Combined additive manufacturing approaches in tissue engineering

Accepted Manuscript Combined additive manufacturing approaches in tissue engineering S.M. Giannitelli, P. Mozetic, M. Trombetta, A. Rainer PII: DOI: Reference:

S1742-7061(15)30004-0 http://dx.doi.org/10.1016/j.actbio.2015.06.032 ACTBIO 3763

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Acta Biomaterialia

Received Date: Revised Date: Accepted Date:

15 January 2015 26 June 2015 26 June 2015

Please cite this article as: Giannitelli, S.M., Mozetic, P., Trombetta, M., Rainer, A., Combined additive manufacturing approaches in tissue engineering, Acta Biomaterialia (2015), doi: http://dx.doi.org/10.1016/j.actbio. 2015.06.032

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Combined additive manufacturing approaches in tissue engineering

S.M. Giannitelli, P. Mozetic, M. Trombetta, A. Rainer*

Tissue Engineering Unit, ―Università Campus Bio-Medico di Roma‖, via Álvaro del Portillo 21, 00128 Rome, Italy.

* Corresponding author: Alberto Rainer, PhD Tissue Engineering Unit Università Campus Bio-Medico di Roma 00128 Rome, Italy Ph +39 06 225419640 Fax +39 06 225411949 e-mail: [email protected] 1

Abstract Advances introduced by additive manufacturing (AM) have significantly improved the control over the microarchitecture of scaffolds for tissue engineering. This has led to the flourishing of research works addressing the optimization of AM scaffolds microarchitecture to optimally trade-off between conflicting requirements (e.g. mechanical stiffness and porosity level). A fascinating trend concerns the integration of AM with other scaffold fabrication methods (i.e. ―combined‖ AM), leading to hybrid architectures with complementary structural features. Although this innovative approach is still at its beginning, significant results have been achieved in terms of improved biological response to the scaffold, especially targeting the regeneration of complex tissues. This review paper reports the state of the art in the field of combined AM, posing the accent on recent trends, challenges, and future perspectives.

Keywords: additive manufacturing (AM); combinational approaches; bi/multimodal scaffolds; biphasic scaffolds; hybrid AM.

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1. Introduction The relevance of additive manufacturing (AM) to tissue engineering (TE) is testified by the increasing number of research works dealing with the production of free-form porous scaffolds with custom-tailored architectures. Depending on their working principle, AM systems have been classified into laser-based, printing-based and nozzle/extrusionbased systems [1]. Laser-based systems benefit from the photopolymerization pathway as a basis to fabricate crosslinked polymeric scaffolds. Printing-based systems can work either depositing a binder on a powder bed, or directly positioning the structural material using inkjet technology. Finally, nozzle-based techniques consist in the microextrusion/dispensation of a small-diameter polymeric filament through a nozzle having an orifice diameter in the range of hundreds of micrometers. According to the type of feedstock (filament, powder, and granulate) and to the extrusion mechanism (mechanical or pneumatic), nozzle-based techniques have been further classified as Fused Deposition Modeling (FDM), Precision Extrusion Deposition (PED), 3D Bioplotter™, and other variants. Advances introduced by AM have significantly improved the ability to control pore volume, pore size distribution, and pore interconnectivity, as well as the mechanical performances of TE scaffolds. This has led to the flourishing of research works addressing the optimization of AM scaffolds to optimally trade-off between often conflicting requirements (e.g. mechanical stiffness and porosity level) [2-4]. As a result, scaffolds mimicking native tissues in terms of shape and mechanical compatibility have been produced [5]. Furthermore, as some AM processes operate at room temperature, hydrogels containing living cells and bioactive molecules have also been successfully fabricated without significantly affecting cell viability. Thus, several research groups have adapted AM techniques to assemble living cell-laden constructs directly from computer-generated design models with high resolution, aiming to demonstrate the feasibility of manufacturing complex tissues [6, 7]. However, due to the rather harsh processing conditions, not all AM techniques are suitable for the generation of viable, self-supporting constructs. Cell encapsulation has been successfully performed with nozzle-based or inkjet techniques, as long as shear stress induced on cells during deposition is sufficiently low [8]. Laser-based techniques (with the exception of selective laser sintering, SLS) might also be suitable for hydrogel processing [9]; however, photoinitiator concentration and light intensity should be minimized to avoid possible adverse effects to cells. Regarding other AM techniques, the use of organic solvents, high temperatures, or crosslinking agents poses significant constraints on cell viability. A comprehensive overview on current trends and limitations of hydrogel-friendly AM techniques has been recently provided by Billiet et al. [9].

