Zn/Sr dual ions-collagen co-assembly hydroxyapatite enhances bone regeneration through procedural osteo-immunomodulation and osteogenesis

Abstract

The immune microenvironment induced by biomaterials played vital roles in bone regeneration. Hydroxyapatite (HA) and its ion-substituted derivates represent a large class of core inorganic materials for bone tissue engineering. Although ion substitution was proved to be a potent way to grant HA more biological functions, few studies focused on the immunomodulatory properties of ion-doped HA. Herein, to explore the potential osteoimmunomodulatory effects of ion-doped HA, zinc and strontium co-assembled into HA through a collagen template biomimetic way (ZnSr-Col-HA) was successfully achieved. It was found that ZnSr-Col-HA could induce a favorable osteo-immune microenvironment by stimulating macrophages. Furthermore, ZnSr-Col-HA demonstrated a procedural promoting effect on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in vitro. Specifically, the osteo-immune microenvironment acted as a dominant factor in promoting osteogenic gene expressions at the early stage through OSM signal pathway. Whereas the direct stimulating effects on BMSCs by Zn²⁺/Sr²⁺ were more effectively at the later stage with Nfatc1/Maf and Wnt signals activated. In vivo study confirmed strong promoting effects of ZnSr-Col-HA on critical-sized cranial defect repair. The current study indicated that such a combined biomaterial design philosophy of dual ion-doping and biomimetic molecular co-assembly to endow HA applicable osteoimmunomodulatory characteristics might bring up a new cutting-edge concept for bone regeneration study.

Keywords: Biomimetic co-assembly; Hydroxyapatite; Osteoimmunomodulation; Strontium; Zinc.

Graphical abstract

Fig. 1

Schematic diagram of ZnSr-Col-HA promoting bone regeneration through procedural osteo-immunomodulation and osteogenesis.

Fig. 2

Characterization of ion-doped hydroxyapatites. (A) XRD analysis and (B) FTIR spectrums showed the typical characteristic peaks of hydroxyapatite. (C) XRF results proved that Zn/Sr individually and jointly incorporated into hydroxyapatite successfully. (D) TGA results of different hydroxyapatites. (E) Zn and Sr cumulative release curves showed continuous-release characteristics in all ion-doped hydroxyapatite. *p < 0.05, indicated that there was a significant difference of released ions content between the two groups. (F) SEM, (G) HR-TEM images of Col-HA, Zn-Col-HA, Sr-Col-HA, and ZnSr-Col-HA, respectively.

Fig. 3

The cytocompatibility and osteoimmunoregulatory ability of ZnSr-Col-HA. Cell viability of (A) RAW and (B) BMSCs cultured with different extractions. (C) Ion concentrations of conditioned medium determined by ICP-OES. (D) FACS results of RAW cells stimulated by different hydroxyapatite extractions for 24 h. (E) Quantitative results of FACS, n = 3, * indicated a significant difference p < 0.05. (F) Expression of M1 macrophages phenotypes genes (CD11c, CCR7) and pro-inflammatory cytokines (IL1β, IL6, TNFα) are significantly upregulated in Zn-Col-HA and ZnSr-Col-HA groups. (G) Expression of osteogenic genes (BMP-2, Wnt10b, OSM) and osteoclastic genes (MMP9, RANK) are remarkably upregulated and downregulated by Zn/Sr-doped hydroxyapatites, respectively. * indicated a significant difference p < 0.05, the cell used in (F) and (G) was RAW264.7. (H) Schematic of osteo-immune response of macrophages cultured with hydroxyapatite extractions.

Fig. 4

Enhanced and procedural osteogenic factors expression of BMSCs by immune microenvironment stimulation. (A) ALP staining and (B) quantitative determination of ALP expression at D3. (C) Osteoblastic genes expression of BMSCs detected by RT-qPCR at D3. (D) Gene expression of OSM pathway mediators. (E) ALP staining and (F) quantitative determination of ALP expression at D7. (G) Osteoblastic genes expression of BMSCs detected by RT-qPCR at D7. (H) Gene expression of Nfatc1/Maf and Wnt pathway. * indicated a significant difference with p < 0.05.

Fig. 5

Micro-CT analysis of rat cranial defect repair in vivo. (A) The surgery drawing of rat cranial defect and film repair. Rat cranial defects were treated with none (Blank), Col-HA, Zn-Col-HA, Sr-Col-HA and ZnSr-Col-HA scaffolds. (B) Micro-CT images of cranial defects showed different bone regeneration at 4 weeks and 12 weeks postoperatively. Upper, sectional view; bottom, 3D reconstruction images.

Fig. 6

H&E and Masson's trichrome staining analysis of cranial defects. Both H&E (A, B) and Masson's trichrome staining (C, D) results verified that ZnSr-Col-HA was able to significantly promote newly bone formation. Upper, overall view; bottom, zoom-in pictures of the rectangular box in the overall view. Black solid triangle, asterisk, arrow, and letter N indicated the border of cranial defects, scaffold, fibrous tissue, and new bone, respectively.