**1. Introduction to Tissue Engineering Sca**ff**olds and Bottom-Up Fabrication**

Traumas, diseases and population ageing are major reasons for damage and failure of human body tissues and organs, which require medical treatments for their restoration or replacement. Despite the intrinsic body capability of repairing small injuries given sufficient time, to date, tissue growth in large (centimeter-size) defects requires complex, expensive and patient-painful autografts, allografts or xenografts [1,2]. In the case of bone, autograft and allograft implantation produces the best clinical results, but it requires secondary surgery and has a limited supply. The main advantage of a xenograft is its abundant supply and no need for secondary surgery, but poor implantation results and problems of infection from donors are critical issues [2]. Besides, the neo-tissue generated within the interstitial spaces of these grafts is often different from the native tissue and requires large remodeling time for the complete biological and biomechanical integration with surrounding tissues. For these reasons, the

development of novel solutions for tissues and organs bioengineering is extremely demanding in the medical field.

Tissue engineering (TE), an important biomedical engineering field, aims to solve this important challenge by combining scaffolds and bioactive molecules for the artificial reconstruction of functional, three-dimensional (3D) tissues and organs [3]. Biomedical scaffolds are porous, implantable biomaterials, shaped to promptly restore the natural tissue anatomy and mechanical functions. The scaffolds must also be capable of controlling foreign body reaction and new-tissue formation by targeted presentation and delivery of key molecules, e.g., anti-inflammatory, growth factors, and proteins. Indeed, these molecules help scaffolds to reduce inflammation, recruit and direct differentiation of stem cells from surrounding tissues and ultimately, promote functional tissue integration in situ [4–6].

Scaffolds design and fabrication have evolved greatly in the past twenty years due to the large knowledge accumulated on materials design, processing and characterization of cell/scaffold interactions. In the natural tissues, cells and extracellular matrix (ECM) organize into 3D structures from sub-cellular to tissue level. Consequently, to engineer functional tissues and organs successfully, scaffolds must capture the essence of this cells/ECM organization and must provide a porous structure able to facilitate cells distribution and guide 3D tissue regeneration [7–9]. Scaffolds pore size and shape, pore wall morphology, porosity, surface area and pore interconnectivity, are probably the most important architectural parameters, as they have been shown to directly impact cells migration and colonization, new ECM biosynthesis and organization, oxygen and nutrients transport to cells, as well as metabolic wastes removal in the whole cell/scaffold construct [10–16]. The scaffold material must be selected and/or designed with a degradation and resorption rate such that scaffold strength is retained until the tissue-engineered transplant is fully remodeled by the host tissue and can assume its own structural role [17,18]. More importantly, controlling mechanical properties at cellular and sub-cellular levels is important to emulate as closely as possible the in vivo cell behavior and tissue growth [19,20]. Nevertheless, controlling the morphological and biomechanical properties of porous scaffolds is not enough for the success of scaffolds-based therapies, as there is also the need to load matricellular and soluble molecules inside scaffolds' matrix as biochemical regulators of cells behavior [21]. Indeed, it is reported that porous scaffolds releasing biochemical signals following a precise dose and time intervals to target sites stimulate cells' functions (e.g., adhesion, proliferation and migration) [22–25], promote the biosynthesis of new ECM [26] and, ultimately, guide tissue growth, morphogenesis [27] and vascularization [28–30].

Increasing scaffolds' design complexity is therefore extremely demanding and scientists face some important challenges, such as: (1) engineering of scaffold microarchitecture to mimic the ECM structure, (2) imprinting topological and biochemical patterns inside scaffolds pores to guide cells' growth and tissue morphogenesis, and (3) developing automated processes for the precise and reliable control of scaffolds' features and geometry.

Porous bioactive scaffolds can be fabricated by combining biomaterials and growth factors through different processing techniques. This review focuses the attention on modular approaches where samples are built by the "bottom-up" assembly of smaller units or "modules", each one specifically designed for distinct tasks [31–33]. Bottom up approaches have the potential to build scaffolds mimicking the complex molecular and structural microenvironment of the native ECM of every kind of tissues by the proper assembly of micro- and nano-structured modules with well-defined morphological and biochemical properties [34].