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One of the main limitations of AM for tissue engineering applications is represented by its low-resolution limits, negatively affecting cell adhesion and tissue regeneration. In fact, surface properties such as roughness and topography have been demonstrated to play a pivotal role in orchestrating cell attachment, migration, proliferation, and differentiation, possibly due to cell–cell interactions occurring on that length scales [10, 11]. On the other side, the plethora of conventional scaffold fabrication techniques reported in the literature [12]—including solvent casting, particulate leaching, gas foaming, electrospinning (ES), phase separation, freeze drying, etc.—albeit providing interesting topographical cues, show severe limitations, which predominantly regard the inability to precisely control scaffold pore size, geometry, and interconnectivity. Instead, the hierarchical structure of native tissues demands for new methods to fabricate scaffolds with pore sizes spanning from micro- to macro-scale, with anisotropic pore distribution, while maintaining tight spatial control over geometry and composition. While macro-scale features within the scaffold are intended to promote tissue ingrowth and vascularization, nanofeatures are designed to better mimic the native extracellular matrix (ECM) arrangement [10]. In this scenario, the combination of AM with other conventional scaffold fabrication techniques arises as a new promising strategy: just like different materials can been integrated into one construct to exploit the intrinsic properties of each of them, converging different technologies to build a single microarchitecture can represent an effective route to overcome shortfalls of the single techniques. The hybrid combination between AM and conventional scaffolding methods is therefore expected to enable the generation of smart hierarchical structures, that can potentially satisfy the clinical demand for complex tissue substitutes and meet the requirements for functional TE constructs (e.g. control over scaffold microstructure and external shape, pore size allowing cell engraftment and migration, as well as adequate mechanical properties). In this framework, surface modification techniques can be easily integrated with the fascinating challenge of instructing tissue regeneration in situ by the addition of physical, chemical, and biological cues. Furthermore, the integration of micro- and nano-features with different mass transfer properties into a single architecture opens new possibilities for the development of sophisticated, highly tunable drug delivery systems [13]. Although several authors have recently pointed out the necessity to combine different processing techniques for the obtainment of biomimetic scaffolds [13-15], a complete overview on recent findings of this cutting edge research trend has not been provided. Thus, the present paper aims to summarize current advances in the combination of AM with other fabrication methods for the production of smart TE scaffolds.

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2. Rationale for combinational approaches Together with the diffusion of additive manufacturing, a paradigm shift toward computer-aided tissue engineering (CATE) and in silico scaffolds optimization has occurred [2, 16]. This approach has led to the successful fabrication of patient-specific 3D structures for several TE applications, such as bone TE [17, 18], repair of osteochondral defects [19, 20] and lumbar interbody fusion implants [21]. As a further evolution, bioreactor knowhow has been integrated with AM technologies, pursuing the development of highly integrated and automated systems [22]. According to this principle, AM techniques have been recently employed to simultaneously generate customized scaffolds readily contained into personalized culture environments (implant-specific bioreactor chambers) with high level of reproducibility [23]. However, although AM methods show a huge potential, they are hampered by low resolution, limited material selection, and smooth-surfaced struts, which affect initial cell adhesion and proliferation. Indeed, due to the resolution limits, AM is not suitable either to directly manufacture sub-micrometer structures that could mimic the features of natural ECM, or to achieve hierarchical porous architectures with multimodal pore size distributions [24]. To overcome these limitations, some attempts have targeted the improvement of existing additive manufacturing setups [25, 26] to obtain highly roughened fiber morphologies for enhanced cell adhesion and proliferation. Alternative solutions have aimed to the development of innovative scaffold architectures [2, 27] acting as a ―sieve‖ for cells, thereby retaining a higher number of cells within the scaffold during seeding procedures. With the same aim, hybrid scaffolds that integrate the advantages of several materials in the same microstructure have been fabricated [28, 29]. In this regard, promising results have been obtained by the addition of nanomaterials (nanoparticles [30, 31], carbon nanotubes [32], etc.) to AM printing media, enabling the creation of novel composites [33]. In this perspective, the combination of AM with other scaffolding fabrication methods, such as electrospinning (ES), freeze-drying, salt leaching, etc., emerges as a growing trend, potentially reaping the advantages of both ―worlds‖. Such integration has been implemented both at the assembly level, by combining substructures obtained through different fabrication technologies, and at the fabrication level, by incorporating multiple-length-scale networks within a single final product. According to the nomenclature introduced by Dalton et al. [34], we will refer to scaffolds containing two or more regions with different topologies as ―multiphasic scaffolds‖, while scaffolds that contain micro/nano-features intertwined within the same architecture will be termed ―bimodal scaffolds‖. As a further evolution, most recent works have tackled integration at the technique level, that has been pursued by fusing the working principles of different fabrication techniques within a single, novel technology. In the following, we will refer to this last approach as ―hybrid 5

AM‖. Figure 1 presents the proposed classification, with an accent on the achieved level of integration. The representative case of the integration between AM and ES has been chosen for sole illustrative purposes.

