Biomimetics, Vol. 7, Pages 227: The Biomimetics of Mg2+-Concentration-Resolved Microenvironment for Bone and Cartilage Repairing Materials Design

4.1. Chemicals

Chemicals and materials (and their purity and manufacture) in the experiment are listed as follows: Magnesium chloride hexahydrate (99%, Aladdin, Shanghai, China), Collagenase Type II (Sigma Aldrich, St. Louis, MO, USA), Trypsin (Sigma Aldrich, St. Louis, MO, USA), Phosphate Buffer Saline (PBS, Hyclone, Cytiva, Logan, UT, USA), Penicillin–streptomycin (Hyclone, Cytiva, Logan, UT, USA), Fetal Bovine Serum (FBS, Biological Industries, Israel), Dulbecco’s modified Eagle’s Medium (DMEM) of High glucose (HG, 4500 mg/dL) (DEME-HG; Hyclone, Cytiva, Logan, UT, USA), Dulbecco’s modified Eagle’s Medium/Nutrient Mixture F12 (DEME/F12; Hyclone, Cytiva, Logan, UT, USA), Cell counting kit-8 (CCK8, Solarbio, Beijing, China), Crystal violet (Solarbio, Beijing, China), Calcein-AM/Propidium Iodide (PI) live/dead cell staining kit (Solarbio, Beijing, China), 4′,6-diamidino-2-phenylindole (DAPI, Solarbio, Beijing, China), FITC-phalloidin (Solarbio, Beijing, China), Lipopolysaccharide (LPS, Solarbio, Beijing, China), L-Ascorbic acid (Sigma Aldrich, A4403, St. Louis, MO, USA), β-Glycerophosphate disodium salt hydrate (Sigma Aldrich, G9422, St. Louis, MO, USA), Dexamethasone (Sigma Aldrich, D4902, St. Louis, MO, USA), Alkaline Phosphatase (ALP) Assay kit (Beyotime Biotechnology, Shanghai, China), BCIP/NBT Alkaline Phosphatase (ALP) Assay Chromogenic Kit (Beyotime Biotechnology, Shanghai, China), Alizarin red (Solarbio, Beijing, China), Trizol reagent (Yeason Biotech, Shanghai, China), Hifair® II First Strand cDNA Synthesis Kit (Yeason Biotech, Shanghai, China), Hieff® qPCR SYBR Green Master Mix (Yeason Biotech, Shanghai, China). Rabbit Collagen Type II ELISA Kit (Cusabio, Wuhan, China), Rabbit Collagen Type X ELISA Kit (MyBioSource, San Diego, CA, USA), Rabbit Collagen Type I ELISA Kit (Cusabio, Wuhan, China), Mouse Collagen Type I ELISA Kit (Cusabio, Wuhan, China).

4.3. Effects of Mg2+ Concentration on the Chondrocytes 4.3.1. Cell Proliferation

The effect of Mg2+ concentration on the proliferation of chondrocytes was assessed by the CCK-8 method. Chondrocytes (1.5 × 103 cells/well) were inoculated into each well of the 96-well plate and cultured for 24 h until updated with the 100 μL of Mg2+-loaded DMEM/F12 culture medium with different Mg2+ concentrations (1.25 mM, 2.5 mM, 5.0 mM, 7.5 mM, 10.0 mM, 12.5 mM, 15.0 mM, 20.0 mM, 25.0 mM, 30.0 mM, 60.0 mM, 90.0 mM. The stock solution of Mg2+ was prepared by magnesium chloride hexahydrate and stored in 4 °C). Five parallel experiments were applied for each Mg2+ concentration and the control group were the pure DMEM/F12 (with the concentration of Mg2+ being 0.7 mM). The culture medium was renewed every two days. For cell proliferation measurement, 10 μL of CCK-8 solution was added to the well and cultured for another 2 h, then the OD value was collected at 450 nm by the microplate reader at the first, third, and fifth day for each Mg2+ concentration and the control group of the culture medium.

The cell proliferation amounts were also observed directly by the crystal violet staining method. The as-cultured cells in the well were washed with PBS solution firstly. Then, the cells were fixed with 100 μL of paraformaldehyde for 30 min, and then re-washed with PBS solution. After that, 100 μL of crystal violet was dripped to stain the cells and re-washed with PBS solution for the image recording by a digital scanner.

