Cell isolation and culture
The study was approved by the local institutional review board of the IRCCS Foundation Neurological Institute ‘C. Besta’, Milan Italy and informed written consent was obtained from all volunteers.
Specimens of fat from the periumbilical regions of four volunteers were collected by a lipoaspirate technique. Ad-MSCs were obtained as previously described . The cells were cultured in stem cell medium (SCM), consisting of (D)MEM/F12 supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA), 1% penicillin and streptomycin solution (Sigma-Aldrich, Basel, Switzerland) and optimized for the growth of stem cells as previously described . The Ad-MSCs were routinely seeded at 2 × 104 cells/cm2 in T75-cm2 flasks and passed weekly. For experiments, the cells were not used after six passages. Cell viability was assessed by the trypan blue dye-exclusion assay.
Flow cytometric analysis
Flow cytometry (FC) was performed on Ad-MSCs to evaluate mesenchymal, hematopoietic, endothelial and immunological markers: CD14, CD31, CD34, CD45, CD73, HLA-DR (BD Pharmingen, San Jose, CA, USA), CD105 (AbDSerotec, Raleigh, NC, USA), CD90 (Millipore, Temecula, CA, USA) and CD19 (Beckman Coulter, Milan, Italy). Briefly, 5 × 104 cells/tube were stained with fluorochrome conjugated monoclonal antibodies (mAbs) and incubated for 30 minutes at 4°C in the dark. Samples were centrifuged at 300 × g for five minutes, washed twice with PBS and fixed with 4% paraformaldehyde (PFA) (Sigma-Aldrich). Analyses were performed in a scan flow cytometer by means of Cell Quest software (BD Pharmingen). Isotype-matched mouse immunoglobulins were used as control. At least 2 × 104 events were acquired for each sample. Non-viable cells were excluded by physical gating.
Multipotent differentiation potential of Ad-MSCs before and after seeding on SF patches
According to the minimal criteria suggested by Dominici et al., Ad-MSCs were tested for their capacity to differentiate into adipocytes, chondrocytes and osteocytes, before and after seeding on SF scaffold. Briefly, to test adipogenic differentiation ability, 3.7 × 104 cells/well were plated onto micro cover glasses (Zeus Scientific, Branchburg, NJ, USA) or onto SF patch in 24 multi-well plates (Sigma-Aldrich) with 0.5 mL/well of SCM. After 48 hours of incubation at 37°C, 5% CO2, the culture medium was replaced with adipogenesis differentiation medium (ADM). After 21 days of culture, cells were washed two times with D-PBS, fixed for 30 minutes at room temperature with 4% PFA and stained with Oil Red O (Sigma-Aldrich) .
Osteogenic differentiation was obtained by incubating cells in osteogenesis differentiation medium (ODM) for 21 days. Cultured monolayers were fixed for five minutes in cooled ethanol at 70% and then processed for Alizarin Red staining (Sigma-Aldrich) .
Chondrogenic differentiation was performed using a Human Mesenchymal Stem Cell Functional Identification kit (R&D System, Minneapolis, USA) following the manufacturer’s instructions. After 21 days of incubation in chondrogenesis differentiation medium (CDM), the pellet was fixed for 30 minutes at room temperature with 4% PFA, cryoprotected with 30% sucrose (Sigma Aldrich) in D-PBS, embedded in optimal cutting temperature (OCT) compound (Sakura Finitek, Torrance CA, USA) and sectioned using a cryostat (Leica). The slides were processed for immunocytochemical detection of aggrecan. Negative controls (cells in SCM culture medium) were performed to evaluate the specificity of the antibody.
Histological and immunohistochemistry stainings were performed on Ad-MSCs-SF after 21 days of adipogenic, osteogenic and chondrogenic differentiation culture conditions in ADM, ODM and CDM, respectively. Briefly, the Ad-MSCs-SF patches were fixed for 30 minutes at 4°C in 4% PFA in 0.1 M D-PBS and cryoprotected with 30% sucrose (Sigma Aldrich) in D-PBS, embedded in OCT compound (Sakura Finitek) and sectioned on a cryostat (Leica). Oil Red O, Alizarin Red (Sigma-Aldrich) and aggrecan (1:100, R&D System) stainings were used to confirm adipogenic, osteogenic and chondrogenic differentiation capabilities.
