Table 1.
The study patient details.
Fig 1.
Effect of processing VB-BM and IC-BM aspirates using LMP versus AC.
A. A representative example of cell pellet extracted from VB-BM and IC-BM using LMP and AC (left panel). The median of total VB-BM and IC-BM cell numbers extracted using LMP or AC (Friedman with multiple comparisons test, n = 18), (right panel). B. The CFU-F assays; a representative example following 14 days of culture) left panel). The median of total colony numbers for VB-BM and IC-BM samples following processing using LMP or AC (Friedman with multiple comparisons test, n = 18), (right panel). IC; Iliac crest, VB: Vertebral Body, BM: Bone Marrow, LMP: Lymphoprep, AC: Ammonium Chloride.
Fig 2.
Abundance, proliferation and phenotype of VB-BM versus IC-BM MSCs.
A. The numbers of CD45low CD271high cells in unprocessed IC-BM and VB-BM aspirates (means are shown, Paired t-test, n = 6). B. The population doubling time (PDT) for VB-BM versus IC-BM MSCs (means are shown, Paired t-test, n = 7). C. The percentage of culture-expanded VB-BM MSCs versus IC-BM MSCs expressing hematopoietic lineage markers (CD34, CD14, CD19, HLA-DR), CD45, CD73, CD90, and CD105 (n = 3). The means of percentages are shown with bars of the standard error of the mean. D. The mean fluorescence intensity of positive markers expressed on VB-BM and IC-BM MSCs (n = 3).
Fig 3.
Tri-lineage differentiation capability of VB-BM versus IC-BM MSCs.
A. A representative example for testing the osteogenic differentiation of MSCs using alkaline phosphatase staining and alizarin red (left panel). The calcium levels of differentiated VB-BM and IC-BM MSCs (Wilcoxon matched pair rank test, n = 4, each in triplicate, right panel). B. A representative example of the chondrogenic differentiation of MSCs tested by the GAG staining of the cell pellet (left panel). The quantitative GAG levels by IC-BM and VB-BM MSCs (Wilcoxon matched pair rank test, n = 4, each in triplicate, right panel). C. A representative example of the adipogenic differentiation of MSCs using Nile Red and Oil Red O staining (left panel). The quantitative measurement of Nile Red/DAPI ratio for IC-BM and VB-BM MSCs (Paired t-test, n = 4, right panel).
Fig 4.
The expression of osteogenic and chondrogenic genes in VB-BM versus IC-BM MSCs.
A. The gene expression levels during the osteogenic differentiation of VB-BM and IC-BM MSCs (4 donor-matched pairs) were assessed using TaqMan real time PCR. The gene expressions of samples collected at 1, 2 and 3 weeks of osteogenesis were calculated relative to undifferentiated cells. B. The gene expression levels during the chondrogenic differentiation of VB-BM and IC-BM MSCs (4 donor-matched pairs) were assessed using TaqMan real time PCR. The gene expressions of samples collected at 1, 2 and 3 weeks of chondrogenesis were calculated relative to undifferentiated cells. *: p<0.05. Dotted lines indicate the basal expression level of genes in undifferentiated cells.
Fig 5.
Attachment and osteogenic differentiation of VB-BM MSCs on Vitoss™.
A. The cell attachment on Vitoss™ after seeding with aspirates of IC-BM (left panel) and VB-BM (right panel) followed by 2-week culture using confocal microscopy. DAPI staining (blue) was used for cell nuclei and Phalloidin (green) for actin expression. Scale Bar: 50μm. B. The flowcytometry gating strategy for CD45-CD90+CD73+ MSCs released from Vitoss™ following 2-week culture (left panel). The percentage of MSCs out of total CD45- cells for IC-BM MSCs and VB-BM MSCs seeded on Vitoss™ (Paired t-test, n = 3). The mean of the data is shown, right panel. C. The ALP activity levels (normalised to DNA content) for VB-BM MSCs and IC-BM MSCs seeded on Vitoss™ and culture in either osteogenic or expansion media (one-way ANNOVA with Tukey's multiple comparison test, n = 6). The mean of the data is shown. E: expansion media, O: osteogenic media.