Preparation and modification of the PLGA scaffold
All the procedure in this experiment was shown in Fig.1. PLGA (specification 50/50, IV: 2.5 dl/g, Daigang Biomaterial Co., Ltd. Jinan, China) was dissolved in 1,4-dioxane (10 wt%, Lingfeng Chemistry Ltd. Shanghai, China) and thoroughly stirred to form a homogenized solution. The molding parameters of the LDM System (Tissue Form II, Tsinghua University, China) were selected as follows: the dimensions were 2.4 × 2.4 × 2.4 cm3, the molding temperature was -25~-35°C, the nozzle diameter was 0.4 mm, the spinning pitch was 1.0 mm, the scanning speed was 15~30 mm/s, and the nozzle speed was 1.0~2.0 mm/s. By using computer-aided design combined with a low-temperature extrusion forming process, PLGA was moulded, solidified and stacked in a low-temperature molding chamber. The scaffolds underwent gas-solid phase separation to obtain three-dimensional porous PLGA scaffolds after freeze-drying in a freeze dryer (LYO-2, Biocool Ltd. Beijing, China) for 48 h.
Scaffolds were placed in a 0.2% NaOH (Sinopharm Chemical Reagent Co., Ltd, Beijing, China) solution in alcohol for 10 min, soaked in distilled water, rinsed, and dried to obtain alkali-treated PLGA (aPLGA) scaffolds. The aPLGA scaffolds were soaked in 0.5% collagen-acetate solution (0.5 g COL I; purchased from Migrang Biotechnology Co., Ltd. Sichuan, China and dissolved in 100 mL 0.3% acetate) for 4 h, soaked and rinsed with distilled water to remove the excess collagen solution on the scaffold, and dried to obtain the PLGA/COL I composite scaffold.
The PLGA scaffold and the PLGA/COL I composite scaffold were immersed in 75% alcohol to sterilize them for 60 min, dried and then sealed tightly prior to further use.
General structure and SEM observation of scaffolds
The PLGA scaffold and the PLGA/COL I composite scaffold were sprayed with gold on their surfaces using a sputter machine and then scanned by scanning electron microscopy (SEM, S-450, Hitachi, Ltd. Japan). The cross section and outer and inner sides of the scaffolds were scanned and observed. Image J software (National Institutes of Health, Bethesda, MD, USA) was used to measure the size of the primary aperture and secondary aperture of the section, and the average values were calculated.
Evaluation of scaffold porosity
The porosity of the scaffold was evaluated by the liquid substitution method. Ethanol can penetrate into the pores between the scaffold fibres without causing expansion or contraction of the scaffold, so it can be used as the substitution fluid for measurement. First, the PLGA scaffold and the PLGA/COL I composite scaffold were cut into cuboids (n=5) of the same size; the volume of the cuboidal scaffold was measured as V1, and then the scaffold was placed in a measuring cylinder containing a specific volume (V2) of ethanol. The measuring cylinder was placed in a vacuum pump, and a vacuum was applied until no air bubbles were emitted from the scaffolds. The volume of the cylinder was measured as V3. The porosity of the scaffold was calculated as ρ (%) = (V1 + V2-V3)/V1.
Evaluation of the scaffold hydrophilicity
The PLGA and PLGA/COL I composite scaffolds were cut into cubes (n=4) of 0.9 × 0.9 × 0.9 cm3, and the mass of the cube was weighed as W1. The scaffold was immersed in distilled water for 3 d until the water had been fully absorbed. Filter paper was used to absorb the surface moisture, and the mass of the scaffold was weighed as W2. The hydrophilicity of the scaffold was determined as hydrophilicity (%)= (W2-W1)/W2 × 100%.
Mechanical testing of the scaffold
The compressive strength and elastic modulus of the scaffold were measured using the Universal Testing System (Instron, Massachusetts, United States). The scaffold was cut into 0.9×0.9×0.9 cm3 cubes (n=4), and the compressive strength of the PLGA and PLGA/COL I composite scaffolds was measured with a load of 100 N and a loading speed of 1 mm/min. The elastic modulus of the scaffold was calculated.
