The Institutional Review Board (IRB) at the Georgia Institute of Technology issued a waiver (H19004) as no studies with human subjects were performed at the Georgia Institute of Technology for the present study. This study used de-identified samples from participants enrolled in the MILES clinical trial (NCT03818737) which was approved by the Western IRB, Emory University IRB, and Duke University IRB (IRB001080460). All participants provided written informed consent, and the clinical trial design for the MILES trial was performed independently of the present study. Participant samples used for the present study were chosen by Marcus Center for Therapeutic Cell Characterization and Manufacturing staff based on the availability of samples that were in excess of the planned investigations of the MILES clinical trial. This study was in compliance with all ethical requirements. The design of the study was exploratory, aimed at identifying potential indicators of knee Osteoarthritis correlated to pre-determined KL scores.

Vials of pdSF (n = 36) and a reference SF were thawed at room temperature and then centrifuged at 1000×g for 10 minutes. For each pdSF, 500 µL of supernatant was transferred to microcentrifuge tubes and incubated with 500 µL 4 mg/mL hyaluronidase (Sigma-Aldrich, St. Louis, MO) solution (50% v/v) for 15 minutes at 37˚C. Samples were vortexed for 5 seconds and then incubated for an additional 15 minutes at 37˚C. Samples were then centrifuged at 1000×g for 5 minutes and supernatants were collected and directly used in Luminex assays.

Hyaluronidase-treated pdSF samples (n = 36) were analyzed using Invitrogen™ Cytokine 30-Plex Human Panel Luminex™ (LHC6003M, Thermo Fisher Scientific, San Jose, CA) according to the manufacturer’s instructions. Briefly, lyophilized standards were reconstituted and serially diluted to prepare 8 working standards. Luminex™ plates containing magnetic antibody-coated beads were washed after which standards (2 technical replicates prepared) or samples of pdSF (4 technical replicates prepared) were added to the wells for overnight incubation at 4˚C under mild agitation and protected from light. Using a magnetic 96-well separator, samples were decanted, washed twice, and incubated with biotinylated detector antibody for 1 hour at room temperature under mild agitation and protected from light. Using a magnetic separator, Luminex™ plates were then decanted, washed twice, and then incubated with streptavidin-RPE solution for 30 minutes at room temperature under mild agitation and protected from light. Next, the Luminex™ plates were washed three times using the magnetic separator and samples were analyzed using Luminex™ xMAP™ technology on a Bio-plex 200 system (Bio-Rad) to quantify cytokines concentrations.

Total protein of neat pdSF samples (n = 36) was measured using Pierce™ BCA Protein Assay Kit according to manufacturer’s instruction using the microplate procedure. Albumin standards were prepared by serial dilutions and added to wells of a 96-well plate (2 technical replicates prepared). pdSF samples were diluted 1:10 in PBS and then added to the wells of a 96-well plate (3 technical replicates prepared). Wells containing standards and pdSF were incubated with working reagent for 30 minutes at 37˚C. Following incubation, plates were allowed to cool for 5 minutes and then read using a standard plate reader under 562 nm absorbance.

Hyaluronidase-treated pdSF samples (n = 6) were analyzed for chondroitin sulfate and keratan sulfate using commercially available ELISAs (Aviva Systems Biology) according to the manufacturer’s protocols.

Neat pdSF samples (n = 17) were thawed and centrifuged to remove any cells or debris. Samples were then directed tested using 50mm cone-and-plate 1˚ attachment (Anton Paar MCR302). An amplitude sweep of pdSF was initially run to determine a strain % within the linear range. At a constant strain, each sample was tested via frequency sweep from 0.1 to 100 rad/sec.

