• View in gallery
    Figure 1

    Box-and-whisker plots of metabolic activity (optical density) of bovine cartilage explants incubated with synovial fluid (SF) alone or incubated with S aureus–inoculated SF (SA) alone or with platelet-rich plasma (PRP; 25% culture medium volume; SA+PRP), ampicillin-sulbactam (AmpS; 2 mg/mL; SA+AmpS), or both PRP (25% culture medium volume) and AmpS (2 mg/mL; SA+PRP+AmpS) for 24 hours. Cartilage explants were obtained from the stifle joints of 6 healthy adult nonlactating Jersey-crossbred cows, and metabolic activity was assessed for 3 explant replicates/treatment group/cow. For each plot, the horizontal line within the box represents the median, the lower and upper edges of the box delimit the interquartile (25th to 75th percentile) range, and the whiskers delimit the range. The median value differs significantly (P < 0.05) between plots annotated with different lowercase letters.

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    Figure 2

    Representative photomicrographs of bovine cartilage explants incubated in SF alone and incubated with SA, SA+PRP, SA+AmpS, or SA+PRP+AmpS for 24 hours as described in Figure 1. Cell viability was assessed for 2 explant replicates/treatment group/cow and was determined by means of fluorescent microscopy with calcein to detect live cells (excitation, 495 nm; emission, 515 nm; green staining) and a high-affinity nucleic acid stain to detect dead cells (excitation, 633 nm; emission, 658 nm; red staining). The explants were incubated in both stains simultaneously for 30 minutes at 37 °C and observed with an inverted, confocal microscope. Notice that explants incubated with SA alone had the highest number of dead (red-stained) cells. Bar = 100 μm in all panels.

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    Figure 3

    Mean percentage of dead cells (A) and glycosaminoglycan content (B) in bovine cartilage explants incubated in SF alone and incubated with SA, SA+PRP, SA+AmpS, or SA+PRP+AmpS for 24 hours as described in Figure 1. Both the mean percentage of dead cells and mean glycosaminoglycan content were determined for 2 explant replicates/treatment group/cow. Error bars represent the SD. The mean differs significantly (P < 0.05) between plots annotated with different lowercase letters.

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Autologous platelet-rich plasma effects on Staphylococcus aureus–induced chondrocyte death in an in vitro bovine septic arthritis model

Andrew J.T. MuirDepartment of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH

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Andrew J. NiehausDepartment of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH

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Joseph W. LozierDepartment of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH

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Sara L. ColeOptical Microscopy Core, Notre Dame Integrated Imaging Facility, University of Notre Dame, Notre Dame, IN

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Zarah A. BelacicDepartment of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH

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Abstract

OBJECTIVE

To investigate the chondroprotective effects of autologous platelet-rich plasma (PRP), ampicillin-sulbactam (AmpS), or PRP combined with AmpS (PRP+AmpS) in an in vitro chondrocyte explant model of bovine Staphylococcus aureus–induced septic arthritis.

SAMPLE

Autologous PRP and cartilage explants obtained from 6 healthy, adult, nonlactating Jersey-crossbred cows.

ProcedureS

Autologous PRP was prepared prior to euthanasia using an optimized double centrifugation protocol. Cartilage explants collected from grossly normal stifle joints were incubated in synovial fluid (SF) alone, S aureus–inoculated SF (SA), or SA supplemented with PRP (25% culture medium volume), AmpS (2 mg/mL), or both PRP (25% culture medium volume) and AmpS (2 mg/mL; PRP+AmpS) for 24 hours. The metabolic activity, percentage of dead cells, and glycosaminoglycan content of cartilage explants were measured with a resazurin-based assay, live-dead cell staining, and dimethylmethylene blue assay, respectively. Treatment effects were assessed relative to the findings for cartilage explants incubated in SF alone.

RESULTS

Application of PRP, AmpS, and PRP+AmpS treatments significantly reduced S aureus–induced chondrocyte death (ie, increased metabolic activity and cell viability staining) in cartilage explants, compared with untreated controls. There were no significant differences in chondrocyte death among explants treated with PRP, AmpS, or PRP+AmpS.

CLINICAL RELEVANCE

In this in vitro explant model of S aureus–induced septic arthritis, PRP, AmpS, and PRP+AmpS treatments mitigated chondrocyte death. Additional work to confirm the efficacy of PRP with bacteria commonly associated with clinical septic arthritis in cattle as well as in vivo evaluation is warranted.