3. Hierarchical integration of modular units at the assembly level A popular research trend regards the use of combinational approaches in terms of hierarchical assembly of modular units to recapitulate complex and multiple tissues with functional interfaces. Scaffolds have been assembled to produce bio-mimicking structures and have been loaded with inductive biological signals and specific cells to ideally promote individual growth of each compartmental layer within a single integrated implant. Thus, bi/multiphasic scaffolds have been developed using different material types, internal architectures (e.g. porosity, pore interconnectivity), cells, biological factors, and—in the majority of cases—more than one fabrication technology [35, 36]. These scaffolds often comprise a hard compartment, generally obtained by AM, and a soft phase, mostly represented by polymeric foams or textile meshes [37-39]. Thanks to the similarity of electrospun matrices with the native ECM, electrospinning represents one of the most common techniques for the production of the soft compartment. Vaquette et al. reported the fabrication of biphasic scaffolds for the regeneration of alveolar bone/periodontal ligament complex using an AM scaffold and an ES membrane for the bone and periodontal compartments, respectively (Table I.a). The ES membrane acted as a support, promoting the adhesion of a periodontal ligament fibroblast cell sheet, while the AM scaffold enabled space maintenance for bone regeneration and provided biomechanical stability. The resulting construct supported bone and ligament tissue formation and promoted the attachment onto an ectopically implanted dentin block in a rodent model [38]. A ―second generation‖ biphasic construct has been successively developed by the same group with the aim to incorporate osteoinductive signals in the bone compartment by means of calcium phosphates (Ca-P) coating [40]. The original design was further modified replacing the solution electrospun membrane with a thin melt electrospun scaffold comprising larger pores for improving cellular and tissue interaction, especially in terms of vascularization. The resulting construct showed significant bone formation, high level of vascularization, and, ultimately, improved integration between bone and periodontal ligament compartments. Furthermore, given the distinctive differences between cartilage and bone, multiphasic scaffolds are currently being sought for osteochondral tissue engineering applications [41]. Indeed, while monophasic scaffolds have for long represented the gold standard in osteochondral repair, there is growing interest toward bi/multi-compartmental architectures, to separately optimize the architecture and composition of the cartilage and bone layers, that can be post6

assembled after chondrogenic and osteogenic in vitro pre-culture [42]. AM structures have been frequently chosen as the bone-mimicking compartment in biphasic constructs for osteochondral regeneration. One of the first attempts in this direction has been made by Schek et al., who fabricated bi-layered composite scaffolds using indirect solid free-form techniques to produce the molds for an hydroxyapatite bone compartment, which was coupled to a poly-L-lactide foamy cartilage section obtained by particulate leaching [37]. More recently, Jeon et al. [43] developed a multiphasic scaffold composed by an alginate hydrogel surmounting a biphasic PCL scaffold (obtained by FDM and ES) (Table I.b). Assembly of the two compartments was achieved via partial de-crosslinking of the alginate layer, which was pressfitted on top of the PCL scaffold and re-crosslinked. Histological analysis of the constructs, following subcutaneous implantation in rats, showed delamination issues, possibly owing to gradual weakening of the interface region. In general, assembling different scaffold units to obtain an interface strong enough to withstand shear stresses and to prevent delamination is not a trivial process. Common bonding strategies include components sintering [44], pressfitting [38], and other lamination techniques [45, 46]. Alternatively, solutions have been proposed to mitigate interfacial bonding issues, including heterogeneous scaffolds composed of a single material displaying differential growth factor enrichment, porosity, or composition in the two integrated layers, or the generation of scaffolds with a continuous interface [47]. As an example, different rapid prototyping (RP) technologies have been integrated by Moroni et al. to create an osteochondral scaffold in which the chondral section was directly deposited onto the bone compartment and finely interlocked through intertwined concentric polymeric fibers [48]. Good cohesion between the two compartments was obtained thanks to the swelling properties of the materials and the optimized interlocking fiber system. As a further advancement in this direction, osteochondral scaffolds with an additional intermediate compartment have been fabricated to guarantee a more functional interface between bone and cartilage. Indeed, the addition of this interface layer should represent a valid strategy to prevent any upward migration of the subchondral bone plate into the chondral layer [47]. In particular, a bio-inspired scaffold with a transitional structure resembling the biological transitional interface from cartilage to bone was fabricated using a combination of stereolithography and gel-casting techniques [49]. Alternatively, a novel multiphasic scaffold with a ―compact layer‖ functioning both as a connector and an insulator was obtained by integrating low-temperature deposition manufacturing (LDM) and a modified temperature gradient-guided thermal-induced phase separation (TIPS) [50] (Table I.c,d). This scaffold showed superior biomechanical properties and led to improved histological scores upon implantation into rabbit knee joints, compared with a compact-layer-free control scaffold. Although these multiphasic osteochondral scaffolds have still room for improvement, they might represent an ideal route to overcome a traditional obstacle of osteochondral regeneration, namely delamination of cartilage and bone compartments. 7

The regeneration of large engineered tissues has been often limited by low cellularity, resulting in poor implant survival and integration with the host tissue in vivo [5]. To address this issue, 3D cell-laden structures have been recently fabricated with various printing technologies and combined with structural synthetic biomaterials, with the aim to mimic the multi-composite features of native complex tissues, guaranteeing high cellularity upon construct fabrication [51, 52]. As a further advancement, biological modules, typically consisting of cell masses or cell/polymer constructs, have been introduced as building units, leading to a new concept of ―modular TE‖ [5]. Following this approach, Kim et al. developed an intriguing multifunctional assembly to hierarchically mimic 3D vascularized adipose tissue [53]. The authors fabricated composite multicellular spheroids composed of mesenchymal stem cells (MSCs) and synthetic ECMmimicking nanofilaments by cellular self-assembly. The spheroids were then packed along the ordered array of void spaces of a porous AM architecture fabricated by direct polymer melt deposition. Ultimately, a 3D cylindrical geometry was produced by the successive stacking of spheroid-embedded scaffold units. This multiscale and multifunctional assembly was demonstrated to enable successful formation of vascularized adipose tissue in vivo and is expected to be further extended to the reconstruction of other multifunctional tissues.

