Three-Dimensionally Printed Ti2448 With Low Stiffness Enhanced Angiogenesis and Osteogenesis by Regulating Macrophage Polarization via Piezo1/YAP Signaling Axis

Abstract

Previous studies have found that the novel low-elastic-modulus Ti2448 alloy can significantly reduce stress shielding and contribute to better bone repair than the conventional Ti6Al4V alloy. In this study, the promotion of osteogenesis and angiogenesis by three-dimensionally printed Ti2448 were also observed in vivo. However, these were not significant in a series of in vitro tests. The stiffness of materials has been reported to greatly affect the response of macrophages, and the immunological regulation mediated by macrophages directly determines the fate of bone implants. Therefore, we designed more experiments to explore the role of three-dimensionally printed Ti2448 in macrophage activation and related osteogenesis and angiogenesis. As expected, we found a significant increase in the number of M2 macrophages around Ti2448 implants, as well as better osteogenesis and angiogenesis in vivo. In vitro studies also showed that macrophages pre-treated with Ti2448 alloy significantly promoted angiogenesis and osteogenic differentiation through increased PDGF-BB and BMP-2 secretion, and the polarization of M2 macrophages was enhanced. We deduced that Ti2448 promotes angiogenesis and osteogenesis through Piezo1/YAP signaling axis-mediated macrophage polarization and related cytokine secretion. This research might provide insight into the biological properties of Ti2448 and provide a powerful theoretical supplement for the future application of three-dimensionally printed Ti2448 implants in orthopaedic surgery.

Keywords: Ti2448; angiogenesis; macrophage; osteogenesis; polarization.

GRAPHICAL ABSTRACT

FIGURE 1

Characterization of Ti6Al4V and Ti2448 scaffolds. Ti6Al4V scaffold in animal experiments (A1) and cellular experiments (A3); Ti2448 scaffold in animal experiments (A2) and cellular experiments (A4). 3D reconstruction results of Ti6Al4V (B1) and Ti2448 (B2) scaffolds by micro-CT (B). Surface topography of the Ti6Al4V (C1) and Ti2448 (C2) scaffolds, Scale bar = 50 µm (C). EDS results of Ti6Al4V and Ti2448 showing their respective elemental compositions and surface morphology (D). Water contact angle of the Ti6Al4V and Ti2448 scaffolds (E). AFM images of Ti6Al4V and Ti2448 surfaces.

FIGURE 2

Micro-CT and histomorphometric analysis in vivo. New bone tissue around each scaffold reconstructed by micro-CT (A). Quantitative analysis of the BV/TV for the different scaffolds at 4, 8 and 12 weeks (B). Histomorphological analysis of inward growth and integration of bone tissue at corresponding time points. Scale bar = 200 µm (C). Quantitative analysis of the bone−implant contact and BV/TV for different scaffolds at 4, 8 and 12 weeks (D,E). Note: #p < 0.05 compared to Ti2448.

FIGURE 3

Assessment of osteogenesis by EDS. Elemental composition of different groups of scaffolds at 4, 8 and 12 weeks detected by SEM-EDS. Scale bar = 500 µm (A). Quantitative analysis of the elemental Ca content of different scaffolds at 4, 8 and 12 weeks (B). Quantitative analysis of the elemental P content of different scaffolds at 4, 8 and 12 weeks (C). Quantitative analysis of the BV/TV of different scaffolds at 4, 8 and 12 weeks (D). Note: #p < 0.05 compared to Ti2448.

FIGURE 4

Analysis of vascularization around and inside the scaffolds. 3D reconstruction of blood vessels around different scaffolds at 2, 4, and 8 weeks (A). Vascularization inside the scaffolds. Scale bar = 200 µm (B). Immunofluorescence staining of CD31 in bone tissue. Scale bar = 100 µm (C). Quantitative analysis of vascular volume around the scaffold (D). Quantitative analysis of the number of blood vessels inside the scaffold in a single field of view (E). Quantitative analysis of the total length of blood vessels inside the scaffold (F). Quantitative analysis of the maximum single vessel length inside the scaffold (G). Quantitative analysis of the maximum blood vessel diameter inside the scaffold (H). Quantitative analysis of the fluorescence intensity of CD31 around the scaffold (I). Note: #p < 0.05 compared to Ti2448.

FIGURE 5

Inflammatory response to scaffolds evaluated and analysed in bone tissue. HE staining of bone tissue at 1, 2, and 3 weeks after implantation. Scale bar = 100 µm (A). Evaluation of inflammation by immunofluorescence staining for the macrophage marker CD68 in bone. Scale bar = 200 µm (B). Immunofluorescence staining for CCR7 (marker for M1 macrophages) and Arg-1 (marker for M2 macrophages) in the bone at 2 weeks. Scale bar = 100 µm (C). Quantitative analysis of the number of nuclei around the scaffolds (D). Quantitative analysis of the fluorescence intensity of CD68 around the scaffolds (E). Quantitative analysis of the fluorescence intensity of CCR7 and Arg-1 around the scaffolds (F). Note: #p < 0.05 compared to Ti2448.