Neat pdSF samples (n = 24) that did were thawed, diluted 1:10 in PBS, and electrophoresed through 1.0% agarose gel. For each agarose gel, non-OA SF from 2 different donors (Articular Engineering, IL) were used as standards for non-degraded HA (e.g. 1.5 MDa and higher). In addition, varying amounts of 2.5 MDa HA (Creative PEGWorks, NC) and MW ladders (Echelon Biosciences, UT) were included on agarose gel to demonstrate linear range and size of HA quantification using 0.0025% Stain-All (Millipore-Sigma, Burlington, MA). All SF were stained and analyzed on 3 different gels and HA scores from each gel were averaged. simSF was prepared and analyzed similar to non-OA SF and pdSF.

Classification of HA was as follows: Score 1, intact HA stained-intensity (1.5 MDa and higher) was similar to non-OA SF with no sign of degradation (less than 1.5 MDa); Score 2, slight reduction of intact HA stained-intensity relative to non-OA SF with some sign of degradation; Score 3, decreased intact HA stained-intensity relative to non-OA SF (50% - 80%) with degradation; Score 4, HA was marginally detected on agarose gel (<20%). Scoring of all samples was done by the same blinded and trained observer to ensure consistency. pdSF samples and simSF were randomly arranged on a gel, except for non-OA SF samples, MW ladders, and 2.5 MDa HA standards. The observer was not privy to other SF assessments and analysis until complete submission of HA analysis for all pdSF and simSF.

Cryopreserved bone marrow aspirate concentrate (BMAC) was collected from four subjects in the MILES clinical trial and used for the isolation and expansion of bone marrow-derived mesenchymal stromal (BM-MSCs). Briefly, cryopreserved BMAC was thawed, washed, and counted to prepare 1 × 10⁶ nucleated cells in 2 mL of isolation media (PRIME-XV MSC Expansion XSFM media (Fujifilm Irvine Scientific, Santa Ana, CA) supplemented with 1% PLTGold human platelet lysate (Millipore-Sigma) and 1% Penicillin-Streptomycin (Thermo Fisher Scientific) and plated into individuals wells in 6-well plates. After 24 hours, each well was washed twice with PBS to remove nonadherent cells, and then 2 mL isolation media was added to the adherent cells. Adherent cells were expanded for up to 14 days in a humidified incubator at 37˚ and 5% CO₂ with media changes every 3 - 4 days. Next, the cells were washed and harvested as passage (P) 0 BM-MSCs by incubation with TrypLE™ Express Enzyme (1X), no phenol red (Thermo Fisher Scientific) for 5 minutes at 37˚C and 5% CO₂. The cells were collected, neutralized with isolation media, and centrifuged at 300×g for 5 minutes. The supernatant was discarded and then cells were resuspended in isolation media and counted. The cells were further expanded to P1 in T-75 culture flasks (Corning^®, Corning, NY) at 1 × 10³ cells/cm² in serum-free media (PRIME-XV MSC Expansion XSFM media) for 4 - 5 days. P1 cells were harvested as previously described. P1 cells, now referred to as BM-MSCs, were cryopreserved in CryoStor10^® (STEMCELL Technologies, Vancouver, Canada) at a concentration of 1.0 × 10⁶ cells/mL. For expansion to P2, P1 BM-MSCs were thawed, counted, and then plated in Corning ^® CellBIND^® T225 flasks (Corning) at 1 × 10³ cells/cm² in serum-free media and cultured no longer than 5 days in a humidified incubator with 5% CO₂. P2 BM-MSCs were washed, harvested, and then cryopreserved as previously described until used for in vitro testing.

P2 BM-MSCs from four subjects (referred to as donors in the assay) were thawed, counted, and then prepared at a concentration of 8.0 × 10⁵ cell/mL in serum-free media (PRIME-XV MSC Expansion XSFM media). Using a 96-well plate, 100 µL of cell suspension was plated into each well (4 technical replicates prepared) and then placed in a humidified incubator set to 37˚C and 5% CO₂. After 24 hours, 20% pdSF or 20% simSF was prepared in serum-free media, and 100 µL of either pdSF or simSF was added to the cells in each well designated for pdSF or simSF exposure, resulting in exposure of the cells to a final formulation of 10% pdSF or 10% simSF. 100 µL of XSFM alone was also added to cells designated as basal media controls. The plate was placed in a humidified incubator for 24 hours, and then conditioned media was collected and stored at −20˚C for Luminex™ analysis. The Invitrogen™ Cytokine 30-Plex Human Panel Luminex™ was used to analyze the conditioned media as described above in the methods for SF characterization using Luminex™ Assay.

