MSC isolation, characterization, and culture
MSCs were isolated from discarded filters of bone marrow harvests of healthy adult human donors based on a protocol approved by the University of Wisconsin Hospital and Clinics Regulatory Committee per our published protocols [12, 26]. Isolated MSCs at passage 4 were characterized for positive and negative markers via flow cytometry, and for multidifferentiation potential as described previously [12, 26, 27]. MSCs were cultured in 75-cm2 tissue culture flasks (TPP, St. Louis, MO, USA) with Dulbecco’s Modified Eagle Medium (DMEM; Cellgro Mediatech, Inc., Corning, NY, USA), 10% fetal bovine serum (FBS), 2 mM l-glutamine, and 2 mM nonessential amino acids (NEAA) with medium changes every 3–5 days. Only MSC passages 4–8 were used in this study.
Minocycline treatment, protein extraction, and western blot
MSCs were cultured in six-well tissue culture plates (CellTreat, Shirley, MA, USA) at 300,000 wells/well in triplicate with 2 ml of MSC culture medium at 0, 50, or 100 μg/ml minocycline (Research Products International, Mt. Prospect, IL, USA) for 48 hours. MSCs were then harvested and protein was extracted using a NE-PER® Nuclear and Cytoplasmic Extraction Kit with Halt™ protease and phosphatase inhibitor cocktail (Thermo Fischer Scientific). Cytoplasmic protein extract concentrations were determined using a DC™ Protein Assay (BioRad, Hercules, CA, USA) and the presence of specific proteins in the extracts was determined using a western blot analysis of 10 μg of protein as described previously . Primary antibodies used were from Cell Signaling Technologies (Danvers, MA, USA): monoclonal rabbit α-phospho-NF-kappaB (catalog no. 3033), monoclonal rabbit α-NF-kappaB (catalog no. 8242), monoclonal rabbit α-phospho-JNK2 (catalog no. 9251), monoclonal α-JNK2 (catalog no. 9258), monoclonal rabbit α-phospho-MAPK (ERK 1/2) (catalog no. 4370), monoclonal rabbit α-MAPK (ERK 1/2) (catalog no. 4695), and monoclonal α-beta-actin (catalog no. 4970) all at 1:1000. The secondary antibody used was alkaline phosphatase-conjugated goat α-rabbit IgG (1:30,000; GE Healthcare, Little Chalfont, UK). Membranes were developed for imaging with ECF substrate (GE Healthcare) and imaged on a Storm 840 Scanner (Amersham Bioscience, Amersham, UK) with ImageQuant TL software version 7.0 (GE Healthcare).
MSCs were cultured overnight in six-well tissue culture plates (CellTreat) with glass coverslips added to the wells at 175,000 cells/well with 2 ml of MSC culture medium overnight. MSCs were then cultured with 0, 50, or 100 μg/ml minocycline for 24 hours. After treatment, media were removed and MSCs were washed three times with PBS. MSCs were then fixed for 10 minutes with 4% paraformaldehyde and washed once with PBS. The slides were blocked for 40 minutes using 4% BSA in TBS and then treated with the same primary rabbit α-phospho-NF-kappaB antibody stated earlier at 1:200 in 4% BSA in TBS at 4 °C overnight. The MSCs were washed four times for 10 minutes with TBS and then treated with goat anti-rabbit IgG Alexa Fluor 488 secondary antibody (ThermoFisher Scientific) at 1:200 in 4% BSA for 30 minutes at room temperature. MSCs were then washed twice for 10 minutes with TBS and stained with DAPI for 2 minutes, covered with a coverslip coated with VECTASHIELD® mounting medium, and imaged on an Olympus Fluoview FV1000.
NFκB phosphorylation and protein production analysis
Phosphorylation of NFκB residues was determined using a FACE™ NFκB p65 profiler (Active Motif, Carlsbad, CA, USA). Briefly, MSCs were plated at 50,000 cells/well overnight and then treated with or without minocycline for 24 hours and the protocol was continued using the manufacturer’s instructions. MSC proliferation was determined using the crystal violet stain included in the kit. VEGF, IL-6, TNF-α, and MCP-1 analysis was carried out using a DuoSet® ELISA kit system (R&D Systems, Minneapolis, MN, USA). LL-37 analysis was performed using an ELISA kit (Hycult Biotech, Uden, the Netherlands). Complement (C5, C5a, C3, C3b) analysis was performed using respective ELISA kits (Abcam, Cambridge, MA, USA). For VEGF analysis, MSCs were cultured in 24-well tissue culture plates (CellTreat) at 100,000 cells/well in triplicate with 1 ml of MSC culture medium at 0, 50, and 100 μg/ml minocycline with or without 10 μM of the NFκB inhibitor PDTC (BioVision, Milpitas, CA, USA) for 24 hours prior to analysis. For LL-37 production, a treatment condition was used where MSCs were stimulated with IFNγ (100 ng/ml; Sigma Aldrich) for 24 hours .