Abstract

OBJECTIVE

To investigate the chondroprotective effects of autologous platelet-rich plasma (PRP), ampicillin-sulbactam (AmpS), or PRP combined with AmpS (PRP+AmpS) in an in vitro chondrocyte explant model of bovine Staphylococcus aureus–induced septic arthritis.

SAMPLE

Autologous PRP and cartilage explants obtained from 6 healthy, adult, nonlactating Jersey-crossbred cows.

ProcedureS

Autologous PRP was prepared prior to euthanasia using an optimized double centrifugation protocol. Cartilage explants collected from grossly normal stifle joints were incubated in synovial fluid (SF) alone, S aureus–inoculated SF (SA), or SA supplemented with PRP (25% culture medium volume), AmpS (2 mg/mL), or both PRP (25% culture medium volume) and AmpS (2 mg/mL; PRP+AmpS) for 24 hours. The metabolic activity, percentage of dead cells, and glycosaminoglycan content of cartilage explants were measured with a resazurin-based assay, live-dead cell staining, and dimethylmethylene blue assay, respectively. Treatment effects were assessed relative to the findings for cartilage explants incubated in SF alone.

RESULTS

Application of PRP, AmpS, and PRP+AmpS treatments significantly reduced S aureus–induced chondrocyte death (ie, increased metabolic activity and cell viability staining) in cartilage explants, compared with untreated controls. There were no significant differences in chondrocyte death among explants treated with PRP, AmpS, or PRP+AmpS.

CLINICAL RELEVANCE

In this in vitro explant model of S aureus–induced septic arthritis, PRP, AmpS, and PRP+AmpS treatments mitigated chondrocyte death. Additional work to confirm the efficacy of PRP with bacteria commonly associated with clinical septic arthritis in cattle as well as in vivo evaluation is warranted.

Introduction

Septic arthritis is a common joint disease and an important source of lameness in cattle that negatively impacts animal welfare and consequently results in economic losses to the industry.1,2,3 Septic arthritis can occur secondary to bacteremia with hematogenous dissemination to joints in calves,2,3 percutaneous infection from trauma, and iatrogenic sources.1,4 Following prompt diagnosis, treatment should involve rapid intervention that includes joint lavage via arthrotomy and systemic and local administration of antimicrobials.2,5,6,7,8,9,10 Despite timely treatment, persistent or recurrent infection and subsequent long-term joint degeneration and associated lameness are common sequelae. There is an unmet need for improved therapies, as existing studies report poor to guarded long-term prognosis (as low as 30%) for complete resolution of lameness and restoring functionality.2,9,10,11

Local administration of antimicrobials is commonly used to treat septic arthritis in large animals12,13,14 and achieves antimicrobial concentrations that exceed the minimum inhibitory concentrations for common bacterial pathogens for a prolonged duration. Local administration of antimicrobials is typically associated with fewer adverse effects than systemic administration and may decrease the risk for antimicrobial residues in meat and milk.15,16,17 In the US, there are currently no antimicrobials approved by the FDA for the treatment of septic arthritis or for local (intravenous regional limb perfusion [IVRLP] or intra-articular) administration in cattle. However, the AMDUCA does permit extralabel administration of ampicillin-sulbactum (AmpS) in cattle.18 In an experimental study,17 IVRLP of AmpS in healthy adult cattle resulted in therapeutic drug concentrations in synovial fluid (SF) samples without any adverse effect. 19 Given the restrictions on the use of antimicrobials in food-producing animals, the identification of a biologic product for treatment of septic arthritis in those species would be advantageous.

Platelet-rich plasma (PRP) is an autologous solution of concentrated platelets that contain anabolic and anti-inflammatory proteins or peptides. In veterinary medicine, PRP is commonly used to treat horses and dogs with inflammatory or degenerative musculoskeletal disorders, such as osteoarthritis and tendon and ligament injuries, because the α-granules of platelets are rich sources of anti-inflammatory, anabolic, and angiogenic growth factors.20 Multiple in vitro studies21,22,23,24 have demonstrated the antimicrobial properties of equine and human PRP. Platelets participate in innate immunity and secrete antimicrobial peptides, such as platelet factor-421,22 and defensins.25 PRP also contains mononuclear leukocytes that, when activated, secrete myeloperoxidate and subsequently hypochlorous acid, which has bactericidal properties.23 In a 2020 study,24 Gilbertie et al demonstrated that PRP has antibiofilm effects against free-floating Staphylococcus aureus biofilms established in SF in vitro and restores the antimicrobial activity of amikacin. Since PRP is an autologous biologic product, it is free from FDA restrictions in food-producing animals and warrants further exploration regarding the efficacy in bovine septic arthritis treatment.