FIGURE 6

Inflammatory response to scaffolds evaluated and analysed in subcutaneous tissue. HE staining of skin specimens at 3 days, 1 and 2 weeks after implantation. Scale bar = 100 µm (A). Evaluation of inflammation by immunofluorescence staining for CD68 in the skin. Scale bar = 50 µm (B). Immunofluorescence staining for CCR7 (marker for M1 macrophages) and Arg-1 (marker for M2 macrophages) in the skin at 1 week. Scale bar = 50 µm (C). Quantitative analysis of the number of nuclei around the scaffolds (D). Quantitative analysis of the fluorescence intensity of CD68 around the scaffolds (E). Quantitative analysis of the fluorescence intensity of CCR7 and Arg-1 around the scaffolds (F). Note: #p < 0.05 compared to Ti2448.

FIGURE 7

Evaluation of the effects of the different scaffolds on angiogenesis in HUVECs in vitro. Immunofluorescence staining for examination of the cytoskeleton and expression of the angiogenic marker CD31 in HUVECs. Scale bar = 100 µm (A,B). Tube formation (C) and Transwell (D) assays to determine the effect of different scaffolds on HUVECs. Scale bar = 200 µm. Quantitative analysis of the cell area/nuclear area in different groups (E). Quantitative analysis of the fluorescence intensity of CD31 in different groups (F). Quantitative analysis of the number of branch points per field in different groups (G). Quantitative analysis of the total capillary tube length per field in different groups (H). Quantitative analysis of the cell quantity in different groups (I). Note: *p < 0.05 compared to the control.

FIGURE 8

Evaluation of the effects of different scaffolds on osteogenesis in MC3T3-E1 cells in vitro. Staining of the cytoskeleton in MC3T3-E1 cells. Scale bar = 100 µm (A). Quantitative analysis of the cell area/nuclear area in different groups (B). Alizarin red staining after 21 days of osteogenic induction to evaluate the effect of different scaffolds on MC3T3-E1 cells. Scale bars: black = 2 mm, white = 100 µm (C). Semiquantitative analysis of mineralization in cells cultured on different scaffolds (D). Observation of the cell morphology on the surface of the scaffolds by SEM. Scale bar = 100 µm (E). Analysis of the expression levels of Runx2 and OPN by western blot (F). Note: *p < 0.05 compared to the control.

FIGURE 9

Effects of different scaffolds on the angiogenic effect on HUVECs by regulating the polarization of macrophages. Immunofluorescence staining for examination of the cytoskeleton and expression of the angiogenic marker CD31 in HUVECs. Scale bar = 100 µm (A,B). Representative images of the invasion, tubule formation and migration assays. Scale bar = 200 µm (C–E). Quantitative analysis of the cell area/nuclear area in different groups (F). Quantitative analysis of the fluorescence intensity of CD31 in different groups (G). Quantitative analysis of the cell quantity in different groups (H). Quantitative analysis of the migration distance in different groups (I). Quantitative analysis of the number of branch points per field in different groups (J). Quantitative analysis of the total capillary tube length per field in different groups (K). Note: *p < 0.05 compared to the control; #p < 0.05 compared to Ti2448.

FIGURE 10

Effects of different scaffolds on the osteogenic effect on MC3T3-E1 cells by regulating the polarization of macrophages. Immunofluorescence staining for examination of the cytoskeleton of MC3T3-E1 cells and evaluation of the cell morphology. Scale bar = 100 µm (A). Quantitative analysis of the cell area/nuclear area in different groups (B). Examination of mineralized nodules on different scaffolds by stereoscopy to evaluate the osteogenic effect. Scale bars: black = 2 mm, white = 100 µm (C). Semiquantitative analysis of mineralization in cells cultured on different scaffolds (D). Observation of the morphology, adhesion and spreading of MC3T3-E1 cells on different scaffold surfaces by SEM. Scale bar = 100 µm (E). Western blot analysis of the expression levels of Runx2 and OPN in different groups (F). Note: *p < 0.05 compared to the control; #p < 0.05 compared to Ti2448.

FIGURE 11

Scaffold stiffness modulates macrophage inflammatory activation. Morphological observation of macrophages on different scaffold surfaces. Scale bar = 100 µm (A). Immunofluorescence staining for iNOS (red, M1 macrophages) and Arg-1 (green, M2 macrophages). Scale bar = 20 µm (B). Quantitative analysis of immunofluorescence intensity (C). Levels of IL-1β and TNF-α secreted by M1 macrophages (D,E), IL-10 and IL-4 secreted by M2 macrophages (F,G), and VEGF, PDGF-BB and BMP-2 (H–J) at 3 and 8 days. Note: *p < 0.05 compared to the control; #p < 0.05 compared to Ti2448.

FIGURE 12

Substrate stiffness regulates Piezo1 expression and YAP nuclear translocation. Piezo1 expression and cell adhesion to the extracellular matrix (focal adhesion) were observed by immunofluorescence. Scale bar = 20 µm (A). Effect of different substrate stiffnesses on YAP localization in macrophages evaluated by immunofluorescence staining. Scale bar = 20 µm (B). Piezo1 and YAP expression in different groups (C). Quantitative analysis of immunofluorescence intensity (D,E). Note: *p < 0.05 compared to the control; #p < 0.05 compared to Ti2448.