A Novel Zwitterionic Hydrogel Incorporated with Graphene Oxide for Bone Tissue Engineering: Synthesis, Characterization, and Promotion of Osteogenic Differentiation of Bone Mesenchymal Stem Cells

Abstract

Zwitterionic materials are widely applied in the biomedical field due to their excellent antimicrobial, non-cytotoxicity, and antifouling properties but have never been applied in bone tissue engineering. In this study, we synthesized a novel zwitterionic hydrogel incorporated with graphene oxide (GO) using maleic anhydride (MA) as a cross-linking agent by grafted L-cysteine (L-Cys) as the zwitterionic material on maleilated chitosan via click chemistry. The composition and each reaction procedure of the novel zwitterionic hydrogel were characterized via X-ray diffraction (XRD) and Fourier transformed infrared spectroscopy (FT-IR), while the morphology was imaged by scanning electron microscope (SEM). In vitro cell studies, CCK-8 and live/dead assay, alkaline phosphatase activity, W-B, and qRT-CR tests showed zwitterionic hydrogel incorporated with GO remarkably enhanced the osteogenic differentiation of bone mesenchymal stem cells (BMSCs); it is dose-dependent, and 2 mg/mL GO is the optimum concentration. In vivo tests also indicated the same results. Hence, these results suggested the novel zwitterionic hydrogel exhibited porous characteristics similar to natural bone tissue. In conclusion, the zwitterionic scaffold has highly biocompatible and mechanical properties. When GO was incorporated in this zwitterionic scaffold, the zwitterionic scaffold slows down the release rate and reduces the cytotoxicity of GO. Zwitterions and GO synergistically promote the proliferation and osteogenic differentiation of rBMSCs in vivo and in vitro. The optimal concentration is 2 mg/mL GO.

Keywords: graphene oxide; osteogenic differentiation; stem cell; tissue engineering; zwitterionic.

Figure 1

(A) Optical images of hydrogels. The color changed gradually from milky white to black with the increase of GO concentration. (B) Pore sizes of different scaffolds increased with the increase of GO concentration (* p < 0.05, ** p < 0.01). (C) SEM images of scaffolds with different concentrations of GO. The red dotted boxes in the upper panels were enlarged in the lower panels. (D) Swelling rate and (E) degradation rate decreased with increasing GO concentration.

Figure 2

Mechanical properties of the zwitterionic hydrogel. (A) Stress—strain curve. The concentration of GO at 1mg/mL showed the strongest stress—strain curves. (B) Young’s modulus of the zwitterionic scaffolds at different concentrations of GO ** p < 0.01, *** p < 0.001, and **** p < 0.0001). FT-IR spectra of (C) CS, MA, Cys, CS/MA, Z-CS, and (D) GO, β-TCP, Z-CS/β-TCP, Z-CS/β-TCP/GO-1, Z-CS/β-TCP/GO-2, Z-CS/β-TCP/GO-4. XRD patterns of (E) GO, β-TCP, Z-CS/β-TCP/GO-1, Z-CS/β-TCP/GO-2, Z-CS/β-TCP/GO-4 of different samples.

Figure 3

Proliferation, viability, and morphology of cells cultured with different scaffolds for one, three, and seven days. (A) CCK-8 assay (** p < 0.01, *** p < 0.001, and **** p < 0.0001). (B) Live/dead staining: green (live), red (dead), Scale bars, 100 μm. (C) Fluorescent images of rBMSCS morphologies: blue (nucleus), red (cytoskeleton), Scale bars, 300 μm.

Figure 4

rBMSc osteogenic activity of rBMSc with different scaffolds. (A) ALP straining staining of rBMSCs cultured with different scaffolds for seven and fourteen days, scale bars, 200 μm. (B) Alizarin red S stained cultured with different scaffolds for 14 and 21 days, scale bars, 200 μm. (C) ALP activity. (D) Calcium nodules (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001).

Figure 5

Expression levels of osteogenic genes β-catenin (A), OPN (B), OCN (C), COL 1 (D), RUNX2 (E), and ALP (F) tested by qRT-PCR. Osteogenic proteins (G) analyzed by western blotting. (H–J) Quantification results of β-catenin, RUNX2, and COL-1 (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001).

Figure 6

Osteogenic differentiation in vivo. Bone repairing model by Micro-CT at (A) six weeks (scale bars, 10 mm) and (B) twelve weeks post implantation (scale bars, 20 mm). (C) Quantitative comparison of bone mineral density (BMD) and (D) bone volume to total volume (BV/TV). Histological analysis of bone regeneration in different zwitterionic hydrogel at six and twelve weeks (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001). (E) H&E staining and (F) Masson’s trichrome staining showed fibrous tissues were regenerated in the control group. The newly formed bone with fibrous tissues was obvious in the Z-CS/β-CTP group. In the Z-CS/β-CTP/GO-2 group, a mount of new bone was observed. (NB: newly formed bone. Magnification ×200.)

Figure 7

Scheme of the synthetic zwitterionic hydrogel.