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MINI-REVIEW Scaffold and Scaffold-Free Self-Assembled Systems in Regenerative Medicine Dilip Thomas,1,2 Diana Gaspar,1,3 Anna Sorushanova,1,3 Gesmi Milcovich,1 Kyriakos Spanoudes,1,3 Anne Maria Mullen,4 Timothy O’Brien,1,2 Abhay Pandit,1 Dimitrios I. Zeugolis1,3 1 Centre for Research in Medical Devices (CURAM), Biosciences Research Building, National University of Ireland Galway (NUI Galway), Galway, Ireland 2 Regenerative Medicine Institute (REMEDI), Biosciences Research Building, National University of Ireland Galway (NUI Galway), Galway, Ireland 3 Regenerative, Modular & Developmental Engineering Laboratory (REMODEL), Biosciences Research Building, National University of Ireland Galway (NUI Galway), Galway, Ireland; telephone: þ353-0-9149 3166; fax: þ353-0-9156 3991; e-mail: [email protected] 4 Teagasc Food Research Centre, Ashtown, Ireland

ABSTRACT: Self-assembly in tissue engineering refers to the spontaneous chemical or biological association of components to form a distinct functional construct, reminiscent of native tissue. Such self-assembled systems have been widely used to develop platforms for the delivery of therapeutic and/or bioactive molecules and various cell populations. Tissue morphology and functional characteristics have been recapitulated in several self-assembled constructs, designed to incorporate stimuli responsiveness and controlled architecture through spatial confinement or field Correspondence to: D.I. Zeugolis Contract grant sponsor: Science Foundation Ireland Contract grant number: 09/SRC/B1794 Contract grant sponsor: Industry-Academia Partnerships and Pathways (IAPP) Contract grant numbers: 251385; EU FP7/2007-2013 Contract grant sponsor: NMP award, Green Nano Mesh Project Contract grant number: 263289 Contract grant sponsor: Health Research Board, Health Research Awards Programme Contract grant number: HRA_POR/2011/84 Contract grant sponsor: Irish Research Council Contract grant number: RS/2012/82 Contract grant sponsor: Irish Research Council, Government of Ireland Postgraduate Scholarship Scheme Contract grant number: GOIPG/2014/385 Contract grant sponsor: European Union 7th Framework Programme ITN AngioMatTrain Contract grant number: 317304 Contract grant sponsor: Teagasc Walsh Fellowship Contract grant number: 2014045 Contract grant sponsor: ReValueProtein Research Project Contract grant number: 11/F/043 Contract grant sponsor: Department of Agriculture, Food, and the Marine (DAFM) Contract grant sponsor: Science Foundation Ireland and the European Regional Development Contract grant number: 13/RC/2073 Received 29 June 2015; Revision received 19 October 2015; Accepted 23 October 2015 Accepted manuscript online 24 October 2015; Article first published online 10 November 2015 in Wiley Online Library (http://onlinelibrary.wiley.com/doi/10.1002/bit.25869/abstract). DOI 10.1002/bit.25869

ß 2015 Wiley Periodicals, Inc.

manipulation. In parallel, owing to substantial functional properties, scaffold-free cell-assembled devices have aided in the development of functional neotissues for various clinical targets. Herein, we discuss recent advancements and future aspirations in scaffold and scaffold-free self-assembled devices for regenerative medicine purposes. Biotechnol. Bioeng. 2016;113: 1155–1163. ß 2015 Wiley Periodicals, Inc. KEYWORDS: tissue engineering; modular engineering; selfassembly; stimuli-responsive polymers; delivery of biologics; cellassembled devices

Introduction Self-assembly is considered as a modular, bottom-up phenomenon that is governed by short-distance interactions (e.g., from Angstrom scale for covalent or hydrogen bonds to nano scale for ionic or electrostatic bonds), various structural motifs forces (e.g., van der Waals, electrostatic, magnetic, molecular, and entropic) and equilibrium conditions (Bishop et al., 2009; Mann, 2009; Walker et al., 2011). In living, self-replicating organisms, self-assembly is a dynamic process, which is triggered by changes in equilibrium or external stimuli. Changes in equilibrium during development, for example, dictate tissue self-organisation, while in response to injury or pathophysiology, self-assembly is recruited to restore physiological morphogenesis and function. Modular tissue engineering technologies utilise natural selfassembly processes (biomimicry) to create functional implantable devices for therapeutic purposes. For example, protein-based approaches (Fig. 1) undergo spontaneous peptide aggregation to form highly ordered supramolecular structures and from there,

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Self-Assembly Within Spatial Confinement

Figure 1.

