Stepwise degradable PGA-SF core-shell electrospinning scaffold with superior tenacity in wetting regime for promoting bone regeneration

Abstract

Regenerating bone in the oral and maxillofacial region is clinically challenging due to the complicated osteogenic environment and the limitation of existing bone graft materials. Constructing bone graft materials with controlled degradation and stable mechanical properties in a physiological environment is of utmost importance. In this study, we used silk fibroin (SF) and polyglycolic acid (PGA) to fabricate a coaxial PGA-SF fibrous scaffold (PGA-SF-FS) to meet demands for bone grafts. The SF shell exerted excellent osteogenic activity while protecting PGA from rapid degradation and the PGA core equipped scaffold with excellent tenacity. The experiments related to biocompatibility and osteogenesis (e.g., cell attachment, proliferation, differentiation, and mineralization) demonstrated the superior ability of PGA-SF-FS to improve cell growth and osteogenic differentiation. Furthermore, in vivo testing using Sprague-Dawley rat cranial defect model showed that PGA-SF-FS accelerates bone regeneration as the implantation time increases, and its stepwise degradation helps to match the remodeling kinetics of the host bone tissue. Besides, immunohistochemical staining of CD31 and Col-1 confirmed the ability of PGA-SF-FS to enhance revascularization and osteogenesis response. Our results suggest that PGA-SF-FS fully utilizing the advantages of both components, exhibites stepwise degradation and superior tenacity in wetting regime, making it a promising candidate in the treatment of bone defects.

Keywords: Bone regeneration; Core-shell; Electrospinning; Stepwise degradation; Tenacity.

Graphical abstract

Fig. 1

Characterization of the morphology and composition of SF-FS, PGA-FS and PGA-SF-FS. (A) SEM images and histograms of fiber diameter distributions of SF-FS, PGA-FS, and PGA-SF-FS (scale bar: 10 μm for low magnification and 5 μm for high magnification). (B) FTIR spectra of SF-FS, PGA-FS, and PGA-SF-FS. (C) SEM image of PGA-SF-FS fracture surface (scale bar: 5 μm). (D) TEM image of PGA-SF-FS (scale bar: 2 μm).

Fig. 2

Tensile mechanical property analysis of SF-FS, PGA-FS and PGA-SF-FS in dry and wet conditions. (A, B) Representative tensile stress-strain curves in dry (A) and wet conditions (B). (C, F) Elongation at break in dry and wet conditions. (D, G) Tensile strength in dry and wet conditions. (E, H) Young's modulus in dry and wet conditions. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 3

Molecular structure simulation of SF, PGA and PGA-SF layered composite polymers under dry and hydrated conditions at uniaxial stress.

Fig. 4

Bond energy evolution during uniaxial tensile test simulation. (A) Total bond energy change. (B) Hydrogen bond energy change.

Fig. 5

Morphological change and fiber diameter distributions of SF-FS, PGA-FS, and PGA-SF-FS incubated in PBS solution at 37 °C after 4, 8, 12, and 16 w (scale bar: 5 mm for photographs, 10 μm for SEM images at low magnification and 5 μm for SEM images at high magnification).

Fig. 6

Degradation of SF-FS, PGA-FS, and PGA-SF-FS after incubation in PBS for 0–16 w. (A) Percentage of weight loss of SF-FS, PGA-FS, and PGA-SF-FS during 16 w degradation. (B) The pH of degradation solution of SF-FS, PGA-FS, and PGA-SF-FS during 16 w degradation. (C) FTIR absorption spectra of PGA-SF-FS from 0 to 16 w degradation.

Fig. 7

SEM micrographs and the corresponding EDS elemental mapping images of the prepared SF-FS and PGA-SF-FS (scale bar: 20 μm for low magnification and 5 μm for high magnification).

Fig. 8

BMSCs attachment and proliferation during 7 d culture on SF-FS, PGA-FS, and PGA-SF-FS. (A) Fluorescent micrographs of BMSCs on SF-FS, PGA-FS, and PGA-SF-FS after 1, 4, and 7 d culture (scale bar: 50 μm). (B) DNA quantification assay after 1, 4, and 7 d culture. *p < 0.05.

Fig. 9

Osteogenic differentiation of BMSCs on SF-FS, PGA-FS and PGA-SF-FS. (A, D) ARS and quantitative analysis of the calcium nodules on SF-FS, PGA-FS and PGA-SF-FS after 14 and 21 d osteogenic differentiation. The red arrow indicates PGA-FS desquamation with cell reduction due to the rapid degradation of PGA-FS. (scale bar: 200 μm for low magnification and 100 μm for high magnification). (B) ALP activity of cells after 7 and 14 d osteogenic differentiation on SF-FS, PGA-FS and PGA-SF-FS. (C) Relative expression of osteoblast-specific genes of BMSCs seeding on SF-FS and PGA-SF-FS after 14 d osteogenic differentiation. *p < 0.05, **p < 0.01, ***p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 10

Bone regeneration of SF-FS, PGA-FS, PGA-SF-FS and control groups in critical cranial defects after 4, 8, and 12 w. (A) Specimen photographs (scale bar: 5 mm). (B) A 3D reconstruction of cranial defects (scale bar: 5 mm).

Fig. 11

Microcomputed tomographical analysis of new bone formed in calvarial defect after SF-FS, PGA-FS and PGA-SF-FS implantation for 4–12 w. (A, C) Bone volume/total volume of defect zone. (B, D) A trabecular number of the defect zone. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 12

Histological images after HE staining of SF-FS, PGA-FS, PGA-SF-FS and control groups implanted in the cranial defect for 4, 8, and 12 w. A white dashed line represents the defect margin, white arrows indicate scaffolds, and pentacle represents the new bone (scale bar: 2 mm for low magnification and 200 μm for high magnification).

Fig. 13

Histological images after MT staining of SF-FS, PGA-FS, PGA-SF-FS and control groups implanted in the calvarium defect for 4, 8 and 12 w. The white dashed line represents a defect margin, the white arrows indicate residual scaffolds, and pentacle represents new bone (scale bar: 2 mm for low magnification and 200 μm for high magnification).

Fig. 14

CD31 immunohistochemical staining of SF-FS, PGA-FS, PGA-SF-FS, and control groups implanted in the calvarium defect for 4, 8, and 12 w. The white dashed line represents the defect margin, and the orange arrows represent the CD31 expression (scale bar: 2 mm for low magnification and 200 μm for high magnification). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 15

Col-1 immunohistochemical staining of SF-FS, PGA-FS, PGA-SF-FS, and control groups implanted in the calvarium defect for 4, 8, and 12 w. A white dashed line represents the defect margin, and blue arrows represent the Col-1 expression (scale bar: 2 mm for low magnification and 200 μm for high magnification). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)