Human THP-1 monocytes (TIB-202, ATCC, Manassas, VA) were thawed, counted and expanded in 20% heat-inactivated (HI) fetal bovine serum (FBS; Hyclone), 1% Penicillin/Streptomycin (P/S; Sigma), 0.05 mM β-mercaptoethanol (BME, Sigma) supplemented RPMI-1640 medium (ATCC) in T-25 suspension flasks, placed upright in a humidified incubator set to 37˚C and 5% CO₂. Using a CellBIND^® 96-well plate (Corning, Corning, NY), harvested cells were plated at a seeding density of 105 cells/well in differentiation media (1% HI FBS, 100 ng/mL phorbol 12-myristate 13-acetate, 1% P/S, RPMI-1640) and left to attach and differentiate to naïve macrophages over 48 hr at 37˚C and 5% CO₂. After this time, the plate was centrifuged at 300×g for 5 min to settle loosely adherent cells and media was aspirated carefully to avoid removing cells. Media was changed to 1% FBS, P/S, RPMI-1640 supplemented with lipopolysaccharide (LPS from E. coli, 100 ng/mL, Sigma-Aldrich) and interferon gamma (IFNγ, 100 ng/mL, Peprotech, Rocky Hill, NJ) with or without the addition of 10% simSF or 10% pdSF. The plate was then incubated for 24 hours at 37˚C and 5% CO₂. Conditioned media supernatant was removed to a 96-well plate and stored at −20˚C prior to Luminex™ analysis as described above in the methods for SF characterization using Luminex™ Assay.

A tabular matrix was created with patient information and associated pdSF characterization data as described in Supplementary Table S1. Symbolic regression was performed to identify top features, or potential predictors, correlative to KL score or HA electrophoresis grade using DataModeler software (Evolved Analytics, Rancho Santa Fe, CA). Using all inputs (i.e., available patient information and pdSF characterization data), DataModeler generated symbolic regression models and selected the best fit models which are those possessing the greatest accuracy (R² value) and lowest complexity as identified by a Pareto front. The fittest models were then analyzed to identify top variables using the Variable Presence and Variable Combinations functions. Predictive features of KL score or HA electrophoresis grade were selected based on the highest percentage of presence in coinciding models identified by Variable Combinations function. This same approach was performed to identify top variables from either Luminex™ (Lumx) data only or patient information only correlated to KL score or HA electrophoresis grade.

Raw data from Luminex™ assays were imported into JMP 15 software for analysis and to generate Z-scores. Heat maps of Z-scores, violin plots, and bar and line graphs were created using GraphPad Prizm software v10. Data was represented as the mean ± standard error of the mean.

The distribution of de-identified OA patient information and pdSF samples data is shown in Supplementary Figures 1(A)-(F), and the record of related patient information and characterization performed for each pdSF sample is compiled in Supplementary Table S1. Ages ranged from 50 to 70 years with an average age of 60.0 ± 5.3 years. The average BMI was 29.4 ± 5.2 which ranged from 21.6 to 42.9. KL scores of OA patients were used as model outputs and were reported as KL 2 (n = 9), KL 3 (n = 18), and KL 4 (n = 14). An unequal distribution of patients’ sex, i.e. females (n = 16) to males (n = 25), resulted from the pdSF sample selection based on available volume for characterization assays. An unequal distribution of KL scores also resulted among each sex. Of the females, the average age was 59.6 ± 5.0 years old, BMI was 27.9 ± 5.5, and KL score distribution was 4 OA patients with KL 2, 9 with KL 3, and 3 with KL 4. For the males, the average age was 60.0 ± 5.3, BMI was 30.4 ± 4.9, and KL score distribution was 5 OA patients with KL 2, 9 with KL 3, and 11 with KL 4 (Supplementary Figure S1(A), Supplementary Figure S1(B), Supplementary Figure S1(C)).

