MeHA synthesis
Excessive molecular weight hyaluronic acid sodium salt (HAs, Mw = 1.5–1.8 106 Da from Streptococcus equi, Sigma Aldrich, Milan, Italy) was modified to graft photoactive polymerizable teams by reacting with methacrylic anhydride (Me, Sigma Aldrich Milan, Italy) as beforehand described [31, 65, 66]. Briefly, 1 g of HAs was dissolved in 10 mL of pure water (H2O, Carlo Erba, Cornaredo, Italy) and stirred at room temperature (RT) for full dissolution. MeHA was obtained by reacting the first hydroxyl teams (-OH) with Me at 4 °C, conserving the pH between 8 and 9 utilizing a sodium hydroxide resolution (NaOH, Sigma Aldrich, Milan, Italy). An extra of 30 mol% ME per (-OH) was used. The response was carried out for 12 h and it was stopped by precipitating MeHA into chilly anhydrous ethyl alcohol (EtOH, Sigma Aldrich, Milan, Italy). The supernatant was recovered by vacuum filtration. The remoted MeHA polymer was solubilized in pure water then dialyzed towards distilled water for five days and freeze-dried (LaboGene’s CoolSafe 55 − 4 PRO, Bjarkesvej, Denmark).
1 H nuclear magnetic resonance (NMR)
1 H Nuclear magnetic resonance (NMR, Bruker AVIII 400HD, Fällanden, Swiss) was employed to evaluate the success of the functionalization response. MeHA (5 mg/mL) was utterly dissolved in deuterium oxide (D2O) by utilizing a vortex mixer, and it was transferred into NMR tubes. The info have been collected at a frequency of 400 MHz. Section and baseline corrections have been utilized earlier than acquiring the areas (integrals) of purely absorptive peaks. The presence of methacrylate moieties on the HAs spine was confirmed by the peaks at 5.7 and 6.2 ppm.
3D printing of MeHA
Freeze-dried MeHA was dissolved in diH2O at a focus of 4% (w/v) containing 0.1% (w/v) 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959, Sigma Aldrich, Milan, Italy), as beforehand reported [31]. Briefly, 3D printing was carried out on “Rokit Invivo 4D2” (Rokit Healthcare Inc., Seoul, Korea) utilizing 1.80 firmware. The enter printing mannequin was sliced with a grid sample utilizing New Creator Okay 1.57.70. The printing velocity was set at 6 mm/s. The dispenser temperature was set at 15 °C, whereas the mattress was set at 0 °C. A needle of 0.6 mm, a layer thickness of 0.4 mm and a fill density of fifty% have been used to construct porous patches measuring 50 mm × 50 mm × 3 mm. Throughout printing, UV mild (λ: 365 nm) was used to crosslink the biomaterial ink enhancing the mechanical properties and avoiding the collapse of the constructions. After printing, 3D porous constructions have been additionally post-crosslinked in a UV cupboard (Analytik Jena UVP crosslinker, CL-1000, λ: 365 nm) for 10 min. Lastly, the patches have been freeze dried for twenty-four h and saved at -80 °C earlier than utilizing.
Dynamic mechanical evaluation
A-Q800 (TA-Instrument, New Fort, DE, USA) was employed to evaluate the mechanical properties of 3D printed constructions. The frequency was various between 0.5 and 5 Hz, utilizing an amplitude of 100 µm in compression, a preload of 0.001 N and a power observe of 125%. The assessments have been carried out in a closed chamber in a moist state at room temperature. Elastic modulus (E’) is reported as imply worth ± SD, n = 5.
Swelling habits
Dried 3D patches have been weighted (w0) scaffolds and positioned in 5 mL of sterile medium that was supplemented with antibiotics and allowed to swell underneath physiological situations for as much as 5 h (pH = 7.4, T = 37 °C). The swollen hydrogels have been then eliminated at particular time intervals, instantly blotted on filter paper to take away the superficially absorbed water, the burden was measured (wt), and the samples have been then put again into the answer. Equation (1) was used to calculate the swelling ratio (Q):
$$Q{textual content{ }} = {textual content{ }}left( {{w_t} – {w_0}} proper)/{w_0}$$
(1)
Outcomes have been reported in Fig. 5G as imply worth ± SD, n = 5.
Scanning electron microscopy
The 3D printed MeHA patch was noticed by scanning electron microscopy (SEM, FEI Quanta 200 FEG, Hillsboro, OR, United States). Earlier than the evaluation, 3D patches have been ready as described earlier than, frozen, lyophilized for 48 h. The lyophilized construction was coated with an ultrathin layer of Au/Pt by utilizing an ion sputter after which noticed by SEM.
