Review on Bioinspired Design of ECM-Mimicking Scaffolds by Computer-Aided Assembly of Cell-Free and Cell Laden Micro-Modules

Abstract

Tissue engineering needs bioactive drug delivery scaffolds capable of guiding cell biosynthesis and tissue morphogenesis in three dimensions. Several strategies have been developed to design and fabricate ECM-mimicking scaffolds suitable for directing in vitro cell/scaffold interaction, and controlling tissue morphogenesis in vivo. Among these strategies, emerging computer aided design and manufacturing processes, such as modular tissue unit patterning, promise to provide unprecedented control over the generation of biologically and biomechanically competent tissue analogues. This review discusses recent studies and highlights the role of scaffold microstructural properties and their drug release capability in cell fate control and tissue morphogenesis. Furthermore, the work highlights recent advances in the bottom-up fabrication of porous scaffolds and hybrid constructs through the computer-aided assembly of cell-free and/or cell-laden micro-modules. The advantages, current limitations, and future challenges of these strategies are described and discussed.

Keywords: CAD scaffold; micro-modules; modular tissue engineering; tissue spheroids.

Figure 1

Synthetic ECM-mimicking scaffold features. Multifunctional scaffolds should provide cells with proper microenvironmental features by displaying arrays of biophysical properties and biochemicals with an on-demand logic following the evolution of new tissue morphogenesis. Clockwise, from top: scaffold architectural features, namely morphology, pore structure and size, must support cell adhesion and organization into the scaffold space to mimic the organization and structure of native tissues. Cell orientation, motility, adhesion, and differentiation can be modulated by a specific surface stiffness and micro- and nano-topography. The degradation of scaffold structure controls the space available for cell migration and tissue growth, while electrical properties are directly correlated to the orientation and growth of aligned tissues, such as nerve and muscles. Biochemical signals, such as GFs and cell adhesive ligands, must be presented with the correct conformation to elicit the desired biological response. By incorporating within scaffold material either micro- or nano-carriers loaded with bioactive peptides, the time and space evolution of these molecules can be programmed to enhance, among others, tissue vascularization and ECM deposition. The 3D structure of scaffolding materials also serves as a sensing platform for the on-time monitoring of cellular metabolic activity, and as an actuating system to promote and guide correct cell and tissue morphogenesis.

Figure 2

Challenges of bottom-up ECM-mimicking scaffolds design, engineering, and fabrication. Design relevant properties of micro- and nano-building block modules encoding a specific function; engineer a virtual scaffold model by using advanced numerical procedures able to match the spatial arrangement of building blocks with any predefined complex molecular and structural microenvironment. Manufacture bottom-up scaffolds by modules printing and/or micro- and nano-positioning. On-time monitor and guide cells and tissue morphogenesis. The integration of these platforms would allow simultaneous but almost independent control of multiple structural features and bioactive signals together with the evaluation and possibly the stimulation of functional tissue growth.

Figure 3

Microscaffolds and tissue spheroids micro-modules. Cell-laden micromodules can be achieved by seeding and culture of cells onto highly interconnected microscaffolds or by engineering multi-cellular spheroids. (A) Microscaffolds provide cells with adequate shape and resistance to mechanical stresses during handling (e.g., printing). Furthermore, microscaffolds may allow to achieve proper drug release during cell expansion and tissue development. (B) Tissue spheroids are formed as a 3D cellular structure with dense cell-cell/cell-ECM interactions. Spheroids can be shaped by mould and maximize cell delivery into the scaffolds. (A), reproduced with permission from [92]; (B) reproduced with permission from [93].

Figure 4

Tissue spheroids patterning by (A) aspiration-assisted freeform bioprinting ((a) The bioprinting setup, where a box was filled with the yield-stress gel in one compartment and cell media in the other) and (B) mould patterning. In the aspiration-assisted freeform bioprinting, the aspiration force was used to pick up spheroids from the spheroid reservoir and transfer them into the yield-stress gel one by one following five layers of circles forming a cylinder. In (B), 3D spheroids were generated using non-adhesive agarose gels to guide self-assembly, and subsequently transferred to non-adhesive troughs. (A), reproduced with permission from [109]; (B), reproduced with permission from [116].

Figure 5

Micromodules patterning within the pores of a custom-made 3D printed thermoplastic cage. (A) Automated 3D bioassembly platform capable of fabricating hybrid constructs via a multistep bottom-up bioassembly strategy. The bioassembly system consisted of a fluidic-based singularisation and injection module that delivers individual micro-tissues to an injection module for insertion into precise locations within a 3D plotted scaffold (sections of assembled micro-tissues and associated tissue fusion in adjacent culture over 28 days (a–f) stained with safranin-O/haematoxylin/fast green (a–c) or Collagen II antibodies (e,f). Bioassembled HAC-laden 9.5% GelMA-0.5% HepMA micro-spheres (g,h) stained with Calcein AM (live cells, green) and Propidium Iodide (dead cells, red) (g) or DAPI (blue) and Aggrecan (purple) antibodies (h) after 35 days culture in chondrogenic differentiation media). (B) The modular capability of the bioassembly platform was used to build biphasic, graduated, and assorted structures of mesenchymal stromal cells (hMSCs) and human nasal chondrocytes (hNCs). (A), reproduced with permission from [24]; (B), reproduced with permission from [120].