Preparation of Ad-MSCs- SF and D-Ad-MSCs-SF patches
Sheets of electrospun SF were produced according to the method of Alessandrino et al. . Five-mm-diameter SF patches were cut by using a sterile 5-mm punch biopsy tool (KLS Martin cod. 28-240-05-07 Umkirch, Germany), placed into Petri dishes, washed with 70% ethanol solution for one hour, sterilized under UV light for six hours and then stored at 4°C until use.
Each patch was placed into a well of a 96-multiwell plate (Corning Incorporated, Corning, NY, USA) and seeded with a suspension of 3 × 104 Ad-MSCs in 200 μL of SCM. Cellularized patches were incubated at 37°C for at least seven days in a humidified atmosphere containing 5% CO2. Concomitantly, control patches with 200 μL of medium without cells were incubated under the same conditions. The medium was changed once. The decellularization procedure for the SF patches was performed by removing the culture medium, washing the patches with sterile distilled H2O at 4°C and incubating them with distilled MilliQ water for 30 minutes at 4°C. The D-Ad-MSCs-SF patches were then transferred into a new well and stored under different conditions: in distilled water at 4°C, under dry conditions at 4°C, frozen in distilled water at −20°C, or frozen under dry conditions at −20°C. D-Ad-MSCs-SF patches stored for more than seven days were not used in vivo.
Conformational analysis of untreated and D-Ad-MSCs-SF patches
Conformational analysis was performed on untreated and decellularized patches to test whether sterilization, decellularization, and different storing conditions could affect the SF structure.
Fourier transform-infrared (FTIR) spectroscopy measurements were performed on a NEXUS (Thermo Nicolet) FTIR spectrometer employing an attenuated total reflectance (ATR) accessory model, Smart Performer. All spectra were obtained with a ZnSe crystal cell as the internal reflection element. Spectra were recorded in the 400 to 4,000 cm-1 wave number range by accumulating 64 scans at a resolution of 4 cm-1, subjected to data smoothing (15 points with the Savitzky-Golay method) and normalized to the 1,452 cm-1 peak before any data processing. Each spectrum was the average of at least three spectra measured in different areas of the sample. The crystallinity index was calculated as the intensity ratio between the two amide III components at 1260 cm-1 and 1230 cm-1 (C.I. = I1260/I1230) .
Differential scanning calorimetry (DSC) measurements were performed with a DSC Q200 instrument (TA Instruments, Waters, Milan, Italy). The scanned temperature ranged from room temperature to 400°C, at a heating rate of 10°C/minute. The analysis was carried out on samples of about 2 to 3 mg weight, in open aluminum pans of 40 μL volume, under N2 gas flux.
Morphological analysis of SF, Ad-MSCs-SF and D-Ad-MSCs-SF patches
The morphology of SF, Ad-MSCs-SF and D-Ad-MSCs-SF patches was investigated by standard histology and by scanning electron microscopy (SEM) to evaluate the efficacy of cellularization and the presence of alterations in fibroin structure induced by sterilization, decellularization and different storing conditions. For histology, the samples were fixed in 10% neutral-buffered formalin, dehydrated and embedded in paraffin wax. Sections 5 μm thick were mounted on glass slides and stained with H&E. For SEM, patches were washed in 0.1 M pH 7.2 cacodylate buffer (CB), fixed with 2.5% glutaraldehyde (Sigma-Aldrich) in CB for two hours at 4°C and dehydrated in a graded series of ethanol up to absolute. Ad-MSCs-SF and D-Ad-MSCs-SF patches were placed on metal stubs, coated with gold to a thickness of 15 nm and viewed using a Philips XL30 scanning electron microscope (Center for Electron Microscopy - CUME, University of Perugia, Perugia, Italy).
Ad-MSCs-SF patches were also subjected to immunofluorescence to confirm the maintenance of the Ad-MSC phenotype after cultivation on SF. Briefly, Ad-MSCs-SF cultured for seven days on SF patches were fixed for 30 minutes at room temperature in 4% PFA. The patches were permeabilized with 0.1% Triton X-100. The following primary antibodies were applied overnight at 4°C: mouse anti-human CD44 (1:500), CD45 (1:400), CD146 (1:400) and rabbit anti-human CD49d (1:500) (all purchased from Chemicon-Millipore, Temecula, California, USA). Cells were then incubated in Cy3-, and/or Cy2-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA) at room temperature for 45 minutes and mounted with Fluorsave™ (Merck-Millipore, Nottingham, UK). The three-dimensional digital images were made using a Leica TCS SP2 AOBS (Leica Microsystems, Wetzlar, Germany) confocal laser scanning microscope.
