Bioceramic akermanite enhanced vascularization and osteogenic differentiation of human induced pluripotent stem cells in 3D scaffolds in vitro and vivo

Abstract

A growing number of studies suggest that the modulation of cell differentiation by biomaterials is critical for tissue engineering. In previous work, we demonstrated that human induced pluripotent stem cells (iPSCs) are remarkably promising seed cells for bone tissue engineering. In addition, we found that the ionic products of akermanite (Aker) are potential inducers of osteogenic differentiation of iPSCs. Furthermore, composite scaffolds containing polymer and bioceramics have more interesting properties compared to pure bioceramic scaffolds for bone tissue engineering. The characteristic of model biomaterials in bone tissue engineering is their ability to control the osteogenic differentiation of stem cells and simultaneously induce the angiogenesis of endothelia cells. Thus, this study aimed at investigating the effects of poly(lactic-co-glycolic acid)/Aker (PLGA-Aker) composite scaffolds on angiogenic and osteogenic differentiation of human iPSCs in order to optimize the scaffold compositions. The results from Alizarin Red S staining, qRT-PCR analysis of osteogenic genes (BMP2, RUNX2, ALP, COL1 and OCN) and angiogenic genes (VEGF and CD31) demonstrated that PLGA/Aker composite scaffolds containing 10% Aker exhibited the highest stimulatory effects on the osteogenic and angiogenic differentiation of human iPSCs among all scaffolds. After the scaffolds were implanted in nu/nu mice subcutaneous pockets and calvarial defects, H&E staining, BSP immunostaining, qRT-PCR analysis and micro-CT analysis (BMD, BV/TV) indicated that PLGA + 10% Aker scaffolds enhanced the vascularization and osteogenic differentiation of human iPSCs and stimulated the repair of bone defects. Taken together, our work indicated that combining scaffolds containing silicate bioceramic Aker and human iPSCs is a promising approach for the enhancement of bone regeneration.

Fig. 1. Scanning electron microscopic evaluation of the scaffold microstructure and the state of cells. (A) Scaffold without cells; (B) 5 days after the human iPSCs were seeded onto the scaffold, cells (arrows) grew tightly to each other and some extracellular matrix deposited on the scaffold.

Fig. 2. The vascularization and osteogenic differentiation of human iPSCs in the 3D scaffolds. (A) Alizarin Red staining of human iPSCs indicating the formation of calcium nodules after culturing in different scaffolds for 21 days. (B) Quantitative real-time PCR was performed for expressions of osteogenic genes, such as ALP, BMP2, COL1, OCN and RUNX2 of human iPSCs in 3D scaffolds. (C) Quantitative real-time PCR was performed for expressions of angiogenic genes, including VEGF and CD31 of human iPSCs in 3D scaffolds. *p < 0.05, **p < 0.01, compared with PLGA. ^#p < 0.05 compared with PLGA + 20% Aker.

Fig. 3. The vascularization of human iPSCs in scaffolds in vivo. (A) (A-a) PLGA + C; (A-b) PLGA + 10% Aker + C; PLGA + 10% Aker + C group at 4 (A-c) and 8 (A-d) weeks after human iPSCs in scaffolds were implanted into subcutaneous pockets, implants were taken out for analysis. (B) H&E staining, functional blood vessels were defined by structures that had a clearly defined lumen containing red blood cells (red circles), blood vessel of different implants at 4 and 8 weeks, bar = 50 μm. (C) Quantitative real-time PCR was performed for evaluating the expressions of angiogenic genes CD31 of the new formation tissue in vivo at 4 and 8 weeks. *p < 0.05, **p < 0.01. NS: no significance.

Fig. 4. The osteogenic differentiation of human iPSCs in scaffolds in vivo. (A) Implants were taken out from subcutaneous pockets at 4 and 8 weeks, the BSP immunostaining (brown structures) was performed to indicate the osteogenic differentiation of human iPSCs in vivo, bar = 100 μm. (B) Quantitative real-time PCR was performed for evaluating the expression of the osteogenic gene BSP in the newly formed bone tissue in vivo at 4 and 8 weeks. *p < 0.05, **p < 0.01. NS: no significance.

Fig. 5. Scaffolds combine Aker and human iPSCs promote the healing of skull defects. (A) Human iPSCs in scaffold complex in vivo for repairing skull defects. (A-a) A non-healing full thickness defect of 4 mm diameter in the either side of the cranial bone was made; (A-b) the skull defect was filled with an implant; (A-c–g) the skull defect was filled with nothing, PLGA, PLGA + C, PLGA + Aker and PLGA + Aker + C respectively after implantation for 8 weeks; (B) BSP secreted by cells was detected by immunostaining (brown structures) at 8 weeks, greater BSP expression was observed in the PLGA + Aker + C group than in the other groups. Bar = 50 μm; (C) the percentage of the brown area surface to the total surface of images taken from groups. The group PLGA + Aker + C secreted more BSP than the other groups. (D) Quantitative real-time PCR was performed for expressions of osteogenic marker genes BMP2, RUNX2, COL1 and OCN in new formation tissue around the defects. *p < 0.05, **p < 0.01. NS: no significance.

Fig. 6. Analysis of new bone formation in skull defects at 8 weeks post-operation. (A) Microcomputer tomography (micro-CT) images of skull defects were detected after implantation at 8 weeks. Scale bar = 1 mm. (B) Local bone mineral density (BMD) analysis by micro-CT of five groups at 8 weeks post-operation. (C) Morphometric analysis (BV/TV) of new bone formation. Quantitative micro-CT analysis revealed that implantation of PLGA + Aker + C group achieved the highest local BMD and the highest amount of new bone formation than other four groups. **p < 0.01. NS: no significance. Scale bar = 1 mm.