Bletilla striata (China Pharmaceutical Corporation-Canton, Guangzhou, China); Bletilla striata polysaccharide (BSP) was prepared following our laboratory established and reported protocol; Fetal bovine serum and DMEM medium were obtained from Life Technologies; Calcium AM/PI kit was purchased from shanghaiyisheng (China); CCK8 kit; GoTaq 2-Step RT-qPCR system was purchased from Promega; TRIzol Reagent was obtained from Sigma-Aldrich; Other chemicals and reagents were purchased from Sigma-Aldrich unless otherwise stated.
The primers of relevant genes were listed as follows:
Mouse beta-actin: Forward: 5′-GCTGGTCGTCGACAACGGCTC-3′.
Mouse Nos2: Forward: 5′-CCAAGCCCTCACCTACTTCC-3′.
Mouse Il1b: Forward: 5′-GCAACTGTTCCTGAACTCAACT-3′.
Mouse Tnfa: Forward: 5′-ACGGCATGGATCTCAAAGAC-3′.
Mouse Mrc1: Forward: 5′-GTGGTCCTCCTGATTGTGATAG-3′.
Mouse Tgfb: Forward: 5′-TGGAGCAACATGTGGAACTC-3′.
Mouse Arg1: Forward: 5′-CAGAAGAATGGAAGAGTCAG-3′.
Mouse Osm: Forward: 5′-AACTCTTCCTCTCAGCTCCT-3′.
Mouse Vegfa: Forward: 5′-GTTCAGAGCGGAGAAAGCAT-3′.
Synthesis of methacrylated BSP
Oxidation of BSP, the C6 primary hydroxyls of Bletilla striata polysaccharides are oxidized to C6 carboxylate groups by TEMPO/NaClO/NaClO2 oxidation system in sodium acetate buffer (0.2 M, pH 5.0). After stirring at 40 °C for 24 h, oxidation was quenched by adding excessive ethanol. The precipitate of oxidated BSP was collected by centrifugation. Then, oxidized products were dialyzed (MWCO: 3500) with milli-Q water, and lyophilized.
Preparation of methacrylated BSP: oxidized BSP was dissolved in a buffer solution (1% w/v, pH 6.5) of 50 mM 2-morpholinoethanesulfonic acid (MES). N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) (molar ratio of NHS:EDC =1:2) were added to the solution to activate the carboxylic acid groups of the oxidized BSP. After activation for 5 min, AEMA (molar ratio of NHS: EDC: AEMA= 1:2:1) was added to the mixture and the reaction was maintained at room temperature for 24 h. The precipitate of methacrylated BSP was collected by centrifugation. Then, oxidized products were dialyzed (MWCO: 3500) with milli-Q water, and lyophilized.
Characterization of methacrylated BSP
Characterization of oxidized BSP: oxidized BSP was dissolved in deuterated dimethylsulfoxide (DMSO-d6), and the 13 C NMR spectra of these glucomannan/DMSO solutions were recorded. Carboxylate content of oxidized BSP was determined by the potentimetric titration method. 0.1 M HCl was added to methacrylated BSP solution and set pH value in the range of 2.5–3.0, then record HCl volume. 0.1 M NaOH solution was added up to pH11 and record NaOH volume.
Characterization of methacrylated BSP: methacrylated BSP was characterized by 1H-NMR analysis and the efficiency of BSP methacrylation was determined from 1H-NMR spectra based on the ratio of the integrals for the internal standard protons to the methylene protons of methacrylate.
The FTIR spectra of lyophilized pure BSP and BSP-MA were obtained on KBr pellet performed on a FTIR spectrophotometer (MAGNA IR560, Nicolet). All spectra were recorded with the resolution of 4 cm−1 in the range 400–4000 cm−1.
Methacrylated BSP scaffolds preparation
GM scaffolds preparation: methacrylated BSP solution (2%–10%) was synthesized using deionized water as a solvent. Then add tetramethylethylenediamine (TEMED) [0.5% (wt/vol)] and ammonium persulfate (APS) [0.25% (wt/vol)] to the methacrylated BSP solution which was precooled to 4 °C to decrease the rate of polymerization. After a complete incubation in −20 °C refrigerator for one night, the cryogels were put at room temperature to remove ice crystals and washed with milli-Q water.
Pore size and rheology measurement of methacrylated BSP scaffolds
GM scaffolds were stained with fluorescent dyes (FITC). A solution of FITC, 1 mM in 20 mM Na-carbonate buffer (pH 9.4) was applied to the stained scaffold for 24 h and thoroughly washed with buffer and water. The stained cryogels were sectioned into slices. Samples were examined by confocal laser scannin microscopy (CLSM) (Leica TCS SP8), using a 20× objective and excitation and emission wavelengths 488 and 530 nm. ImageJ software (http://rsb.info.nih.gov/ij/) was used to analyze images to obtain the pore size and pore size distribution.