4. Multi-feature integration at the fabrication level As previously underlined, cell seeding efficiency often represents a critical factor for optimal tissue regeneration, due to the limited resolution of additively manufactured features. Therefore, high initial cell concentration and substantial in vitro pre-conditioning are required to attain sufficient adherent cells to engineer functional tissue constructs [54]. This issue has been tackled by introducing a secondary submicrometer-scale network inside the AM architecture to mimic the hierarchical organization of natural tissues. Such an approach ensures that a stable support is available through the AM compartment, while the superimposed microenvironment creates additional sites for cell anchorage, and may provide distinct biochemical cues to guide cell behavior. In this contest, a significant contribution has been provided by Moroni et al., who firstly integrated AM scaffolds with electrospun matrices in a layer-by-layer fashion (Table II.a) demonstrating enhanced biological activity in terms of cell entrapment, proliferation and ECM production [54]. This work paved the way for new studies on hybrid scaffolds obtained alternating macro-sized struts and micro/nano-sized ES sheets [55-58]. Kang et al. provided another significant contribution by developing a complex hierarchical arrangement close to the native architecture of annulus fibrosus. Aligned poly(ε-caprolactone) (PCL) nanofibers sheets were inserted into FDM microfibers to build a multi-lamellar structure in which the angle of fiber alignment in each adjacent layer was maintained to 60° [59]. In such a way, the 8

fibrous layers not only provided a suitable structure for the colonization of the inter-filament gap, but also offered additional ECM-like cues as a contact guidance to cell morphology. Multilayer hierarchical scaffolds with topographical cues at the nanoscale have also been developed for muscle tissue engineering [60]. However, although the addition of this secondary submicrometer-scale network aids in overcoming various disadvantages of conventional AM scaffolds, there are still some limitations, such as low cell migration along the thickness direction and inhomogeneous cell proliferation. This issue has been addressed by Yeo et al., who developed a novel cell-laden hydrogel-based hierarchical scaffold produced by assembling micro-sized PCL struts, with both electrospun PCL nanofibers and cell-laden alginate struts to induce continuous cell release and promote cell migration, proliferation and ECM synthesis [61, 62]. Another innovative approach to obtain bimodal structures foresees the combination of AM techniques with electrohydrodynamic (EHD) direct writing processes. Differently from electrospray and electrospinning, EHD exploits the initial jet of an electrospinning process to work in a stable jet region. Hierarchical 3D structures were obtained by alternating layers of PCL microsized melt plotted struts and highly roughened microsized treads (Table II.b). A significant increment of cell viability and bone mineralization compared to bare AM scaffolds (~2 and 2.5-fold increase, respectively) was registered after in vitro cell culture in combination with osteoblast-like cells [63]. As a further advancement, the authors further optimized the EHD process, using an ethanol bath as the target medium. The optimized AM/EHD scaffold resulted in enhanced in vitro activity compared with both bare AM scaffolds and bimodal scaffolds consisting of AM struts interlayered with electrospun microfibers. This improvement was ascribed to higher cell infiltration registered in the AM/EHD scaffold respect to the AM/ES one [64]. Alternatively, additional native-like microenvironments have been integrated inside previously developed AM architectures by conventional freeze drying technologies [65] or unconventional layer-by-layer electrostatic selfassembly (E-LbL) [66]. According to the former approach, a pre-fabricated AM porous scaffolds was dipped into a gelatin solution, that underwent freeze-drying and cross-linking, providing a supplementary cell entrapment system, with a potential for drug delivery [65, 67] (Table II.c). In the latter, fibrillar structures were introduced across the pores wall of an AM scaffold by the use of unconventional E-LbL self-assembly. E-LbL involves the alternating deposition of polyanions and polycations to produce self-assembled multilayer coatings on scaffold surfaces. Differently from conventional approaches, where intensive washing steps should be used to remove non-reacted or weakly bonded polyelectrolytes, the developed ―unconventional‖ E-LbL exploited incomplete washing steps to leave excess quantities of polyelectrolytes giving rise to small complexes. The process was used to generate multilayer alginate-chitosan fibrillar structures inside the pores of AM scaffolds. The inner structure, increasing the available surface area for cell 9

