Supplies
RHC was purchased from Jiangsu JL and Biotech Company (China). The cell culture-related supplies, together with Dulbecco’s modified Eagle’s medium (DMEM), Fetal Bovine Serum (FBS), Penicillin-Streptomycin (PS), and trypsin have been from Gibco (USA). Calcein AM (KGMP012-1) and propidium iodide (PI, KGA214) have been obtained from KeyGEN BioTECH. The cell Stay-Useless assay equipment, CCK-8 have been suplied by Thermo fisher (USA). IL-1β (Gb11113), IL-6 (Gb11117) and CD31 (Gb113151) antibodies have been obtained from Servicebio expertise Co., Ltd. All chemical supplies not talked about have been procured from Sigma-Aldrich (USA). 3T3 cells and HUVECs have been supplied by the Cell Financial institution (Shanghai). Six-week-old male Sprague–Dawley (SD) rat was sourced from the Mannequin Animal Analysis Heart of Nanjing College. Animal experiment acquired approval from the Animal Experiments Moral Committee of Nanjing drum tower hospital.
Preparation of RHCMA
Freeze-dried human recombinant collagen was dissolved in DPBS (Hyclone) to realize a ten% focus, adopted by stirring for 1 h at room temperature. Subsequent, MA (2 mL) was incrementally added into 40 mL of this 10% collagen answer, with steady stirring at 26 °C for an extra hour to advertise methacrylation. The response was terminated by the introducing 30 mL of DPBS. After that, the ensuing answer was dialyzed in deionized water for five–7 days. Eventually, dialyzed answer was lyophilized to provide RHCMA sponge. RHCMA and the photoinitiator, lithium 2-hydroxy-2-methyl-1-phenylpropane (HMPP), have been dissolved in DPBS after which photopolymerized below UV mild for 60 s to organize RHCMA hydrogels at completely different concentrations (10%, 12.5%, 15%, and 17.5%). To guage gelation properties, optical footage of the hydrogel was captured, together with these of the ten% RHCMA previous to and following gelation.
MSC-exos isolation and identification
MSCs have been sourced from human adipose tissue, bone marrow, and umbilical wire, supplied by the Stem Cell Heart of Jiangsu Province. These MSCs have been cultured at roughly 70% confluency. Subsequent, they have been transferred to exosome-depleted media and cultured for 48 h. The supernatants from these cultures have been collected and subjected to ultracentrifugation to isolate exosomes. The ensuing exosome pellets have been resuspended in PBS and saved at -80 ℃ for additional use. The freshly ready exosome pattern have been noticed utilizing TEM. Dimension distribution and zeta potential was assessed utilizing dynamic mild scattering (Malvern).
Western blot evaluation
MSCs and their purified exosomes of three distinct varieties have been first diluted 1:4 with 5× protein loading buffer, then heated to 99.9 °C for roughly 6 min. The protein samples have been separated utilizing 10% SDS-PAGE, transferred to PVDF membranes. Following switch, which have been blocked with nonfat milk after which washed thrice with TBST buffer, every wash lasting 10 min. The membranes have been then incubated in a single day at 4 °C with major antibodies: rabbit anti-TSG101, mouse anti-CD63, and GAPDH for loading management. Detection was carried out utilizing HRP-conjugated secondary antibodies particular to both mouse or rabbit, protein bands have been visualized with an Odyssey infrared imaging system.
MSC-exos loading and launch
In our examine, MSC-exos labeled with DID have been employed. The preparation concerned combining DID-labeled MSC-exos (1 mg/mL) with RHCMA answer, and photoinitiator, which was then positioned right into a cylindrical mould and subjected to ultraviolet mild polymerization. Subsequently, the DID-labeled MSC-exos@RHCMA have been submerged in PBS buffer at room temperature. At specified intervals—1, 3, 6, 9, 12, 18, 24, 36, 48, 60, and 72 h—the buffer was sampled, with an equal quantity of contemporary medium added. The discharge kinetics of the DID-labeled MSC-exos have been tracked at every interval, with the preliminary content material within the hydrogel set to 1.
Cell cultures and mobile uptake
3T3 and HUVEC cells have been cultured in high-glucose DMEM supplemented with 10% fetal bovine serum. Cells have been maintained in a 37 °C incubator with a 95% air and 5% CO2 environment, and the tradition medium was changed each two days till the cells reached the specified confluence. For the evaluation of mobile uptake, MSC-exos have been labeled with DID (crimson, 1 mM). HUVECs have been plated on 24-well glass plates and subsequently uncovered to the DID-labeled MSC-exos at 10, 20, and 40 µg/mL concentrations. After incubation, these cells have been washed with PBS and glued with paraformaldehyde at room temperature for 10 min. The cytoskeleton was visualized utilizing Phalloidin-iFluor 488, whereas cell nucleus was counterstained with DAPI for 10 min. Exosome uptake was assessed by way of confocal microscopy.