Additional pdSF information recorded at the time of collection was the total volume of pdSF collected and the nucleated cell concentration in pdSF (Supplementary Table S1). Less than 20 mL was collected from afflicted OA knees for most of the pdSF samples (Supplementary Figure S1(D)) and most pdSF collected had little to no cells present (Supplementary Figure S1(F)). The color of pdSF was evaluated using a color key (Supplemental Figure S1(G)) which was recorded

Figure 1. Characterization of pdSF composition. (A) The cytokine profile of pdSF samples (n = 36) included 24 cytokines present in all pdSF samples. FGF-2 which was measured with the highest concentration in 18 of 36 pdSF samples. Violin plots showed the distribution and standard deviation of pdSF samples for each cytokine. (B) The total protein concentration of each pdSF sample (n = 36) arranged by KL score. (C), (D) Chondroitin sulfate and keratan sulfate concentrations measured in pdSF (n = 7) and arranged by KL score.

after a freeze-thaw cycle in which a close to parabolic distribution around color 4 was seen among all pdSF samples (Supplementary Figure S1(E)).

Twenty four (out of 30) cytokines were detected in all 36 pdSF by Luminex™ analysis. The most abundant cytokine detected was basic fibroblast growth factor, or FGF-2, which was measurable in 18 of the 36 pdSF with the remaining samples missing data as a result of being above the range of detection. Of these pdSF, the variability of FGF-2 among the sample measured was represented by the wide range in concentration from 3321.4 to 81,509.7 pg/mL averaging 14,401.6 ± 18,082.2 pg/mL. The top cytokines present in all pdSF samples included 6 cytokines with markedly high average concentrations including hepatocyte growth factor (HGF; 1657.3 ± 1182.4 pg/mL), granulocyte-colony stimulating factor (G-CSF; 1746.2 ± 1261.4 pg/mL), monocyte chemoattractant protein-1 (MCP-1; 1433.5 ± 634.0 pg/mL), monokine induced by interferon-gamma (MIG; 1130.9 ± 1027.2 pg/mL), interleukin-2R (IL-2R; 778.9 ± 365.1 pg/mL), and interleukin-6 (IL-6; 719.0 ± 653.9 pg/mL; Figure 1(A)).

The total protein content of pdSF (n = 31) was measured with a combined average of 13.9 ± 3.9 mg/mL. The total protein content ranged between 7.13 to 23.6 mg/mL with no apparent trend related to KL score (Figure 1(B)). Similarly, CS and KS were glycosaminoglycans measured in pdSF (n = 7) with average combined concentrations of 26.7 and 2.2 ng/mL, respectively. CS ranged from 22.0 to 31.5 ng/mL, and KS ranged from 1.4 to 2.8 ng/mL (Figure 1(C) and Figure 1(D)). Rheological properties were measured as the viscosity, storage modulus, and loss modulus for pdSF (n = 18). As the shear rate increased from 0.1 to 100 s⁻¹, the viscosities of all pdSF samples decreased with corresponding viscosity curves. At 0.1 s⁻¹, the pdSF with the highest viscosity measured 3622.8 ŋ, the lowest viscosity of 69.4 ŋ, and combined average of all pdSF was 1552.4 ŋ (Figures 5(A)-(C)).

The HA content of 24 pdSF samples was assessed using Stained-All agarose electrophoresis (Bowman et al., 2011) (Figure 2(A)). To provide semi-quantification assessment of HA, a classification system was developed to score HA content from 1 (normal) to 4 (severe degradation of HA). This classification system relied on using non-OA SF as reference on the agarose gel and HA molecular weight ladders to delineate degraded (<1.5 MDa) from intact (>1.5 MDa) HA. Intact HA in pdSF was ~4 MDa as assessed on agarose gel, since pdSF HA band ran slower than the 2.5 MDa HA standard (from Creative PEGWorks) and faster than the 6 MDa band (Select-HA MegaLadder from Echelon Biosciences) (Figure 2(B)). Of the 24 pdSF samples assessed, 6 showed a slight decrease in HA content (score of 2), 11 exhibited decreased HA content and evidence of degradation (score of 3), and 7 had almost no HA in their SF (score of 4), and non-OA controls were classified as a score of 1 (Figure 2(C)).