4.3.2. Cell ActivityThe living activity of the cells was assessed via the dead-live staining method. The ultrahigh Mg2+ concentration groups (30 mM, 60 mM, and 90 mM) were excluded based on the CCK-8 results (due to the high inhibition effect on cell proliferation). The chondrocytes (1.5 × 104 cells/well) were inoculated in 48-well plates for the Mg2+ concentration groups of 1.25–25.0 mM with DMEM/F12 as the control group. The culture medium was updated every 2 days. The dead and live cells were stained after being cultured for 72 h [60]. The results were recorded by a fluorescence microscope. 4.3.3. Cell MorphologyMorphology of the chondrocytes was observed by a combination of FITC-phalloidin and DAPI staining method. The Mg2+ concentration sets and cell culture method were the same as that in Section 4.3.2. After cultured for 48 h, FITC-phalloidin and DAPI were applied to stain the F-actin and nucleus, respectively, to illustrate the morphology changes of the chondrocytes. The results were recorded by a fluorescence microscope. 4.3.4. Chondrogenic-Related Gene ExpressionsThe effect of Mg2+ concentration on chondrogenic-related gene expressions (SOX9, COL II, AGC, COL X, and COL I) was evaluated by real-time quantitative PCR technique. Briefly, chondrocytes were seeded on 6-well plates with a density of 2 × 105 cells/well. After cultured for 24 h, the culture media was replaced with fresh culture media containing different concentrations of Mg2+ (2.5 mM, 5 mM, 7.5 mM, 10 mM, 12.5 mM, 15 mM, 17.5 mM, and 20 mM). The total RNA of each group was extracted by Trizol reagent (Invitrogen, USA) after cultured for 7 days. Then reverse transcription was performed to synthesize cDNA from purified RNA using Oligo(dT) primers (Promega, San Luis Obispo, CA, USA) and SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA) in accordance with the instructions of the suppliers. Finally, cDNA was subjected to real-time PCR (Applied Biosystems 7300, Foster City, CA, USA) using SYBR Green detection (PerfeCTa SYBR Green FastMix, ROX; Quanta Biosciences, Gaithersburg, MD, USA) with custom-designed primers (Takara Bio, Dalian, China; Table S1). All genes were quantified by comparison with the internal reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for standardization. The 2−ΔΔCt method was used to analysis of relative gene expression levels according to a reference previously published [61]. The experiment was repeated three times with three replicate wells for each sample. 4.3.5. Chondrogenic-Related Protein Expressions

ELISA was performed to evaluate the protein levels of COL II, COL X, and COL I secreted by the chondrocytes cultured in varied Mg2+ concentrations. Briefly, chondrocytes were seeded on 6-well plates with a density of 2 × 105 cells/well. After cultured for 24 h, the culture media was replaced with fresh culture media containing different concentrations of Mg2+ (2.5 mM, 5 mM, 7.5 mM, 10 mM, 12.5 mM, 15 mM, 17.5 mM, and 20 mM). After cultured for 7 days and 14 days, the supernatant was collected and centrifuged at 3000 rpm for 10 min at 4 °C, then stored at −80 °C for use. ELISA was performed according to the manufacturer’s protocol. The absorbance of each sample was detected using a microplate reader at the wavelength of 450 nm. Protein concentrations were calculated using a standard curve according to the instructions.

4.3.6. Inflammatory-Related Gene ExpressionsThe effect of Mg2+ concentration on inflammatory-related gene expressions (IL-1β, MMP13, ADAMTS5, TIMP3, and HIF-1α) of chondrocytes was also evaluated by real-time quantitative PCR technique. We built the chondrocytes inflammatory models by the lipopolysaccharide (LPS)-induced method. Briefly, chondrocytes were seeded on 6-well plates with a density of 2 × 105 cells/well. After cultured for 24 h, the culture media was replaced with fresh culture media containing different concentrations of Mg2+ (5 mM, 7.5 mM, 10 mM, 12.5 mM, 15 mM, 17.5 mM) and 10 μg/mL of LPS. The real-time quantitative PCR technique was used after being cultured for 24 h. The specific experimental process was the same as that in Section 4.3.4.

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