Coating preparation and characterization

Tissue culture plates substrates (24 well plates) were coated with 1 ml of hyaluronic acid solutions (ACROS Organics™) of concentrations 0.1, 0.2, 0.5, and 1 mg/ml in PBS overnight at 4 °C (respectively called HA 0.1, HA 0.2, HA 0.5, HA 1). Uncoated wells were used as negative controls (CTRL). Before the use, the wells were washed with sterile PBS in order to remove the aggregates. In order to verify the presence of HA coating, Alcian blue 8 G (Sigma-Aldrich) was used to label hyaluronic acid (blue stain). Briefly, after coating the plate, 100 μl of Alcian Blue 1% in 3% acetic acid was added to the plate for 30 min and then washed with PBS. Images of plates’ surface were taken by Nikon inverted microscope and examined by Nikon Element.

The infrared spectra of TCP coated with different HA solution were obtained using a Fourier transform infrared (FTIR) using a Nicolet 6700 spectrometer (Thermo-Fisher Scientific, Waltham, MA). The samples were analyzed in attenuated total reflection (ATR) mode at 2 cm−1 resolution 256 times over the range of 500–2,000 cm−1 using the ATR/FTIR spectra following background subtraction, baseline correction, and binomial smoothing (11 points).

AFM images of the coated samples were collected in tapping™ mode by a Nanoscope V using single-beam silicon cantilever probes (RTESP: resonance frequency 300 KHz, nominal tip radius of curvature 10 nm, force constant 40 N/m). If necessary, data sets were subjected to a first-order flattening. Roughness (Ra), an arithmetic value that describes the absolute height of a surface in comparison to a two-dimensional plane represented by the average sample height was calculated using Nanoscope 6.13R1 software (Digital Instruments, NY, USA). Mean values from 4 random fields per sample in 3 independent experiments are reported. AFM tip scratch was performed on HA 0.1. A defect 1 um × 1um was introduced using AFM operating in contact mode with a Tap525 probe (Bruker, Sb (n) doped Si, nominal spring constant 200 N/m, nominal resonance frequency of 525 kHz). A set point of + 10 V (maximum applied force) was used to scan the selected area at 2 Hz.

Tissue collection

BALB/c mice were sacrificed by carbon dioxide inhalation after isofluorane sedation in accordance with the regulatory guidelines of the Institutional Animal Care and Use Committee. A 70% (v/v) ethanol solution was applied liberally to the lower half of the animal to achieve a level of sterility prior to harvesting the tissues. Tibias and femurs were removed and cleaned rigorously with a sterile scalpel to remove excess muscle, tendons, periosteum, and connective tissues. The cleaned bones were kept on ice-cold PBS containing 2% (v/v) fetal bovine serum (FBS) for further processing.

Cell isolation and culture

Mesenchymal stem cells were isolated from the bone marrow of healthy BALB/c as previously described2, 55. Briefly, bone marrow was flushed and plated into a large petri dish with alpha MEM supplemented with 20% fetal bovine serum (FBS, Thermo Scientific). For maintenance of cultures, cells were plated at up to 1 × 104 cells/cm2 and incubated at 37 °C in a humidified atmosphere (90%) with 5% CO 2 , 5% O 2 . The number of viable cells was counted by the trypan blue dye exclusion method, using a Burker chamber. Adherent cells were serially passaged using TrypLETM Express (Invitrogen) upon reaching near confluence (80%) and then reseeded for culture maintenance. For in vitro and in vivo studies MSC were used at passage 3.

MSC characterization before HA-treatment

Upon reaching confluence, cells were collected and characterized for the expression of MSC-associated markers (CD44, CD90, and CD105) and for the absence of the hematopoietic associated marker (CD45) with a FortessaTM cell analyzer (Beckton Dickinson), under the assistance of the HMRI Flow Cytometry Core. Antibodies used (PE anti-mouse/human CD44, APC/Cy7 anti-mouse CD45, PE/Cy7 anti-mouse CD105, and AlexaFluor 700 anti-mouse CD90) were purchased from BioLegend. Briefly, MSC were recovered using trypsin and spun down at 500 g for 5 min. They were then washed with FACS buffer labeled with directly conjugated antibodies according to manufacturer’s indications.

