Multifunctional biomimetic hydrogel based on graphene nanoparticles and sodium alginate for peripheral nerve injury therapy

https://doi.org/10.1016/j.bioadv.2022.212727Get rights and content

Highlights

  • A hydrogel based on graphene and alginate was first designed with multifunctional ability for peripheral nerve healing.

  • The novel hydrogel could mimic the microenvironment of regeneration and promote the release of nerve growth factors.

  • This easy-made hydrogel improved the single mechanical support function of the traditional single ingredient scaffolds.

Abstract

Peripheral nerve injury (PNI) caused by injury may influence the patients' lifelong mobility unless there is an appropriate treatment. Tissue engineering has become a hot field to replace traditional autologous nerve transplantation due to its low surgical damage and easy-to-industrial advantages. Graphene (GR) is a kind of carbon nanomaterial with good electrical and mechanical properties that satisfy the demand for a good tissue scaffold for nerve regeneration. Herein, a novel and biosafe hydrogel is fabricated by using graphene and sodium alginate (GR-SA) together. This hydrogel not only can mimic the nerve growth microenvironment but also can promote the expression of neurotrophic substances and growth factors. Additionally, GR-SA hydrogel can significantly reduce inflammatory factors. Moreover, the results of both in vitro and in vivo tests demonstrate that GR-SA hydrogel has a promising prospect in PNI regeneration.

Introduction

Peripheral nerve injury (PNI) is a kind of serious and direct mechanical nerve injury. It often occurs in people with fractures, injuries in car accidents, or mechanical injuries. People who suffer from PNI always have limited abilities in daily life and even at work [1]. The most common PNIs are the radial nerve of the upper limbs and common personal injury of the lower limbs. The incidence of PNI is on the rise and nearly 20 million people were suffering from PNI in 2017 [2]. Though peripheral nerve has the ability to self-repair and to reactivate the inner growth mechanism in theory, there are only 3% of patients who can recover sensibility, and motor function is recovered by less than 25% of patients [3]. Consequently, it is hard for patients who undergo serious PNIs to recover by themselves. If there is no surgical or medical intervention, PNI will lead to permanent loss of nerve function, including nerve transplantation failure, chronic pain, and muscle atrophy.

Nowadays, the primary clinical therapy for PNI is autologous nerve transplantation (ANG) [4], [5]. ANG is usually used in PNI with long gaps between the nerve ends caused by traumatic injuries instead of direct suture surgery that can only repair short gaps. However, this “gold standard” method [6] also has many disadvantages: limited nerve donors [7], mismatched size [8], and donor tissue injury [9]. Consequently, it is meaningful and important to find alternative methods to improve neural regeneration after PNI.

To reduce the side effects of ANG, tissue engineering strategy has been widely researched and shown prominent potential prospects [10], [11], [12]. The purpose of a tissue scaffold is to construct a biological material to repair tissue defects or replace organ functions. Recently, there is an increasing number of tissue scaffolds have been reported for repairing PNI. Some natural antioxidants, like melatonin, are proved to reduce oxidative stress and inflammation, and their degradation products are conductive to PNI repair [13], [14]. While polycaprolactone (PCL) is also a common artificial polymer scaffold for PNI repairing because of its good biodegradability [15], [16], [17]. However, both natural and artificial polymer scaffolds have shortcomings. For natural polymer scaffolds, their poor mechanical strength should be aware [18]. And for artificial polymer scaffolds, the cell viability and biocompatibility are often poor [19].

Among them, the hydrogel is a kind of cross-linked system with a three-dimensional network structure and has high water content. It can simulate the extracellular matrix system and act as a cell carrier to promote nerve regeneration [20], [21]. Many studies have previously reported that hydrogel made up of alginate (ALG), a natural polysaccharide extracted from brown seaweed, has good biocompatibility [22], mammalian extracellular matrix structure [23], and the characteristic of being chemically modified easily [24]. These are important properties for promising regenerative biomaterials. Meanwhile, it has been proved that the hydrogel itself can be suitable for the adhesion of nerve cells and can also improve the efficiency of regeneration [25]. Although pure alginate hydrogel can repair PNI theoretically, its weak mechanical resistance will be insufficient to bear physiological loading conditions [26]. Therefore, it is wise to blend alginate with other biomaterials to enhance its mechanical characteristics.