Surgical procedure: engraftment of Ad-MSCs-SF and D-Ad-MSCs-SF patches in diabetic (Leprdb/db) mice
Procedures involving animals were conducted in conformity with institutional guidelines in compliance with the Italian guidelines for animal care (DL 116/92), the European Community Council Directive (86/609/EEC) and the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. The protocol for the use of laboratory animals was approved by the Italian Ministry of Health and by an internal ethics committee. All surgery was performed under chloral hydrate anesthesia and all efforts were made to minimize suffering.
Eight-week old Lepr db/db male mice (Charles River Laboratories, Calco, Lecco, Italy) were housed for at least one week in their home cages at a constant temperature. Mice were randomized into four groups of six each. Control no-graft mice received only saline and treated mice received SF, Ad-MSCs-SF and D-Ad-MSCs-SF patches, respectively.
Briefly, mice were anesthetized with chloral hydrate (4%, 400 mg/kg i.p) and one wound on each side of the mouse’s back midline extending through the panniculus carnosus was made with a sterile 5-mm punch biopsy tool (KLS Martin cod. 28-240-05-07 Germany). Patches were applied on the wounds and fixed to the skin with 30 μL of Hydrogel (ExtracelTM -X Maxi Hydrogel Kit, Glycosan BioSystems Inc. Alameda, CA, USA) ensuring prolonged adhesion to the wound. Mice were placed in individual home cages.
Mice were photographed postoperatively at three and ten days using a Canon high-resolution digital camera. Wound closure areas were measured as previously described . Briefly, a metric ruler was placed adjacent to the wound limit and three individual photomicrographic measurements were taken for each mouse. The percentage of wound closure (contraction) was calculated with the formula: area at postoperative day 0 - area at postoperative day x/ area at postoperative day 0 × 100. All measurements were independently evaluated by two investigators blinded to the randomization.
Specimens of mouse skin treated with control SF, Ad-MSCs-SF and D-Ad-MSCs-SF patches were collected after transplants. Skin samples were fixed in 10% neutral buffered formalin, paraffin embedded, stained with H&E and examined histologically for the following main parameters: re-epithelialization, neovascularization and dermal fiber organization.
Immunohistochemical analysis of Ad-MSCs-SF and D-Ad-MSCs-SF patches after transplantation
Tissue samples were collected 14 days after transplantation, fixed in 10% neutral buffered formalin and paraffin embedded. Immunohistochemistry was performed on 5 μm thick serial sections mounted on poly-l-lysine coated glass slides. After deparaffinization, endogenous peroxidase activity was quenched with 3% hydrogen peroxide in water. Sections were then microwaved for 15 minutes in 10 mM citric acid (pH 6.0) for antigen retrieval and treated with normal goat serum (Dako Corporation, Carpinteria, CA, USA) 1:10 for 30 minutes to prevent non-specific binding of primary antibodies. Subsequently, sections were incubated overnight at room temperature with the following primary antibodies: rabbit polyclonal anti-collagen IV antibody diluted 1:100 (Abcam, Cambridge, UK), mouse monoclonal anti-VEGF antibody diluted 1:100 (Abcam, Cambridge, UK), mouse monoclonal anti-human nuclei (HuNu) antibody diluted 1:50 (Merck Millipore, Billerica, MA, USA). The next day, after washing in PBS, the sections were incubated for 30 minutes at room temperature with the corresponding secondary biotin-conjugated antibody: goat anti-mouse immunoglobulin G (IgG) (for anti-HuNu and anti-VEGF) and goat anti-rabbit IgG (for anti-collagen IV antibody). Bound antibodies were revealed with avidin–biotin–peroxidase complex (Vector Elite kit, Vector Laboratories, Burlingame, CA, USA). The reaction was finally visualized by exposure to fresh diaminobenzidine chromogen substrate solution (Vector Laboratories). Negative controls without the primary antibody were run in parallel. Tissue observation was carried out using a light microscope (Nikon Eclipse E800, Nikon Corporation, Tokyo, Japan) connected to a digital camera (Dxm 1200 Nikon digital camera).