Spontaneous aggregation of proteins gives rise to supramolecular structures, which through subsequent engineering interventions create threedimensional hierarchical structures, such as fibres.

through engineering interventions, structures of great complexity, and highly sophisticated function that closely mimic the diverse properties of native tissues are created (Cui et al., 2010; Lai et al., 2012; Sengupta and Heilshorn, 2010). Spatially confined (Gentili et al., 2014; Jiang et al., 2014; Zhong et al., 2014) or field-directed (Feng et al., 2014; Gopinadhan et al., 2014; Tousley et al., 2014) approaches are often utilised to control morphological features. To enhance further the bioactivity of the device, very elegant chemical(Azevedo and Pashkuleva, 2015; Kesharwani and Iyer, 2015), biological- (Park et al., 2012; Zhang et al., 2015a) or biomaterial(Jayaraman et al., 2015; Meyer et al., 2015) based functionalisation strategies are also frequently recruited to allow incorporation of therapeutic and/or bioactive molecules. Degradation or responsiveness of the carrier to exogenous stimuli (e.g., temperature, pH, enzymatic activity, presence of redox species) offer controlled and localised release of the cargo, promoting that way functional repair and regeneration (Dumont et al., 2015; Kim and Tabata, 2015). An alternative strategy is based on the principles of in vitro organogenesis, where the inherent capacity of cells to create highly sophisticated, self-organised, three-dimensional tissue-like assemblies is utilised (Ader and Tanaka, 2014; Lancaster and Knoblich, 2014; Li et al., 2014; Owaki et al., 2014; Sasai et al., 2012). The clinical relevance of this technology has been well documented for skin (Gallico et al., 1984; Phillips et al., 1989), cornea (Nishida et al., 2004) and blood vessel (L’Heureux et al., 2007), while very promising pre-clinical data are available for bone (Pirraco et al., 2011), cartilage (Ebihara et al., 2012), heart (Dube et al., 2014), kidney (Takasato et al., 2014), and liver (Baimakhanov et al., 2015; Takebe et al., 2013). Herein, we discuss recent advancements (Table I) and future aspirations in spatial confined, field-directed, stimuli-responsive, and scaffoldfree self-assembly for regenerative medicine purposes.

