentage of live cells per explant was calculated by dividing green-staining cells over the sum of green- and red-staining cells (minimum, 1,200). This percentage was averaged across all 4 explants attained at the same time point. Additionally, a full visual scan of the explant under fluorescent microscopy was performed to note any qualitative findings based on comparisons to negative controls from pilot work, including blunted villous tips, high adiposity in the fibrous region, or patterns of necrosis.
Histologic evaluation and immunohistochemistry
Four formalin-fixed explants from each time point were dehydrated and embedded in paraffin, then cut into 4-µm sections for microscope slide mounting. Sections were either routinely stained with H&E or underwent immunohistochemical staining for macrophagic character using antibodies to CD14, expressed on granulocytes and macrophages and playing a role in phagocytosis and complement recognition, or CD11b, a glycoprotein preferentially expressed on macrophages. Paraffin sections were deparaffinized and subjected to heat-induced epitope retrieval in sodium citrate buffer (pH, 6). Following an hour-long block with universal blocking reagent (UltraCruz sc-516214), monoclonal mouse anti-human/porcine/equine CD14 (433423; 1:250; Novus Biologicals) or monoclonal mouse anti-human/equine CD11b (MAB16991; 1:100; R&D Systems Inc) were applied overnight at 4 °C in a humidified chamber sequentially followed by the quenching of endogenous peroxidases with H2O2 and the application of horseradish peroxidase–conjugated anti-mouse binding protein (1:500; Santa Cruz Biotechnology), and then 3,3'diaminobenzine as chromogen. Slides were counterstained with Gill II hematoxylin and coverslipped. Equine lymph node sections from 1 donated horse fixed and prepared in the same way were used as positive controls for CD11b and CD40 staining. For the negative control, isotype antibody (Cancer Diagnostics) replaced primary antibody. Day 4 explant sections were evaluated for chromogenic staining at the villous periphery and surrounding microcapillaries in the sections, with deposited chromogen suggestive of the retention of macrophagic character associated with resident macrophage-like synoviocytes.
For explants stained with H&E, grading was performed as described in similar explant work. The percentage of the synovial villous surfaces covered by nondegenerate cells as well as the appearance of intact, long villi were semiquantitated through blinded grading. Both parameters were scored from 0 to 3, with 0 meaning > 75% villous surface covered with nondegenerate cells or > 75% of villi appearing normal. A score of 1 was assigned to between 25% and 75% healthy appearance in both parameters. A score of 2 was assigned to < 25% normal explants and 3 to explants with complete degenerative changes. Both parameters were then added together for the total score. This scoring strategy was established following a review of previous literature and previously confirmed with control tissues fixed immediately after harvest (positive control; expected score of 0 for both parameters) or contaminated tissue that did not receive standard media changes (negative control; expected score of 6).
Enzyme-linked immunosorbent assay
After a primary media change (day 0), explants were placed into tissue culture as described above. On day 2 of culture, 3 explants were blotted and weighed, and their supernatant was collected, spun at 275 times gravity for 10 minutes, aliquoted, and stored at −80 °C until analysis. The remaining 3 explants underwent an additional media change and were cultured until day 4. Protein quantification was performed on day 2 and day 4 using validated anti-equine IL-1β, anti-equine tumor necrosis factor-α (TNFα), and hyaluronic acid (HA) ELISA kits (R&D). Polyclonal antibody (capture antibody) was used to coat 96-well plates, which were then incubated with a bovine serum albumin blocking buffer for 1 hour. The plates were then serially washed 3 times with a 0.05% Tween-20 and PBS buffer solution. Standard dilutions or samples were added to wells, and plates were incubated for 2 hours at room temperature prior to further washes and the addition of a detection antibody. After another 2-hour incubation, streptavidin–horseradish peroxidase was added, then washed 3 times before a peroxide substrate solution was added. Each plate was verified to have an appropriately fitted standard curve (R2 ≥ 0.97) and acceptable replicate agreement (coefficient of variation ≤ 15%) across both across standards and samples. Sample dilutions were determined previously with the aim of diluting them to fall onto the most linear portion of the curve, between approximately 100 and 1,000 pg/mL.