Incorporation and launch of MSC-sEVs from MeHA dressing
The 3D printed MeHA patches have been positioned right into a 24-well tissue tradition plate and loaded with 20 or 120 µg of MSC-sEVs resuspended in 100 µL PBS. Management situation (0 µg) was MeHA patches loaded with 100 µL PBS. The discharge profile was evaluated by inserting the loaded dressing in a 24-well plate containing serum-free tradition medium for as much as 11 days. Then, samples have been withdrawn at chosen time factors and saved at -80 °C till all samples have been collected. The discharge of sEVs have been quantified utilizing the BCA Protein Assay Package (Thermo Fisher Scientific) in response to the producer’s directions. Knowledge reported as imply worth ± SD, n = 3.
MSCs tradition in bioreactor
Commercially out there MSCs (Human Adipose-derived Mesenchymal Stem Cells; ScienCell Analysis Laboratories, Inc., CA, USA) have been seeded at a density of 5 × 105 cells into handheld 3D bioreactors (VITVO®; Rigenerand Srl, Medolla, MO, Italy) [27] as much as 14 days. The bioreactor is a closed system with two optical clear membranes that permit gasoline alternate. Internally, a 400 μm matrix of biocompatible polyester separates two chambers. Every chamber has a port performing as enter or output relying on the media stream. By enter port, liquid can enter the primary chamber, cross via the 3D matrix, fill the second chamber, and exit via the outlet port. Thus, the 3D matrix works as a filter that retains cell and as substrate for cell development. MSCs at passage 3 have been resuspended in 1.4 mL of tradition medium (DMEM, excessive glucose, no glutamine, no phenol crimson; Thermo Fisher Scientific, Waltham, MA, USA) accomplished with exosome-depleted fetal bovine serum (Capricorn Scientific GmbH, Ebsdorfergrund, Germany) and injected into bioreactors by a syringe via one of many two ports offered. Then, bioreactors have been positioned in an incubator set at 37 °C and 5% CO2. Recent medium (1.5 mL) was injected via each ports each 48 h and an equal quantity of CM was collected. In 14 days, about 20 mL CM was collected for every bioreactor. At every harvest, CM was centrifuged at 300xg for 10 min at RT to remove lifeless cells, filtered with a 0.22 μm filter to take away cell particles.
Calcein AM staining
The expansion and viability of MSCs into bioreactors have been detected by staining with 1 µM Calcein AM (Life Applied sciences, Chagrin Falls, OH, USA) for 20 min at 37 °C and 5% CO2. After eradicating the surplus probe, a z-stack (z-step 2.5 μm) was acquired with a confocal microscopy (Nikon A1 confocal microscope, Nikon Company, Tokyo, Japan) outfitted with a 10X goal.
Cell stream cytometry
MSCs have been incubated with the next fluorescent monoclonal mouse anti-human antibodies: CD44 FITC (BD Biosciences, San Jose, CA, USA); CD73 APC (eBioscienceTM, Thermo Fisher Scientific); CD90 PE (eBioscienceTM); CD105 PE (BD Biosciences); CD14 PE (eBioscienceTM); CD34 FITC (eBioscienceTM); CD45 APC (eBioscienceTM); HLA-DR FITC (eBioscienceTM). After two washes, fluorescent cells have been detected with Attune NxT stream cytometer (Thermo Fisher Scientific). Knowledge have been analyzed utilizing Attune NxT software program (Thermo Fisher Scientific). Experiments have been carried out in triplicate.
sEV isolation
The clarified CM was loaded onto Amicon Extremely-15 100 kDa centrifugal gadgets (Amicon Extremely-15 centrifugal ultrafilters with Ultracel-PL PLGC membrane, 100 kDa; Millipore, MA, USA), beforehand sterilized with 70% ethyl alcohol (Sigma-Aldrich, Saint Louis, MA, USA), and centrifuged at 2000xg for 20 min at 4 °C. The filtrate was washed with sterile PBS (Euroclone, Milan, Italy) via an additional centrifugation at 2000xg for 20 min at + 4 °C. The hMSC-SEVs have been lastly recovered from the filtering unit, quantified by Pierce™ BCA protein assay equipment (Thermo Fisher Scientific), and saved in small aliquot at -80 °C instantly.
Nanoparticle monitoring evaluation
Nanoparticle monitoring evaluation (NTA) makes use of laser mild scattering and Brownian movement to find out EVs measurement and focus. Measurement of particle measurement and particle measurement distribution was carried out with Nanosight NS300 (Malvern, UK) instrument outfitted with a 488 nm laser. All samples have been diluted in filtered PBS to a ultimate quantity of 1 mL. Ideally suited measurement concentrations have been discovered by pre-testing the best particle per body worth (20–100 particles/body). For every measurement, 5 1-min movies have been captured underneath temperature 25 °C and syringe pump velocity 30. Knowledge are represented as averaged finite observe size adjustment (FTLA) focus / measurement.
