Osteogenic differentiation and staining of BMSCs

Osteogenic differentiation and staining of BMSCs

Authors

  Weizhou Wang     wangweizhou@kmmu.edu.cn

  Fei He                       drhefei@126.com

Shengyu Long        1043873657@qq.com

Abstract

Bone marrow mesenchymal stem cells(BMSCs) possess multilineage differentiation potential and can differentiate into osteoblasts under the influence of specific biofactors. The quantification of osteoblast-specific products can be achieved through alkaline phosphatase (ALP) and Alizarin Red S (ARS) staining, thereby evaluating the osteogenic differentiation capacity of biofactors/materials. This paper provides a brief overview of the two commonly used staining methods for assessing osteogenic differentiation: ALP and ARS.

Materials and Reagents

Equipment and software

Procedure

1. ALP. Staining and activity determination

①. ALP Staining

1.) Prepare each group of fibrous membranes into circles with a diameter of 1.6 millimeters, using sterile coverslips, cell sieves, or stainless steel rings. Gently press the edges of the membranes to fix them at the bottom of a 24-well plate, ensuring they do not curl or float. Then, soak all the fibrous membranes in 75% ethanol for 2 hours, followed by 1 hour of ultraviolet irradiation for disinfection.

2.) Select P3 generation BMSCs in good growth condition, and inoculate them onto the culture plate at a cell density of 5 × 10^4 cells/ml per well.

3.) Cultivate the BMSCs using a standard medium (45 ml of α-MEM cell culture medium + 5 ml of FBS + 0.5 ml of penicillin-streptomycin solution, adjusted to a final concentration of 50 ml of α-MEM medium containing 10% FBS and 1% antibiotics). After culturing for one day, once the cells have been seeded at the bottom of the plate, replace the standard medium with an osteogenic induction medium (50 ml of α-MEM medium containing 10% FBS and 1% antibiotics, plus 10 μl of dexamethasone, 100 μl of vitamin C, and 500 μl of sodium glycerophosphate). Replace the medium every 2 days.

4.) On the 7th and 14th days, remove the osteogenic induction medium and wash the cells three times with PBS.

5.) Fix the cells with 4% paraformaldehyde for 30 minutes, then wash them three times with PBS.

6.) Prepare the BCNP/NBT alkaline phosphatase staining solution according to the protocol: 10 ml of alkaline phosphatase staining buffer + 33 µl of BCIP solution (300X) and 66 µl of NBT solution (150X). Add 300 µl of the staining solution to each well.

7.) Stain the cells at 37°C in the dark for 30 minutes, then terminate the reaction with PBS. Observe and photograph the cells under a fluorescence microscope with white light. (n = 3 experiments).

②. ALP activity determination

1.) Cells were cultured under the same conditions as mentioned above, and the ALP activity was measured on the 7th and 14th days of incubation.

2.) After washing the cells three times with PBS, 1 ml of 1% Triton-X100 was added to each well and the cells were lysed for half an hour.

3.) The lysate was collected and centrifuged in a high-speed centrifuge for 15 minutes to obtain the supernatant.

4.) The ALP activity was detected using the ALP assay kit, with the absorbance measured at a wavelength of 405 nm using a microplate reader.

5.) The protein content was determined using the BCA assay kit, with the absorbance measured at a wavelength of 562 nm using a microplate reader. Finally, the alkaline phosphatase activity in the sample was calculated based on the definition of enzyme activity. (n=3)

2. Calcium nodule staining and semiquantitative analysis of Alizarin Red S (ARS).

①. ARS staining

1). Cultivate the cells under the aforementioned identical conditions.

2). On days 14 and 21, remove the osteoinductive medium, and wash three times with PBS.

3). Fix with 4% polyformaldehyde for 30 minutes.

4). Wash three times again with PBS.

5). Add 500 µl of Alizarin Red staining solution to each well.

6). Incubate at 37℃ for 2 hours.

7). Wash three times with PBS, and observe and photograph under an upright fluorescence microscope (n=3).

②. Alizarin Red S Semiquantitative Analysis

1). Cultivate the cells under the same conditions as mentioned above.

2). On the 14th and 21st days, remove the osteogenic induction medium and wash the cells three times with PBS.

3). Fix the cells with 4% paraformaldehyde for 30 minutes.

4). Wash the cells three more times with PBS.

5). Add 500 µl of alizarin red staining solution to each well.

6). Incubate at 37°C for 2 hours, then slowly wash with PBS solution after rinsing with warm water until no obvious red staining solution is observed.

7). Add an additional 350 µl of 10% hexadecylpyridinium chloride solution to each well.

8). Incubate at 37°C for another 2 hours.

9). Pipette 100 µl of the cell culture into a 96-well plate and measure the absorbance at a wavelength of 562 nm using a microplate reader (n=3).

Statistical analysis 

The data were analysed via Origin 2021 (OriginLab, Massachusetts, USA). All experimental data are presented as the mean ± standard deviation (SD). Univariate analysis of variance (ANOVA) and Tukey’s multiple comparison test were used for analysis.

Interests.

The authors declare that they have no competing interests.