Sequential gastrodin release PU/n-HA composite scaffolds reprogram macrophages for improved osteogenesis and angiogenesis

Abstract

Wound healing is a highly orchestrated process involving a variety of cells, including immune cells. Developing immunomodulatory biomaterials for regenerative engineering applications, such as bone regeneration, is an appealing strategy. Herein, inspired by the immunomodulatory effects of gastrodin (a bioactive component in traditional Chinese herbal medicine), a series of new immunomodulatory gastrodin-comprising biodegradable polyurethane (gastrodin-PU) and nano-hydroxyapatite (n-HA) (gastrodin-PU/n-HA) composites were developed. RAW 264.7 macrophages, rat bone marrow mesenchymal stem cells (rBMSCs), and human umbilical vein endothelial cells (HUVECs) were cultured with gastrodin-PU/n-HA containing different concentrations of gastrodin (0.5%, 1%, and 2%) to decipher their immunomodulatory effects on osteogenesis and angiogenesis in vitro. Results demonstrated that, compared with PU/n-HA, gastrodin-PU/n-HA induced macrophage polarization toward the M2 phenotype, as evidenced by the higher expression level of pro-regenerative cytokines (CD206, Arg-1) and the lower expression of pro-inflammatory cytokines (iNOS). The expression levels of osteogenesis-related factors (BMP-2 and ALP) in the rBMSCs and angiogenesis-related factors (VEGF and BFGF) in the HUVECs were significantly up-regulated in gastrodin-PU/n-HA/macrophage-conditioned medium. The immunomodulatory effects of gastrodin-PU/n-HA to reprogram macrophages from a pro-inflammatory (M1) phenotype to an anti-inflammatory and pro-healing (M2) phenotype were validated in a rat subcutaneous implantation model. And the 2% gastrodin-PU/n-HA significantly decreased fibrous capsule formation and enhanced angiogenesis. Additionally, 2% gastrodin-PU/n-HA scaffolds implanted in the rat femoral condyle defect model showed accelerated osteogenesis and angiogenesis. Thus, the novel gastrodin-PU/n-HA scaffold may represent a new and promising immunomodulatory biomaterial for bone repair and regeneration.

Keywords: Angiogenesis; Gastrodin-delivery; Immune/inflammatory response; Osteogenesis; Tissue repair.

Graphical abstract

Fig. 1

(A) FTIR spectral characterization of n-HA, gastrodin, and gastrodin-PU/n-HA scaffolds. (B) XRD characterization of n-HA and gastrodin-PU/n-HA scaffolds. (C) Digital photo of scaffolds. (D, E) Effects of gastrodin concentration on mechanical properties: (D) Elastic modulus and (E) Compression strength. (F) In vitro degradation study of scaffolds in 0.5 M NaOH solution. (G) Gastrodin release profiles from scaffolds in 0.5 M NaOH solution. Error bars represent standard deviation from mean (n = 5). ***p < 0.001; **p < 0.01; *p < 0.05.

Fig. 2

In vitro viability and osteogenic differentiation of rBMSCs cultured on scaffolds. (A) Live/dead staining of rBMSCs on scaffolds after 4 and 7 days: (a, e) PU/n-HA, (b, f) 0.5% gastrodin-PU/n-HA, (c, g) 1% gastrodin-PU/n-HA, and (d, h) 2% gastrodin-PU/n-HA. Live cells are stained green, dead cells are stained red. (B) Images showing rBMSC morphology after 7 days of culture. F-actin is stained green, cytoplasmic mitochondria are stained red, and nuclei are stained blue. (C) CCK-8 assay for proliferation after 4 and 7 days. (D–F) Relative expression of osteogenic genes (ALP, RUNX2, COL1) in rBMSCs cultured on composite scaffolds for 14 days. Error bars represent standard deviation from mean (n = 5). ***p < 0.001; **p < 0.01; *p < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Assessment of immunomodulatory effects of scaffolds on macrophages after 3 days of culture. (A) Viability of RAW 264.7 cells using CCK-8. (B) Relative gene expression levels of Arg-1, CD206, and iNOS. (C) Immunofluorescence staining of Arg-1 (green), iNOS (red), and nuclei (blue). (D, E) Quantitative analysis of optical density of (D) Arg-1 and (E) iNOS staining. (F) Arg-1, CD 206, iNOS, and TNF-α protein expression levels in RAW 264.7 cells measured by western blotting, and (G) calculated corresponding protein levels. Error bars represent standard deviation from mean (n = 3). ***p < 0.001; **p < 0.01; *p < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