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Non-planar spatial confinement of synthetic polymers and biopolymers gives rise to macroscopic architectures (e.g., fibres, tubes, spheres) defined by the boundaries of the template. For example, extrusion of a collagen solution (Fig. 2) through small diameter laboratory tubing induces parallel to the direction of flow molecular orientation. Upon subsequent contact of the collagen solution with the neutral buffers maintained at physiological temperature, macroscopic fibres are spontaneously formed with high levels of structural alignment (Zeugolis et al., 2008). Such constructs have been shown to induce bidirectional cell growth and could potentially used as in vitro model systems for cell migration (Cornwell et al., 2004). However, as the natural cross-linking pathway of collagen does not occur in vitro, exogenous crosslinks are introduced to induce mechanical and enzymatic resilience (Sanami et al., 2015). The quest of the ideal cross-linking method still continues to achieve balance between cytotoxicity, foreign body response, mechanical strength, and resorption rate in vivo (Delgado et al., 2015; Enea et al., 2013). Rotary shearing of collagen gel bundles at physiological temperature allows uniaxial elongation of fibrils by resisting destruction of fibrillar network due to stress relief and also affords bidirectional cell elongation (Yunoki et al., 2015). However, this scaffold fabrication technique is relatively slow compared to other fibre spinning technologies and controlling cell distribution over volume remains challenging. Template-based approaches, of variable complexity, have also been exploited for the development of multiple implantable devices for diverse clinical indications. Macroscopic single or multi-channel collagen conduits, for example, can be fabricated through coating of a collagen solution onto a cylindrical mandrel, evaporation of collagen and finally removal of the mandrel. Such elegant structures, with or without biologics, have been used extensively for peripheral nerve and spinal cord repair (Dienstknecht et al., 2013; Yao et al., 2010, 2013). Given the importance of anisotropy in peripheral nerve repair (Bellamkonda, 2006; Hoffman-Kim et al., 2010), extruded collagen fibres have been incorporated within the channels as intraluminal guidance structures for Schwann cell migration and axonal regeneration, while resulted in significantly decreased axonal dispersion and axonal mismatch of distal nerve targets (Daly et al., 2012). The optimal however number and dimensionality of the channels and the fibrous fillers has yet to be identified. Modern bioengineering has evolved from medical devices that imitate architectural features and mechanical properties of tissues to be repaired/regenerated to biofunctional devices that aspire to interact with the host and through their sustained and localised release of their cargo promote functional repair and regeneration. To this end, extracellular matrix-based hollow microscopic spheres (Fig. 3), derived after coating of spherical micro-templates and subsequent dissolution of the template, was a significant milestone in the field of regenerative medicine, as allowed spatiotemporal delivery of bioactive and/or therapeutic molecules (Kraskiewicz et al., 2013; Saul et al., 2007). As we understand more and more the complexity of injuries and pathophysiologies, it became apparent that multi-cargo release at different reparative stages is required. However, we are still at early stages of engineering devices with

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Living substitutes

Stimuli responsive

Field-directed

Spatial confinement

Self-assembly principle

Neonatal cardiac cell sheet grown on temperature responsive culture dishes Osteogenic matrix cell sheets grown on temperature responsive culture dishes

Cardiac cell sheet grown on temperature responsive culture dishes

Engineered three-dimensional hepatic tissue sheets on PIPAAm-grafted dishes

IGF-1 tethered tetravalent streptavidin and biotinylated self-assembled peptide scaffold Photointitated methacrylated chitosan-collagen type II hydrogel composite loaded with TGF-b1 H2O2 sensitive gelatin-phenolic hydroxyl hydrogel with ECFCs

Peptide amphiphiles based nano-fibres with IKVAV epitope

In situ static magnetic field treated BMP2 collagen sponge

Orthogonal magnetically aligned collagen lamellae in a gel

Magnetically aligned type I collagen tubes

Collagen fibres and multi-channel conduits fibres

Collagen tube with collagen filaments

Multi-channel collagen conduit (NeuraGen1)

In situ gelation of fibrin matrix and human MSCs

Elastin hollow spheres loaded with eNOS plasmid DNA

Conformation/functionality

Increased concentration of H2O2 resulted in lower proteolytic degradability and reduction in vascular density in subcutaneous mouse model Hepatic tissue sheets engineered in the subcutaneous space of mice showed maintenance of functional volume and restoration of stratified structures after hepactomy Transplantation of cardiac cell sheet improved fractional shortening and inhibited left ventricular dilation in rat myocardial infarction model Transplantation of cell sheets into subcutaneous rat model resulted in rapid vasculature formation which led to mature blood vessels Transplantation of cell sheets into subcutaneous mouse model resulted in tissue mineralisation with high levels of bone matrix protein and osteopontin

Improved angiogenesis and reduced inflammation in a hind-limb ischemia model Histological, ultrastructural and biomechanical restoration in a rat patellar defect model Digital nerve repair across 10–20 mm defect with improvement in sensory functions in over 80% of patients Sciatic nerve repair across 30 mm defect in dogs with higher distribution of myelinated nerve fibres Provided guidance for Schwann cell migration and increased guidance of regenerating axons towards their distal nerve targets Regeneration of myelinated nerves and higher fascicular organisation over a 6 mm peripheral nerve defect Hemi-corneal reconstruction was achieved in a rabbit model supported by re-epithelialisation, transparency, and ultra-structural organisation Increased bone tissue formation and bone marrow was observed in an ectopic bone formation mouse model Inhibition of glial scar formation and regeneration of descending motor fibres and ascending sensory fibres in a mouse spinal cord injury model Increased cardiomyocyte maturation due to interaction between tethered IGF-1 and cardiomyocytes in rat myocardial infarction model Significantly enhanced chondrogenesis in a subcutaneous mouse model

Significant advancement

Table I. Indicative examples of recent advancements in the field of self-assembly for regenerative medicine purposes in preclinical models.

