A sericin/ graphene oxide composite scaffold as a biomimetic extracellular matrix for structural and functional repair of calvarial bone

Abstract

Bone defects affect millions of people worldwide each year, leading to severe disabilities. Biomimetic scaffolds mediated tissue regeneration represents a promising alternative for bone repair. However, the major problem associated with most currently clinical available artificial bone substitutes (scaffolds) is that they mainly possess filling function but lack of osteo-induction abilities. Therefore, development of biomaterials with osteo-induction property for effective bone regeneration is highly desired. Methods: We report the design and fabrication of a photo-crosslinked sericin methacryloyl (SerMA)/ graphene oxide (GO) hydrogel (SMH/GO) as a biomimetic scaffold for the functional repair of the bone. The mechanical strength, degradation and biocompatibility behavior of SMH/GO hydrogel were measured in vitro. The effect of SMH/GO hydrogel on BMSCs proliferation, migration, osteogenesis differentiation was assessed. After that, SMH/GO-2 was used as an artificial bone substitute for bone regeneration after calvarial defects and effect on bone repair was evaluated by histological, X-Ray and microCT analysis. Furthermore, the potential mechanism of SMH/GO hydrogel regulating BMSCs migration and differentiation was investigated by RNA sequencing. Results: This scaffold has good biocompatibility, cell adhesive property, proliferation- and migration-promoting effects, and osteogenic induction property. After being implanted in a rat calvarial defect model, this SMH/GO scaffold effectively promotes new bone regeneration and achieves structural and functional repair within 12 weeks by inducing autologous bone marrow-derived mesenchymal stem cells (BMSCs) differentiation. By utilizing cell-biological assays and RNA sequencing, we reveal its possible regeneration mechanisms: the SMH/GO hydrogel regulates BMSCs migration and osteo-differentiation via activating MAPK, TNF, and chemokine signaling for bone regeneration. Conclusion: Aiming to meet clinical demands and overcome current limitations of existing artificial bones, we have developed a new type of sericin/ graphene oxide composite scaffold and provided histological, functional, and molecular evidence demonstrating that it is capable of effectively repairing defective bones by inducing autologous BMSCs directional migration and osteogenic differentiation.

Keywords: Bone marrow-derived mesenchymal stem cells; Bone regeneration; Graphene oxide; Migration and osteogenesis differentiation; Sericin.

Scheme 1

SMH/GO hydrogels as an artificial bone substitute for bone regeneration in a rat calvarial defect model via regulating BMSCs migration and osteogenesis differentiation.

Figure 1

Characterizations of SMH/GO hydrogels. (A) Preparation of SMH/GO hydrogels via UV light at 365 nm. (B) FTIR spectra of SMH/GO hydrogels. (C) Mechanical properties of SMH/GO hydrogels. (D) Degradation profiles of SMH/GO hydrogels in PBS (pH7.4, 37 ^oC). (E) Scanning electron micrographs of SMH (left), SMH/GO-1 (middle) and SMH/GO-2 (right). Scale Bars, 500 μm (upper panel) and 10 μm (lower panel). Red arrowheads indicate graphene oxide, *P<0.05, **P<0.01, ***P<0.001. n = 3 per group per condition.

Figure 2

SMH/GO hydrogels as an artificial bone substitute for bone regeneration after calvarial defect. (A) The experimental procedure using SMH/GO hydrogels for rat calvarial defect treatment. (B) The establishment of the rat calvarial defect model. (C) Photographs of the wounds in the animals receiving no treatment (control), Osteobone, SMH, and SMH/GO-2 treatments, respectively. Scale bars, 1 cm. n = 6 per group per condition.

Figure 3

Radiological evaluation of bone regeneration after treatment. (A) Radiographs of defective area 4, 8, 12 weeks after treatment. (B) Radiopacity of calvarial defects 4, 8, 12 weeks after treatment was quantified using Image J. (C) MicroCT reconstruction of defective area 4, 8, 12 weeks after treatment. (D-E) Micromorphometric bone parameters of the calvarial defects after treatment including bone surface density (bone surface area of regenerated tissue (µm²) / total bone volume of wound site (µm³)) (D) and relative bone volume (bone volume of regenerated tissue/ total bone volume of wound site×100%) (E). *P<0.05, ** P<0.01; n=6 per group per condition. Scale bars, 5 mm.

Figure 4

Histological evaluation of bone regeneration after treatment. (A) Hematoxylin-eosin (H&E) and (B) Masson's trichrome staining of newly generated bone tissue 4, 8 and 12 weeks after treatments. The black dotted boxes in the upper panels were enlarged in the lower panels. The area marked by the dotted green line in (A) was new bone tissue. n=6 per group. Scale bars, 200 μm.

Figure 5

Osteoblasts in wound site after SMH/GO hydrogel's treatment. (A) The immunohistological staining of Collagen I, Ocn and Runx 2 at the wound sites at 12 weeks after treatment. The black dotted boxes in the upper panels were enlarged in the lower panels. The black triangle indicates positively stained cells. (B-D) Quantification of the numbers of Collagen I (B), Ocn (C) and Runx 2 (D) positive cells in (A) and Fig. S3. *P<0.05, **P<0.01, ***P<0.001. n=6 per group per condition, six random fields per slide and 3 slides per animal. Scale bars, 20μm.

Figure 6

BMSCs in wound sites after treatments. The immunohistological staining of (A) CD44 (BMSCs marker) and (B) CD73 (BMSCs marker) in wound site 4, 8 and 12 weeks after treatments. The black dotted boxes in the upper panels were enlarged in the lower panels. The black triangles indicate positive cells. (C) Quantification of CD44 positive cells in (A). (D) Quantification of CD73 positive cells in (B). *P<0.05, **P<0.01, N.S, not significant. n=6 animal per group per condition, six random fields per slide and 3 slides per animal. Scale bars, 20μm.

Figure 7

BMSCs osteogenesis differentiation after treatment. (A) BMSCs were immunofluorescently stained with (Ocn, Col 1 and Runx 2) after being co-cultured with SMH/GO-2 or SMH hydrogels for24 hours. (B-D) Quantification of red fluorescence intensity of BMSCs in (A). (E-G) The relative mRNA expression of three osteogenesis genes (Ocn (E), Col1 (F) and Runx 2(G)) in BMSCs co-cultured with SMH/GO-2 or SMH hydrogels for 24 hours. *P<0.05, ***P<0.001, N.S: not significant. n=3 per group. Three random fields per group were quantified. Scale bars, 50 μm.

Figure 8

RNA sequencing of BMSCs co-cultured with SMH/GO hydrogels. (A) The scheme of RNA sequencing of BMSCs co-cultured with SMH/GO-2 hydrogel. (B) Volcano plots for all the genes of the SMH/GO-2 hydrogel group compared with those of the untreated group. The red and green dots indicate up- and down-regulated DEGs with padj< 0.05, and blue dots indicate unchanged genes. (C-D) Heat map of genes associated with BMSCs migration (C) and osteogenic differentiation (D) in the SMH/GO-2 hydrogel group and the untreated group. (E) Top 20 GO terms associated with biological processes (padj<0.05) involving up-regulated genes in the SMH/GO-2 hydrogel group. (F) Top 20 enriched KEGG pathways with up-regulated genes (padj<0.05) in the SMH/GO-2 hydrogel group. (G-H) Gene Set Enrichment Analysis (GSEA) of gene sets for positive regulation of cell migration (G) and positive regulation of osteoblast differentiation (H). NES, normalized enrichment score. FDR, false discovery rate. Three samples were measured per group.