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Nanotopography presents an effective physical approach for biomaterial cell manipulation mediated through material-extracellular matrix interactions. The extracellular matrix that exists in the cellular microenvironment is crucial for guiding cell behaviours, such as determination of integrin ligation and interaction with growth factors. These interactions with the extracellular matrix regulate downstream mechanotransductive pathways, such as rearrangements in the cytoskeleton and activation of signal cascades. Protein adsorption onto nanotopography strongly influences the conformation and distribution density of extracellular matrix and, therefore, subsequent cell responses. In this review, we first discuss the interactive mechanisms of protein physical adsorption on nanotopography. Secondly, we summarise advances in creating nanotopographical features to instruct desired cell behaviours. Lastly, we focus on the cellular mechanotransductive pathways initiated by nanotopography. This review provides an overview of the current state-of-the-art designs of nanotopography aiming to provide better biomedical materials for the future.

Collagen type I lacks affinity for growth factors (GFs) and yet it is clinically used to deliver bone morphogenic protein 2 (BMP-2), a potent osteogenic growth factor. To mitigate this lack of affinity, supra-physiological concentrations of BMP-2 are loaded in collagen sponges leading to uncontrolled BMP-2 leakage out of the material. This has led to important adverse side effects such as carcinogenesis. Here, we design recombinant dual affinity protein fragments, produced in E. Coli, which contain two regions, one that spontaneously binds to collagen and a second one that binds BMP-2. By adding the fragment to collagen sponges, BMP-2 is sequestered enabling solid phase presentation of BMP-2. We demonstrate osteogenesis in vivo with ultra-low doses of BMP-2. Our protein technology enhances the biological activity of collagen without using complex chemistries or changing the manufacturing of the base material and so opens a pathway to clinical translation.

Mesenchymal stem cell (MSC)-based tissue engineering strategies are of interest in the field of bone tissue regenerative medicine. MSCs are commonly investigated in combination with growth factors (GFs) and biomaterials to provide a regenerative environment for the cells. However, optimizing how biomaterials interact with MSCs and efficiently deliver GFs, remains a challenge. Here, via plasma polymerization, tissue culture plates are coated with a layer of poly (ethyl acrylate) (PEA), which is able to spontaneously permit fibronectin (FN) to form fibrillar nanonetworks. However, vitronectin (VN), another important extracellular matrix (ECM) protein forms multimeric globules on the polymer, thus not displaying functional groups to cells. Interestingly, when FN and VN are co-absorbed onto PEA surfaces, VN can be entrapped within the FN fibrillar nanonetwork in the monomeric form providing a heterogeneous, open ECM network. The combination of FN and VN promote MSC adhesion and leads to enhanced GF binding; here we demonstrate this with bone morphogenetic protein-2 (BMP2). Moreover, MSC differentiation into osteoblasts is enhanced, with elevated expression of osteopontin (OPN) and osteocalcin (OCN) quantified by immunostaining, and increased mineralization observed by von Kossa staining. Osteogenic intracellular signalling is also induced, with increased activity in the SMAD pathway. The study emphasizes the need of recapitulating the complexity of native ECM to achieve optimal cell-material interactions.

The tumor microenvironment plays an important role in cancer development and the use of 3D in vitro systems that decouple different elements of this microenvironment is critical for the study of cancer progression. In neuroblastoma (NB), vitronectin (VN), an extracellular matrix protein, has been linked to poor prognosis and appears as a promising therapeutic target. Here, we developed hydrogels that incorporate VN into 3D polyethylene glycol (PEG) hydrogel networks to recapitulate the native NB microenvironment. The stiffness of the VN/PEG hydrogels was modulated to be comparable to the in vivo values reported for NB tissue samples. We used SK-N-BE (2) NB cells to demonstrate that PEGylated VN promotes cell adhesion as the native protein does. Furthermore, the PEGylation of VN allows its crosslinking into the hydrogel network, providing VN retention within the hydrogels that support viable cells in 3D. Confocal imaging and ELISA assays indicate that cells secrete VN also in the hydrogels and continue to reorganize their 3D environment. Overall, the 3D VN-based PEG hydrogels recapitulate the complexity of the native tumor extracellular matrix, showing that VN-cell interaction plays a key role in NB aggressiveness, and that VN could potentially be targeted in preclinical drug studies performed on the presented hydrogels.

Embryonic stem cells (ESCs) possess remarkable abilities, as they can differentiate into all cell types (pluripotency) and be self-renewing, giving rise to two identical cells. These characteristics make ESCs a powerful research tool in fundamental embryogenesis as well as candidates for use in regenerative medicine. Significant efforts have been devoted to developing protocols to control ESC fate, including soluble and complex cocktails of growth factors and small molecules seeking to activate/inhibit key signaling pathways for the maintenance of pluripotency states or activate differentiation. Here we describe a novel method for the effective maintenance of mouse ESCs, avoiding the supplementation of complex inhibitory cocktails or cytokines, e.g., LIF. We show that the addition of zinc to ESC cultures leads to a stable pluripotent state that shares biochemical, transcriptional and karyotypic features with the classical LIF treatment. We demonstrate for the first time that ESCs maintained in long-term cultures with added zinc, are capable of sustaining a stable ESCs pluripotent phenotype, as well as differentiating efficiently upon external stimulation. We show that zinc promotes long-term ESC self-renewal (>30 days) via activation of ZIP7 and AKT signaling pathways. Furthermore, the combination of zinc with LIF results in a synergistic effect that enhances LIF effects, increases AKT and STAT3 activity, promotes the expression of pluripotency regulators and avoids the expression of differentiation markers.