The objective of the in vitro study reported here was to evaluate the chondroprotective effects (assessed by chondrocyte viability, metabolic activity, and cartilage glycosaminoglycan [GAG] content) of autologous bovine PRP used alone or in combination with AmpS (PRP+AmpS) in cartilage explants inoculated with S aureus aggregated in SF (SA). A previously validated S aureus–induced in vitro explant model of bovine septic arthritis was used.26,27,28 We hypothesized that the chondroprotective effects of PRP and PRP+AmpS would not differ significantly from those of AmpS alone in an in vitro explant model of bovine S aureus–induced septic arthritis.

Materials and Methods

Study overview and experimental groups

The autologous PRP and matched cartilage explants used for the assays were obtained from 6 healthy, adult, nonlactating Jersey-crossbred cows. Synovial fluid was obtained from the same 6 cows and pooled for use in the study. Cartilage explants were inoculated in vitro with S aureus strains ATCC 29213 and 43300 aggregated in SF as described.24,29 The treatment groups investigated in the study included SF alone, SA, and SA supplemented with PRP (25% culture medium volume [ie, 125 to 500 µL]; SA+PRP), AmpS (Unasyn; 2 mg/mL; SA+AmpS), and both PRP (25% culture medium volume) and AmpS (2 mg/mL; SA+PRP+AmpS).

Preparation of PRP

From each of the 6 study cows, blood was aseptically collected by means of jugular venipuncture with a 14-gauge, 1.5-inch needle. Approximately 240 mL of blood was collected from each cow over 3 minutes into four 60-mL syringes, each of which contained 6 mL of acid citrate dextrose A solution as an anticoagulant. A standard automated CBC, which included a platelet count that was verified by means of a manual platelet count estimation, was performed on each blood sample. Then the blood was immediately transferred to 50-mL conical polypropylene tubes and centrifuged at 250 X g for 5 minutes to separate the erythrocytes from plasma containing leukocytes and platelets. From each conical tube, the resultant plasma layer (approx 100 mL) above the grossly visible buffy coat layer was transferred to clean 15-mL conical tubes and centrifuged again at 500 X g for 10 minutes. The resulting supernatant, consisting of platelet-poor plasma (PPP), was removed and saved. The residual pellet was resuspended in 8 to 10 mL of PPP to create PRP, which had a platelet concentration that was 3 to 5 times that of whole blood. In other words, 8 mL of PRP was processed from each 240-mL sample of whole blood. Automated CBCs were performed on all PRP and PPP samples. This protocol for PRP preparation from bovine whole blood was optimized in our laboratory on the basis of previously reported techniques.30 The PRP sample from each cow was freeze-thawed in liquid nitrogen twice, divided into 3-mL aliquots, and stored frozen at –80 °C until experimental setup and while cartilage explant equilibration occurred.24,29,31

SF collection and processing

Following collection of the blood sample, each cow was euthanized with IV injection of pentobarbital sodium (150 mg/kg). Synovial fluid was aseptically collected from each cow within 2 hours after euthanasia. Both carpi and tarsi were clipped and aseptically prepared. About 3 to 4 mL of SF was collected from the radiocarpal, middle carpal, and tibiotarsal joints bilaterally and pooled for each cow. Samples were assessed for clarity and lack of blood contamination. The collected SF samples were centrifuged at 1,500 X g for 15 minutes to separate the cellular debris and filtered through a 40-μm cell strainer.24,29 The filtered SF samples were separated into 10-mL aliquots and stored at –20 °C until experimental setup.

Cartilage explant harvest and equilibration

Immediately following euthanasia of each cow, both hind limbs were disarticulated at the level of the hip joint. The stifle region of both limbs was cleaned, the hair was clipped, and the skin was aseptically prepared. An aseptic stifle arthrotomy technique was used to expose the femoral condyles, trochlear ridges, and patella for harvest of cartilage explants. The articular cartilage surfaces were visually assessed for evidence of injury or disease and were determined to be grossly normal in all cows prior to explant harvest. A 4-mm-diameter dermal biopsy punch (Uni-Punch; Premier Medical Products) was used to obtain 2-mm-thick cartilage explant specimens from the weight-bearing aspect of the medial femoral condyle, axial medial trochlear ridge, and medial articular facet of the patella. Approximately 60 to 80 cartilage explants were harvested from each cow. During explant harvest, the explants were placed in sterile 50-mL conical polypropylene tubes with Dulbecco modified Eagle medium (DMEM) containing glucose (4.5 g/L) and l-glutamine (300 µg/mL) and supplemented with sodium penicillin (200 U/mL) and streptomycin sulfate (200 µg/mL).

