Effects of macrophages on the osteogenic differentiation of adipose tissue-derived stem cells in two-dimensional and three-dimensional cocultures

Figure 1 The isolation, extraction and characterisation of adipose tissue-derived stem cell and THP1. A: Cell culture micrographs of adipose tissue-derived stem cells; B: Cell culture micrographs of THP1; C: Flow cytometry showed adipose tissue-derived stem cells positive for CD29 and CD44, negative for CD31 and CD34; D-F: Osteogenic, adipogenic, and chondrogenic differentiations measured by Alizarin Red staining, Oil Red O staining, and Alcian Blue staining.

Figure 2 Three-dimensional culture of adipose tissue-derived stem cells. A: Macro photographs of three-dimensional culture cell; B: Immunofluorescence staining of alive and dead stem cells; C: Three-dimensional cultured cell spheroids recorded on day 0, 7, 14, and 21, and the cells are gradually detached from the and migrated to the culture plate with the prolongation of culture time. 3D: Three-dimensional; ADSC: Adipose tissue-derived stem cell.

Figure 3 Osteogenic differentiation of adipose tissue-derived stem cells. A: Alizarin red staining of adipose tissue-derived stem cell (ADSC) and ADSC-THP1 in two-dimensional culture at 14 and 28 days; B: Semiquantitative measurement of calcium deposits formed during osteogenic differentiation of ADSCs in each group by alizarin red staining colorimetry. ^aP < 0.05 vs day14 two-dimensional-adipose tissue-derived stem cell, ^bP < 0.001 vs day14 three-dimensional-adipose tissue-derived stem cell, ^cP < 0.05 vs day28 two-dimensional-adipose tissue-derived stem cell, ^dP < 0.001 vs day28 three-dimensional-adipose tissue-derived stem cell. 3D: Three-dimensional; 2D: Two-dimensional; ADSC: Adipose tissue-derived stem cell.

Figure 4 Expression of osteogenic gene markers in adipose tissue-derived stem cells. A: Relative expression of alkaline phosphatase in each group on day 7 and 14 (^aP < 0.05 vs day 7 two-dimensional-adipose tissue-derived stem cell (2D-ADSC), ^bP < 0.001 vs day 7 three-dimensional (3D)-ADSC, ^cP < 0.001 vs day 14 2D-ADSC, ^dP < 0.01 vs day 14 3D-ADSC); B: Relative expression of Runt-related transcription factor 2 mRNA in each group on day 7 and 14 (^aP < 0.05 vs day 7 2D-ADSC, ^bP < 0.01 vs day 7 3D-ADSC, ^cP < 0.01 vs day 14 2D-ADSC, ^dP < 0.01 vs day 14 3D-ADSC); C: Relative expression of osteocalcin in each group on day 7 and 14 (^aP < 0.001 vs day 7 2D-ADSC, ^bP < 0.001 vs day 7 3D-ADSC, ^cP < 0.05 vs day 14 2D-ADSC, ^dP < 0.01 vs day 14 3D-ADSC). ALP: Alkaline phosphatase; RUNX2: Runt-related transcription factor 2; OCN: Osteocalcin; 3D: Three-dimensional; 2D: Two-dimensional; ADSC: Adipose tissue-derived stem cell.

Figure 5 Quantification of pathway proteins. A: Measurement of N-cadherin and Wnt1, β-catenin signaling pathway protein expression in each group using western blotting; B: Quantitative analysis of western blotting in each group. ^aP < 0.01 vs two-dimensional-adipose tissue-derived stem cell (N-cadherin), ^bP < 0.001 vs two-dimensional-adipose tissue-derived stem cell-THP1 (N-cadherin), ^cP < 0.001 vs two-dimensional-adipose tissue-derived stem cell (β-catenin), ^dP < 0.001 vs two-dimensional-adipose tissue-derived stem cell-THP1 (β-catenin), ^eP < 0.01 vs two-dimensional-adipose tissue-derived stem cell (Wnt1), ^fP < 0.001 vs two-dimensional-adipose tissue-derived stem cell-THP1 (Wnt1). 3D: Three-dimensional; 2D: Two-dimensional; ADSC: Adipose tissue-derived stem cell.