In vitro immunoregulation of osteogenesis of MSCs

In vitro immunoregulation of MSCs towards osteogenesis

1. Macrophages response on different sample surfaces.

      RAW 264.7 macrophages were cultured in DMEM (Hyclone, USA) containing 1% penicillin–streptomycin solution (Gibco, USA) and 10% fetal bovine serum (Gibco), in a 5% CO₂ atmosphere in an incubator at 37 ℃ with the medium changed every day.

       To collect supernatant containing macrophage cytokines, the RAW 264.7 macrophage cell line were cultured on different testing surfaces for 48 h. The details are described as follows: All samples were first sterilized using 75% ethanol and then kept under ultraviolet light for 30 min before use. A total of 2 ml of cell suspension RAW 264.7 macrophages (1 × 10⁴ cells/ml) were cultured on different groups of samples (n = 3) in 6-well plates (ϕ32 samples). After culturing for 48 h, the supernatant was collected and stored at 4 ℃ for further use.

2. Immunomodulation of MSCs cultured with the collected supernatant.

2.1. MSCs cell culture.

       MSCs (under a fifth passage) were cultured in α-MEM (Hyclone, USA) containing 1% penicillin–streptomycin solution (Gibco, USA) and 10% fetal bovine serum (Gibco), in a 5% CO₂ atmosphere in an incubator at 37 ℃ with the medium changed every 3 days.

2.2. MSCs cultured with the supernatant extracted from macrophages.

       200 μL of MSCs (1 × 10⁴ cells/ml) were cultured on different samples (ϕ6 samples, n = 3) in 96 well-plates with mixed culture medium (above mentioned culture medium and supernatant from macrophages at a ratio of 1 :1).

3. Evaluations of the immunoregulation of MSCs towards osteogenesis

3.1. ALP activity

       MSCs were cultured for 7 and 14 days on different samples with or without the addition of collected macrophage supernatants. After cultured for designated time points, the cells on different samples were lysed by adding 100 μl 1% Triton X-100 and cultured in a 37 °C water bath for 1 h. Subsequently, the ALP activity of cells and total protein content were measured using a commercial ALP activity kit (Nanjing Jiancheng Bioengineering Institute, China) and BCA protein assay kit (Solarbio, China) according to the manufacturer’s protocols. The relative ALP activity of cells on each well was then normalized to the corresponding total protein content.

3.2. Osteogenesis-related gene expression. 

       A real-time quantitative polymerase chain reaction (RT-PCR) was used to evaluate the expression of level of typical osteogenic genes (ALP, Runx2, and OCN) of MSCs at days 7 and 14. MSCs were cultured for 7 and 14 days on different samples with the addition of collected macrophage supernatants. The cellular total RNA was isolated using a commercial total RNA kit (OMEGA) and reverse-transcribed to cDNA with a PrimeScript RT Master Mix (TaKaRa, Japan) according to the manufacturer’s instructions. RT-PCR was performed with a Bio-Rad RT-PCR system. The results of relative gene expression were normalized to the expression level of GAPDH.

3.3. Alizarin red staining 

       Alizarin Red staining was performed to evaluate the mineralization tendency of MSCs on sample surface when cultured with the collected supernatants. After culturing cells for 7 and 14 days, the cells were fixed with 4% paraformaldehyde and then stained with 2% Alizarin Red solution (10 min for each step). The cells were then rinsed thoroughly with PBS to remove excess stain. The images of stained calcium nodules were captured using a digital camera. Lastly, the mineralized nodules were dissolved in 10% hexadecyl pyridinium chloride and quantified using a microplate reader at 562 nm.

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