The combination of a 3D-Printed porous Ti-6Al-4V alloy scaffold and stem cell sheet technology for the construction of biomimetic engineered bone at an ectopic site

Abstract

Cell sheet technology has been widely used in bone tissue engineering and regenerative medicine. However, controlling the shape and volume of large pieces of engineered bone tissue remains impossible without additional suitable scaffolds. Three-dimensional (3D) printed titanium (Ti) alloy scaffolds are mostly used as implant materials for repairing bone defects, but the unsatisfactory bioactivities of traditional Ti-based scaffolds severely limit their clinical applications. Herein, we hypothesize that the combination of bone marrow mesenchymal stem cell (BMSC) sheet technology and 3D porous Ti-6Al-4V (PT) alloy scaffolds could be used to fabricate biomimetic engineered bone. First, various concentrations of BMSCs were directly cocultured with PT scaffolds to obtain complexes of osteoblastic cell sheets and scaffolds. Then, as an experimental control, an osteoblastic BMSC sheet was prepared by continuous culturing under osteogenic conditions for 2 weeks without passaging and used to wrap the scaffolds. The BMSC sheet was composed of several layers of extracellular matrix (ECM) and a mass of BMSCs. The BMSCs exhibited excellent adherent, proliferative and osteogenic potential when cocultured with PT scaffolds, which may be attributed to the ability of the 3D microstructure of scaffolds to facilitate the biological behaviors of cells, as confirmed by the in vitro results. Moreover, the presence of BMSCs and ECM increased the angiogenic potential of PT scaffolds by the secretion of VEGF. Micro-CT and histological analysis confirmed the in vivo formation of biomimetic engineered bone when the complex of cocultured BMSCs and PT scaffolds and the scaffolds wrapped by prepared BMSC sheets were implanted subcutaneously into nude mice. Therefore, the combination of BMSC sheet technology and 3D-printed PT scaffolds could be used to construct customized biomimetic engineered bone, offering a novel and promising strategy for the precise repair of bone defects.

Keywords: 3D-printed porous titanium alloy scaffold; Angiogenesis; Biomimetic engineered bone; Cell sheet technology; Osteogenesis.

Graphical abstract

Fig. 1

In vitro adhesion, proliferation, and viability of BMSCs on PT scaffolds. (A–O) Phalloidin-DAPI staining of BMSCs attached to PT. Scale bar ​= ​50 ​μm. In detail, (A–E) FITC phalloidin was used to stain the cytoskeleton. (F–J) DAPI was used to stain cell nuclei. (K–O) Merged views of the cytoskeleton and cell nuclei. (P–Y) SEM of BMSCs attached to PT. (P–T) Low-magnification SEM images show the adhesion and proliferation of BMSCs on PT scaffolds. In the PT/LC group, a few cells adhered to the surface of the PT scaffolds (Q, red arrow). In the PT/MC group, more cells adhered than in the PT/LC group, and a small amount of ECM was observed on the surface of the scaffolds (R, green arrow). In contrast, in the PT/HC group, a large number of BMSCs adhered to the surface and inner sides of scaffolds (S, yellow arrow and blue arrow, respectively) and were embedded in their abundant secreted ECM. (U–Y) High-magnification SEM images show the adhesion and proliferation of BMSCs on PT scaffolds. An osteoblastic cell sheet was observed in the PT/CS group (T), made up of cells (Y, red pentacle) and their secreted ECM (Y, black arrow). (Z) CCK-8 assay of BMSCs on PT on days 7 and 14. ∗ indicates significant differences (p ​< ​0.05).

Fig. 2

In vitro analysis of osteogenesis of the combination of BMSCs and PT scaffolds. (A) Schematic of the experiment. BMSCs were collected from each group after 7 days and 14 days of coculturing with PT scaffolds in osteogenic induction medium. (B) Alizarin red staining images. (C) Quantification of Alizarin red staining based on the relative absorbance value. (D–G) The RT‒PCR results of osteogenesis-related gene expression of OCN, OPN, COL-I, and Runx2 in the PT, PT/LC, PT/MC, PT/HC, and PT/CS groups on days 7 and 14. These results suggested that increasing the number of initial seeding cells improved the capability of osteogenic differentiation. (H) Western blot (WB) analysis presented results similar to those of RT‒PCR. ∗ indicates significant differences (p ​< ​0.05).

Fig. 3

The migration potential of HUVECs when cultured in BMSC-CM. (A–O) Wound healing assay results indicated that the more BMSCs were seeded, the faster the wound healed. Scale bar ​= ​100 ​μm. (U) At 24 ​h, the wound healing rates in the PT and PT/LC groups were markedly lower than those in the other groups (p ​< ​0.05), and there was no statistically significant difference between the PT/HC and PT/CS groups, which both had significantly higher rates than the PT/MC group. (P–T) Representative fluorescent images demonstrated that there were more CD31-positive cells in the PT/HC and PT/CS groups than in the other groups. Scale bar ​= ​50 ​μm ∗ indicates significant differences (p ​< ​0.05).

Fig. 4

In vitro analysis of angiogenesis of the combination of BMSCs and PT scaffolds. (A–E) A tube formation assay was used to verify angiogenesis with the combination of PT scaffolds and BMSCs. (F–M) Images were captured and analyzed quantitatively using AngioTool software. The tube formation results showed that a capillary-like network with closed lumen morphology formed when HUVECs were cultured in BMSC-CM from the PT/HC and PT/CS groups and led to a longer network of tube-like structures with more junctions than in the other groups. The vessel percentage area (K), total number of junctions (L), and total vessel length (M) were calculated based on AngioTool software to quantitatively compare tube formation among the different groups. (N and O) The RT‒PCR results of angiogenesis-associated gene expression of VEGF and bFGF in the PT, PT/LC, PT/MC, PT/HC, and PT/CS groups. (P) WB analysis displayed results similar to those of RT‒PCR. These results indicated that BMSCs have to reach a sufficient number to produce enough VEGF to promote angiogenesis. ∗ indicates significant differences (p ​< ​0.05).

Fig. 5

Evaluation effect of the combination of PT scaffolds and BMSCs on the construction of biomimetic engineered bone at an ectopic site. (A–C) Macroscopic images of the subcutaneous implantation sites at 12 weeks of implantation. Scale bar ​= ​1 ​cm. (D–F) Micro-CT reconstruction of the bone regeneration of the complex of PT scaffolds with BMSCs. The newly formed bones were brownish yellow in colour. (G–L) Masson's trichrome (MTC) staining of the PT scaffolds with BMSCs was performed to assess osteogenesis. Only a small quantity of regenerated bone was present in the PT/LC scaffolds (G and J), while more woven bone with more mature trabeculae was observed in the PT/HC (K, blue arrow) and PT/CS groups (L, white arrow). (G–I) Scale bar ​= ​500 ​μm. (J–L) Scale bar ​= ​200 ​μm. (M ​− ​O) CD31 immunofluorescence staining images of the newly formed vessels around PT scaffolds demonstrated that the newly formed vessels were more abundant in the PT/HC and PT/CS groups than in the PT/LC group (yellow arrow). Scale bar ​= ​200 ​μm. (P) Bone volume/total volume (BV/TV) analysis of the complex of PT scaffolds with BMSCs at 12 weeks after implantation. (Q) Bone mineral density (BMD) analysis of the complex of PT scaffolds with BMSCs at 12 weeks after implantation. (R) Percentages of newly formed mineralized tissues among different groups. The percentages in the PT/HC and PT/CS groups were much greater than that in the PT/LC group. ∗ indicates significant differences (p ​< ​0.05).