Metabolic enzyme evaluation
To determine the metabolic capacity of key cellular and mitochondrial enzymes in the explants, pyruvate or malate were dosed into tissue homogenates and subsequent NADH production measured using a fluorescent assay. For this, 40 mg of villi from each synovial explant was dissected using a dissection microscope and mixed at a 1:4 weigh-to-weight ratio with a 33-mM phosphate buffer solution. A mechanical homogenizer was used for 60 seconds at the highest speed (150,000 times gravity; tissue homogenizer probe and autoclavable hard tissue homogenization tips; OMNI International) while over ice to release cells into the buffer. The homogenate was placed into the −80 °C freezer for 5 to 10 minutes, then removed for 10 to 15 minutes. This freeze-thaw cycle was repeated a total of 3 times. Then, in a crystal cuvette, 10 μL of homogenate was added to 190 μL of assay buffer, which was made from 15 mL zymogen buffer, 250 μL of 1 mg/mL alamethicin, 75 μL of 1 mM rotenone, and 60 μL of 500 mM NAD+. To read fluorescence from NADH synthesis, a flea stirrer was added to the cuvette, which was then capped with a stopper and placed into a chamber at 30 °C and set to stir at 265 times gravity in a fluorescence spectrometer (HORIBA Scientific). Excitation was set to 340 nm and emission to 450 nm to be read every 10 seconds. After a baseline reading for 5 to 10 minutes, 2.2 μL of 500 mM pyruvate, or 10 μL of 1M malate, was added to the sample, then readings continued for 1 hour, by which time the fluorescence readings from each of the samples had plateaued. Fluorescence measured by the detector was plotted and exported as counts per second into the program HJYMulti FluorEssence (Keyence INC), then compiled and plotted for data extraction in Origin Viewer, version 9.6.5 (OriginLab).
Statistical analyses
Statistical analyses were performed in Excel (Microsoft Corp) and RStudio (R Foundation for Statistical Computing version 4.4.2). Two-tailed paired Student t tests were performed to confirm that the means of day 0 or day 2 analyses (average of the technical replicates of n = 5 separate horses) were statistically different from those of day 4, except for H&E grading, for which a Wilcoxon signed-rank test of the paired comparison between the median scores (between 1 and 6) of day 0 and day 4 explants was performed. Sample size estimation (n = 5) was determined using a power of 0.8, significance level of 0.05, and desired detection of a 20% SD in viability (signal-to-noise ratio, 2); thus, these were the parameters used in statistical analyses.
Results
Due to the lack of cohesive literature suggesting the optimal culture conditions for synovial explants, we initially employed LIVE/DEAD staining, dissection microscopy, and histologic techniques to qualitatively assess parameters important to overall explant viability and improvement over monolayer culture techniques. This pilot work informed our 3-D suspension culture (Figure 1), which maximizes the surface area of explants to reduce medium underperfusion, a finding which can be recapitulated by configuring explants within wells of low diameters and total media volume (Supplementary Figure S1). Importantly, in our pilot work, we observed cell death at cut surfaces, which was subsequently minimized using round, sharp biopsy punches and excising tissues under moderate stable tension with copious PBS washes during dissection. Dissection microscope evaluation of explants cultured in this fashion in 3-D suspension suggested the maintenance of abundant, free-flowing villi through 4 days. For the formal study, 4 representative explants were harvested on days 0 and 4 from each horse for fluorescent viability staining, which demonstrated that explant cells remained viable at between 85% and 90% calcein uptake through the duration of the study (day4 to day0, 4.1%; CI, −4.6% to 12.99%; P = .307; Table 1).
Mean percentage of viability and metabolism (95% CI) did not significantly change by day 4 of explant culture (n = 5 horses).
| Analysis | Percentage of changeday4 over initial measurements (n = 5 horses) | Significance (paired t test) |
|---|---|---|
| Viabilitya | 104.73% (40.70%–168.80%) | P = .31 |
| Pyruvate NADH spikeb | 96.18% (85.78%–106.58%) | P = .34 |
| Malate NADH spikeb | 90.64% (69.09%–112.18%) | P = .56 |
We employed histologic staining and semiquantitative scoring to further assess viability and morphologic parameters not recapitulated in 2-D nor 3-D synovial spheroid cultures such as in vivo–like cell organization and villous morphology. Staining revealed the multiple retained features of synovial tissue, including fibrous connective tissue regions, localization of ciliated synovial cells to villous borders, subintimal microvasculature and adiposity, and complex villous structures (Figure 2). Semiquantitative summed scores (scoring; Supplementary Figure S2) for surface covered by degenerate cells (none = 0; > 75% = 3) and appearance of clubbed/shortened villi (villi > 1 mM = 0; villi ≤ 50 µm = 3) were found not to differ significantly between day 0 and day 4 explants (mean ± SD: d0 = 1.54 ± 0.81; d4 = 1.81 ± 0.55; Figure 2). Findings were nonsignificant with and without continuity correction. Importantly, synovium in vivo is comprised of fibroblastic and macrophagic synoviocytes, which contribute to homeostatic and inflammatory responses in the tissue. Therefore, we employed immunohistochemical staining to verify the presence of CD11b and CD14. We were indeed able to elucidate positive staining of these surface markers at the villous periphery on day 4 explants (Figure 3).