The scratch assay was set up with an Ibidi Culture-Insert (Ibidi GmbH, Münich, Germany). Briefly, Ibidi Culture-Inserts were placed on the bottom of wells in a 24-multiwell plate coated with 0.2 mg/mL of collagen type I solution (Sigma-Aldrich). Human KCs, DFs and HUVECs (all purchased from Centro Substrati Cellulari, ISZLER, Brescia, Italy) were seeded to a final density of 5 × 103 cell/cm2 onto an Ibidi Culture-Insert in SCM. The cells were maintained for 24 hours at 37°C and 5% CO2 to allow cell monolayer formation. Thereafter, the Ibidi Culture-Insert was removed, cells were gently washed with PBS, and 0.5 mL of SCM (without additional growth factors) was added. Next, transwells 8 μm polycarbonate membrane inserts filter (Corning Life Sciences) were placed on the well and then SF, Ad-MSCs-SF and D-Ad-MSCs-SF patches were located on the transwells and rapidly filled with 200 μL of SCM. After 24 hours or 48 hours, transwells were removed and cells were observed under a Zeiss Axiophot-2. To evaluate the reparative effects of SF patches, cells migrated into the inter-space were counted under microscopy at 20 × magnification in five random fields. Each migration test was run in triplicate.
At the end of the HUVECs scratch test, aliquots of conditioned medium (CM) were collected and, according to the manufacturer’s instructions, human FGF2, EGF (Mini ELISA Development kit purchased from Peprotech, Rocky Hill, New York, USA), TGFβ (Demeditec, Kiel-Wellsee, Germany) and VEGF (Ray Biotech, Inc, Georgia, USA) were quantified by ELISA in accordance with the standard guideline protocol supplied with the kits. The background value (<10%) of each growth factor contained in the SCM medium was assessed. Absorbance was measured at 450 nm or 405 nm with a microplate photometric reader DV990BV4 (GDV, Milan, Italy). The results were normalized with the CM obtained by the co-culture of HUVEC with SF patches and were expressed as mean ± SD of the secreted factor. The assay was repeated twice and each sample was run in triplicate.
Quantitative reverse transcriptase–polymerase chain reaction
Reverse transcriptase–polymerase chain reaction (RT-PCR) was performed on Ad-MSCs cultured on plastic and on SF patches. For the analysis of mRNA levels, 500 ng of total RNA isolated using the RNeasy kit (Qiagen, Milan, Italy) was reverse-transcribed using an iScript cDNA Synthesis Kit (Bio-Rad Laboratories). Triplicate PCRs were carried out on an iCycler iQ Real Time PCR Detection System (Bio-Rad Laboratories, Milan, Italy). Relative gene expression was calculated by a comparative method (2-ΔΔCt) using GAPDH as a housekeeping gene. Primer sequences were designed using Primer3 software.
Specimens of skin tissue were excised and analyzed for gene profile. Briefly, 500 ng of total RNA was used for each plate and was reverse-transcribed using RT2 First Strand Kit (SABioscience Corporation, Frederick, MD, USA), according to the manufacturer’s instructions. The RT2 Profiler™ PCR array for mouse wound healing (PAMM-121, SABioscience Corporation) was performed in triplicate for each sample to compare quantitative PCR (Q-PCR)–validated murine cDNA samples. Plates were run in Applied Biosystem AB 7300 real-time PCR, according to the manufacturer’s instructions. Relative gene expression was calculated using RT2 Profiler™ PCR array data analysis (SABioscience).
Rat aortic ring assay
The rat aorta ring assay was also performed to investigate the angiogenic potential of Ad-MSCs-SF- and D-Ad-MSCs-SF-derived CMs during HUVECs scratch test. The aorta ring assay was performed as previously described . Briefly, the dorsal aorta was excised from six-week-old Sprague–Dawley rats (Charles River Laboratories). Around 1 mm-thick rings were prepared. The rings were placed in a collagen solution prepared as described . Around 40 μL of collagen solution was placed in each well and then one aortic ring/well was placed into the collagen solution. The plates were then incubated in a humidified incubator at 37°C for 30 minutes to obtain collagen jellification. At the end of incubation, the wells were filled with 500 μL of endothelial basal medium (EBM, LONZA, Walkersville, MD, USA) containing control medium, or SF, Ad-MSCs-SF and D-Ad-MSCs-SF derived CMs. The plates were incubated for 10 to 12 days. Quantification of angiogenesis was obtained by taking photographs with a Zeiss microscope at 10 × magnification every three days and by counting the number of microvessels arising from the aorta rings .
The results are expressed as mean ± SD in replicate with n ≥3. One way analysis of variance (ANOVA) two-tailed test was utilized for all statistical analyses and performed with GraphPad Prism software, version 4.0. P values of less than 0.05 were considered to be significant.