Effects of three-dimensionally printed polycaprolactone/β-tricalcium phosphate scaffold on osteogenic differentiation of adipose tissue- and bone marrow-derived stem cells

Abstract

Background: Autogenous bone grafts have several limitations including donor-site problems and insufficient bone volume. To address these limitations, research on bone regeneration is being conducted actively. In this study, we investigate the effects of a three-dimensionally (3D) printed polycaprolactone (PCL)/tricalcium phosphate (TCP) scaffold on the osteogenic differentiation potential of adipose tissue-derived stem cells (ADSCs) and bone marrow-derived stem cells (BMSCs).

Methods: We investigated the extent of osteogenic differentiation on the first and tenth day and fourth week after cell culture. Cytotoxicity of the 3D printed PCL/β-TCP scaffold was evaluated by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay, prior to osteogenic differentiation analysis. ADSCs and BMSCs were divided into three groups: C, only cultured cells; M, cells cultured in the 3D printed PCL/β-TCP scaffold; D, cells cultured in the 3D printed PCL/β-TCP scaffold with a bone differentiation medium. Alkaline phosphatase (ALP) activity assay, von Kossa staining, reverse transcription-polymerase chain reaction (RT-PCR), and Western blotting were performed for comparative analysis.

Results: ALP assay and von Kossa staining revealed that group M had higher levels of osteogenic differentiation compared to group C. RT-PCR showed that gene expression was higher in group M than in group C, indicating that, compared to group C, osteogenic differentiation was more extensive in group M. Expression levels of proteins involved in ossification were higher in group M, as per the Western blotting results.

Conclusion: Osteogenic differentiation was increased in mesenchymal stromal cells (MSCs) cultured in the 3D printed PCL/TCP scaffold compared to the control group. Osteogenic differentiation activity of MSCs cultured in the 3D printed PCL/TCP scaffold was lower than that of cells cultured on the scaffold in bone differentiation medium. Collectively, these results indicate that the 3D printed PCL/TCP scaffold promoted osteogenic differentiation of MSCs and may be widely used for bone tissue engineering.

Keywords: Adipose tissue; Bone marrow; Cell differentiation; Mesenchymal stromal cells; Polycaprolactone; Stem cells; Tricalcium phosphate.

Fig. 1.

Collection of adipose tissue-derived stem cells (ADSCs) and bone marrow-derived stem cells (BMSCs), and seeding inside three-dimensionally (3D) printed polycaprolactone (PCL)/β-tricalcium phosphate (TCP) scaffold. RBC, red blood cell; PBS, phosphate-buffered saline.

Fig. 2.

Toxicity test results of three-dimensionally printed polycaprolactone/β-tricalcium phosphate scaffold 24 hours (A, B) and 4 weeks (C, D) after cell culture. ADSC, adipose tissue-derived stem cell; BMSC, bone marrow-derived stem cell.

Fig. 3.

Interaction between the three-dimensionally (3D) printed polycaprolactone (PCL)/β-tricalcium phosphate (TCP) scaffold and cells. Alkaline phosphatase assay was performed 1 and 10 days after cell culture, followed by von Kossa staining at 4 weeks. Group C, only cultured cells; M, cells cultured in the 3D printed PCL/β-TCP scaffold; D, cells cultured in the 3D printed PCL/β-TCP scaffold with a bone differentiation medium; ADSC, adipose tissue-derived stem cell; BMSC, bone marrow-derived stem cell.

Fig. 4.

Alkaline phosphatase assay of osteogenic differentiation. (A) Day 1, ×5 magnification. (B) Day 10, ×5 magnification. Group C, only cultured cells; M, cells cultured in the three-dimensionally (3D) printed polycaprolactone (PCL)/β-tricalcium phosphate (TCP) scaffold; D, cells cultured in the 3D printed PCL/β-TCP scaffold with a bone differentiation medium; ADSC, adipose tissue-derived stem cell; BMSC, bone marrow-derived stem cell.

Fig. 5.

von Kossa staining of osteogenic differentiation. (A) Week 4, ×5 magnification. (B) Week 4, ×10 magnification. Group C, only cultured cells; M, cells cultured in the three-dimensionally (3D) printed polycaprolactone (PCL)/β- tricalcium phosphate (TCP) scaffold; D, cells cultured in the 3D printed PCL/β-TCP scaffold with a bone differentiation medium; ADSC, adipose tissue-derived stem cell; BMSC, bone marrow-derived stem cell.

Fig. 6.

Reverse transcription-polymerase chain reaction analysis of the expression levels of ossification genes (COL I, Osteocalcin, and RUNX2). (A) adipose tissue-derived stem cell (ADSC). (B) Bone marrow-derived stem cell (BMSC). Group C, only cultured cells; M, cells cultured in the three-dimensionally (3D) printed polycaprolactone (PCL)/β-tricalcium phosphate (TCP) scaffold; D, cells cultured in the 3D printed PCL/ β-TCP scaffold with a bone differentiation medium; COL 1, type I collagen; RUNX2, runt-related transcription factor 2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Fig. 7.

Western blotting analysis of the expression of ossification-related proteins (COL I, Osteocalcin, and RUNX2). (A) Adipose-derived stem cell (ADSC). (B) Bone marrow-derived stem cell (BMSC). Group C, only cultured cells; M, cells cultured in the three-dimensionally (3D) printed polycaprolactone (PCL)/β-tricalcium phosphate (TCP) scaffold; D, cells cultured in the 3D printed PCL/β-TCP scaffold with a bone differentiation medium; COL 1, type I collagen; RUNX2, runt-related transcription factor 2.