Transmission electron microscopy (TEM)
For TEM acquisition, the protocol described elsewhere was comply with [67]. Briefly, the sEVs have been fastened in a 2% glutaraldehyde resolution in phosphate buffer (ratio 1:1). The sEVs have been then deposited, rinsed, and stained with heavy metallic compounds onto a gridded slide in response to the usual protocols. The slide was visualized with a TEM Zeiss EM 910 instrument (Zeiss, Oberkochen, Germany).
Exosome antibody array
The immunoblotting evaluation of sEV particular markers have been carried out utilizing the business Exo-Examine™ exosome antibody array (Methods Biosciences, USA) in response to the producer’s directions. The array accommodates eight recognized sEV markers, together with CD63, CD81, ALIX, FLOT1, ICAM1, EpCam, ANXA5, and TSG101; 4 controls, together with two optimistic controls (HRP Detection), clean spot (background management) and GM130 cis-Golgi marker, which screens for any mobile contamination. Briefly, 50 µg of MSC-sEVs was lysed and labeled for 30 min with fixed mixing. The labeled samples have been washed and blocked with the blocking buffer. Array membrane was incubated with labeled lysate/blocking buffer combination at 4 °C in a single day on a rocker. The following day, the membrane was washed and incubated with a detection buffer for 30 min at RT on a shaker. The membrane was washed and the chemiluminescence was developed with Readability Western ECL substrate (Bio-Rad, USA). Membrane array was developed on the chemiluminescence imaging system (ChemiDoc, Bio-Rad). Experiments have been carried out in triplicate.
sEV stream cytometry
sEVs have been harvested with exosome-Human CD81 Movement Detection (from cell tradition) (Thermo Fisher Scientific), in response to the producer’s directions. Intimately, 50 µL of sEV suspension was added to a tube containing 20 µL of CD81 magnetic beads, beforehand washed with 500 µL of Assay Buffer containing 0.1% bovine serum albumin (BSA, Sigma-Aldrich) in PBS, and incubated at 4 °C in a single day underneath stirring (650 rpm/min). After incubation, the bead-bound sEVs have been remoted with the MagnaRack magnetic separator (Thermo Fisher Scientific) and washed twice with of Assay Buffer. Remoted CD81-positive sEVs have been then labeled with mouse anti-human CD63 PE (eBioscience™), CD81 PE (BD Biosciences), CD73 APC (eBioscience™), or CD90 PE (eBioscience™). After 1-h incubation at RT protected against mild on an orbital shaker (1000 rpm/min), the bead-bound sEVs have been washed twice and suspended in Assay Buffer. Two controls have been carried out: PBS (car) and ultrafiltrated exosome-depleted medium have been stained as an alternative of sEVs. Knowledge have been collected with Attune NxT stream cytometer (Thermo Fisher Scientific) and analyzed utilizing Attune NxT Software program v2.5 (Thermo Fisher Scientific). Experiments have been carried out in triplicate and the info characterize the common.
sEV internalization
sEVs or PBS (adverse management) have been stained with PKH67 (PKH67 Inexperienced Fluorescent, Sigma-Aldrich) for 20 min at 37 °C, as beforehand described [18]. The surplus unincorporated dye was faraway from the labeled options by utilizing Exosome Spin Columns (MW 3000) (Thermo Fisher Scientific), following the producer’s directions. Then, 1 × 104 dermal fibroblasts/cm2 (ATCC, MA, USA) or 1 × 104 endothelial cells/cm2 (HUVEC; ThermoFisher Scientific) have been incubated with the labeled sEVs or PBS for 3, 6, and 24 h in DMEM (EuroClone) or in Medium 200 PRF (M200PRF, ThermoFisher Scientific) with out dietary supplements), respectively. After incubation, cells have been washed, and the nuclei have been stained with Hoechst 33,342 (ThermoFisher Scientific) for 10 min at RT. Lastly, cells have been fastened with 4% paraformaldehyde, mounted with ProLong™ Glass Antifade Mountant (Thermo Fisher Scientific), then noticed with a laser scanning confocal microscopy system (Nikon A1 confocal microscope, Nikon Company, Tokyo, Japan) outfitted with a 63X goal. Experiments have been carried out in triplicate.
