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.

Background Immune responses and osteogenesis differentiation induced by implants are crucial for bone tissue regeneration. Consideration of only one of those properties is not sufficient. To investigate the synergistic actions, we designed alginate/graphene oxide/sericin/nanohydroxyapatite (Alg/GO/Ser/nHAP) nanocomposite hydrogels with both osteoimmunomodulatory and osteoinductive activities. This study aimed to explore the effect of hydrogel with osteoimmunomodulatory properties on promoting osteogenesis of bone marrow stem cells (BMSCs). Methods Alg/GO/Ser/nHAP nanocomposite hydrogel was fabricated and was characterized by SEM, FTIR, XRD, stress-strain, rheology, swelling and degradation. After the impact of sericin on M2 macrophage polarization was identified, the BMSCs viability and adhesion were evaluated by CCK8 assay, live/dead staining, cytoskeleton staining. The cell osteogenic differentiation was observed by ALP/ARS staining, immunofluorescence staining, RT-PCR, and Western blotting, respectively. Rat cranial defect model was used to assess osteoimmunomodulatory effects of scaffolds in vivo by micro‑computed tomographic, histological, and immunohistochemical analyses after 8 weeks of healing. Results In vitro experiments revealed that the hydrogel presented desirable mechanical strength, stability, porosity, and biocompatibility. Significantly, sericin and nHAP appeared to exert synergistic effects on bone regeneration. Sericin was observed to inhibit the immune response by inducing macrophage M2-type polarization to create a positive osteoimmune microenvironment, contributing to improving osseointegration at the bone-implant interface to promote osteogenesis. However, the osteogenic differentiation in rat BMSCs was further enhanced by combining nHAP and sericin in the nanocomposite hydrogel. Eventually, the hydrogel was implanted into the rat cranial defect model, assisting in the reduction of local inflammation and efficient bone regeneration. Conclusion The nanocomposite hydrogel stimulated bone formation by the synergistic effects of immunomodulation of macrophage polarization by sericin and direct osteogenic induction by nHAP, demonstrating that such a scaffold that modulates the osteoimmune microenvironment to promote osteogenesis is a promising approach for the development of bone tissue engineering implants in the future.

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