growth, resulted in enhanced cell seeding efficiency [66] (Table II.d). The potential of this approach has been further demonstrated by the addition of cell-instructive signals within the fibrillary structure obtained by E-LbL. In particular, osteogenic induction of human adipose-derived stem cells was achieved by incorporation of human platelet lysate within E-LbL fibrils [68]. From a totally different perspective, few bimodal scaffolds in which the larger structural component is not represented by the AM compartment should also be mentioned. In these cases, AM architectures are superimposed to previously fabricated soft scaffolds to give a custom-designed structural support [69] or to ameliorate the mechanical properties of the final construct [70]. A remarkable example has been obtained combining electrospinning and fused deposition modeling technique to increase the mechanical strength of tissue engineering vascular grafts [70]. In particular, an electrospun poly-L-lactide (PLLA) tubular scaffold was reinforced with a single-layer helical PCL coil produced by FDM technique. Such a hybrid scaffold showed better mechanical properties compared to to pristine electrospun grafts, preserving the optimal fibrillar arrangement for initial cell attachment. Similarly, AM hydrogel constructs have been mechanically reinforced by the integration with secondary thermoplastic compartments, such as electrospun polymer networks [71, 72] and rigid thermoplastic AM architectures [73, 74]. However, the reinforcing network and the hydrogel often show insufficient adhesion, mostly because of their different physicochemical properties, leading to construct delamination upon the application of mechanical stresses. To overcome this limitation, covalent grafting of a 3DP thermoplastic polymer network to a gelatin hydrogel has been recently proposed to enhance interface-binding strength of reinforced hydrogel constructs [75]. 4.1.

Surface modification of pre-fabricated scaffolds

Another route to improve scaffold performances through the integration of different technologies relies on scaffold surface engineering to confer desirable physical, chemical, and biological functionalities to regulate and accelerate tissue repair and regenerative processes. In particular, plasma treatments have been recently proved to enhance cell attachment and proliferation on several AM polymeric scaffolds [76, 77]. Plasma functionalization is a well-established technique to alter the surface properties of a material introducing physical and chemical cues through the creation of micro-scale roughness and the formation of chemical functional groups (e.g. hydroxyl and carboxylic groups) on the scaffold surface. Thanks to its high homogeneity and the possibility to alter the surface properties without changing the bulk behavior of the treated substrates, plasma functionalization is often preferred to other surface modification techniques used to enhance the bioactive potential of the final construct [78]. Furthermore, the synergistic effect of

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physico-chemical and biological cues obtained by combining plasma surface modification with protein immobilization has been demonstrated to significantly increase cell differentiation [78, 79]. Alternatively, AM scaffolds have frequently undergone surface modification with hydroxyapatite (HA) [80], collagen/HA [81, 82], hyaluronic acid [83], and several signaling molecules (e.g. bone morphogenetic proteins [84, 85]) to improve their functional performances, especially in the case of bone TE. Indeed, although composite polymer/ceramic scaffolds are the gold standard in this field, difficulties in their processing often limit the amount of ceramic phase that can be incorporated into the scaffold. Furthermore, in a conventional blend, the majority of the ceramic particles are masked by the polymeric phase, thus reducing the accessibility of the bioceramic to the physiological environment and hindering its direct contact with the cells [86]. One way to overcome this shortfall consists in the replacement of these ―conventional‖ composite scaffolds with assembled scaffolds, consisting of distinct polymer and ceramic phases integrated within a single final construct in attempt to retain the mechanical properties of the AM component, as well as the bioactivity of the ceramic phase. With this aim, assembled scaffolds have been obtained fitting ceramic HA pillars into the pores of a polymeric 3D structure fabricated using 3D fiber deposition (3DF) [87]. However, considering that the bioactivity of calcium phosphates is believed to lie in the dissolution/re-precipitation processes occurring on the scaffold surface [88], coating the scaffold with a bioactive layer of ceramic has emerged as a simple yet challenging approach to improve its osteoinductive properties. In this regard, different methods have been tested to obtain a homogeneous, adherent, and functional bioactive layer throughout the entire surface without compromising the scaffold shape [81, 86, 89]. Among them, immersion in aqueous solutions of inorganic salts simulating physiological fluids [86, 89] represents the most frequently exploited technique. As a further advancement toward multi-combinational approaches, such a coating process has been recently applied to coat bimodal scaffolds obtained integrating AM and ES, in order to create a construct encompassing the required physical and chemical cues for bone TE applications: 3DF was used to create a mechanically stable structure, ES to provide an ECM-like substrate, and Ca-P coating to increase the bioactivity of the whole structure [90].

5. Integration at the technique level: toward “hybrid” AM technologies With the aim to reach a more sophisticated level of integration, the combination of two or more scaffolding technological platforms into a single apparatus has been pursued. Hence, starting from existing AM equipment, modifications have been introduced in order to meet some essential requirements for biomimetic scaffolds design. 11

