2.4 In vitro experiments
2.4.1 Cell culture
RAW264.7 cells (murine macrophage cell line) were purchased from the Typical Culture Preservation Commission Cell Bank, Chinese Academy of Sciences (Shanghai, China), and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma-Aldrich, USA) containing 10% fetal bovine serum (FBS, Hyclone, USA) at 37℃ in a humidified incubator with 5% CO2. Rat bone marrow-derived mesenchymal stem cells (rBMSCs) were isolated, cultured, and identified utilizing a well-established technique as previously described [22, 23].
The COL and DP scaffolds were placed individually in 96-well plates, and cells were seeded over the scaffolds at a density of 2×104 cells/mL. Cells seeded on plates without scaffolds served as controls. Cell proliferation was quantified using the Cell Counting Kit-8 (CCK-8; Sigma, USA) on days 1, 3, and 5 after seeding.
2.4.2 Evaluation of the effects of DP scaffolds on macrophage polarization
Cells seeding on different scaffolds were harvested after 3 days, and the impact of DP on macrophage polarization was evaluated by flow cytometry (BD Accuri C6, USA) using antibodies against CD86 (1:50 dilution, BioLegend, USA) and CD206 (1:400, BioLegend, USA) along with Dylight 488-anti-mouse secondary antibodies. The relative levels of inducible nitric oxide synthase (iNOS) and arginase 1 (Arg-1) gene mRNA transcripts relative to the control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA in the different groups of RAW264.7 cells were quantified by qRT-PCR. The primers sequences used for qRT-PCR are presented in Table 1. For enzyme-linked immunosorbent assay (ELISA) evaluation, the supernatants of RAW264.7 cultures were collected. The concentrations of the cytokines interleukin (IL)-10 and TNF-α in the supernatant samples were quantified using ELISA kits (Dakewe Bioengineering, China) according to the manufacturer’s instructions.
For RNA-sequencing analysis, total RNA was extracted from the tissue samples using TRIzol reagent (Invitrogen, USA), according to the manufacturer’s instructions. cDNA libraries were then constructed for pooled RNA samples using the VAHTSTM Total RNA-Seq (H/M/R) Library Prep Kit (VAHTSTM, China). Differentially expressed genes (DEGs) were identified using TopHat and Cufflinks, and their expression levels were determined by the fragments per kilobase of transcript per million mapped reads method. The DESeq algorithm was used to screen DEGs between groups. The biological functions of the DEGs were then investigated by Gene Ontology (GO) enrichment analysis (http://www.geneontology.org/).
2.4.3 Evaluation of the effects of polarized macrophages on the proliferation, migration, and differentiation of rBMSCs
To analyze the impact of macrophage polarization on the proliferation, migration, and differentiation of BMSCs, supernatant samples were collected from wells containing macrophages seeded on scaffolds after 3 days of culture to prepare conditioned medium (CM). BMSCs were cultured in 96-well plates and then treated with a mixture of DMEM and CM at a ratio of 1:1. After 1 and 3 days in culture, the CCK-8 assay was performed to evaluate the proliferation of BMSCs.
Wound scratch assays and Transwell assays were performed to evaluate the effects of polarized macrophages on the migration of BMSCs. In the wound scratch assay, BMSCs were seeded in six-well plates with basal medium, once they reached 90–100% confluency, a scratch with the head of 200-μL pipette tip (Axygen, USA) was made through the cell layer. Then, serum-free CM was added, and the cells were further incubated for 12 h and 24 h. Cell migration was observed under a microscope (Olympus, Japan), and the healing area was calculated using ImageJ software (NIH, USA). Boyden chambers were used for the Transwell assay. BMSCs were plated in the upper chamber with basal medium, and RAW264.7 cells were seeded on different materials in the lower chambers. After culture for 24 h, the penetrating cells were fixed with 4% paraformaldehyde, stained with crystal violet solution, and counted under an optical microscope (Olympus, Japan).
Alizarin Red staining (ARS) was conducted to evaluate calcium nodule deposition by BMSCs cultured in CM with osteogenic components for 21 days. The rinsed BMSCs were fixed in 4% paraformaldehyde and stained by exposure to 2% ARS solution at room temperature for 20 min. The stained calcium deposits were dissolved by application of 10% cetylpyridinium chloride (Sigma-Aldrich, USA) for 15 min, and dye release was quantified by spectrophotometry at 562 nm (Thermo Fisher Scientific, USA).
The mRNA expression levels of bone morphogenetic protein-2 (BMP2) and runt-related transcription factor 2(RUNX2) in BMSCs cultured with CM for 3 days were measured using qRT-PCR. The relevant primers sequences are presented in Table 1.