Cartilage explants were cultured in 24-well plates in DMEM containing glucose (4.5 g/L) and l-glutamine (300 µg/mL) and supplemented with 2% fetal bovine serum, 1% insulin-transferrin-selenium, sodium penicillin (100 U/mL), streptomycin sulfate (100 µg/mL), and l-ascorbic acid (50 µg/mL). Each explant was individually placed in a well (1 explant/well) with 1 mL of culture medium and orientated so that the articular surface faced upwards throughout the experiment. Prior to experimental setup, all cartilage explants were allowed to equilibrate to in vitro culture conditions (37 °C with 5% CO2 at 95% humidity) for 36 to 48 hours in the aforementioned medium.32,33 Prior to experimental setup, the explants were grossly evaluated to ensure that they were of uniform size and thickness.

Staphylococcus aureus strain, culture, and preparation

All protocols were based on previously published studies.24,27,28,29 Staphylococcus aureus strains ATCC 29213 (methicillin susceptible, mecA negative) and 43300 (methicillin-resistant, mecA positive) were used for the experiments. In vitro antimicrobial susceptibility testing was performed with a semiautomated broth microdilution system (Sensititre GPN3F; ThermoFisher Scientific) in accordance with the manufacturer’s instructions and interpreted by use of Clinical and Laboratory Standards Institute breakpoint guidelines. Breakpoint-associated MICs of the S aureus strains were summarized (Supplementary Table S1). Bacteria were stored at –80 °C in a 1:1 ratio of tryptophan broth and 60% glycerol solution. When required, bacteria were thawed and streaked onto tryptone soy broth (TSB) agar plates infused with 5% sheep blood and incubated for 24 hours at 35 °C. A single isolate was inoculated in 5 mL of TSB and incubated at 35 °C overnight to generate stock cultures. Stock cultures were made fresh for each experiment. The stock cultures were vortexed, and a 10-µL inoculate was transferred to 5 mL of fresh TSB. This inoculate was incubated at 35 °C with shaking at 225 rpm to a 0.5 McFarland standard, which required approximately 3 hours. MacFarland standards were diluted in fresh TSB to achieve bacterial concentrations of 1 X 106 CFUs/mL for further experimentation.

Experimental groups, S aureus SF inoculation, and experimental setup

The groups established and evaluated in this study included SF alone, SA alone, SA+PRP, SA+AmpS, and SA+PRP+AmpS. The concentration of PRP was set at 25% volume during in vitro culture on the basis of results of existing studies.24,29,31 The AmpS concentration used in the study corresponded with the reported maximum AmpS concentration achieved in SF following IVRLP in a study by Depenbrock et al.17

Staphylococcus aureus aggregates in SF were prepared from S aureus cultured in TSB as described.24,29 Briefly, SF samples were thawed, and 500 µL was added to each well of a 24-well ultralow attachment plate inoculated with prepared 1 X 106 CFUs of S aureus/mL. The plate was incubated for 6 hours at 37 °C in a microaerophilic chamber on a shaker at 120 rpm to facilitate S aureus aggregation.

Cartilage explants equilibrated to in vitro culture conditions were then transferred and incubated in SF alone and the 4 experimental treatments (SA, SA+PRP, SA+AmpS, and SA+PRP+AmpS) at 37 °C with 5% CO2 and 95% humidity for 24 hours. In other words, cartilage explants were exposed to S aureus aggregates and treatments simultaneously. One randomly allocated cartilage explant was incubated per well containing 500 μL of the aforementioned SA. Similar uninfected SF cartilage explant cultures (SF+PRP, SF+AmpS, SF+PRP+AmpS) were also established.