First-generation approaches to build scaffolds with both locally and globally porous inner architectures implied the integration of AM with traditional porogen methods [91]. In particular, several studies have reported the implementation of AM techniques (such as 3D printing [92]) with different porogen leaching systems, and, more recently, the use of SLS to create highly porous structures with embedded flow channel networks [93]. However, most AM approaches are not compatible with local-pore fabrication processes (i.e. gas foaming), and several difficulties have been experienced also with standard porogen leaching. Due to these limitations, recent research trends have pursued the development of novel, AM-compatible porogen methods [94], as well as the use of indirect AM techniques [95-97]. More integrated solutions have been implemented by modifying standard manufacturing technologies in order to simultaneously combine sub-millimeter and micrometer sized pores within 3D customized architectures through freeze drying methods. This combination of rapid prototyped and lyophilized structures, which provides a multi-level pore arrangement to a 3D microstructured architecture, ensures a larger surface area for cell adhesion and proliferation [98]. According to this approach, a polymer solution is dispensed at low temperature, where the strands are frozen and then lyophilized to remove the solvent [99]. As an example, poly(lactic-co-glycolic acid) (PLGA) scaffolds with different surface topography on stacking fibers were obtained extruding PLGA solutions of different concentrations by liquidfrozen deposition manufacturing (LFDM) [100]. Similar structures were obtained by Xiong et al. using low temperature deposition manufacturing (LDM) [101]. Other variants based on the same working principle have been reported in the literature, namely cryogenic direct-plotting [102] and rapid freeze prototyping (RFP) [103, 104]. In particular, the innovation of these methods relies in the fact that controlled microporosity levels can be integrated within 3D AM scaffolds avoiding the addition of porogens, by simply tuning process temperature. Furthermore, low viscosity and highly hydrophilic polymers, such as natural biopolymers, can be processed with a level of precision unobtainable with direct printing methods. As an example, 3D collagen scaffolds with designed pore structures were obtained and successfully integrated with micro/nano-sized ES collagen fibers to create hierarchical 3D scaffolds with highly porous and rough surface [105]. A representative scaffold microstructure achievable with rapid freeze prototyping techniques has been reported in Table III.a. As an alternative route to freeze drying, a novel computer-assisted wet-spinning (CAWS) system was recently used to manufacture 3D structures with both locally and globally porous inner microarchitectures by collecting a solidifying filament of polymer solution into a coagulation bath with predefined layer-by-layer patterns [106, 107]. Differently from what commonly observed in scaffold by melt extrusion AM, the produced fibers showed a ―spongy‖ morphology originated by a phase inversion process, and resulted in an enhanced biological response (Table III.b).

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However, most of published studies describe the fabrication of 3D ordered structures through the combined use of additive manufacturing and electrospinning techniques. In this vein, recent studies have begun to use AM techniques to fabricate non-standard collector plates for the electrospinning process. Thus, micropatterned ES scaffolds consisting of random fibers with a defined 3D surface microtopography or vascular-like branching networks [108, 109], as well as optimized grafts for coronary artery bypass [110] were obtained thanks to the use of additively manufactured sacrificial targets. Alternatively, targeting a closer integration between AM and ES, novel hybrid equipment have been successfully developed by exploiting the electrospinning working principle. In particular, the most interesting results have been obtained by modification of electrospinning—and, more in general, of electrohydrodynamic processes—by the addition of a fast-motion automated collecting system. Melt electrospinning writing represents a leading example in this sense and, although still in its infancy, it is starting to spark the interest of the TE community as a novel AM approach ideally derived from the integration of FDM and melt ES [34, 111, 112]. The predictable deposition of several melt electrospun fibers has been combined with an automated collection stage to create a direct writing process, which enables the fabrication of scaffolds with controllable architectures and patterns (Table III.c). Thus, fibers can consistently be laid on top of each other to create scaffolds in a manner comparable to many of the melt-extrusion-based direct writing processes used in AM. However, in this case, the achievable filament resolution and fiber-to-fiber distances approach the micrometer scale and, in this sense, melt electrospinning writing can be seen to bridge the gap between solution electrospinning and direct writing AM [113, 114]. Recently, the 1 micron barrier has been broken by Hochleitner et al. [115], who reported the obtaining of uniform, sub-micron thermoplast filaments of 817 ± 165 nm. EHD jet plotting technology is also evolving in this direction by employing melt biodegradable polymers for the fabrication of periodic 3D structures with a resolution below 10 µm [116]. In addition, to overcome the necessity of high processing temperatures, improved EHD jet printing (E-jetting) techniques have been developed to fabricate highly porous 3D scaffolds with desired controlled filament orientation and large pore size at room temperature [117, 118]. In particular, 3D fibrous scaffolds consisting of a microsized mesh of randomly interwoven micro/nano–fibers were obtained using ethanol media as a target bath [118, 119] (Table III.d). In vitro results showed improved seeding efficiency, with ca. 3.5-fold better cell adhesion if compared with an extrusion-based AM scaffold with similar geometry. Figure 2 summarizes the key features of hybrid AM technologies, with details on porosity levels and resolution limits.