At passage 3 cells were also assessed for their capability to undergo osteogenic and adipogenic differentiation following a previously reported procedure2. Briefly, to induce osteogenesis MSC were seeded at the density of 5,000 cells/cm2 in 12-well plates. Osteogenic induction was performed over 14-day period using a StemPro Osteogenesis Differentiation Kit (Gibco). To confirm differentiation, conventional von Kossa and alkaline phosphatase staining were performed (VECTOR Blue Alkaline Phosphatase Substrate Kit; Vector Labs). For adipogenesis, cells were seeded at the density of 104 cells/cm2. Induction was performed using the StemPro Adipogenic Differentiation Kit (Gibco) for a 21-day period. Intracellular lipid droplet accumulation was visualized by conventional Oil red O staining.

Effect of HA treatment on cell viability, morphology, and surface markers expression

After ensuring the protocol used allowed for the isolation of a homogeneous MSC population positive for MSC-associated markers and negative for hematopoietic markers (Supplementary Figure 1A) and confirming its differentiative potential towards two mesodermal lineages (Supplementary Figure 1B), the effect of HA on their viability, morphology and phenotype was assessed. Proliferation of cells grown onto HA-coated TCP at concentrations ranging from 1 to 0.2 mg/ml was evaluated by Alamar Blue assay (Invitrogen, ThermoFisher Scientific) according to manufacturer’s instructions during a 7-day period. Cells were seeded onto HA-coated plates at the concentration of 5,000 cells/cm2. Optical density was measured at wavelengths of 570 and 600 nm. Because the culture medium was not changed during this period, the calculated percentage of Alamar blue reduction (%AB) is a cumulative value. Data are shown as mean of 3 independent biological replicates. Values are reported as %AB over time, which is associated with the presence of metabolically active cells. Data obtained from MSC grown in standard conditions are used for comparison. Cells were also monitored and imaged for morphology at the same time points. Fluctuation in the MSC-associated surface markers following the HA-treatment was assessed at different time points (1 and 3 days) by flow cytometry as reported above.

Hyaluronic acid receptor (CD44) expression

Gene expression

Quantitative RT-PCR analysis was performed to evaluate CD44 expression following HA treatment at 1, 3 and 7 days in vitro. Total RNA was isolated from HA-treated cells and explanted tissues using TRIzol reagent (Invitrogen). DNAse (Sigma) treatment followed the reaction. RNA concentration and purity were measured using a NanoDrop ND1000 spectrophotometer (NanoDrop Technologies). The cDNA was synthesized from 1 μg total RNA, using the iScript retrotranscription kit (Bio-Rad Laboratories), and quantitative PCR was run in the ABI 7500 Fast Sequence Detection System (Applied Biosystems) using commercially available master mix. The following target probe (Applied Biosystems) was used to evaluate the expression of CD44: Mm01277161_m1. Gene expression was normalized to the level of glyceraldehyde 3-phosphate dehydrogenase (Gapdh; Mm99999915_g1).

Western blotting assay

For the analysis of CD44 expression at a protein level following the treatment with HA at different concentrations (HA 1, HA 0.5, and HA 0.2) for 3 and 7 days, 1 × 106 MSC were lysed using RIPA buffer (Thermo) mixed with halt protease inhibitor cocktail (Thermo). Untreated cells acted as control. Protein concentration was determined by Bradford assay (Bio-Rad) using BSA at known concentration (1 µg/µl) to build the standard curve. Forty μg of whole cell extract were loaded onto 12% acrylamide SDS-PAGE gel (Bio-Rad) and run for 40 minutes at 25 mA constant. Proteins were then transferred to a nitrocellulose membrane for Western blot analysis. Blots were incubated with primary anti-CD44 antibody (Abcam, 1:2000) and anti-GAPDH (Abcam, 1:20,000) overnight. Then, horseradish peroxidase (HRP)-conjugated secondary antibodies were used to incubate the membrane for 1 hr. Bands were visualized using a SuperSignal West Dura Chemiluminescent Substrate (Thermo) and images were acquired with ChemiDoc XRS + System and Image Lab software (Bio-Rad). Densitometry analysis was performed using ImageJ software (NIH).

Immunocytochemistry

After 1 day from exposure, cells were fixed with 4% formaldehyde in phosphate-buffered and stained for actin cytoskeleton with FITC-Phalloidin (Life Technology) and for CD44 with Alexa Fluor® 647 anti-mouse/human CD44 Antibody (Biolegend). After washing in PBS, samples were stained with anti-fading 4’,6-diamidino-2-phenylindole (DAPI; Life Technology) for 1 minute and captured on a confocal laser microscope (A1 Nikon Confocal Microscope). Images were acquired and analyzed with NIS-Element Nikon.