Explant metabolic activity assay

The metabolic activity of cells in individual cartilage explants (3 randomly allocated replicate explants/treatment group/cow) was assessed with a resazurin-based assay as described.33,34 This is a fluorescent metabolic assay that detects the conversion of resazurin to the fluorescent compound resorufin by metabolically active cells. After the 24-hour incubation was complete, cartilage explants were rinsed twice with PBS solution and transferred to new wells in 24-well plates in 1 mL of DMEM containing glucose (4.5 g/L) and l-glutamine (300 µg/mL) and supplemented with 2% fetal bovine serum, 1% insulin-transferrin-selenium, sodium penicillin (100 U/mL), streptomycin sulfate (100 µg/mL), and l-ascorbic acid (50 µg/mL). Resazurin (100 µL) was added to each well and incubated in the dark at 37 °C for 8 hours. A 200-µL aliquot of the medium from each well was transferred, in duplicate, to 96-well fluoromicrotiter plates, and fluorescence was measured at 570 nm (excitation) and 585 nm (emission) with a plate reader (Infinite M1000PRO; Tecan). The optical densities (median ± range of fluorescence) of cartilage explant groups (SF, SA, SA+PRP, SA+AmpS, and SA+PRP+AmpS) incubated for 24 hours were measured. The metabolic activities of harvested cartilage explants prior to equilibrating to in vitro culture, and explants maintained in SF+PRP, SF+AmpS, and SF+PRP+AmpS groups, were also measured with a similar protocol, and results were expressed as an absolute fluorescence (median ± range) value.

Assessment of live-dead cell staining by confocal microscopy and quantitative image analysis

The numbers of live and dead cells were determined among cartilage explants (2 randomly allocated replicates/treatment group/cow) after 24 hours of incubation was determined by use of live-dead cell staining as described.27,28,33 The explants were sectioned in half longitudinally prior to staining to view cells through the thickness of the explant. Cell status (live or dead) was assessed by means of fluorescent microscopy with fluorescent stains, namely calcein (15 µM) for live cells (excitation, 495 nm; emission, 515 nm; green staining) and a high-affinity nucleic acid stain (50 nm) for dead cells (excitation, 633 nm; emission, 658 nm; red staining). The explants were incubated in both stains simultaneously for 30 minutes at 37 °C, in accordance with the manufacturer’s instructions. The explants were secured so that the articular cartilage surface faced toward the microscope objective in a 35-mm glass-bottomed cell culture dish and observed with a 20X objective lens of an inverted, confocal microscope. Digital images of the respective cartilage explants were acquired in double-channel sequential scans (green fluorescence: excitation, 488 nm, and emission, 498 to 544 nm; red fluorescence: excitation, 514 nm, and emission, 563 to 663 nm). A minimum of 3 image stacks/explant group was obtained for a thickness of 1 mm, and imaging was performed with the same settings throughout the study.

Quantitative image analysis was conducted on the image stacks to determine the percentage of dead cells in cartilage explants groups.33 The numbers of live and dead cells and total number of cells in each image were determined with a software program (Imaris version 9.5; Bitplane Inc). The data were thresholded on the basis of pixel intensity; individual cells were then identified and counted on the basis of cell volume. In each image, the percentage of cell death was calculated as the number of dead cells (red channel) divided by the total number of cells (live and dead) detected in both the green and red channels. Cell death in explant groups SF, SA, SA+PRP, SA+AmpS, and SA+PRP+AmpS was expressed as the percentage of dead cells among all cells counted within the cartilage explant. The metabolic activities of freshly harvested cartilage explants before equilibration in in vitro culture, and explants maintained in SF+PRP, SF+AmpS, and SF+PRP+AmpS, were also measured. The final reported values for percentage of cell death represented the mean of at least 3 image stacks from each explant group.

Explant GAG content

Total explant GAG content was determined in cartilage explants (2 randomly allocated replicates/treatment group/cow) after 48 hours of incubation by use of a dimethylmethylene blue dye–binding colorimetric assay as described.32,35 Cartilage explants were first digested in papain (0.5 mg/mL in sodium phosphate buffer) adjusted for explant weight (1 mL of papain digest was used for every 10 mg of cartilage). All samples were compared against a chondroitin sulfate standard curve to estimate the total GAG content of digested explants. All samples were run in duplicate.

Statistical analysis

A sample size calculation was performed prior to study initiation. That calculation used preliminary data from our laboratory that suggested there would be a predetermined effect difference of 20% between SF- and SA-treated explants. Results indicated that a minimum of 6 cows was necessary to detect a difference of that magnitude with 80% power and 95% confidence (α = 0.05).