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6. Conclusions Although an impressive number of scaffold fabrication technologies have been developed, the challenge remains to define a proper combination of biomaterials and manufacturing techniques meeting all the requirements for a successful TE approach. Recently, computer-aided rapid prototyping techniques have demonstrated a great potential to generate complex 3D micro- or millimeter-scale structures with a highly reproducible architecture in terms of pore size, geometry, and interconnectivity. Material composition in the different layers of the scaffold can also be highly controlled, leading to compositional gradients within a single microarchitecture. However, resolution and minimum feature size are usually constrained by processing parameters and material properties. Thus, the combination of AM with other scaffolding technologies holds great promise to generate bio-inspired, multi-scale, 3D structures with unmatched properties and multi-functionality. This continuous research of new routes to include biomimetic features within AM architectures is also contributing to the development of innovative, hybrid AM technologies, and to the transfer of laboratory-level manufacturing techniques into real-world processes. Although still at their beginning, these combinational approaches are starting to give positive results not only in the recapitulation of complex tissues, but also in the enhancement of biological processes, such as constructs vascularization. Furthermore, the incorporation and controlled release of biologically active molecules through surface modification is giving a substantial contribute to the creation of a new generation of ‗smart‘ scaffolds, with a strong potential in regenerative medicine applications. However, although some general trends have been drawn, significant research efforts are still needed to fully appreciate the potential of these combinational approaches. In the last years, only a few of the described architectures have been deeply characterized though extensive in vitro and in vivo studies, and results are mostly limited to a restrict number of biomaterials. Furthermore, milder processing conditions should be sought to enable a closer integration with cell encapsulation during manufacturing.

Disclosures The authors declare no conflicts of interest.

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List of Figures Fig.1: Classification of combined additive manufacturing approaches, with the accent on the achieved level of integration. Details on advantages and limitations of each approach, together with the key scaffold architectural characteristics are provided. Combination of AM with ES has been chosen as a representative example for the illustration of manufacturing equipment and obtained microstructures. In the drawings: a) bonding between AM and ES scaffolds (assembly level); b) deposition of AM and ES layers within a single scaffold (fabrication level); c) melt electrospinning direct writing as a de novo, single technique integrating the working principles of the two processes (technique level). Fig.2: Schematic representation of representative ‗hybrid AM‘ technologies, with details on characteristic features. All example figures are reprinted with permission from the respective publishers.

List of Tables Table I: Integration at the assembly level. Example of bi-multiphasic scaffolds obtained by combining AM with conventional techniques. Table II: Integration at the fabrication level. Example of bimodal scaffolds obtained by combining AM with conventional techniques.

19

Table III: Integration at the technique level. Examples of single multifunctional architectures obtained by hybrid AM techniques.

20

EXAMPLE

ARCHITECTURE

APPROACH

INCREASING INTEGRATION

ASSEMBLY LEVEL

FABRICATION LEVEL

TECHNIQUE LEVEL

PROS: Optimization of the single compartment prior to in vivo implantation. CONS: Possible difficulties in achieving proper bonding between layers.

PROS: Dual/multi-scale porosity. High cell seeding efficiency. CONS: Often complex experimental setup. Multi-step process.

PROS: Integrated processes. Fully interconnected inner architectures. CONS: Limited set of biomaterials. Not compatible with cell microencapsulation. No nano-sized features.

Bi/multiphasic scaffolds with compartmented architectures

Bimodal scaffolds with fullyintegrated architectures

Single multi-functional architecture

Key architectural features: • Fiber/pore size: hundreds of µm range (AM layer); tens of nm to hundreds of µm (conventional technique layer) • Thickness: high (cm scale)

Key architectural features: • AM fibers (hundreds of µm range) intertwined with conventional scaffold features (tens of nm to hundreds of µm) • Thickness: high (cm scale)

Key architectural features: • Fiber/pore size: tens to hundreds of µm. Possible micron-sized porosity within fibers • Thickness: medium (mm to cm range)

a

b

c

AM hybrid technologies

LFDM, LDM, RFP, cryogenic direct-plotting (Table III.a). Image source: [102] Porosity: 78-98% Resolution (min. fiber Ø): 250-300 µm Pore wall: micron-size porosity Pore size: > 100 µm

Melt electrospinning direct writing Table (III.c). Image source [112] Porosity: up to 87% Resolution (min. fiber Ø): < 1 µm Pore wall: smooth Pore size: > 20 µm

Computer-aided wet spinning system Table (III.b). Image source [106] Porosity: < 60% Resolution (min. fiber Ø): 200-250 µm Pore wall: micron-size porosity Pore size: > 200 µm

Direct EHD-jet process Table (III.d). Image source [119] Porosity: 91-97% Resolution (min. fiber Ø): 200-300 µm Pore wall: fibrous structure (Ø: 3-9 µm) Pore size: around 350 µm

Table I. Integration at the assembly level. Example of bi-multiphasic scaffolds obtained by combining AM with conventional techniques.

Description

3D AM bone compartment (FDM)

a

Fibrous ES compartment (solution or melt ES) [38, 40]

Advantages/Limitations

Materials

Applications

Example*

 AM scaffold provides biomechanical stability while ES membrane mimics soft tissue 

Potential adhesion issues between layers demand for carefully optimized bonding strategies (e.g. partial melt technique reported in [38])

AM compartment: PCL/ β-tricalcium Phosphate (β-TCP) Fibrous compartment: PCL

Alveolar bone/periodontal ligament complex

ASSEMBLY LEVEL

source: [38]

b

Bone compartment: biphasic construct (FDM compartment and ES)

 Separate optimization of the single compartment prior to in vivo implantation

Cartilage compartment: hydrogel casting [43]



Bone compartment: PCL Cartilage compartment: Alginate

Osteochondral TE

Gradual weakening of the interface region

source: [43]

