Patients

The present study was approved by the Medical College of Wisconsin and Froedtert Hospital Institutional Review Board (IRB) for Human Subject Research (PRO00035381, “Arthritis research at MCW”). Written informed consent was obtained from 66 patients undergoing elective, primary TKA with one of the senior authors (AE). None of the patients had a prior history of knee infection, prior knee surgery, or had an intra-articular injection within three months of surgery. We also enrolled four patients undergoing debridement and component explant due to periprosthetic joint infections to serve as positive controls and to optimize immunostaining. Ten of the OA patient samples were excluded due to excessive, nonspecific background staining. Of the remaining 56 quality samples, 53 patients had been diagnosed with degenerative arthritis.

Clinical evaluation

Patient demographics and comorbidities were collected during the pre-operative appointment. Knee injury and Osteoarthritis Outcome Score for Joint Replacement (KOOS JR) and Veterans Rand-12 Health Survey (VR-12) scores were collected at baseline and again at 3, 6, and 12 months postoperatively. All patients were followed clinically for at least one-year post-operatively to monitor recovery and occurrences of complications. Pain and functional recovery were assessed by patient reported outcome measures; occurrence of any infectious complications or reoperations were recorded.

Specimen collection

At the beginning of each patient’s TKA procedure, synovial fluid was aspirated from the operative knee using an 18-gauge needle following sterile prepping and draping and skin incision but prior to arthrotomy. Following arthrotomy, synovial tissue was harvested from the suprapatellar pouch and the medial and lateral gutters. Specimen was stored in sterile specimen containers and prepared for various analyses within 6 h of collection.

Specimen preparation and isolation

All synovial fluid and synovial tissue samples were collected and processed under sterile conditions and stored in -80 °C freezer or liquid nitrogen until further use. If available, synovial fluid was flash-frozen and stored for downstream cytokine analysis. Synovium was isolated from collected synovial tissue using sterile surgical scissors and forceps and sectioned into small (1–2 mm3) tissue fragments. Two sections from each patient were embedded within optimal cutting temperature compound. Samples were stored in a -80 °C freezer overnight, then transferred to liquid nitrogen for long term storage prior to histopathologic analysis.

Histopathology

Two sections from each patient were used to assess inflammation by hematoxylin and eosin (H&E) stain and fibrosis by Masson’s trichrome stain. H&E-stained sections were qualitatively evaluated and blindly scored for markers of inflammatory synovitis on a scale of 0 to 3, with 3 being most severe. Three separate scores for overall inflammatory infiltrate, number of inflammatory foci, and synovial lining thickness were summed together to produce an overall inflammatory synovitis score for each patient sample. Each trichrome-stained section was scored in a blinded fashion using a scale of 0 to 3, with 3 being most severely fibrotic. All scores were independently reviewed prior to unblinding of the coded samples.

Anti-peptidoglycan antibody generation

B. burgdorferi B31-A3 was cultured in complete BSK-II media supplemented with 6% rabbit serum. Escherichia coli strain K12; Bacillus subtilis strain 168; and Staphylococcus aureus (FDA 209); were propagated in Lysogeny Broth (LB), Streptococcus mutans strain Clark in Brain Heart Infusion (BHI) broth, and Deinococcous radiodurans strain 13,939 in Tryptone Yeast (TY) media supplemented with 10% glucose.

All bacteria were grown to mid-exponential phase, harvested at 4,000 x g for 15 min, and then washed twice with PBS. For peptidoglycan purification, bacterial pellets were resuspended in PBS and added dropwise into boiling SDS (5% w/v, final concentration) and boiled for 1 h as previously described [17]. All Gram-positive bacteria were bead-beat (BeadBug, Benchmark Scientific) prior to SDS boiling for 3 cycles of 60 s on, 60 s on ice. After boiling, all samples were cooled to 30ºC, and the pellets washed with autoclaved H2O four times using ultracentrifugation at 283,346 x g for 60 min at 30ºC. The pellets were then resuspended in H2O and treated with lipase (1 mg/ml) for 3 h, benzonase nuclease (4 µl/ml) for 2 h, and overnight with chymotrypsin (0.3 mg/ml), all with shaking at 37ºC. The next day 0.5% SDS was added to each pellet and heated to 80ºC for 30 min. The pellets were washed 3 times with autoclaved H2O at the same centrifugation conditions listed above. The Gram-positive samples were treated with a final concentration of 1 M HCl while continuously rotating at 4ºC for 48 h and centrifuged/washed 3 times, as described above. The dry weight was measured to quantify the amount of PG purified. To create the anti-peptidoglycan antibody, 5 BALB/cJ mice purchased from Jackson Laboratories were injected subcutaneously with 200 µg total of peptidoglycan from the bacteria listed above and mixed with equal parts of Freund’s Complete adjuvant (Thermo Scientific Ref: 77,140) (2 mg/ml final of PG). After 3 weeks all mice received a 265 µg booster injection of the same PG mixture. The mice were euthanized 2.5 weeks later and blood was collected. The blood was incubated at room temperature for 30 min prior to spinning at 1,500 x g for 10 min at 4ºC. The serum was then removed, pooled together, and frozen at -20ºC. The specificity of the antibody was tested using immunofluorescence and was found to bind S. mutans, D. radiodurans, S. aureus, and E. coli PG (data not shown) using methods described elsewhere [25, 26].