The histologic appearance of tissues described in Figure 1 showed villous structure and cellular integrity after 4 days. A—Representative villous H&E staining of day 0 explant. Scale bar = 300 µm. B—Representative villous H&E staining of day 4 explant. Scale bar = 300 µm. C—Blinded grading was performed, with 0 representing minimal and 6 representing maximal degradative changes. Day 4–Day 0 = not significant. D—Chromogenic immunohistochemical staining for CD14 (top panels) and CD11b (bottom panels) was also performed on day 4 tissues for macrophage-like character using 3,3’diaminobenzine chromogen stain with hematoxylin counterstain. Scale bars = 300 µm.
Citation: American Journal of Veterinary Research 2025; 10.2460/ajvr.24.11.0357
Enzyme-linked immunosorbent assay quantification was performed on explant supernatant immediately prior to media changes on days 2 and 4 of culture after supernatant was spun down to be free of cellular debris. Results from each plate were used with a standard curve from the same plate to generate concentrations across technical duplicates of n = 4 explants per time point per horse. Samples with unacceptable replicate disagreement (coefficient of variation ≥ 15%) were rerun. Data depict mean ± 1 SD across n = 5 horses and are plotted on a log10 scale. HA = Hyaluronic acid. TNFα = Tumor necrosis factor-α.
Citation: American Journal of Veterinary Research 2025; 10.2460/ajvr.24.11.0357
To corroborate the evident high viability of the tissue, we next performed a mitochondrial functionality assay on minced, lysed explant homogenates, reflecting the activities of pyruvate and malate dehydrogenase by measuring the fold change in NADH production after spiking their substrates into the homogenate. The NADH response after both pyruvate and malate spikes, though somewhat variable, remained high (mean ± SD; pyruvate: d0 = 1.85 ± 0.72, d4 = 1.73 ± 0.52; malate: d0 = 6.40 ± 4.21, d4 = 5.76 ± 4.16; Cohen d, 0.184 and 0.153, respectively; Table 1). Additionally, concentrations of TNFα, IL-1β, and HA released into the medium over 2 days when measured by ELISA were lower but not significantly altered in the second half of the culture period (Figure 3). Encouragingly, we were also able to demonstrate a dose-responsive TNFα secretion in explants upon 14 to 18 hours of stimulation with lipopolysaccharide, a potent immune stimulant derived from gram-negative bacteria immunogen (Supplementary Figure S3).
Discussion
Clinical trials of therapeutics focusing on joint degradation have a high rate of failure due to limited understanding of the many mechanisms driving disease pathogenesis.19 Even though the synovium is critical to the maintenance of joint homeostasis, limited efforts are made to support synovial tissue health specifically. Maintenance of the villous architecture, surface structures, mechanical signaling, and cell-matrix arrangements of cultured synovium is crucial in the use of this system as a model to the aim of filling knowledge gaps in synovial biology and disease treatment.6,20 For example, previous work has suggested that the transcriptional signatures of culture-expanded synoviocytes are dependent on their positional location within the synovial tissue, and these critical features are lost in monolayer culture.21,22 This and other studies23–25 corroborate the modulated biology of monolayer joint cells in vitro.
Our 3-D, short-term explanted tissue culture minimized the degradative changes known to occur in cultured tissue, including necrosis,26 loss of immune cell character,27 and morphologic changes to villous structures.28 Though limited, the time frame chosen for this validation is appropriate for the evaluation of inflammatory cytokine secretion,29 cytotoxic or metabolic degeneration,30 or events associated with joint sepsis,31 all examples of clinical phenomena that, with better therapeutic approaches, could be treated and simultaneously modulated to avoid subsequent joint degeneration.32
Importantly, these data do not extrapolate to longer culture periods, the feasibility of which should be validated to ensure that culture alone continues to not have a significant effect on tissue health and function after 4 days.