MSC cocultures with Staphylococcus aureus
MSCs were cultured overnight in 48-well tissue culture plates (CellTreat) at 50,000 cells/well with 0.5 ml of MSC culture medium. After overnight culture, media were removed and replaced with either fresh MSC media alone, fresh MSC media at 100 μg/ml minocycline, or fresh MSC media at 80 μg/ml vancomycin and cells were cultured for 24 hours. SA (strain #29213; ATCC, Manassas, VA, USA) culture vials stored at −80 °C were thawed, plated on trypticase soy agar (TSA; Becton Dickinson, Sparks, MD, USA) Petri dishes (Fischer Scientific), and cultured as described previously . The SA culture was allowed to proliferate in a suspension of tryptic soy broth (TSB; Becton Dickinson) on a rotator at 37 °C until the optical density at 600 nm reached 0.1 (1 × 104 CFU/ml) as measured by the spectrophotometer (Nanodrop 2000x UV-vis Spectrophotometer; Thermo Scientific). After MSC treatments, the media were removed and each well was washed three times with 0.5 ml of PBS and each well was replaced with fresh MSC media without minocycline. Immediately following, 20 μl of the SA suspension (200 CFU) was added to the MSC cultures and incubated at 37 °C for 6 hours. Cultures of MSC media alone and TSB alone with 200 CFU SA were included as controls. After 6 hours of culture, 50 μl of the coculture supernatant was removed and diluted serially (1:10) for colony forming ability enumeration as described previously . Immediately following cocultures, supernatants were collected and spun at 12,000 × g for 10 minutes. Supernatants were removed from bacterial cell pellets and placed in a clean Eppendorf tube and stored at −20 °C until cytokine analysis. LIVE/DEAD® stain was applied to the MSCs remaining on the wells, quantified using a FLUOstar Omega plate reader, and imaged using a Nikon Eclipse TE300 microscope. Cytokine analysis of supernatants was performed using ELISA kits as already mentioned. Another experiment included MSC/SA coculture incubation with a mouse anti-human IL-6 antibody at 100 μg/ml (PeproTech, Rocky Hill, NJ, USA) during SA cocultures and were analyzed similarly . MSCs were also cultured against SA in a transwell system to prevent cell-to-cell contact. MSCs were plated at 175,000 cells/well in the bottom of a six-well 0.4-μm transwell plate (Corning) with 2 ml of culture medium and cultured overnight. MSCs were then treated with or without minocycline for 24 hours and washed three times with PBS. SA was grown as already stated and 600 CFU (60 μl) of SA was added to 1 ml of culture medium in the top transwell and cocultured for 6 hours. After coculture, SA colony forming analysis was done as already stated and SA attachment to the top transwell surface was carried out using a gentian violet stain protocol as described previously .
Internalization of Staphylococcus aureus by MSCs
MSCs were cultured overnight in six-well tissue culture plates (CellTreat) with glass coverslips added to the wells at 175,000 cells/well with 2 ml of MSC culture medium. MSC treatment with or without minocycline and SA culture preparation was performed as already stated, and then 60 μl of the SA suspension (600 CFU) was added to the MSC cultures and incubated at 37 °C for 4 hours. After coculture, media were removed and the wells were washed three times with PBS. Cells were fixed with 4% paraformaldehyde for 10 minutes, and then washed with PBS. Cells were then stained with Vacnomycin BOPIDY® FL Conjugate (ThermoFisher Scientific) at 1 μg/ml with ActinGreen™ 488 ReadyProbes™ (ThermoFisher Scientific) in PBS for 10 minutes at room temperature and washed with PBS. Coverslips were removed from the wells and stained with DAPI for 2 minutes, covered with a coverslip coated with VECTASHIELD® mounting medium, and imaged on an Olympus Fluoview FV1000. Total present SA and MSC-internalized SA were counted using ImageJ software (National Institutes of Health, MD, USA). MSCs were also cocultured in 48-well plates as already stated but including conditions with MSCs treated with minocycline in addition to the internalization inhibitors cholorquine (20 μM) and 3-methyladenine (5 mM) .