Pirraco et al. (2014)

Takeuchi et al. (2014)

Sekine et al. (2011)

Ohashi et al. (2007)

Chuang et al. (2015)

Choi et al. (2015)

Tysseling-Mattiace et al. (2008) Davis et al. (2006)

Kotani et al. (2002)

Ceballos et al. (1999); Eguchi et al. (2015) Builles et al. (2010)

Daly et al. (2012)

Okamoto et al. (2010)

Bushnell et al. (2008)

Hankemeier et al. (2009)

Dash et al. (2015)

Reference

Figure 2. Flow diagram of production of extruded collagen nano-textured micro-fibres (a). Transmission electron microscopy reveals that the produced fibres exhibit the characteristic quasi quarter-staggered alignment pattern of collagen and they are structurally aligned (b). The high order of sub-fibrillar alignment (b) is responsible of undulations and crevices running parallel to the longitudinal fibre axis, as revealed by scanning electron microscopy (c). Such surface characteristics facilitate bidirectional cell growth, elongation and migration (d).

spatial features down to the nano-meter scale, with large surface area for conjugation of specific chemical and/or biochemical moieties and with compartmentalisation capacity for delivery of multiple cargos. Thus, biotemplating is at the forefront of scientific research. For example, highly sophisticated natural structures (e.g., diatoms) are used as templates to build novel functional materials (Bariana et al., 2013; Belegratis et al., 2014; Chao et al., 2014; Liu et al., 2015). Of significant importance are recent studies, where cyanobacteria cells (Zhang et al., 2015b) and microalgae (Tao et al., 2014) were used as bio-templates, however, not for self-assembled materials as yet.

Field-Directed Self-Assembly Field-directed self-assembly allows development of fibrous and membrane implantable devices with highly ordered substructure. Ordered self-assembly has a significant impact in the creation of biomimetic scaffolds, due to close imitation of the hierarchy encountered in native tissues. Predominantly, field-controlled self-assembly is achieved by applying electric or magnetic fields to a polymeric solution resulting in spatial organisation of the electric- and magnetic- field sensitive domains of the polymer. Isoelectric focusing (Fig. 4), for example, induces migration and focusing of collagen monomers at their isoelectric focusing point, where the global charge is neutral, giving rise to anisotropically

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ordered fibrous hydrogels. Such systems have been shown to promote bidirectional neurite outgrowth for spinal cord repair (Abu-Rub et al., 2011) and to differentiate more effectively than growth factors mesenchymal stem cells towards tenogenic lineage (Younesi et al., 2014). Strong magnetic fields resulted in collagen structures resembling the native architecture of corneal stromal, inducing alignment of keratocytes by contact guidance (Torbet et al., 2007). An alternative approach is based on the use of magnetic beads to produce nano-fibrillar structures of fibrin in a geodesic pattern (Alsberg et al., 2006) and directed spatial patterning of stem cell aggregates across multiple length and time scales (Bratt-Leal et al., 2011). In a more recent study, magnetoceptive polymeric subunits were self-assembled into a 3D conformation, allowing fabrication of tunable blocks of encapsulated living cell (Tasoglu et al., 2014). Smooth muscle cells were shown to expand and proliferate rapidly under the direction of the magnetic field due to the alignment of the cell’s longest axis and the magnetic flux (Iwasaka et al., 2003). In comparison to spatial confined self-assembly, field-directed selfassembly has not been extensively investigated for scaffold fabrication, primarily due to technical limitations associated with the required infrastructure. For example, fabrication of magnetically aligned collagen scaffolds requires high strength magnets (3 to 8 Tesla) (Builles et al., 2010; Eguchi et al., 2015), frequently encountered in MRI equipment.