Graphene (GR) was used to solve this problem. GR is an important carbon nanomaterial, and it has huge potential in PNI repair [27]. GR is a two-dimensional nanomaterial in which carbon atoms are arranged in a honeycomb pattern in sp2 hybridization. Each carbon atom in GR is connected to three adjacent carbon atoms by a σ bond, and the remaining π electrons form a large π bond with the remaining carbon atoms [28]. Thus, owing to freely moving electrons in this area and the strong σ bond, GR has excellent electrical conductivity and good mechanical properties [26]. Many studies have confirmed that GR and its derivates can enhance the mechanical characteristics of the scaffolds which can overcome the shortcomings of the single natural biological material [29], [30], [31]. For example, Golafshan et al. [32] developed a GR-PVA scaffold and found that the scaffold with GR has superior tensile strength, elongation, and toughness than the scaffold with just PVA and sodium alginate. Meanwhile, GR also has considerable electrical properties because of the unpaired π electron among each carbon atom which can move freely [33]. Qian et al. [34] reported that a member of the GR family, graphene oxide, blended with PCL by integration molding technology could induce the bioelectricity of the cell itself without external electrical stimulation. The electrical signal conduction was restored and the nerve motor and sensory abilities were promoted.

In this study, a biocompatibility and biodegradable scaffold was prepared by co-cross-linking GR/sodium alginate (SA) method to manufacture nerve-repair synergistic hydrogel (Scheme 1). In theory, compared with single SA hydrogel, the improved GR-SA hydrogel will be much more efficient for PNI repair due to the respective advantages of the two materials. Herein, the low cytotoxicity and good biocompatibility properties of the GR-SA hydrogel were characterized. To demonstrate the effectiveness and biosafety of this novel biological scaffold, a clamp-defect SD rat model was established to further assess the scaffold's therapeutic effect in vivo. Besides, GR-SA hydrogel characterization and in vitro performances had also been studied.

Section snippets

Synthesis of GR-SA hydrogel

The concentration of sodium alginate hydrogel (SA, Sigma-Aldrich, China) and single-layer graphene (GR, Sigma-Aldrich, China) were 4% (w/v) and 1%, 0.5%, 0.1% (w/wSA). SA and GR were dissolved in water and stirred overnight. Avoid air bubbles in the solvent during this process. 1 mL pipette was used to draw SA-GR solution (100 μL for SEM and in vitro cytotoxicity, 900 μL for western blot and immunofluorescence staining, 1000 μL for in vivo test) into 6-well or 24-well plate (24-well plate for

Synthesis and characterization of GR-SA hydrogel

In this experiment, different concentrations of graphene (GR) and SA were co-cross-linked (Fig. 1a). This method was drawn lessons from previous common methods of sodium alginate (SA) hydrogel fabrication [36], [37]. Due to the excessively fast chemical crosslinking itself, the speed of chemical cross-linking was controlled by two aspects. Firstly, the concentration of calcium chloride was 0.1 M which was proved a better concentration for hydrogel making in the nerve regeneration field [38].

Conclusions

In this study, a novel, biocompatible and biosafe Graphene-Sodium Alginate hydrogel was designed to make up for the inefficient PNI recovery by single sodium alginate hydrogel. Biologically sourced SA and GR both can significantly improve the sciatic nerve growth-related factors and reduce the expression of inflammation factors. Meanwhile, the good mechanical and biocompatibility properties help GR-SA hydrogel imitate the micro-environment in the body to let the enrichment and release of

CRediT authorship contribution statement

W. Yuan conceived the conceptualization. W. Yuan and Y. Qian conceived the study design and participated in data extraction and analysis. Y. Jin, W. Zhang, Y. Zhang, Y. Yang, Z. Fang, J. Song, Y. Qian, and W. Yuan searched databases and performed studies. Y. Jin, W. Zhang, Y. Zhang, Y. Yang, and Z. Fang drafted the manuscript. W. Yuan and Y. Qian revised the manuscript. All authors read and approved the final manuscript.

Declaration of competing interest

The authors declare no conflict of interest.

Acknowledgements

The study was sponsored by National Key R&D Program of China (No. 2021YFC2400801), the Interdisciplinary Program of Shanghai Jiao Tong University (YG2019QNA24), National Natural Science Foundation of China (Grant No. 82002290), Young Elite Scientist Sponsorship Program by Cast (No. YESS20200153), the Shanghai Sailing Program (No. 20YF1436000), and the Interdisciplinary Program of Shanghai Jiao Tong University (YG2019QNA24). We appreciate the support from faculties of the Instrumental Analysis

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