RNA isolation, cDNA synthesis and real-time PCR
Complete RNA was remoted from MSC-sEVs-treated cells with the RNeasy Mini Package (Qiagen, Hilden, Germany). Complete RNA from pores and skin biopsies with RNeasy Fibrous Tissue Mini Package (Qiagen, Hilden, Germany). The RNA high quality and focus of the samples was measured with the NanoDrop™ 2000 (Thermo Fisher Scientific). For the first-strand cDNA synthesis, 500 ng of RNA have been reverse-transcribed utilizing the QuantiNova™ Reverse Transcription Package (Qiagen) in a SimpliAmp™ Thermal Cycler (Thermo Fisher Scientific). Actual-time PCR was carried out utilizing the designed primers (Desk S1 and Desk S2) at a focus of 700 nM and QuantiNova SYBR Inexperienced PCR (Qiagen) on a StepOnePlus™ Actual-Time PCR System (Thermo Fisher Scientific). Knowledge evaluation was carried out utilizing the 2ΔΔCt methodology [68], and introduced as imply fold change of six measurements.
Animal experiments
Genetically diabetic male mice db/db (pressure C57BL/KsJ-m+/+Leprdb, Charles River Laboratories -Calco-Lecco) have been housed underneath normal situations. Blood glucose was measured (Contour XT, Bayer, Basel, Switzerland), and solely mice with blood glucose ≥ 250 mg/dL have been included within the examine. Underneath anesthesia (isoflurane 3% plus 2 L/min O2), stress ulcers have been induced within the dorsal again utilizing two magnetic disks of 12 mm diameter (anisotropic ferrite) and a thickness of 5.0 mm, with a mean weight of two.4 g and 1000 G magnetic power (Algamagnetic, Italy), as beforehand described [33, 69]. Briefly, a pores and skin fold was raised and positioned between the 2 magnets to generate a compressive stress of fifty mm Hg [34]. Three ischemia-reperfusion (I/R) cycles have been used, every single I/R cycle consisting of a interval of 12 h of magnet placement adopted by a relaxation interval of 12 h with out magnet. A surgical wound curettage was carried out on day 3 to take away the ischemic pores and skin and eschar. After curettage, mice have been randomly assigned to the therapy teams (N = 5 in every group). Topical software of the drugs started after curettage, weekly renewed and dressed with Tegaderm (3 M Well being Care; Tegaderm Roll, St Paul, MN, USA). After curettage and earlier than every dressing renewal, each wounds have been photographed and wound areas measured utilizing Nis-Parts AR 3.2 software program (Nikon Company, Tokyo).
Histology, immunofluorescence and picture evaluation
Mice have been sacrificed at 7, 14 or 21 days in response to the experimental goal. Pores and skin samples have been rapidly dissected and stuck in 4% (v/v) paraformaldehyde resolution and picric acid–saturated aqueous resolution in 0.1 M Sörensen’s phosphate buffer (pH 7.4). Proper wounds have been embedded in paraffin, sectioned at 4 μm, and stained with hematoxylin and eosin (H&E). For immunofluorescence, left wounds have been fastened as above for twenty-four h, washed for 48 h in 0.1 M phosphate buffer 5.0% sucrose and rapidly frozen. Cryostat Sect. (14 μm thick, HM550 Microm, Bio-Optica) have been incubated in a single day with main antibody anti-laminin (rabbit, 1:200 dilution; SIGMA Aldrich) and anti PGP-9.5 (rabbit, 1:350 dilution; Proteintech) at 4 °C in a damp chamber. After rinsing, sections have been incubated with secondary antiserum Cy2 Donkey anti-Rabbit IgG (Jackson Immunoresearch), rinsed and mounted in glycerol containing 1,4-phenylendiamine (0.1 g/L). Immunofluorescence pictures have been taken by a Nikon Eclipse E600 microscope outfitted with the Q Imaging Retiga-2000RV digital CCD digital camera (Q Imaging, Surrey, BC, Canada) and a motorized z-axis stage. Evaluation was carried out utilizing the Nis-Parts AR 3.2 software program, by making use of the identical process to all pictures underneath comparability. The immunoreactive space was calculated as space/fraction (proportion of immunoreactivity over 400 × 300 μm space). For morphological evaluation, 5 pictures and two ranges/animal have been sampled on the heart of the repaired ulcer. All analyses have been carried out blindly. Epidermal thickness was decided on the equator of the lesioned space by H&E staining on histologic sections in the identical space. The imply worth of 5 measurements/part and three sections per animal was used for the statistical evaluation.
Statistical evaluation
The outcomes have been expressed as imply ± SD and analyzed by GraphPad Prism software program. One-way evaluation of variance (ANOVA) and Pupil’s t-test have been used to guage the statistical significance (p < 0.05).
