A sustained release of BMP2 in urine-derived stem cells enhances the osteogenic differentiation and the potential of bone regeneration

Abstract

Cell-based tissue engineering is one of the optimistic approaches to replace current treatments for bone defects. Urine-derived stem cells (USCs) are obtained non-invasively and become one of the promising seed cells for bone regeneration. An injectable BMP2-releasing chitosan microspheres/type I collagen hydrogel (BMP2-CSM/Col I hydrogel) was fabricated. USCs proliferated in a time-dependent fashion, spread with good extension and interconnected with each other in different hydrogels both for 2D and 3D models. BMP2 was released in a sustained mode for more than 28 days. Sustained-released BMP2 increased the ALP activities and mineral depositions of USCs in 2D culture, and enhanced the expression of osteogenic genes and proteins in 3D culture. In vivo, the mixture of USCs and BMP2-CSM/Col I hydrogels effectively enhanced bone regeneration, and the ratio of new bone volume to total bone volume was 38% after 8 weeks of implantation. Our results suggested that BMP2-CSM/Col I hydrogels promoted osteogenic differentiation of USCs in 2D and 3D culture in vitro and USCs provided a promising cell source for bone tissue engineering in vivo. As such, USCs-seeded hydrogel scaffolds are regarded as an alternative approach in the repair of bone defects.

Keywords: BMP2; bone tissue engineering; chitosan microspheres; urine-derived stem cells.

Graphical Abstract

Figure 1.

The characterizations of USCs. (A) The morphology of USCs. Scale bar = 200 μm. (B) The growth curve of P3 USCs. (C) The osteogenic, adipogenic and chondrogenic differentiation of USCs. (D) The cell surface markers of USCs. Positive markers: CD73, CD29, CD44, CD105 and CD90; negative markers: CD34, CD14, CD31, CD45 and HLA-DR

Figure 2.

Characterizations of CSM and different hydrogels. (A) The morphology of CSM. Scale bar = 30 μm. (B) The particle size distribution of CSM. (C) The cumulative release of BMP2 from BMP2-CSM/Col I hydrogels. (D) Inverting study showed hydrogels were fabricated successfully. (E) The morphology of freeze-dried hydrogels. Arrows represent microspheres. Scale bar in the left column of images of each hydrogel is 100 μm. Scale bar in the right column of images of each hydrogel is 10 μm

Figure 3.

(A) Cell proliferation using a CCK-8 kit in 2D culture. (B) Cell proliferation using a CCK-8 kit in 3D culture. (C) Scratch wound healing assay and quantification. Scale bar = 200 μm. *P < 0.05, **P < 0.01

Figure 4.

(A) Live/dead staining in 2D culture. Scale bar = 100 μm. (B) Live/dead staining in 3D culture. (C) Cytoskeleton staining in 2D culture. Scale bar = 50 μm. (D) Cytoskeleton staining in 3D culture. Scale bar = 50 μm

Figure 5.

SEM Images of cell adhesion and proliferation at Days 1 and 4. Arrows indicate cell adhesion on microspheres. Scale bar = 50 μm

Figure 6.

Osteogenic differentiation in 2D co-culture. (A) ARS staining and quantification at Days 7, 14 and 21. Scale bar = 100 μm. (B) ALP staining and quantification at Days 4, 7, 14 and 21. Scale bar = 100 μm. *P < 0.05, **P < 0.01

Figure 7.

The expression levels of osteogenic genes and proteins. (A) The relative mRNA expression of COL1A1, ALP and RUNX2 at Days 4, 7, 14 and 21 after osteogenic induction. (B) Western blot analysis showed the protein expression levels of COL1A1, ALP, p-Smad 1/5/9 and Smad 1/5/9 at Days 4, 7, 14 and 21 after osteogenic induction. (C) Quantification analysis of protein expression. *P < 0.05, **P < 0.01, ***P < 0.001

Figure 8.

Evaluation of antibacterial capacity of hydrogels. S.aureus and E.coli survival rates at 12 h

Figure 9.

Results of in vivo experiments. (A) Representative micro-CT images and quantification using the ratio of new bone volume to total bone volume (BV/TV). *P < 0.05, **P < 0.01, ***P < 0.001. (B) HE staining. The second row represents magnification images of the corresponding black rectangles in the upper row. Scale bar = 200 µm

Figure 10.

(A) COL1A1 immunohistochemical staining. (B) OCN immunohistochemical staining. (C) CD31 immunohistochemical staining. The second row represents magnification images of the corresponding black rectangles in the upper row. Scale bar = 200 µm

Figure 11.

Implanted USCs were traced in vivo by immunofluorescent staining using anti-NuMA antibodies. Positive group: paraformaldehyde-fixed paraffin-embedded tissue sections of human femoral condylar cartilage. Negative group: paraformaldehyde-fixed paraffin-embedded tissue sections of SD rat cranial bone. Scale bar = 100 μm