Synovial viability is an important readout for therapeutic testing as the functionality of this tissue has been shown to influence the long-term function of the joint, making synovial dysfunction an important facet of early disease.33 Unlike in monolayer studies, viability staining of whole explants reveals stable fibroblastic, macrophagic, and even adipose and endothelial cell populations. Qualitatively, this method can also show localized regions of cell death. Though there are many approaches to viability staining, we were able to attain a robust readout with the extensively utilized LIVE/DEAD kit approach already common to in vitro literature.34 Histologic analysis of synovial explants allows for key insight into cell-specific degradative changes, tissue reorganization, and specific degenerative events, including fibrosis and edematous changes, which were not induced by explant culture conditions alone. This is an additional improvement over other models that are not able to recapitulate these aspects of synovial pathophysiology as evidenced by extensive work correlating decreased hematoxylin update and decondensed intimal cell nuclei as biomarkers of clinical degenerative joint disease.35
Cytokine production by synovial explants is highly translatable to the in vivo scenario, in which TNFα and IL-1β levels are mechanistically important in both clinically healthy and inflamed joints. Though often described as purely proinflammatory, IL-1 is thought to be a key contributing factor to homeostasis in normal cartilage tissue.36 In normal joints, IL-1β has been shown to vary between 21 and 5,501 pg/mL in synovial fluid, whereas TNFα ranges from 6 and 35 pg/mL.37 In our culture system, explants secreted more of both cytokines in the first 2 days of culture than in the final 2, though this decline was not significant, yielding levels that persisted within the same order of magnitude throughout the study (102 pg/mL for TNFα and 104 pg/mL for IL-1β). Still, this change could be reflective of the inherent inflammation associated with cell damage during tissue harvest and dissection, and thus it may be important to allow for at least 2 days of acclimation before applying inflammatory stimulants to these explants. Additionally, previous explant studies38 showed between 1,000 and 10,000 pg/mL (1 and 10 ng/mL) of HA secretion in normal equine synovia after 48 hours in culture when analyzed by ELISA, which we were also able to replicate at both days 2 and 4 of culture. Changes in the molecular weight or clustering pattern of HA secreted by synovial explants may be an interesting area of future study.
Metabolic integrity, which is reflected by mitochondrial enzyme function, is a useful and specific measure in the study of disease processes in both in vivo and cultured tissues. Its utility is heightened in the validation of culture scenarios where relatively thick pieces of tissue move from extensive vascularization in vivo to requiring nutritional maintenance solely by diffusion. Pyruvate and malate dehydrogenase are 2 key metabolic enzymes specifically important to the homeostatic functions of synoviocytes. Pyruvate dehydrogenase activity is known to regulate metabolic switch in inflammatory macrophages, wherein it diverts substrates toward heightened LDH activity. The “Warburg effect” can also be appreciated using this method, wherein activated synovial cells divert from pyruvate dehydrogenase to alternate pathways when ATP is needed more rapidly.39 This metabolic pathway is thus representative of inflammation, or dysfunction, in the whole explant, and in our protocol was limited to representing synovial cells in the tissue villi due to careful excision of villi under dissection microscopy prior to metabolic assay. Synovial tissues highly metabolized malate, indicating an interesting possible metabolic preference that warrants further investigation. In addition to shunting back into the citric acid cycle, malate is involved in stress signaling in osteoarthritis: the catabolism of glutamine into lactate produces NADPH via the activity of NADP+-specific malate dehydrogenase (malic enzyme). This has already been cited as a possible therapeutic target in osteoarthritis, with a monoclonal antibody to LDH already being assessed in preclinical trials.40
The use of standardized, viable synovial tissue explants in the study of synovial inflammation and cell death is highly warranted as therapeutics for inflammatory and infectious joint pathologies have begun to branch into biologic products supportive of tissue health and recovery.41,42 These therapeutics include blood-derived products, such as platelet-rich plasma, which may be associated with trophic and immunomodulatory effects. Improving our understanding of these therapies using the synovial explant such as the one described here may be beneficial since this model includes functional macrophage-like synoviocytes, an extensive extracellular matrix important to normal tissue function, and the complex macroscopic structure of synovium. Our in-depth validation of this culture platform sets up future work to assess its utility for the study of synovial disease states, both natural and experimental, in a standardized fashion.
Supplementary Materials
Supplementary materials are posted online at the journal website: avmajournals.avma.org.
Acknowledgments
The authors would like to sincerely thank Dr. Jarrod Call and his trainee, Dr. Jennifer Figueroa, for assisting in the metabolic analysis of the tissues; Dr. Elizabeth Howerth and the University of Georgia histology lab for preparing tissue sections and consulting for antibody staining; and finally, Dr. Roy Berghaus for offering advice on the statistical analyses performed in this study.
Disclosures
The authors have nothing to disclose. No AI-assisted technologies were used in the composition of this manuscript.
Funding
Supported by the Morris Animal Foundation, grant No. D21EQ-048, and by internal funding from the University of Georgia Summer Research Grant Program. This material is based upon work supported by the National Science Foundation under grant No. DGE-1545433.
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