Figure 3. Flow diagram of production of hollow, collagen-based micro-spheres (a). Scanning electron micrograph of hollow collagen micro-spheres (b). Scanning electron micrograph of hollow fibrin micro-spheres (c). Hollow collagen spheres loaded with fluorescent-labelled plasmid (d).

Figure 4. Flow diagram of production of isoelectric focused collagen hydrogels (a). Polarised light microscopy indicates high levels of structural alignment of the isoelectricfocused collagen hydrogels (b). Scanning electron microscopy reveals high levels of surface alignment (c). This high level of structural alignment enables bidirectional rat dorsal root ganglia elongation and growth (d).

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Stimuli-Responsive Self-Assembly The dynamic and spontaneous naturally occurring self-assembly process is influenced by various extrinsic conditions, such as pH, temperature, light, ion nature, and concentration, redox potential, overexpression of a chemical or biological signal. More recently, responsiveness to electrical signals (Qazi et al., 2014) and radio frequency (Rejinold et al., 2015) are under intense investigation for regenerative medicine and cancer therapeutics delivery respectively. From a physicochemical point of view, these naturally occurring extrinsic signals can be readily recapitulated artificially by incorporating responsive sequences into natural and synthetic structures. Numerous stimuli-responsive self-assembled peptides (Maude et al., 2012), polysaccharides (Alvarez-Lorenzo et al., 2013), glycopolypeptides (Krannig and Schlaad, 2014) and synthetic polymers (He et al., 2008) have been explored over the years, enabling materialisation of more tunable implantable devices, revolutionising that way modern biomedicine. Of significant importance are recent developments in the field of multi-domain responsive (e.g., chemical, physical or biological responsiveness) materials (Zhuang et al., 2013) that by careful selection of the individual building blocks, controlled

is offered over dimensionality, viscoelastic properties, cargo delivery capacity, sensitivity to local stimuli (e.g., proteolytic degradation) (Galler et al., 2010) and even programmable transformation capacity (Estephan et al., 2013). Advanced in chemistry and engineering have enabled the development of dynamic four-dimensional hydrogels, which allow spatial and temporal patterning of ligands that guide cell behaviour in the three-dimensional space and in the fourth dimension, time (DeForest et al., 2009). (Bakarich et al., 2015; Luo et al., 2015; Tibbitt and Anseth, 2012). Overall most stimuli responsive systems are limited to in vitro applications, which allow dissection of cellular mechanisms such as growth, differentiation, and proliferation under controlled conditions. Until toxicity issues are addressed effectively in a regulatory acceptable manner, such stimuli responsive approaches are expected to thrive in diagnostic applications (Hoffman, 2013), given that in this case direct contact with the body is not necessary.

Self-Assembled Living Substitutes Traditional tissue engineering approaches rely on the use of synthetic or natural biomaterial to provide initial structural

Figure 5. In customary cultures, the development of an implantable device takes several days or even months. Under macromolecular crowding conditions, the development of an implantable device is a matter of hours to days. Macromolecular crowded (carrageenan, þCR) dramatically accelerates collagen type I deposition in human adult dermal fibroblast culture as early as 2 days in culture, as revealed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Immunocytochemistry analysis further corroborates the enhanced collagen type I deposition in human adult dermal fibroblast, human bone marrow stem cell and human corneal fibroblast cultures. ‘‘Ctrl’’ indicates bovine Achilles tendon collagen 1mg/mL concentration in 0.5 M acetic acid. ‘‘CR’’ indicates without macromolecular crowder (carrageenan in that case). ‘‘þCR’’ indicates with macromolecular crowder (carrageenan in that case).