Fourier transform infrared spectroscopy (FTIR)
The PLGA scaffold, COL I, and the PLGA/COL I composite scaffold were detected by FTIR, and the changes in the chemical group absorption peaks were recorded.
Isolation, culturing and identification of rabbit chondrocytes
The animal experiment was carried out in accordance with the relevant guidelines and regulations and was approved by the Medical Ethics Committee of the First Affiliated Hospital of Shenzhen University (grant number: LL2015028). Six-week-old New Zealand White Rabbits (Guangdong Animal Experiment Center, Guangzhou, China) (n=5) were used for chondrocyte isolation. All rabbits were euthanized by air embolism. The knee joint articular cartilage was stripped in aseptic conditions. After rinsing and cutting it into small pieces, the cartilage was placed in 5 ml collagenase solution and shaken in an oscillator at a constant temperature for 6-8 h. The cell suspension was obtained after filtration with a 100 μm cell strainer and centrifugation at 1500 r/min for 10 min (Centrifuge 5804/5804 R, Eppendorf, Germany). The supernatant was discarded, and the cells were resuspended by pipetting and adding them into the chondrocyte medium solution. Cells were seeded into a culture dish and placed in a 37°C cell culture incubator (Heracell 150i, Thermo Fisher Scientific, United States) with 5% CO2. The culture medium (DMEM/F12 with 10% FBS, all purchased from Gibco, United States) was changed every 2 to 3 d, and subculturing was performed at 90% confluency, as determined under a microscope.
The second-passage (P2) chondrocytes were subjected to toluidine blue staining (Sigma-Aldrich, Missouri, United States) to identify chondrocytes. The second and fifth passages (P5) underwent immunofluorescence staining for the detection of COL I and type II collagen (COL II).
RNA isolation and RT-qPCR
Total RNA was extracted from cartilage tissues using TRIzol reagent (Invitrogen, Life Technologies, Carlsbad, CA). For real-time quantitative polymerase chain reaction (RT-qPCR) analysis, RNA was reverse-transcribed into cDNA using iScript Reverse Transcription Supermix (Bio-Rad, Hercules, CA). qPCR was carried out using SsoAdvanced™ Universal SYBR® Green Supermix (Bio-Rad) and the CFX Connect™ Real-Time PCR Detection System (Bio-Rad). 18S rRNA was used as an internal reference for each sample. Primer sequences are listed in Table 1. Target gene mRNA expression was normalized to 18S rRNA expression and is shown as the fold change.
Combinational culture of rabbit chondrocytes and scaffolds and cell adhesion and proliferation assays
The PLGA scaffold and the PLGA/COL I composite scaffold were cut into disc-shaped scaffolds with a diameter of 10.2 mm and a thickness of 1 mm, sterilized, dried, and placed into a 48-well culture plate. The P2 chondrocytes from rabbits were adjusted to a density of 5×104/ml using a cell counting analyser. One millilitre of cell suspension was inoculated onto the disk-shaped PLGA and PLGA/COL I composite scaffolds in a 48-well culture plate with an appropriate amount of chondrocyte culture medium. The plate was placed in a 37°C cell culture incubator with 5% CO2.
After the combinational culture of the cells and the scaffolds for 1, 3, 5, 7, and 10 d, the medium was discarded, 500 μl culture medium and 100 μL of the MTT solution (Life Technologies, Shanghai, China) were added to each well, and the plate was cultured in a 37°C incubator with 5% CO2 for 4 h. The solvent on the surface of the scaffold was discarded, and 200 µl dimethyl sulfoxide (DMSO) was added to each well. The plate was shaken on a shaker for 10-20 min to completely dissolve the crystals, 100 μl of the solution from each well was transferred to a 96-well plate, and cell proliferation was measured by a microplate reader by determining the optical density (OD) at 490 nm.
Statistical analysis
Data are presented as the mean ± SD. The software SPSS 16.0 (Chicago, IL, USA) was applied for statistical analysis and management. One-way analysis of variance (ANOVA) and the SNK-q and Dunnett’s T3 tests were applied for comparisons of the means of multiple samples and the determination of the heterogeneity of variance. Any difference for which p < 0.05 was considered statistically significant.