(A) Schematic of osteogenic differentiation using conditioned medium from RAW 264.7 cells cultured with scaffolds for 3 days. (B) Cell migration in scratch assay. (C) Quantitative analysis of migratory ability of rBMSCs. (D, E) Osteogenic gene expression of ALP (D) and BMP-2 (E) analyzed by RT-qPCR. (F, G) Immunofluorescence staining of BMP-2 (F) and corresponding optical density (G). (H, I) Western blotting (H) and relative intensity of bands (I). Error bars represent standard deviation from mean (n = 3). ***p < 0.001; **p < 0.01; *p < 0.05.

Fig. 5

(A) Schematic of angiogenic differentiation using conditioned medium from RAW 264.7 cells cultured with scaffolds for 3 days. (B) Cell migration in scratch assay. (C) Quantitative analysis of migratory ability of HUVECs. (D, E) Angiogenic gene expression of BFGF (D) and VEGF (E) analyzed by RT-qPCR. (F, G) Immunofluorescence staining of VEGF (F) and corresponding optical density (G). (H–J) Western blotting (H) and relative intensity of BFGF (I) and VEGF (J) bands. Error bars represent standard deviation from mean (n = 3). ***p < 0.001; **p < 0.01; *p < 0.05.

Fig. 6

Histological analysis of host response to gastrodin-PU/n-HA scaffolds following 2-week subcutaneous implantation. (A) Schema of subcutaneous implantation of scaffolds. (B, C) Content of IL-1β protein measured by ELISA assay at 3 (B) and 14 (C) days. (D) Immunofluorescence staining of PU/n-HA (a, e), 0.5% gastrodin-PU/n-HA (b, f), 1% gastrodin-PU/n-HA (c, g), and 2% gastrodin-PU/n-HA (d, h) at 3 (a–d) and 14 days (e–h). iNOS was used to label M1 macrophages with red signals, and Arg-1 was used to label M2 macrophages with green signals. (E, F) Quantitative analysis of (E) Arg-1 (M2) and (F) iNOS (M1) macrophage populations. (G) Masson and H&E staining of subcutaneous implanted scaffolds after 14 days. (H) Thickness of fibrous capsules surrounding implants. (I) CD31 staining. (J) Images and (K) quantitative analysis of vascular connections from ex vivo experiment to analyze gastrodin effects on HUVECs. S represents scaffolds; Two-way black arrows mark span of fibrous capsule at scaffold-tissue interface; White arrows indicate newly formed vessels. Error bars represent standard deviation from mean (n = 3). ***p < 0.001; **p < 0.01; *p < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7

In vivo bone regeneration at 4, 8, and 12 weeks after implantation of scaffolds in rat femoral condyle defects. (A) Micro-CT 3D images of new bones. (B) Bone volume/Total volume (BV/TV). (C) Trabecular thickness (Tb. Th). (D) 3D images and (E) Volume fraction of residual scaffold in defect areas. (F) Masson staining of PU/n-HA (a, d, g), 1% gastrodin-PU/n-HA (b, e, h), and 2% gastrodin-PU/n-HA (c, f, i). (G) Immunofluorescence and immunohistochemical staining of RUNX2 at 8 (a–f) and 12 weeks (g–l). (H) Quantitative analysis (immunohistochemical optical density within defect) of RUNX2 expression. (I) Immunofluorescence and immunohistochemical staining of CD31 at 8 (a–f) and 12 weeks (g–l). (J) Quantitative analysis (immunohistochemical optical density within defect) of CD31 expression. Green arrows mark “F- fibrous tissue”; Red arrows mark “HB-host bone”; Black arrows mark “NB-new bone”; Yellow dashed lines mark interface between HB and NB; Yellow arrows mark newly formed vessels. Error bars represent standard deviation from mean (n = 5). ***p < 0.001; **p < 0.01. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 8

Mechanism of macrophage-modulating gastrodin-PU/n-HA composite scaffold improved osteogenesis and angiogenesis.