Outcomes of interest were explant metabolic activity, cell death percentage, and GAG content. The data distribution for each outcome of interest was assessed for normality by use of the Shapiro-Wilk test. One-way ANCOVAs were used to compare cell death percentage and GAG content among the 5 treatment groups (SF, SA, SA+PRP, SA+AmpS, and SA+PRP+AmpS) with pairwise comparisons performed with the Tukey post hoc test when necessary. A ranked ANCOVA with the Holm-Sidak method used for post hoc pairwise comparisons was used to assess the explant metabolic activity among the 5 treatment groups. All models included a covariate for cow to account for analysis of multiple explants of each animal (ie, repeated measures). Values of P ≤ 0.05 were considered significant. All analyses were performed with commercially available statistical software (SigmaPlot version 14; Systat Software Inc).

Results

Verification of PRP preparation

The WBC and platelet counts verified that leukocyte-poor PRP was generated and used for the study. The mean ± SD platelet count in the PRP samples (942,500 ± 492,300 platelets/µL) was significantly (P = 0.01) greater than the platelet count in the whole blood samples (232,600 ± 70,000 platelets/µL). The mean ± SD platelet enrichment percentage was 258.8 ± 157.2%. The mean ± SD WBC count in the PRP samples (900 ± 800 WBCs/µL) was significantly (P = 0.02) less than the WBC in the whole blood samples (8,530 ± 3,800 WBCs/µL).

Metabolic activity

In vitro explant culture during equilibration and incubation in SF+PRP, SF+AmpS, or SF+PRP+AmpS did not affect the metabolic activity of cartilage explants. The median (range) fluorescence (optical density) did not differ significantly (P = 0.20) among freshly harvested cartilage explants, explants following in vitro equilibration, and explants maintained in SF, SF+PRP, SF+AmpS, or SF+PRP+AmpS for 24 hours.

The median (range) fluorescences (optical density) of cartilage explants incubated in SF, SA, SA+PRP, SA+AmpS, and SA+PRP+AmpS were summarized (Figure 1). Explant inoculation with S aureus inoculation significantly (P = 0.001) decreased the explant metabolic activity. Metabolic activity did not differ significantly among cartilage explants maintained in SF and those maintained in SA+PRP (P = 0.8), SA+AmpS (P = 0.7), and SA+PRP+AmpS (P = 0.13).

Figure 1
Figure 1

Box-and-whisker plots of metabolic activity (optical density) of bovine cartilage explants incubated with synovial fluid (SF) alone or incubated with S aureus–inoculated SF (SA) alone or with platelet-rich plasma (PRP; 25% culture medium volume; SA+PRP), ampicillin-sulbactam (AmpS; 2 mg/mL; SA+AmpS), or both PRP (25% culture medium volume) and AmpS (2 mg/mL; SA+PRP+AmpS) for 24 hours. Cartilage explants were obtained from the stifle joints of 6 healthy adult nonlactating Jersey-crossbred cows, and metabolic activity was assessed for 3 explant replicates/treatment group/cow. For each plot, the horizontal line within the box represents the median, the lower and upper edges of the box delimit the interquartile (25th to 75th percentile) range, and the whiskers delimit the range. The median value differs significantly (P < 0.05) between plots annotated with different lowercase letters.

Citation: American Journal of Veterinary Research 83, 2; 10.2460/ajvr.21.01.0007

Live-dead cell staining

Live-dead cell staining demonstrated that dead cells were highest in cartilage explants maintained in SA, compared with those maintained in SF, SA+PRP, SA+AmpS, and SA+PRP+AmpS (Figure 2). There were no dead cells in cartilage explants incubated in SF, SF+PRP, SF+AmpS, and SF+PRP+AmpS (ie, explants incubated without S aureus).

Figure 2
Figure 2

Representative photomicrographs of bovine cartilage explants incubated in SF alone and incubated with SA, SA+PRP, SA+AmpS, or SA+PRP+AmpS for 24 hours as described in Figure 1. Cell viability was assessed for 2 explant replicates/treatment group/cow and was determined by means of fluorescent microscopy with calcein to detect live cells (excitation, 495 nm; emission, 515 nm; green staining) and a high-affinity nucleic acid stain to detect dead cells (excitation, 633 nm; emission, 658 nm; red staining). The explants were incubated in both stains simultaneously for 30 minutes at 37 °C and observed with an inverted, confocal microscope. Notice that explants incubated with SA alone had the highest number of dead (red-stained) cells. Bar = 100 μm in all panels.

Citation: American Journal of Veterinary Research 83, 2; 10.2460/ajvr.21.01.0007

The percentages of cell death determined via quantitative image analysis were consistent with live-dead cell staining findings (Figure 3). Incubation of explants with SA alone significantly increased the percentage of cell death (P < 0.001). The mean percentages of cell death in explants maintained in SA+PRP, SA+AmpS, and SA+PRP+AmpS groups did not differ significantly (P = 0.8) but were significantly (P < 0.001) less than that in explants maintained in SA explants.

