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.