PG staining and scoring

Two sections of tissue from each patient were coded and stained by immunohistochemistry using the mouse anti-PG antiserum to label PG in synovial tissue, followed by incubation with horseradish peroxidase-conjugated goat anti-mouse IgG (Sigma-Aldrich) as detection antibody. Non-immunized mouse serum was used as a negative control. Following staining optimization for the custom anti-serum, all sections were processed at one time by staff at our core facility to control for technical variability. Slides that had nonspecific edge staining artifacts were excluded from further analysis. For each Sect. (2 per patient), five 1mm2 fields were randomly selected from the tissue section and number of stained foci, corresponding to individual PG occurrences, were counted and summed across both Sect. (10 mm2 total area analyzed per patient sample). Samples were then scored from 0 to 4 based on the number of PG occurrences in tissue: 0 = no PG occurrences; 1 = 1–9 PG occurrences; 2 = 10–19 PG occurrences; 3 = 20–29 PG occurrences; 4 = 30 + PG occurrences.

Immunofluorescence microscopy

Sections of synovial tissue were stained with mouse anti-PG antiserum (custom), goat anti-human CD90/Thy1 antibody (TA318808, Origene), rabbit anti-human CD68 (76,437, Cell Signaling) and DAPI (Sigma). Samples were then incubated with secondary (detection) antibodies: AF488-conjugated anti-mouse IgG (A10037, Invitrogen), Cy3-conjugated anti-rabbit IgG (715-166-152, Jackson Immuno), and AF647-conjugated anti-goat IgG (A21447, Invitrogen). Serial sections from the same patient stained with secondary antibodies only were performed for each sample to control for nonspecific staining. Slides were imaged using an Olympus VS120 slide scanner and images were analyzed using (LAUREN H ADD INFO HERE). Samples with high nonspecific staining of negative controls (primary antibodies omitted) were excluded from analysis.

Primary human fibroblast isolation and stimulation

Fibroblasts were isolated from the human synovial tissue samples described above. A portion of the tissue fragments were transferred to a 15 ml conical centrifuge tube containing 5 ml of collagenase D (Sigma Aldrich 11,088,858,001) at a concentration of 1 mg/ml (dissolved in Hank’s balanced salt solution (HBSS) [Sigma Aldrich 55,037 C] + 1% Penicillin/Streptomycin (Pen/Strep) [Fisher Scientific 15,140,122]). The tube was kept in a 37 °C bead bath for 1 h and was shaken vigorously every 5 min to release cells. Large tissue fragments were removed using sterile forceps and discarded. Remaining liquid was centrifuged at 1100 rpm for 10 min at room temperature. Supernatant was discarded and cell pellet was resuspended in 5 ml of enriched human fibroblast medium (High glucose DMEM [Sigma Aldrich D5671] + 20% fetal bovine serum (FBS) [BioWest S1690] + 1% Pen/Strep + 1% glutamine [Fisher Scientific 35,050,061] + 1% non-essential amino acids (NEAA) [Fisher Scientific 11,140,050] + 5 ng/ml recombinant human FGF-basic [BioLegend 792,504]). Cells were then transferred to a T25 tissue culture flask and placed in a 37 °C incubator with 5% CO2. Cell culture medium was replaced every 3–4 days, and cells were passaged at ∼ 90% confluency. Primary fibroblasts were frozen at passage 4 and stored in liquid nitrogen.

Fibroblast stimulation

Samples were passaged at least 6 times prior to use to enable isolation and expansion of fibroblasts. Cells were plated in 24-well plates at 2.5 × 10^5 cells per well in 500 µl of medium. Each patient sample was plated in two wells, and one of the wells was stimulated with 10 µg/ml of the muramyl dipeptide fragment from Staphylococcus aureus peptidoglycan (Sigma Aldrich 77,140) for 24 h. Cell culture supernatants were collected and stored at -80 °C until further analysis.

Cytokine analysis

Cytokine analysis was performed using the LEGENDplex Human Inflammation Panel (Biolegend) to quantify 13 human inflammatory cytokines/chemokines (IL-1β, IFN-α2, IFN-γ, TNF-α, MCP-1 (CCL2), IL-6, IL-8 (CXCL8), IL-10, IL-12p70, IL-17 A, IL-18, IL-23, and IL-33). Bead populations conjugated with antibodies specific to the mentioned cytokines/chemokines were incubated with neat synovial fluid samples allowing the target analytes to bind to the specific capture bead. Biotinylated detection antibodies were then combined with the analyte bound beads and each detection antibody formed a bond with their specific analyte. Thereafter, Streptavidin-phycoerythrin (SA-PE) was added to bind to the biotinylated detection antibodies generating a fluorescent signal with an intensity proportionate to amount of the specific cytokine/chemokine bound to the capture bead. Each sample was run through a flow cytometer where SA-PE fluorescence intensity was converted to cytokine/chemokine concentration based on a standard concentration curve.

Statistical analysis

Statistical associations between PG severity scores and synovial inflammation, accumulation of fibrotic tissue, cytokine levels, population demographics, and patient reported outcome scores were assessed using Pearson correlations and regression analysis (p value cutoff = 0.05). Statistically significant differences in cytokine secretion levels between stimulated vs. unstimulated fibroblasts were determined by paired two-tailed t test (p value cutoff = 0.05). All statistical analyses were performed using GraphPad Prism (v.9).