Figure 3
Figure 3
Figure 3

Mean percentage of dead cells (A) and glycosaminoglycan content (B) in bovine cartilage explants incubated in SF alone and incubated with SA, SA+PRP, SA+AmpS, or SA+PRP+AmpS for 24 hours as described in Figure 1. Both the mean percentage of dead cells and mean glycosaminoglycan content were determined for 2 explant replicates/treatment group/cow. Error bars represent the SD. The mean differs significantly (P < 0.05) between plots annotated with different lowercase letters.

Citation: American Journal of Veterinary Research 83, 2; 10.2460/ajvr.21.01.0007

GAG content

The GAG content did not differ significantly (P = 0.57) among cartilage explants maintained in SF, SA, SA+PRP, SA+AmpS, and SA+PRP+AmpS (Figure 3).

Discussion

The present study tested the hypothesis that the chondroprotective effects of PRP and PRP+AmpS would be similar to AmpS in an in vitro explant model of bovine S aureus–induced septic arthritis. Explant metabolic activity and live-dead cell staining measurements at the 24-hour time point demonstrated that PRP significantly reduced S aureus–induced chondrocyte death in vitro, and the effects of SA+PRP, SA+AmpS, and SA+PRP+AmpS treatments were equivalent. The explant culture system (SF alone) and SF+PRP, SF+AmpS, and SF+PRP+AmpS treatments did not impact chondrocyte viability.

Existing in vitro studies24,29 investigating PRP antimicrobial effects for septic arthritis applications have only included the bacterial pathogen and SF. Therefore, to test our hypothesis, we employed a previously validated S aureus in vitro model of septic arthritis with bovine cartilage explants.27,28 As reported by Smith et al,27,28 we utilized chondrocyte viability 24 hours following S aureus inoculation as our experimental endpoint to investigate the efficacy of PRP, AmpS, and PRP+AmpS treatments. The S aureus–derived toxin α-hemolysin is the key toxin responsible for rapid chondrocyte death with this model,26,28 and both S aureus strains used in this study, ATCC 29213 and 43300, secrete α-hemolysin toxin.36,37,38 The S aureus inoculum size used in this study was consistent with other in vitro studies24,29 and an in vivo direct joint-inoculation study.27

Concomitant with existing studies21,22,24,29 that demonstrated the antimicrobial properties of PRP, results of the present study indicated that PRP treatment significantly reduced chondrocyte death in bovine cartilage explants inoculated with S aureus, compared with untreated controls, as determined via increased explant metabolic activity and live cell staining. The possible mechanisms linked to antimicrobial properties of PRP have been explored in a few in vitro studies.24,39,40,41 Cation-rich, low-molecular-weight antimicrobial peptides isolated via protein fractionation have been identified as the bioactive component responsible for the antimicrobial properties of equine PRP and PRP lysates.24,39,40,41 Lopez et al21,22 indicated that the transient in vitro bacteriostatic effects of equine PRP against methicillin-resistant S aureus are due to plasma complement proteins and platelet factor-4 activity. Further, from an S aureus bacterial pathogen standpoint, the secreted α-hemolysin toxin stimulates platelet activation and subsequent platelet lysis-dependent and independent procoagulant release, which negatively affects bacterial growth and proliferation.42 Although bovine PRP likely has similar properties, continued investigations are warranted to identify the specific platelet-derived antimicrobial peptides and the related mechanisms protective against the deleterious effects of septic arthritis.

Due to the lack of FDA-approved antimicrobials labeled for clinical treatment of septic arthritis, we used AmpS in the present study because it can be used in an extralabel manner for regional use in cattle under AMDUCA. Ampicillin is a semisynthetic penicillin with a narrow spectrum of activity, but when combined with sulbactam, a β-lactamase inhibitor, the spectrum of the combination product expands to include β-lactamase–producing bacteria such as S aureus. Penicillins do not exhibit an inhibitory effect on bovine platelets in vitro and in vivo, whereas they impair functional responses of human and canine platelets by stimulating platelet aggregation and subsequently decreasing thromboxane synthesis.43 The ampicillin concentration used in the present study exceeded therapeutic concentrations achieved in SF when administered through IVRLP in healthy cows17 and was well above the breakpoints established by the Clinical Laboratory Standards Institute for ampicillin-susceptible bacterial isolates of human44 and large animal45,46 origin (0.5 and 0.25 µg/mL, respectively). Results of the present study indicated that the chondrocyte protective effects of AmpS and PRP were similar and were equivalent to the PRP+AmpS combination treatment.