3D AM bone compartment (low-temperature deposition manufacturing (LDM) or indirect AM)

 Simultaneous integration of bone and cartilage in discrete regions of the same construct

Sponge-like chondral phase (salt leaching or TIPS) [37, 50]



c

Poor integration between neo-formed cartilaginous and bone tissues

AM compartment: PLGA/β-TCP Spongy phase: decellularized cartilage ECM [50] AM compartment: hydroxyapatite (HA) Spongy phase: poly-L-lactic acid [37]

Osteochondral TE

source: [50] 3D AM bone compartment (LDM or ceramic stereolithography)

d

Sponge-like chondral phase (freeze drying or TIPS)

 The transitional structure between bone and cartilage ameliorates their integration

Intermediate connection layer [49, 50]

AM compartment: PLGA/β-TCP Spongy phase: decellularized cartilage ECM Intermediate layer: PLGA/β-TCP [50] Osteochondral TE AM compartment: β-TCP Spongy phase: type-I collagen Intermediate layer: β-TCP/collagen [49] source: [50]

*All example figures are reprinted with permission from the respective publishers (indicated in the source)

1

Table II. Integration at the fabrication level. Example of bimodal scaffolds obtained by combining AM with conventional techniques.

Description

Advantages/Limitations

Materials

 ES membrane enhances seeding efficiency

FABRICATION LEVEL

a

b

AM (3D fiber deposition [54], 3D plotting [55,57], direct polymer melt deposition [56], 3D bioprinting [58], FDM technique [70]) and Electrohydrodynamic processes (e.g. electrospinning)

AM (melt-plotter) and Electrohydrodynamic processes (e.g. electrohydrodynamic direct writing, EHD) [63,64]



Possible delamination in case of bi-layered assembly



Complex experimental setup

 The highly roughened microsized threads provide large cell-anchorage area 

Applicable to a limited set of biomaterials

Applications

Example*

AM and ES: Poly(ethylene oxide terephthalate)/poly(butylene terephtalate) (PEOT/PBT) copolymers [54] AM and ES: PCL [55] AM: PCL + ES: PCL/collagen [56] AM: starch–polycaprolactone + ES: PCL [57] AM: gelatin/sodium alginate + ES: PCL [58] AM: single-layer helical PCL + ES: tubular PLLA scaffold [70]

AM compartment: PCL Microsized threads: PCL Type-I collagen biomimetic coating [64]

Bone, cartilage, muscle, annulus fibrosus repair and vascular grafts

source: [56]

Bone

source: [63]  Porous foam structure acts as a cell entrapment system

c

AM (screw extrusion system) and Conventional freeze drying technology [65,67]

AM compartment: PCL/TCP Fibrillar compartment: Gelatin

 Simple equipment 

Bone

Laborious post-processing source: [65]

d

AM (Bioplotter®) and Non-conventional Layer-By-Layer (LbL) Electrostatic Self-Assembly [66,68]

 Fibrillar elements introduce surface for cell growth 

extended

Scarce control over thickness morphology of the inner structure

AM compartment: PCL Fibrillar compartment: Alginate and Chitosan

Bone

and source: [66]

*All example figures are reprinted with permission from the respective publishers (indicated in the source)

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Table III. Integration at the technique level. Examples of single multifunctional architectures obtained by hybrid AM techniques.

Description

a

Liquid-frozen deposition manufacturing (LFDM), low temperature deposition manufacturing (LDM), rapid freeze prototyping (RFP), cryogenic directplotting and robocasting [99-104]

Advantages/Limitations  High porosity with smaller pore size on the surface of the scaffold  Control over microporosity levels 

Two-step process

Materials

Chitosan/nanobioactive glass [99], Poly(D,L-lactide-coglycolide) (PLGA) [100], poly(L-lactic acid)/(tricalcium phosphate) [101], Type-I collagen [102], poly(Llactide)/chitosan [103], chitosan [104],

Applications

Example*

Bone, cartilage, nerve

TECHNIQUE LEVEL: HYBRID AM

source: [99]

 Spongy fiber morphology

b

Computer assisted wet-spinning system (CAWS) [106,107]

 One-step process 

PCL, PCL/hydroxyapatite [106] and star poly(εcaprolactone)/hydroxyapatite [107]

Bone

Applicable to a limited set of biomaterials source: [106]

c

Melt electrospinning direct writing (AM + melt ES) [112-115]

 Filament resolution and fiber-to-fiber distances in the low micron region PCL 

Sophisticated experimental setup (heating + electrical insulation)

Skin, neural and vascular TE

source: [113]  Microscale threads consisting of randomly interwoven micro/nanofibers

d

Direct Electro-hydrodynamic jet process (AM + wet electrospinning) [118,119]

 Different strut morphology depending on the viscosity of target solution 

Difficult control of micro/nanosized strut size

PCL

Bone

source: [118]

*All example figures are reprinted with permission from the respective publishers (indicated in the source)

3

Bi/multiphasic scaffolds with compartmented architectures

Bimodal scaffolds with fully-integrated architectures

Single multifunctional architecture