Migration assay in vitro

Cell Migration Kit (including 24 well migration plate with well inserts, cotton tips, cell stain solution, and extraction solution) was purchased from Cell Solutions, Inc. HA-treated MSC (HA 1, HA 0.5, and HA 0.2) were seeded on the well inserts (1 × 105 cells/insert) and incubated for 24 hrs. Untreated cells were used as positive control. Negative controls were represented by cells kept in FBS-free media with the addition of cytochalacin D (2.5 μg/μl) to inhibit MSC migration. Migration was calculated 24 hrs after cell seeding. Media was then aspirated from the well inserts and the interior of each insert was swabbed with wet cotton tips to remove any non-migratory cells. The inserts were then kept in 400 μl of cell stain solution, incubated for 10 minutes, washed with sterile water, and allowed to air dry. Cells were imaged before 300 μl of extraction solution was added to the bottom well and incubated for 10 minutes on an orbital shaker. 100 μl of each sample from the bottom well was transferred to a 96-well plate to measure absorbance on the spectrophotometer.

Inflammation ear in vivo model

BALB/c mice ears (n = 17) were removed of hair using a depilatory cream (Nair®) and inflamed using E. coli LPS (Sigma Aldrich) in the right ear as previously described56. Animal studies were conducted following approved protocols (AUP-0515–0031) established by Houston Methodist Research Institute’s Institutional Animal Care and Use Committee (IACUC) in accordance with the guidelines of the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals. Briefly, mice were sedated and administered a single injection of 30 µg of LPS in PBS (1 mg/mL) into the base of the right ear. Similarly, 30 µL of PBS was injected into the base of the left ear. Before injection, MSC were plated onto HA 1-coated plates or in standard conditions and cultured for 24 hrs. Untreated and HA-treated MSC were stained using 10 μM DiD lipophilic tracer following manufacturer suggestions (Invitrogen) and filtered with a 40 µm cell strainer (BD). Next, a one-time, retro-orbital injection of 6 × 105 MSC per mouse (i.e., untreated and HA-treated) was administered. The mice were then imaged 24, 48, and 72 hrs following MSC injection using a Nikon A1 intravital microscope. Just prior to imaging, 40 µL of FITC-dextran (70 kDa; Sigma Aldrich) was injected retro-orbitally for delineation of vasculature. Images were then acquired using a 50-µm z-stack with a step size of 5 µm and analyzed for the MSC present in each section using NIS Elements (Nikon).

Histology and Immunofluorescence analysis

Explanted mice ears where cut by a biopsy pouch centered in the middle zone (diameter 8 mm), washed twice with PBS, embedded at the hedge in O.C.T. (Tissue-Tek O.C.T. Compound, Sakura Finetek), and instantly frozen in liquid nitrogen. 8-μm thick slides were obtained cutting ears block with a cryostat at −20 °C. For H&E staining, slides were thawed, hydrated, washed and stained with hematoxylin and eosin (Sigma-Aldrich). Immunofluorescence staining has been performed on consecutive ear sections. Briefly, slides were thawed and blocked with goat serum 5% (Sigma-Aldrich) PBS-T 1 × solution. After washing, they were incubated overnight at 4° with anti-macrophage antibody and anti- CD206 (Alexa Fluor 488 anti-mouse F4/80 and Brilliant Violet 605 anti-mouse CD206 Biolegend). Excess of the anti-neutrophil antibody was washed out with PBS 1X. Cells nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). Slides were sealed with ProLong Gold antifade reagent (Life Technologies). Images were captured with a Nikon Eclipse Ti Inverted Fluorescent Microscope.

Inflammatory genes expression

The final outcome of the system, by means the reduction in ear inflammation mediated in vivo by HA-treated MSC (HA-MSC) compared to untreated MSC (CTRL MSC) was also evaluated on the explants. The following target probes (Applied Biosystems) were used to evaluate the expression of the following markers. Pro-inflammatory markers include TNF-α (Μm00443258_μ1), IL-6 (Mm00446190_m1), and Cox2 (Mm03294838_g1). Anti-inflammatory markers include Tgf-β (Mm00441727_g1), arginase (Mm00475988_m1) and Il-10 (Mm01288386_m1). The mRNA levels of Tgf-β and Il-10 were also assessed in HA-treated MSC before injection to exclude the possibility that the changes observed in anti-inflammatory markers expression were induced by the treatment itself. Gene expression was determined by qPCR following the procedure reported above.

Statistical analysis

Statistical analysis was performed using GraphPad Instat 3.00 for Windows (GraphPad Software). Three replicates for each experiment were performed and the results are reported as mean ± standard deviation. p ≤ 0.05 was considered as significant, p < 0.01 highly significant. One-way ANOVA for multiple comparisons by Student-Newman-Keuls multiple comparison test was used.