Several processing techniques are available for modules fabrication, including fluidic emulsion [35], electrofluidodynamic processes [36], and advanced computer aided manufacturing [37,38]. All of these approaches offer, nowadays, a wide library of materials, each one characterized by different composition, shape, nano- and micro-topography, and porous architecture. The assembly of individual modules, such as microparticles (μPs) or patterned layers, by packing, stacking, and printing, allows for achieving multifunctional scaffolds for tissue and organ bioengineering. As will be discussed in the next sections, cell-free or cell-laden μPs can be packed together in a mold giving rise to a sintered

matrix by contact points union [39]. Sintering can be obtained by heat or proper plasticizers, in the case of cell-free samples, and by promoting cells/cells and cells/ECM interlocking to obtain hybrid structures [34]. μPs can also be used as cell and/or drug carriers to be loaded inside hydrogel pastes for printing more ordered and complex structures [40]. Layer-by-layer scaffolds' fabrication uses medical imaging combined with computer-aided design (CAD) and automated scaffolds' manufacturing processes to produce customized cell-free or cell-laden scaffolds characterized by a highly controlled structure and reliable properties. This broad category of fabrication techniques includes discontinuous processes, based on the assembly (stacking/sintering) of layered structures obtained by mold replication methods [37]. Alternatively, continuous processes, named additive manufacturing (AM), are used to construct scaffolds by joining/printing biomaterials and cells [38].

The focus of this review is to describe and discuss the advancement of current bottom-up techniques for creating adaptive scaffolds built from μPs or prepared using layer-by-layer assembly techniques, focusing on cell-free and cell-laden strategies. The advantages of each approach to controlling scaffolds' microstructural properties and drug release capability will be discussed, outlining some of the most promising results achieved for regenerating different tissues and organs, such as bone and cartilage, blood vessels, and derma.

#### **2. Microparticles (**μ**Ps) as Building Blocks for Modular Tissue Engineering Sca**ff**olds**

Nowadays, μPs are essential elements of clinical and regenerative medicine applications such as cell culture μ-scaffolds for in vivo cell delivery and in vitro tissue biofabrication, and drug delivery carriers for biosensing and diagnostic purposes [41–43]. Both synthetic and natural polymeric biomaterials have been investigated for μPs design and engineering. Indeed, chemical and physical polymers properties can be easily manipulated to design and fabricate μPs with tailored morphological properties, size-shape distribution, and degradation rate. Furthermore, scaffolds prepared from synthetic polymeric μPs offer better chemical stability and mechanical properties than those prepared by using natural polymers, especially for load bearing applications. Common examples of the main synthetic polymers for μPs' fabrication which can be mentioned are PCL, poly-lactic acid (PLA), polylactic-co-glycolic acid (PLGA), poly-ethylene glycol (PEG), and their composites with ceramic fillers like calcium phosphate, alumina, and hydroxyapatite [4,44–48]. Natural polymers, conversely, have a chemical composition and structure resembling that of native biological tissues. This aspect is extremely fascinating for μPs' fabrication as it makes it possible to achieve materials faithfully replicating the ECM microenvironment functions. Natural polymeric μPs can be classified into two main groups: protein-based, such as silk, collagen, and fibrin, and polysaccharide-based, like agarose, chitosan, and hyaluronic acid. These kinds of μPs have advantages like excellent biocompatibility, immunogenicity, and degradation rate that can be tuned by varying μPs' materials composition, molecular weight, and crosslinking degree. In the next section, an overview will be provided about μPs' fabrication, highlighting the most advanced techniques to control μPs' composition, structure, and size-shape distribution. Furthermore, the use of μPs as building blocks for cell-free and cell-laden scaffolds fabrication and their use as μ-scaffolds for in vitro cell culture and tissue production will be described in detail.