Cytotoxicity evaluation
Cytotoxicity was evaluated utilizing a dwell/lifeless staining equipment. RHCMA extracts at concentrations of 10%, 12.5%, 15%, and 17.5% have been ready by immersing the supplies in sterile PBS for 3 days. 3T3 cells, grown to roughly 70% confluence, have been then uncovered to those extracts, in addition to to PBS as a management, in cell incubator for twenty-four h. The cell therapies included PBS, and RHCMA extracts at 10%, 12.5%, 15%, and 17.5% concentrations. After incubation, cells have been stained with Calcein-AM and propidium iodide (PI) to evaluate cell viability. Fluorescence microscopy (Olympus) was used to visualise the staining patterns, and ImageJ software program was utilized to quantify cell survival charges throughout the completely different remedy teams.
Cell proliferation analysis with CCK-8 equipment
Cell proliferation on hydrogels was assessed using Cell Counting Package-8 (CCK-8). Hydrogels, every with a diameter of 6.5 mm and a peak of 1 mm, have been fabricated utilizing a customized mould and subsequently positioned right into a 96-well plate. 3T3 cells have been seeded on both the hydrogel surfaces or customary cell tradition plates (n = 6) and cultured for 1, 2, and three days. Following these durations, cells have been uncovered to CCK-8 answer for two h in cell incubator till the answer turned orange. Absorbance at 450 nm was then measured with a microplate reader to quantify the extent of cell proliferation, with the clean management subtracted. Every pattern was examined in triplicate.
cDNA synthesis and quantitative RT-PCR evaluation
Complete RNA was extracted from the cells utilizing the TRIzol reagent in keeping with the producer’s protocol. The RNA focus and purity have been decided, and primers have been designed utilizing Primer Premier 5.0 software program (sequences supplied in Desk 1). cDNA was synthesized utilizing a One-Step RT-PCR equipment following the producer’s directions. GAPDH was used as an inner management gene. Quantitative PCR (qPCR) was carried out on an ABI Vii7 real-time PCR system. The response situations have been as follows: 95 °C for 30 s (preliminary denaturation); 95 °C for 10 s (denaturation), 60 °C for 30 s (annealing), for 40 cycles; adopted by a ultimate extension at 72 °C for 10 min. Relative gene expression ranges have been calculated utilizing the two−ΔΔCT methodology.
Tube formation assay
To guage angiogenesis inside a co-culture system, a tube formation assay was performed [41]. Initially, 50 µL of Matrigel have been distributed into every nicely of a 96-well plate and allowed to solidify in a cell incubator for 30 min to permit gel solidification. Subsequently, 1 × 104 HUVECs have been seeded on every nicely. After an preliminary medium aspiration, the wells have been replenished with serum-free DMEM. Every nicely acquired therapies of PBS, RHCMA loaded with BMSC-exos, RHCMA loaded with ADSC-exos, or RHCMA loaded with ucMSC-exos. After 12 h of co-incubation, capillary-like tube formation was visualized and documented utilizing an optical microscope.
Migration assay
To guage mobile migration, a wound scratch assay was employed as beforehand described [42, 43]. HUVECs have been plated in six-well tradition plates at a density of 1 × 105/mL and allowed to succeed in confluence. Following a 6-hour hunger interval, a scratch was launched into the cell monolayer utilizing a 200 µL pipette tip. Subsequent to the elimination of particles, these wells have been replenished with contemporary DMEM containing 0.25% FBS. Cells have been handled with the required situations, and after 12 h of incubation, pictures of the HUVECs in every group have been captured utilizing a microscope outfitted with a digicam. The preliminary wound diameter was measured, and wound closure charges have been quantified utilizing ImageJ software program.
In vivo therapeutic take a look at
For the in vivo therapeutic analysis, we established a full-thickness excisional wound mannequin in Sprague-Dawley rats as beforehand described [44]. Beneath anesthesia, a 1 cm round wound was created on the dorsal pores and skin of every rat. After that, the rats have been randomly divided into 5 teams of 4 every [45]. The primary group was handled with PBS utilized on to the wound; the second group acquired RHCMA movies positioned on the wound beds; and the remaining three teams acquired the BMSC-exos@RHCMA, ADSC-exos@RHCMA and ucMSC-exos@RHCMA hydrogel, respectively. Every hydrogel dressing comprises roughly 100 µg of exosomes. These hydrogels have been instilled onto the wound beds and subjected to UV mild for 60 s to realize photopolymerization. Wounds have been photographed on days 5, 7, and 9, with dressings replenished each 72 h. Wound therapeutic was quantified utilizing ImageJ software program. On day 9, granulation tissues have been harvested, sectioned, and evaluated utilizing immunofluorescence staining (for IL-1β, CD31, and IL-6) and histological staining (H&E and Masson’s trichrome). Moreover, main organs have been harvested and analyzed histologically.
Statistical evaluation
All experiments have been carried out in triplicate. Outcomes are expressed as means ± customary error of the imply. Statistical comparisons between teams have been made utilizing Pupil’s t-test, one-way ANOVA, and two-way ANOVA, analyzed with Origin 2019b software program. Significance ranges have been set at *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Non-significant outcomes are denoted as ns.
