A silk fibroin/chitosan/nanohydroxyapatite biomimetic bone scaffold combined with autologous concentrated growth factor promotes the proliferation and osteogenic differentiation of BMSCs and repair of critical bone defects

Abstract

Purpose: With the goal of increasing the translational efficiency of bone tissue engineering for practical clinical applications, biomimetic composite scaffolds combined with autologous endogenous growth factors for repairing bone defects have become a current research hotspot. In this study, we prepared a silk fibroin/chitosan/nanohydroxyapatite (SF/CS/nHA) composite biomimetic scaffold and then combined it with autologous concentrated growth factor (CGF) to explore the effect of this combination on the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and the efficiency of repairing critical radial defects.

Methods: Three kinds of SF/CS/nHA composite biomimetic scaffolds with mass fractions of 3%, 4%, and 5% were prepared by vacuum freeze-drying and chemical cross-linking methods, and the characteristics of the scaffolds were evaluated. In vitro, BMSCs were seeded on SF/CS/nHA scaffolds, and then CGF was added. The morphology and proliferation of BMSCs were evaluated by live-dead staining, phalloidin staining, and CCK-8 assays. ALP staining, alizarin red staining, cellular immunofluorescence, RT-PCR, and Western blotting were used to detect the osteogenic differentiation of BMSCs. In vivo, a rabbit radius critical bone defect model was constructed, and the SF/CS/nHA-BMSC scaffold cell complex combined with CGF was implanted. The effect on bone defect repair was evaluated by 3D CT scanning, HE staining, Masson staining, and immunohistochemistry.

Results: The characteristics of 4% SF/CS/nHA were the most suitable for repairing bone defects. In vitro, the SF/CS/nHA combined CGF group showed better adhesion, cell morphology, proliferation, and osteogenic differentiation of BMSCs than the other groups (P < 0.05 for all). In vivo imaging examination and histological analysis demonstrated that the SF/CS/nHA scaffold combined with CGF had better efficiency in bone defect repair than the other scaffolds (P < 0.05 for all).

Conclusions: A SF/CS/nHA composite biomimetic bone scaffold combined with autologous CGF promoted the proliferation and osteogenic differentiation of BMSCs in vitro and improved the repair efficiency of critical bone defects in vivo. This combination may have the potential for clinical translation due to its excellent biocompatibility.

Keywords: Biomimetic bone scaffold; Bone marrow mesenchymal stem cells (BMSCs); Bone regeneration; Concentrated growth factor (CGF).

Graphical abstract

Fig. 1

Characterization of the SF/CS/nHA scaffold (A) Appearance of the SF/CS/nHA scaffold (B–C) SEM image of SF/CS/nHA scaffold (D–G) relevant parameters of the SF/CS/nHA scaffold (H–I) energy dispersive spectrometry analysis of the SF/CS/nHA scaffold. SEM, scanning electron microscopy.

Fig. 2

Preparation of CGF, cell culture and cell adhesion in scaffolds (A–C) Preparation of CGF (D) BMSC culture in the SF/CS/nHA scaffold (E) light microscopy image of BMSCs (F–H) SEM image of BMSCs in the SF/CS/nHA scaffold; SEM, scanning electron microscopy; Red Arrow, BMSCs; Green Arrow, calcium crystals.

Fig. 3

BMSCs proliferation and morphology detection (A) Live/dead staining of BMSCs (B) the percentage of living BMSCs among the three groups (n = 3) (C) microfilament skeleton of BMSCs stained with phalloidin (D) the CCK-8 assays showing the proliferation ability of BMSCs among the three groups (n = 4). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared with the BM–SCN–CGF group.

Fig. 4

ALP staining and Alizarin red staining (A) ALP staining images of BMSCs under a microscope after 7 d of osteoinduction (B) Quantitative results of ALP staining (n = 4) (C) Alizarin red staining images of BMSCs under a microscope after 21 d of osteoinduction (D) Quantitative results of Alizarin red staining (n = 4). ALP, alkaline phosphatase; IOD, integrated optical density; ∗p < 0.05, ∗∗p < 0.01 compared with the BM group, ^#p < 0.05 compared with the BM-SCN group.

Fig. 5

The fluorescence of Runx-2 and Col-1 in the three groups (A) The fluorescence of Runx-2 after 7 d of osteoinduction (B) semi-quantitative fluorescence results of expression of Runx-2 (n = 3) (C) The fluorescence of Col-1 after 14 d of osteoinduction (D) semi-quantitative fluorescence results of expression of Col-1 (n = 3).∗∗p < 0.01, ∗∗∗p < 0.001 compared with the BM group, ^##p < 0.01, ^###p < 0.001 compared with the BM-SCN group.

Fig. 6

Osteogenic differentiation of BMSCs at the gene and protein levels (A–D) The gene expression of Runx-2, OCN, Col-1, and VEGF after 14 d of osteoinduction (n = 3) (E) A Western blot assay was used to detect the protein expression levels of Runx-2, OCN, Col-1, and VEGF after 14 d of osteoinduction (F–I) Quantitative results of Western blot assays (n = 3). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared with the BM group, ^#p < 0.05, ^##p < 0.01 compared with the BM-SCN group.

Fig. 7

Surgical procedure (A) The radial bone defect was constructed, the defects were treated with BMSCs (BM group) (B–C) The defects were treated with the scaffold-BMSC complex (BM-SCN group) (D) The defects were treated with CGF membrane combined with the scaffold-BMSC complex (BM–SCN–CGF group) (E) CGF membrane was completely coated onto the surface of the SF-CS-nHA scaffold (F) The incision was sutured (G–I) General observation of gross samples; Yellow arrow, CGF membrane; Red arrow, defects of the radius.

Fig. 8

The repair of bone defects was evaluated by 3D CT (A) 3D reconstruction and sagittal image at 4, 8, and 12 weeks (B) Lane-Sandhu radiology score evaluated the repair of radial bone defects among the three groups (n = 6) (C) bone mineral density of the radial bone defect area quantified by 3D-CT (n = 6); BMD, bone mineral density; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared with BM group, #p < 0.05, ##p < 0.01 compared with the BM-SCN group.

Fig. 9

Histological analysis of bone regeneration in vivo (A) Hematoxylin–eosin (HE) staining, Masson staining, immunohistochemistry of Col-1 and CD31 and immunohistofluorescence of Col-1 (B) New bone formation area quantified by Masson staining (n = 6) (C–D) Expression of COL-1 and CD31 quantified by immunohistochemistry (n = 6) (E) Expression of COL-1 quantified by immunohistofluorescence (n = 6). F, fibrous tissue; C, type I collagen; TB, trabecular bone; NB, new bone; Black arrow, remaining scaffold; Black star, transition zone between the remaining scaffold and the new bone. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared with the BM group, #p < 0.05, ##p < 0.01 compared with the BM-SCN group.