Biomimetic construction of oriented lamellar Col/nHAP composite scaffolds and mediation of macrophages to promote angiogenesis and bone regeneration

doi:10.1007/s11706-023-0666-4

Front. Mater. Sci.

2023, Vol. 17

Issue (4) : 230666 https://doi.org/10.1007/s11706-023-0666-4

RESEARCH ARTICLE

Biomimetic construction of oriented lamellar Col/nHAP composite scaffolds and mediation of macrophages to promote angiogenesis and bone regeneration

Tingting Huang¹, Tao Huang¹, Pin Luo¹, Di Xiao¹, Yiping Huang¹, Shenyu Yang², Rong Zeng¹, Mei Tu¹(

)

¹. College of Chemistry and Materials Science, Jinan University, Huangpu Road 601, Guangzhou 510632, China
². Medical 3D Printing Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450001, China

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Abstract

Pore characteristics have been identified as key design parameters for osteoimmunomodulation. The strategy reported here is to create an appropriate immune microenvironment by regulating pore characteristics of scaffolds, thereby promoting early angiogenesis and enhancing osteogenesis. A series of collagen/nano-hydroxyapatite (Col/nHAP) composite scaffolds with ordered lamellar structures and different layer spacings were prepared by mimicking the ordered lamellar topology of the bone matrix. Our research indicated that the layer spacing and ordered topology of the scaffold exerted an important influence on phenotype transformation of macrophages and the secretion of angiogenic factors. The Col/nHAP-O(135) with large layer spacing not only supported cell attachment and diffusion in vitro, but also promoted early angiogenesis by timely switching from M1 to M2 macrophage phenotype. In vivo data showed that the layer spacing and the ordered structure of the scaffold synergistically regulated the inflammatory response and triggered macrophages to secrete more angiogenesis related cytokines. Col/nHAP-O(135) considerably promoted the neovascularization and new bone formation in the defect site, indicating that Col/nHAP-O(135) could significantly enhance the osteogenic activity of stem cells with the involvement of macrophages.

Keywords pore characteristic macrophage vascularization bone regeneration

Corresponding Author(s): Mei Tu

About author:

Peng Lei and Charity Ngina Mwangi contributed equally to this work.

Issue Date: 06 November 2023

Cite this article:

Tingting Huang,Tao Huang,Pin Luo, et al. Biomimetic construction of oriented lamellar Col/nHAP composite scaffolds and mediation of macrophages to promote angiogenesis and bone regeneration[J]. Front. Mater. Sci., 2023, 17(4): 230666.

URL:

https://academic.hep.com.cn/foms/EN/10.1007/s11706-023-0666-4
https://academic.hep.com.cn/foms/EN/Y2023/V17/I4/230666

Tab.1 Primer sequences for related genes

Fig.1 Characterization of the Col/nHAP scaffolds: (A) SEM image of the scaffolds; (B) layer spacing distribution of the scaffolds; (C) porosity of the scaffolds; (D) compressive strength of the scaffolds; (E) degradation rate of scaffolds at different degradation time points; (F) SEM images of the stent at different degradation time points.

Fig.2 The cell growth in scaffolds: (A) cell proliferation; (B) live/dead staining of cells after culture for 72 h. * Represents a significant difference compared with Col/nHAP-R, ** p < 0.01, *** p < 0.001.

Fig.3 Cytokines in the supernatant of M1 or M0 Mφ co-culturing with the scaffold for 1, 3, and 7 d (M1 Mφs related cytokines: TNF-α and VEGF; M2 Mφs related cytokines: TGF-β1 and PDGF-BB): (A) the time course of the concentration of each cytokine after inoculating M0 Mφs on the scaffold; (B) the time course of the concentration of each cytokine after inoculation of M1 Mφs on the scaffold. * Represents a significant difference between groups, * p < 0.05, ** p < 0.01.

Fig.4 The effect of the conditioned medium after co-cultivation of scaffolds with different pores/layer spacing and macrophages on the scratch healing of HUVECs: (A) representative images of the gap areas at 0, 6, 12, and 24 h after treating HUVECs with CM_φ-Col/nHAP in scratch wound healing assay (scale bar = 50 μm); (B) quantification of the scratch wound areas. Data are expressed as the percentage of the remaining area to the initial scratch area. * Represents a significant difference between groups, * p < 0.05, ** p < 0.01.

Fig.5 Tube formation experiment: (A) the structure and network image of the tube formed after HUVECs was cultured in each group of CM for 2 h (scale bar = 50 μm); (B) the grid formed after HUVECs was cultured in each group of CM for 2 h (mesh), quantitative analysis data of tube length (length) and branch (branch) quantitative analysis. * Represents a significant difference between groups, * p < 0.05, ** p < 0.01.

Fig.6 (A) The expression of CCR7 (M1-related markers) and CD206 (M2-related markers) in the bone defect area after scaffolds were implanted into the bone defect for 3, 7, and 30 d. (B) The expression of VEGF and PDGF-BB in the bone defect area after scaffolds were implanted into the bone defect for 3, 7, and 30 d. * Represents a significant difference between groups, * p < 0.05, ** p < 0.01, *** p < 0.001.

Fig.7 VEGF and PDGF-BB immunohistochemical staining of the bone defect area after scaffolds were implanted into the bone defect at Days 7 and 30 (scale bar = 50 μm; triangles represent the positive area).

Fig.8 CD31 immunohistochemical staining of the bone defect area after scaffolds were implanted into the bone defect at Days 30 and 60 (scale bar = 50 μm; triangles represent the positive area).

Fig.9 (A) The expressions of Col-1, OCN, and BMP-2 in the bone defect area after scaffolds were implanted into the bone defect for 30 and 60 d. * Represents a significant difference between groups, * p < 0.05, ** p < 0.01, *** p < 0.001. (B) OCN immunohistochemical staining of the bone defect area after scaffolds were implanted into the bone defect for 30 and 60 d (scale bar = 50 μm).

Fig.10 Micro-CT analysis of the effect of scaffolds on bone repair in vivo (the identified scaffolds (Col/nHAP-R, Col/nHAP-O(135), Col/nHAP-O(75), and Col/nHAP-O(35)) were implanted into the skull bone defects of rats): (A) representative 3D micro-CT images at Days 30 and 60 of the post-implantation of scaffolds; (B) summarized data showing the micro-architectural parameters of the new formed bone tissue at Days 30 and 60 by analyzing 3D reconstructed micro-CT images using the image analysis software (BV/TV and Tb.Th were shown in the panels). * Represents a significant difference between groups, * p < 0.05, ** p < 0.01.

Fig.11 Histological images and quantitative analyses of the new bone formation (scale bar = 50 μm): (A) H&E staining images of the bone defect sections of implanted Col/nHAP-R scaffold and Col/nHAP-O scaffold after the surgery for 30 and 60 d (the nuclei and cytoplasma were stained blue and red in the H&E staining, respectively (Bk: blank region; NB: new bone)); (B) Masson trichrome staining images of bone defect sections implanted with Col/nHAP-R scaffold and Col/nHAP-O scaffold after the surgery for 30 and 60 d (the osteoid matrix and muscle fibers were stained blue and red in masson trichrome staining, respectively).

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