Figure 2. Electrophoresis analysis categorized the degradation of HA in pdSF. (A) Image of an agarose gel stained with Stains = all to represent HA and degradation. (B) An HA classification system was developed to differentiate between intact and degraded HA. (C) HA electrophoresis grade results of pdSF analysis using the HA classification system arranged by KL score.

THP-1 macrophages (THP-1M) were induced with LPS (+LPS) to a pro-inflammatory M1-like phenotype to mimic local macrophages in OA knees. Groups of THP-1M + LPS were then exposed to either simSF (+simSF) or pdSF (+pdSF) to elicit secretory responses for comparisons among all macrophage groups. THP-1M + LPS group (no exposure) secreted the highest levels of detected cytokines including IL-1β, IL-12, IL-1RA, IL-6, IL-7, MIP-1α, MIP-1β, and VEGF compared to those exposed to simSF (THP-1M + LPS + simSF) or pdSF (THP-1M + LPS + pdSF). Macrophages exposed to simSF or pdSF secreted comparably less IL-1β, IL-7, MIP-1β, and VEGF than those not exposed (THP-1M + LPS). Differences in cytokine secretion between the macrophage exposure groups were measured between IL-10, IL-12, IL-6, IL-8, IP-10, MCP-1, MIG, MIP-1α, and TNF-α and comparability was most evident in the secretion of IL-1RA, IL-2R, RANTES, and VEGF. As expected, the media control groups that did not contain macrophages had no presence of detected cytokines (Figure 7(B)).

SR models were used to identify the top variables of KL score when all of the patient information and Luminex (Lumx) analysis of cytokines were allowed as potential inputs. From the 8903 models generated, 103 models achieved R² ≥ 85% and were further evaluated to identify the variables most present in the top performing models (highest accuracy and lowest complexity). Three variables were present in greater than 80% of the top performing models: SF cell concentration (100%), SF volume collected (97%), BMI (84%), and two were greater than 50%, IL-6 (66%) and IL-17A (55%) (Figure 3(A)). Following a similar approach, SR models were used to identify the top variables of HA electrophoresis grade when all inputs were allowed. From the 8053 models generated, 1643 achieved R² ≥ 95%. Interestingly, SF cell concentration (100%), SF volume collected (100%), and total protein concentration (98%) were identified as top variables along with MIG (85%) and GM-CSF (74%) (Figure 3(B)).

To target key cytokines in pdSF that are indicative of OA severity, SR models were generated with only Luminex data (Lumx) to identify top cytokines correlative to either KL score or HA electrophoresis grade. The cytokines identified as top variables correlative to KL score (R² ≥ 60%) were Eotaxin (100%), IL-8 (100%), IL-13 (100%), and MCP-1 (87%) (Supplementary Figure S2(A)). Top cytokines identified as top variables correlative to HA electrophoresis grade (R² ≥ 90%) were IL-6 (100%), IL-17A (87%), MIG (72%), HGF (67%) and GM-CSF (55%) (Supplementary Figure S2(B)). Using only patient information and pdSF attributes (i.e., sex, age, BMI, color SF volume collected, SF cell concentration), SR models (R² ≥ 70%) identified SF cell concentration (100%), color (100%), BMI (98%), and SF volume collected (94%) as the top variables correlated to KL score (Supplementary Figure S3(A)). Using these same inputs to identify top variables correlative to electrophoresis grade, SR models (R² ≥ 90%) identified age (100%), BMI (100%), SF Volume collected (100%) and SF cell concentration (100%) (Supplementary Figure S3(B)).