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support, to act as a carrier of a trophic molecule and/or a viable cell population and to modulate host cell behaviour and neotissue growth. Shortfalls (e.g., foreign body response, delayed remodelling) and limitations (e.g., the vast number of permutations on polymers, extracellular matrix molecules and biologics concentrations, combinations and timings) of biomaterials have triggered investigations into cell sheet technology (Matsuura et al., 2014) or modular tissue engineering (Nichol and Khademhosseini, 2009) or tissue engineering by self-assembly (Peck et al., 2011). This approach is based on the principles of in vitro organogenesis, where phenomena such as cell-cell and cellextracellular matrix interactions precisely control molecular fusion and direct tissue organisation to form complex, highly sophisticated, and highly ordered tissue-like structures and establishing compartments and boundaries (Gjorevski et al., 2014; Kinney et al., 2014; Sasai, 2013). This approach puts forward the notion that should cells be exposed to tissue-specific biophysical, biochemical, and biological signals, the same phenomena as in vivo would occur and give rise to cellassembled tissue equivalents, with unmet by traditionally used biomaterials level of biomimicry. Despite the promising preclinical and even clinical data to-date, the major obstacle that hinders wide clinical translation and commercialisation of this technology is the prolonged ex vivo culture time required to develop an implantable device (e.g., 196 days for blood vessel), which is frequently associated with loss of cell phenotype and therapeutic potential. To this end, macromolecular crowding (Fig. 5) has been shown to significantly enhance extracellular matrix deposition (up to 80-fold increase) in permanently differentiated (Kumar et al., 2015a; Kumar et al., 2015b; Satyam et al., 2014) and stem cell (Ang et al., 2014; Prewitz et al., 2015; Zeiger et al., 2012) culture, by imitating the localised density of native tissues, significantly reducing the development of an implantable device. Recent advances in micro-patterned thermoresponsive substrates have enabled the development of anisotropic tissue-like assemblies (Takahashi et al., 2015). Although issues associated with automation (Kikuchi et al., 2014) have now been addressed, we are still away from recapitulating completely the in vivo milieu in vitro. Multi-factorial approaches (simultaneous control over biophysical, biochemical, and biological signals) are expected to achieve that in the near future offering control over in vitro expansion of permanently differentiated cells and/or lineage commitment of stem cells. We will then be in position to develop more accurate cell-assembled bioengineered tissues.

Conclusions Scaffold or scaffold-free self-assembly approaches in the field of tissue engineering and regenerative medicine mimic biological processes by recapitulating spatiotemporal events taking place during tissue morphogenesis in vivo, responsible for the hierarchical and functional complexity of living tissues. It is evidenced that significant strides have been achieved to-date, contributing greatly in the development of targeted, contextresponsive, and customised therapies for with a wide range of clinical indications and diagnostic purposes. Nonetheless, further

advancements are necessary to address scalability issues and to comply with current and future regulatory requirements for the delivery of safe and efficacious therapies to patients. The authors would like to thank Mr Maciek Doczyk for the preparation of the figures in this manuscript. This work is supported by: the Science Foundation Ireland (Grant Agreement Number: 09/SRC/B1794) to AP; the EU FP7/20072013, Marie Curie, Industry-Academia Partnerships and Pathways (IAPP) award, part of the People programme, Tendon Regeneration Project (Grant Agreement Number: 251385) to DZ; the EU FP7/2007-2013, NMP award, Green Nano Mesh Project (Grant Agreement Number: 263289) to DZ; the Health Research Board, Health Research Awards Programme (Grant Agreement Number: HRA_POR/2011/84) to DZ; the Irish Research Council, EMBARK Initiative, Postgraduate Scholarship (Grant Agreement Number: RS/2012/82) to DG and DZ; the Irish Research Council, Government of Ireland Postgraduate Scholarship Scheme (Grant Agreement Number: GOIPG/2014/385) to KS and DZ; European Union funding under the 7th Framework Programme ITN AngioMatTrain (Grant Agreement Number: 317304) to AP. Further, this work forms part of the Teagasc Walsh Fellowship (Grant Agreement Number: 2014045) and the ReValueProtein Research Project (Grant Agreement Number: 11/F/043) supported by the Department of Agriculture, Food, and the Marine (DAFM) under the National Development Plan 2007–2013 funded by the Irish Government to AMM and DZ. This publication has also been supported from Science Foundation Ireland and the European Regional Development Fund (Grant Agreement Number: 13/RC/2073) to AP.

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