**1. Introduction**

Tissue engineering is a combination of techniques and materials for the fabrication of scaffolds and devices that, interacting with the cells, can lead to the formation of an analogue of tissues and organs that can improve, assist, or replace those already existing in the human body [1–4]. The biomaterials to be used in tissue engineering should exhibit the most convenient combination of mechanical properties, macro-scale architecture, and nanoscale geometry, to influence the collective behavior of cells and induce cells to form efficient structures. Those structures should be biocompatible, energetically efficient, autonomous, computationally efficient, and should be organized in a way to optimize the exchange of biochemical signals, nutrients, and oxygen between the several different parts of the structures and the external environment [1,2,4,5]. Thus, an ideal scaffold should present details over different hierarchical length scales to enable cell colonization, migration, and organization, and should be preferentially porous to enable the transport of bio-molecules.

At the nanoscale, cell behavior is strongly influenced by their interaction with the surrounding microenvironment [6–11]. Nanomaterials can interact without intermediation with the those adhesion molecules (integrins, cadherins, selectins, the immunoglobulin superfamily of cell surface proteins) involved in several different cell functions, including recognition, binding, adhesion, migration, apoptosis, differentiation, survival, and transcription [7–9,11,12]. Due to this unmediated interaction, nanomaterials can be fine-tuned to manipulate cellular function [13]. Materials with a controlled design at the nanoscale have been demonstrated in applications such as stem cells differentiation [14], the activation of the immune synapse [15], the shaping and signaling in neuronal networks [16], cell adhesion [17–19] and growth [20,21], the manipulation and control of neural polarity [22]. While many of the reported works have focused on bi-dimensional geometries, recent advances in additive manufacturing technologies [23], such as light assisted photopolymerization techniques [24], stereolithography [25], digital light projection, and two-photon polymerization [26], allowed a smooth transition from a 2D to a 3D design of the intended nano-structures [27]. The resulting cell culture models have an increased degree of complexity, an increased number of degrees of freedom, and exhibit a more faithful adherence to the 3D complex architectures of cells in living tissue and organs. This in turn enables to reproduce with an increased level of fidelity the natural microenvironment of cells [28].

Nevertheless, despite important advances in the production of scaffolds for tissue engineering applications, the development of characterization techniques has lagged behind the progress of fabrication. Recently, the demand for new devices for the analysis of the behavior of cells at the length scale of cell receptor is increasing. Those devices may reveal the fundamental biological mechanisms behind cell adhesion, migration, and organization at the cell-adhesion-molecule level, disclosing precious information for those interested in designing the scaffolds in the most efficient way.

In this paper, we present a mesoporous silicon device with details over multiple scales for cell culture, growth, and assembly. The device is functionalized with gold nanoparticle clusters that can be exploited to amplify the Raman signal measured at the cell interface. Thanks to the pores in the silicon matrix, the device can release drugs, growth factors, or other biomolecules to the cells over time. Thus, the device combines the ability of a scaffold to support cell growth with the ability of a drug delivery system to vehicle active-molecules to the cells adhering to the scaffold. The gold nanoparticles on the device enable to examine the combined effects of surface nano-topography and the delivery of drugs on cell adhesion and proliferation. In experiments in which we put in culture cancerous MCF-7 cells on the device, we measured the simultaneous effect of the pore size and of the delivery of an anti-tumor drug on the adhesive and proliferation properties of cells. Moreover, using Raman spectroscopy and a multivariate analysis of data, we mapped the spatial distribution of receptors expressed over the cell surface, and correlated that distribution to the nanoscale architecture of the device. We found that cells exhibit an increased ability to grow and to form clusters on substrates with smaller pore size (PS ≈ 11 nm) and roughness (Ra ≈ 7 nm), compared to substrates with larger pore size (PS ≈ 21 nm) and roughness (Ra ≈ 13 nm), in line with previous studies [29,30]. Both substrates deliver their payload efficiently, up to 10 days from the initial release, demonstrating high anti-cancer efficacy and killing up to 90% of cancerous cells on the smaller mesoporous substrate after 72 h from cell culture.

The multi-functional device that we developed can be used to evaluate the coincidental effects of (i) a timely administrated drug or nutrient and of the (ii) nanoscale characteristics of a surface on the

efficacy of a therapeutic treatment, the functionalities of a sca ffold, or a combination of the two. The device can be potentially used in applications that bridge traditional drug delivery, traditional tissue engineering and regenerative medicine, and diagnostics.