Cartilage catabolism and degradation that occur in the early stages of septic arthritis can result in permanent joint damage in the form of degenerative joint disease. Given the cartilage anabolic effects of PRP in inflammatory and degenerative arthritis, we quantified cartilage explant GAG content following S aureus inoculation and with treatments to determine the extracellular matrix protective effects of PRP and AmpS. In in vitro and in vivo experimental studies47,48,49 of S aureus–induced septic arthritis, cartilage catabolism manifests as rapid loss of GAG during the first 48 hours and is subsequently followed by collagen breakdown. This cartilage extracellular matrix catabolism is mediated, in part, owing to increased protease-inducing factor secretion by S aureus and induction of matrix metalloproteinase and collagenase activities in chondrocytes.50,51 In the present study, the GAG content did not decrease in cartilage explants following inoculation with S aureus inoculation or treatment with PRP, AmpS, and PRP+AmpS. Our evaluation was restricted to total explant GAG content at a single time point (48 hours), and we observed large intersample variability in the data. Variable bacterial activity and colonization of cartilage explants and platelet concentrations used between individual experiments are potential sources for this large intersample variation, and subsequently obscured apparent differences among treatment groups. Analyzing GAG released into culture SF and measuring 35SO4- GAG turnover and proteoglycanases–matrix metalloproteinases were not undertaken in this study and are warranted for comprehensive assessment of extracellular matrix protective properties of PRP, AmpS, and PRP+AmpS treatments.

The present in vitro study had several limitations. The in vitro model used only cartilage tissue and SF and did not include other articular tissues, synovium, and subchondral bone, which are critical for biologic responses to inflammation and infection and treatments.52 Frozen PRP was utilized in this study to accommodate experimental setup (ie, while explant equilibration and S aureus aggregation in SF took place). Although a single freeze-thaw cycle is routinely employed to activate or lyse platelets during clinical use, evaluation of the biological activity of freeze-thawed and freshly processed bovine PRP is warranted. Even though we reported the chondrocyte death–mitigating effects of PRP and AmpS, it must be noted that S aureus inoculation and PRP+AmpS treatments were initiated simultaneously in this study, and this does not mimic clinical scenarios. Inoculating explants with S aureus prior (ie, 12 to 14 hours) to adding treatments and subsequently quantifying chondroprotective indices relative to untreated controls as described by Miller et al26 would have been more reflective of clinical septic arthritis. Platelet concentrations in PRP treatments between experiments from individual cattle were not standardized to control for intersubject variability of quantified outcome measures. Quantifying posttreatment bacterial load in control and treatment groups to assess the direct antimicrobial effects of PRP and AmpS is necessary. In addition, assessing S aureus biological activity and explant colonization via histologic examination would have provided insight on the severity of this in vitro model and is warranted in subsequent investigations. Although Staphylococcus sp–induced septic arthritis has been reported in cattle,2 it is not a commonly encountered pathogen, and follow-up studies with clinically relevant pathogens to confirm the PRP and AmpS efficacy are necessary.

Results of the present in vitro study suggested that autologous PRP mitigated S aureus–induced chondrocyte death in bovine cartilage explants in a comparable manner to AmpS. From a clinical standpoint, further investigations dissecting the antimicrobial properties of autologous bovine PRP as a potential treatment for septic arthritis in cattle are warranted because PRP circumvents the restrictions associated with systemic and local antimicrobial use in food-producing species. This study serves as a foundation for further in vitro evaluations that address the above-listed limitations, as well as in vivo investigations that will facilitate clinical treatment recommendations.

Supplementary Materials

Supplementary materials are posted online at the journal website: avmajournals.avma.org

Acknowledgments

Supported by The Ohio State University College of Veterinary Medicine Consortium of NeuromusculoSkeletal Regeneration/Recovery and Locomotion (CANSL).

Presented in part in abstract form at the American College of Veterinary Surgeons 2020 Virtual Surgical Summit.

The authors thank Dr. Lauren V. Schnabel and Dr. Jessica M. Gilbertie (North Carolina State University College of Veterinary Medicine) for their expertise and technical advice provided during study design.

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Supplementary Materials

Contributor Notes

Corresponding author: Dr. Durgam (durgam.3@osu.edu)