Flow and deformation of materials in response to applied force can be studied by rheology. Elastic modulus and elastic nature of the material is defined as storage modulus (G′). The dissipation (viscous) of the flow is represented by loss modulus (G″). The visco-elasticity behavior or phase angle is the difference between the storage and loss modulus. The cryogels used in experiments were 1.5 cm in diameter and 2 mm in thickness cylindrical shape. Amplitude sweep (strain sweep) test was applied from 0.01 to 100% at the constant frequency of 1 Hz to determine linear viscoelastic region. Then, frequency sweep measurement was performed from 0.01 to 100 Hz at a controlled strain of 0.2% to investigate the modulus change related to the oscillatory frequency.
RAW 264.7, a murine monocyte/macrophage cell line, and human umbilical vein endothelial cells (HUVECs) were purchased from the ATCC (American Type Culture Collection). Cells were cultured in DMEM high glucose medium and RPMI-1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin under 5% CO2 at 37 °C. Cells were passaged after reaching 80% confluence.
Live/dead staining of cells in scaffolds
RAW 264.7 macrophages and HUVECs were washed by PBS. After cell counting, the cells were centrifuged and re-suspended in culture medium solution at a final concentration of 5 × 106 cells per milliliter. 3D scaffolds were sterilized in 75% ethanol for one night. Next, the cell solution was added to the 3D scaffolds and cells can be absorbed into the 3D scaffolds. The cell-laden cryogels were then placed in the atmosphere of 37 °C with 5% CO2 for 6 h to allow cells attachment inside the scaffold.
A live/dead assay was performed to test cell viability in cryogels. Scaffolds loaded with cells in triplicate were incubated with the mixture dye solution containing 1µL of Calcein-AM and 0.5µL propidium iodide (PI) in 1 mL of PBS. After 30 min incubation, the cryogels were rinsed with PBS and cells were imaged by confocal laser scanning microscopy (CLSM) (Leica TCS SP8). Green fluorescence represents live cells and red fluorescence was related to dead cells.
Cell proliferation assay in scaffolds
RAW 264.7 macrophages and HUVECs were seeded in scaffolds as previously mentioned. Scaffolds were placed in a 96-well plate at the density of 2 × 105 cells/scaffold. 6 h after seeding, the cell-laden scaffolds were rinsed with PBS and transferred to another new well to remove the unattached cells. To evaluate cell proliferation, at day 1 and 3, the culture medium was replaced with the cell counting kit-8 (CCK-8) working solution and incubated at 37 °C for 3 h. The CCK-8 solution was collected and the absorbance value was measured with the multi-plate reader at wavelength of 450 nm.
Cell infiltration and distribution in scaffolds
RAW 264.7 macrophages were seeded in scaffolds as previously mentioned. Calcein-AM solution was added to the sample. After 30 min incubation at 37 °C, the cell-laden cryogels were observed by CLSM. All images were generated by optical sectioning in the z-direction. Optical sections each of 10 μm were taken to produce a 250 μm z-stack for image processing.
RAW 264.7 macrophages were seeded in scaffolds at a seeding density of 5 × 104 cells/scaffold. Cell-laden scaffolds were rinsed with PBS and transferred to another new well to remove the unattached cells and 1.5 ml of new culture medium was added. Then, samples were incubated in CO2 incubator for 24 h.
RNA was isolated by kit. RNA was reverse-transcripted into cDNA. Quantitative real-time PCR (q-PCR) measurements were performed using a SYBR Green RT-PCR kit. Marker genes including Tnfa, Il1b, Nos, Mrc1, Tgfb, Arg1, Vegfa and Osm were selected for analysis with the primer sequences using the 2 −ΔΔct relative quantification method.
Implantation of cryogel scaffolds
Male C57BL/6J mice (6–8 weeks) were used with cryogel scaffolds subcutaneous implanted in the back for assessing the host response and the biocompatibility of cryogel scaffolds. All procedures were approved by the Animal Ethics Committee, University of Macau. We divided 18 mice into three groups and treated them with hydrogel and BA2 and BA8 respectively embedded. The mice in each group were divided into 2 days and 14 days.
Before surgery, the mice were anesthetized with an intraperitoneal injection of sodium pentobarbital (70 mg/kg) and then the dorsal hair was shaved. After the sterilization of skin with 75% ethanol, two independent incisions were made on the back, and the scaffolds were embedded in and then the wound was sewn up.
After two days and 2 weeks of housing, the mice were sacrificed and the implants along with the 2 cm × 2 cm skin tissue samples were collected and immediately fixed in 4 vol% formalin and dehydrated by gradient ethanol before embedding in paraffin wax. These samples were cross-sectioned into 6 μm for histological analysis. The sections were deparaffinized and rehydrated for Hematoxylin–eosin (H&E), Masson’s trichrome staining (M&T). Besides, the deparaffinized and rehydrated sections were blocked and stained by anti-VEGF, anti-CD31 and anti-CD86 for immunohistochemistry. The images were recorded by a light microscope (BX51; Olympus). ImageJ software was utilized to quantify the fibrous capsule thickness, immunohistochemistry staining (with the assistant of IHC Toolbox plugin), and obtain the cell coordinate datasets followed by calculating the minimum cell distance in R language.
Statistical differences among samples were studied through t-test or the one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test. The data presented as the mean ± standard deviation was obtained based on at least three independent replicates. Significance was set to p < 0.05. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).