Identification of mesenchymal stem cells and osteogenic factors in bone marrow aspirate and peripheral blood for spinal fusion by flow cytometry and proteomic analysis

Abstract

Background: An in vivo animal study and a prospective clinical study have indicated that bone marrow aspirate (BMA) augments spinal arthrodesis. However, there is no quantified data to explain why fusion rate can be augmented by BMA in lumbar posterolateral fusion.

Methods: To analyze the proportion of mesenchymal stem cells (MSCs) and osteogenic factors in human BMA and peripheral blood (PB) of the same patient. Autologous BMA and PB from the patients were analyzed by flow cytometry (FACS) using cell markers for MSCs. The osteogenic potential of MSCs was determined by alkaline phosphatase (ALP) activity and calcium level quantification. Proteomics were used for the qualitative and quantitative mapping of the whole proteome from BMA and PB plasma. The mass-to-charge ratio was calculated by time-of-flight mass spectrometry (TOF-MS). The overexpression of protein was confirmed using Western blot analysis.

Results: The proportion of MSCs (CD34-/CD29+/CD105+) was higher in the BMA than that in the PB. Colony-forming cell (CFC) assays suggested that fewer colonies were formed in PB cultures than in BMA culture. There was no significant difference in the osteogenic potential of the MSCs between the PB and BMA. Proteomic mass spectrometry assays suggested that the levels of catalase (osteoclast inhibitor) and glutathione peroxidase 3 (osteogenic biomarker) were higher in the BMA than those in the PB, and this was confirmed by Western blot analysis.

Conclusions: The proportions of MSCs and osteogenic factors were higher in the BMA than in the PB. This may explain why fusion rate can be augmented by BMA in lumbar posterolateral fusion.

Figure 1

Flow cytometry analysis of MSC-like cells from PB and BMA. The proportion of CD34⁻/CD29⁺/CD105⁺-nucleated cells in the PB was significantly lower than that in the BMA (t test, p < 0.01, n = 4).

Figure 2

Fibroblast-colony-forming units (CFU-F) assay. Fewer colonies were formed in the PB cultures than in BMA cultures (t test, p < 0.01, n = 4).

Figure 3

Alkaline phosphatase (ALP) activity measurement. There was higher ALP activity in the induction medium culture than in the complete medium culture in both PB and BMA (t test, p < 0.01, n = 3). There were no significant differences in ALP activity between the PB and BMA in complete medium or induction medium culture in each time point shown (t test, p > 0.05, n = 3).

Figure 4

Calcium level quantification. There was no significant difference in calcium level between the PB and BMA (t test, p > 0.05, n = 3).

Figure 5

Protein profiling of BMA and PB specimens. (A) Proteins (150 mg) were subjected to 2-DE (pH 3 to 10) and detected by silver staining. Spots that had more than a 1.5-fold increase or decrease in silver staining were excised and in-gel digested with trypsin. At least 11 protein spots were detected at higher expression levels and 5 at lower expression levels in the BMA than in the PB. (B) Cropped images showing the two selected proteins (catalase and Gpx) from 2-D gels.

Figure 6

Mass spectrometric analysis of the protein spots. Protein spots of catalase (A) and Gpx (B) were picked, in-gel digested with trypsin, and analyzed by MALDITOF MS. The matched peptides from mass spectrometric analysis of both spots to the amino acid sequence of human catalase and Gpx are underlined and in bold.

Figure 7

Western blot analysis of catalase and Gpx in BMA and PB. The protein levels of catalase and glutathione peroxidase in the BMA were significantly higher than those in the PB. The level of β-actin in each sample detected by Western blot was used as a loading control.