The characterization of pdSF identified key components, e.g. cytokines, that were present in high abundances in all samples and provided relative physiological concentrations that were used to inform development of prototypes that were tested to create the final simSF product (Figure 4(A)). A stepwise procedure was

Figure 3. pdSF sample information and characterization data were correlated to KL score to identify top features correlative to KL score.

Figure 4. Development of simSF. (A) Key components determined by pdSF characterization were chosen and included in the simSF at relative physiological concentrations. (B) Stepwise protocol for developing simSF.

performed to create a solution with comparable viscoelastic properties as pdSF (Figure 4(B)). Rheological measurements demonstrated equivalent viscosity, storage modulus, and loss modulus between the final simSF product and the pdSF samples with the highest and lowest measurements (Figure 5).

The OA-targeted potency assay was developed to evaluate donor-specific secretory responses of BMAC when exposed to an OA-like environment. Moreover, this assay’s purpose was to demonstrate the use of pdSF and simSF in an in vitro assay to help predict possible effects of BM-MSCs following therapeutic administration of BM-MSCs into OA knees. The assay was performed according to the schematic shown in Figure 6(A) in which BM-MSCs were exposed to either pdSF or simSF for 24 hours prior to collection and analysis of the conditioned media containing secreted analytes.

Figure 5. Similar viscoelastic properties of simSF and pdSF. A. Viscosity, B. Loss Modulus, and C. Storage Modulus results of pdSF (n = 18; left) and simSF. For simSF results, the maximum (green line) and minimum (blue line) results from pdSF measurements are shown for comparison to simSF (red line).

BM-MSCs derived from the BMAC from four OA donors were evaluated in the OA-targeted potency assay and designated to 3 conditions: 1) exposed to media only, 2) exposed to media containing simSF, or 3) exposed to media containing pdSF (representative sample from one OA donor). Results show measurable responses in the secretion from all BM-MSCs based on fibroblast growth factor-β (FGF-β), hepatocyte growth factor (HGF), interleukin-2-receptor (IL2-R), interleukin-6 (IL-6), interleukin-8 (IL-8), MCP-1, and vascular endothelial growth factor A (VEGF-A). Heatmap results generated from the Z-scores suggest similar secretory responses from donor BM-MSCs exposed to simSF compared to those exposed to pdSF (Figure 6).

THP-1 macrophages (THP-1M) were induced with LPS (+LPS) to a pro-inflammatory M1-like phenotype to mimic local macrophages in OA knees. Groups of

Figure 6. BM-MSC responses to pdSF and simSF in OA-targeted potency assay. A. Schematic of the assay workflow. B. Results shown as heatmap of the Z-scores from testing 3 donors of BM-MSCs in basal culture conditions, with exposure to simSF, and with pdSF. Media controls were included.

THP-1M + LPS were then exposed to either simSF (+simSF) or pdSF (+pdSF) to elicit secretory responses for comparisons among all macrophage groups. THP-1M + LPS group (no exposure) secreted the highest levels of detected cytokines including IL-1β, IL-12, IL-1RA, IL-6, IL-7, MIP-1α, MIP-1β, and VEGF compared to those exposed to simSF (THP-1M + LPS + simSF) or pdSF (THP-1M + LPS + pdSF). Macrophages exposed to simSF or pdSF secreted comparably less IL-1β, IL-7, MIP-1β, and VEGF than those not exposed (THP-1M + LPS). Differences in cytokine secretion between the macrophage exposure groups were measured between IL-10, IL-12, IL-6, IL-8, IP-10, MCP-1, MIG, MIP-1α, and TNF-α and comparability was most evident in the secretion of IL-1RA, IL-2R, RANTES, and VEGF. As expected, the media control groups that did not contain macrophages had no presence of detected cytokines (Figure 7(B)).

Figure 7. simSF mimics cellular responses of macrophages to pdSF in vitro. (A) Schematic of the assay workflow. (B) Results shown as heatmap of the Z-scores from testing THP-1 macrophages in basal culture conditions, with exposure to simSF, and with pdSF. Media controls were included.