Graphene oxide-functionalized nanocomposites promote osteogenesis of human mesenchymal stem cells via enhancement of BMP-SMAD1/5 signaling pathway

Abstract

Biomaterials that can harness the intrinsic osteogenic potential of stem cells offer a promising strategy to accelerate bone regeneration and repair. Previously, we had used methacrylated gelatin (GelMA)-based scaffolds to achieve bone formation from human mesenchymal stem cells (hMSCs). In this study, we aimed to further enhance hMSC osteogenesis by incorporating graphene oxide (GO)-based nanosheets into GelMA. In vitro results showed high viability and metabolic activities in hMSCs encapsulated in the newly developed nanocomposites. Incorporation of GO markedly increased mineralization within hMSC-laden constructs, which was further increased by replacing GO with silica-coated graphene oxide (SiGO). Mechanistic analysis revealed that the nanosheet enhanced the production, retention, and biological activity of endogenous bone morphogenetic proteins (BMPs), resulting in robust osteogenesis in the absence of exogenous osteoinductive growth factors. Specifically, the osteoinductive effect of the nanosheets was abolished by inhibiting the BMP signaling pathway with LDN-193189 treatment. The bone formation potential of the technology was further tested in vivo using a mouse subcutaneous implantation model, where hMSCs-laden GO/GelMA and SiGO/GelMA samples resulted in bone volumes 108 and 385 times larger, respectively, than the GelMA control group. Taken together, these results demonstrate the biological activity and mechanism of action of GO-based nanosheets in augmenting the osteogenic capability of hMSCs, and highlights the potential of leveraging nanomaterials such as GO and SiGO for bone tissue engineering applications.

Introduction

As the median age of the world's population increases, the need for bone repair and regeneration is trending steeply upward [1]. Conventional bone defect management includes bone transplants, with autografts being the “gold standard” [2]. Although these natural grafts usually lead to increased osteoinduction and osteoconduction, they have a number of drawbacks, such as limited availability and donor site morbidity, as well as risks of infection and disease transmission [3]. As an alternative, stem cell-based tissue engineering offers a promising approach to treating bone damage. In particular, human mesenchymal stem cells (hMSCs), which are multipotent cells present in a number of tissues throughout the body, are among the most widely studied cell sources for bone tissue engineering [4]. For example, we recently seeded hMSCs in a gene-activated, methacrylated gelatin (GelMA)-based scaffold and achieved robust reparative capacity in cranial bone defects in mice [5].

To induce osteogenic differentiation of hMSCs, growth factors such as bone morphogenetic proteins (BMPs) are usually required. Among the different BMPs, BMP2 and BMP7 are both highly osteoinductive and have been used in commercially available medical products [6]. However, as is the case with other growth factors, BMP application in tissue engineering is generally hindered by its low stability, short half-life, high cost of production, and undesirable potential side effects, including carcinogenesis [7]. Therefore, growth factor-free approaches to bone tissue engineering has attracted increasing attention [8,9]. Previous studies by our group and others have shown that delivery of osteogenic genes to hMSCs could lead to a sustained production of osteogenic growth factors such as BMPs at physiologically relevant concentrations, resulting in enhanced osteogenic differentiation and mineralization of hMSCs in vitro and in vivo [5,10,11]. However, the safety of such gene delivery strategies warrants further investigation to reach clinical trials.

Discoveries and advances in nanomaterials and nanocomposites in recent decades has greatly advanced technological developments in tissue engineering and regenerative medicine [12,13]. The importance of the nano-dimension and -topography of the components of the native cellular niche provides the conceptual rationale driving these developments. For example, Reznikov et al. [14] identified the hierarchical assembly of bone minerals and collagen at the nanoscale. Recently, the application of graphene and graphene derivatives in tissue engineering has been enthusiastically pursued [15]. These two-dimensional (2D) nanomaterials possess remarkable electrical, mechanical, and thermal properties [16,17]; in addition, the large specific surface area makes graphene nanomaterials effective drug delivery carriers [18,19]. Nayak et al. [20] reported that hMSCs seeded on graphene-coated surfaces had accelerated osteogenic differentiation. Similar results were reported in another study, which demonstrated graphene's ability to preconcentrate β-glycerolphosphate and dexamethasone, two important osteogenic inducers [21]. Lu et al. fabricated free-standing, multi-layered graphene membranes, which facilitated in vivo bone formation via enhanced protein adsorption [22,23]. These studies suggest that graphene nanosheets can independently induce robust osteogenesis, thus overcoming the limitation of using osteogenic growth factors in bone tissue engineering. However, the mechanism governing the responses of cells/tissues to nano-dimensional materials such as graphene is incompletely understood. Future success in clinical applications of nanomaterials requires a more in-depth understanding of how nanomaterials influence cell behavior. In this study, we have characterized BMP signaling in hMSCs encapsulated in 3D nanocomposite scaffolds, in order to gain insights into the nature of biomolecular interactions between hMSCs and 2D nanomaterials.

Hydrogels, such as GelMA, represent an important type of scaffold for bone regeneration. With their high water content, hydrogels provide a tissue-like, biocompatible, 3D environment for cell culture. In particular, injectable and degradable hydrogels can conform to the shape of irregular bone defects and can be gradually replaced via natural biodegradation by the new, growing tissue. However, compared with commonly used biometals (e.g., Ti and Mg alloys) and bioceramics (e.g., tricalcium phosphate, hydroxyapatite), hydrogels usually have reduced osteoinductivity and substantially lower mechanical strengths [24,25]. With the incorporation of nanomaterials such as graphene, hydrogel-based nanocomposites can serve as a versatile bone tissue engineering platform that allows for the generation of 3D cell-laden constructs with tunable mechanical, structural, and biological characteristics [26]. For example, our previous study reported that graphene oxide (GO), a graphene derivative with high aqueous dispersity, increased the Young's modulus of a hydrogel-based cartilage scaffold [27]. GO incorporation in hydrogel scaffolds was also reported to enhance osteogenic differentiation of the encapsulated hMSCs [28,29]. However, the underlying mechanisms need to be further explored to pave the road for their future clinical applications.

In this study, we aimed to incorporate GO-based nanosheets to enhance hMSC osteogenesis within GelMA. We hypothesized that the nanosheet-functionalized hydrogels could induce robust osteogenesis of hMSCs without the supplementation of osteoinductive growth factors. To test this hypothesis, we first prepared 3D constructs consisting of GO-encapsulated, hMSC-laden hydrogel scaffolds, and osteogenesis was assessed after 28 days of culture in growth factor-free osteogenic medium (OM). Moreover, in light of the reported benefits of silicon in bone metabolism [30], we tested the potential of a novel GO derivative—silica-coated GO (SiGO)—in supporting hMSC osteogenesis, which has not been investigated previously. To explore the mechanism responsible for nanosheet-induced osteogenesis, we first assessed the presence of endogenous osteogenic growth factors, such as BMP2 and BMP7, and examined their interactions with the carbonaceous nanosheets. Next, the BMP antagonist, LDN-193189, was used to test the involvement of the BMP signaling pathway in the pro-osteogenesis effect of the nanosheets. Finally, a mouse subcutaneous implantation model was employed to assess the ability of constructs of hMSC-laden, GO- or SiGO-functionalized GelMA scaffolds for in vivo bone formation.