Genetic expression analysis
MSCs were washed with PBS three times after MSC/SA cocultures in 48-well plates. MSC RNA was isolated by lysing MSCs in culture with 0.5 ml of TRIzol reagent (ThermoFisher Scientific) per well and the protocol was continued as described previously . RT-qPCR was performed on synthesized cDNA using a StepOnePlus Real-Time PCR System (ThermoFisher Scientific) using TaqMan Expression Assay Probes for IL-6 and GAPDH. Data pertaining to mRNAs were collected quantitatively and the CT cumber was corrected by CT readings of the corresponding internal control of GAPDH.
In-vivo application of hydrogels in Staphylococcus aureus-inoculated full-thickness wounds
All animal experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Wisconsin-Madison. Female Sprague–Dawley rats (250–300 g, 12 weeks old) were grouped into three different cohorts (n = 3 rats per treatment type for each time point). Sprague–Dawley bone marrow-derived MSCs were similarly isolated, cultured, and characterized accordingly [26, 27]. MSC passages 4–6 were used for this in-vivo study. A day before the experiment, silicone O-rings (McMaster-Carr, Atlanta, GA, USA) were placed under ultraviolet light in a biosafety cabinet for at least 20 minutes and planktonic SA was grown in TSB to OD600 = 0.1 as stated earlier, aliquoted in 1 ml volumes in Eppendorf tubes, and frozen at −80 °C for overnight storage. On the day of the experiment, rat eyes were lubricated with Lubrifresh™ (Amazon, Seattle, WA, USA) and treated with buprenorphine (0.05 mg/kg). The rat dorsum was shaved and scrubbed with Betasept (4%; Amazon) and sterile saline. Two silicone O-rings were glued to the shaved dorsum of the rats using CrazyGlue Advanced Gel (Elmer’s Products Inc., Westerville, OH, USA) by firmly applying pressure for 2 minutes. The silicone O-rings were then sutured (Nylon 4.0; Ethicon, Sun Prairie, WI, USA) to inhibit healing by contraction, which is the accepted method for examining the healing of full-thickness wounds in rats . Full-thickness wounds were created (two per rat) on the dorsum of the shaved rat using 8-mm biopsy punches (Miltex GmbH, York, PA, USA) while under isoflurane anesthesia (1.5% isoflurane, 1.5% oxygen). The wounds were imaged and then inoculated with 10 μl of thawed planktonic SA (OD600 = 0.1) and allowed to dry for 15 minutes. Hydrogels were formulated as described previously, applied to the wounds (100 μl), and subsequently polymerized as stated earlier with either: no antimicrobials or MSCs (no encapsulation); 1 × 106 MSCs/ml (MSCs); or 1 × 106 MSCs/ml + 50 μg/ml minocycline + 40 μg/ml vancomycin + 10 μg/ml linezolid (MSCs + mino. + vanco. + lin.) . Hydrogels loaded with one or two drug combinations were not included to minimize the number of animals while maximizing the utility of the data obtained, and the direct effect of each entity promoting tissue healing via induction of MSC mechanisms was investigated in our proceeding in-vitro analysis. Each rat was randomly assigned to a treatment, with both wounds on the same animal receiving the same treatment. The treated wounds were allowed to dry for 2 minutes, then covered with Tegaderm™ film (3 M Healthcare, Neuss, Germany) and wrapped with cling gauze followed by Vetrap™ (3 M) tape. At 1 and 3 days after treatment application, rats were euthanized with Beuthanasia (0.2 ml/kg; Schering-Plough Animal Health Corp., Union, NJ, USA). The wounds were then imaged, before one wound bed per rat was harvested and put into 1 ml of sterile PBS with sterile beads for SA CFU enumeration, and one wound bed per rat was harvested, fixed in 10% neutral buffered formalin for 48 hours, paraffin embedded, and H&E stained [14, 33]. Wound beds collected for SA CFU enumeration were homogenized using a bullet blender (three 5-minute cycles; Next Advanced, Averill Park, NY, USA) and then diluted serially and cultured on agar plates for colony counting as already stated. Photomicrographs were taken of histological sections from H&E staining and the epidermal thickness of the healing wound tissue with clearly demarcated epidermal and dermal tissue boundaries was measured by averaging five epithelialized keratinocyte widths from randomized locations including the central region of the wound and areas adjacent to the wound margin.
All assays were conducted with at least three replicates per condition. Two-way ANOVA for multiple comparisons was used with a Bonferroni test assessment to determine p values for all assays with three or more conditions, and an unpaired two-tailed Student’s t test was used to determine p values for all assays with only two conditions. p < 0.05 was considered statistically significant. All analyses were performed with GraphPad Prism 5.0. Error bars represent the standard deviation of the specific group mean.