Calcium silicate bioactive ceramics induce osteogenesis through oncostatin M

Abstract

Immune reactions are a key factor in determining the destiny of bone substitute materials after implantation. Macrophages, the most vital factor in the immune response affecting implants, are critical in bone formation, as well as bone biomaterial-mediated bone repair. Therefore, it is critical to design materials with osteoimmunomodulatory properties to reduce host-to-material inflammatory responses by inducing macrophage polarization. Our previous study showed that calcium silicate (CS) bioceramics could significantly promote osteogenesis. Herein, we further investigated the effects of CS on the behavior of macrophages and how macrophages regulated osteogenesis. Under CS extract stimulation, the macrophage phenotype was converted to the M2 extreme. Stimulation by a macrophage-conditioned medium that was pretreated by CS extracts resulted in a significant enhancement of osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), indicating the important role of macrophage polarization in biomaterial-induced osteogenesis. Mechanistically, oncostatin M (OSM) in the macrophage-conditioned medium promoted osteogenic differentiation of BMSCs through the ERK1/2 and JAK3 pathways. This in vivo study further demonstrated that CS bioceramics could stimulate osteogenesis better than β-TCP implants by accelerating new bone formation at defective sites in the femur. These findings improve our understanding of immune modulation of CS bioactive ceramics and facilitate strategies to improve the in vitro osteogenesis capability of bone substitute materials.

Keywords: Calcium silicate; Macrophage; Macrophage polarization; Osteogenesis; Osteoimmune.

Graphical abstract

Fig. 1

(A) XRD patterns of the β-TCP and CS particles. (B) and (C) SEM morphologies of (B) β-TCP and (C) CS particles, respectively.

Fig. 2

Schematic illustration of the experiment. (A) Schematic illustration of the culture of BMDMs and the stimulation of BMDMs and BMSCs. (B) FACS analysis of CD11b and F4/80 expression in BMDMs with or without M-CSF induction.

Fig. 3

CS extracts promote the polarization of BMDMs. (A) FACS analysis and (B) quantification of mean fluorescence intensity for CD11c and CD206 expression of BMDMs treated with CS or the β-TCP extract. (C) mRNA expression levels of IL-1β, TNF-α, IL-10, and TGF-β relative to β-actin in BMDMs with the stimulation of CS and β-TCP extract for 0, 4, 8, and 12 h. (D) Protein expression levels of IL-1β, TNF-α, IL-10, and TGF-β were determined by ELISA. *P < 0.05. The result was normalized to “Blank.”

Fig. 4

mRNA expression levels of (A) M-CSF, (B) VEGF, and (C) OSM relative to β-actin by BMDMs with the stimulation of CS and β-TCP extract (conditioned medium) for 0, 6, 12, and 24 h. (D) OSM protein expression levels were determined by ELISA. *P < 0.05. The result was normalized to “Blank.”

Fig. 5

mRNA expression levels of (A) ALP, (B) OCN, (C) OPN, and (D) COL1 relative to β-actin in BMSCs stimulated by the CS extract, β-TCP extract, or BMDM-conditioned medium treated with these two extracts. *P < 0.05, “NS” represents “not significant.” The result was normalized to “Blank.”

Fig. 6

(A) Protein levels and (B and C) quantification results of ALP and OPN, as determined by immunoblotting. β-actin was used as a control. *P < 0.05, “NS” represents “not significant.” The result was normalized to “Blank.”

Fig. 7

ALP staining results. (A) BMSCs cultured in cultured medium and with osteogenic supplement (OS). (B) BMSCs cultured in BMDM-conditioned medium treated with the β-TCP extract and with OS. (C) BMSCs cultured in BMDM-conditioned medium treated with the CS extract and with OS. (D) Quantification of ALP staining by ImageJ. The result was normalized to “Blank.”

Fig. 8

(A) mRNA expression levels of OSM relative to β-actin in BMDMs stimulated by the CS extract with or without silencing of OSM. (B and C) mRNA expression levels of ALP and OPN relative to β-actin in BMSCs stimulated by the CS-conditioned medium with or without silencing of OSM. (D) Protein levels and quantification (E and F) results of ERK1/2 and JAK3 and related phosphorylation, as determined by immunoblotting. β-actin was used as a control. Images are shown of one representative experiment. Quantification data are presented as the mean ± SD of three independent experiments. *P < 0.05.

Fig. 9

(A) Coronal section of 3D micro-CT reconstruction of bone regeneration in the femoral bone defect in animals with new bone formation; original bone is shown in white, new bone is shown in yellow, implanted material is shown in blue. (B) The defect sites were analyzed to calculate the BMD. (C) Percentage of new bone relative to total tissue volume (BV/TV) (n = 10 rats/batch). *P < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 10

(A) Histological images of newly formed bone in the femoral bone defect at 4, 8, and 12 weeks after operation; the implants materials are shown in black, the newly formed bone tissues are shown in blue. (B) The percentage of new bone area assessed at 4, 8, and 12 weeks after implantation by histomorphometric analysis. We normalized all new bone area data to the percentage of maximum new bone area value. *P < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)