Tissue Engineering and Regenerative Medicine 2019:The Role of Biofabrication-A Year in Review

Despite its relative youth, biofabrication is unceasingly expanding by assimilating the contributions from various disciplinary areas and their technological advances. Those developments have spawned the range of available options to produce structures with complex geometries while accurately manipulating and controlling cell behavior. As it evolves, biofabrication impacts other research fields, allowing the fabrication of tissue models of increased complexity that more closely resemble the dynamics of living tissue. The recent blooming and evolutions in biofabrication have opened new windows and perspectives that could aid the translational struggle in tissue engineering and regenerative medicine (TERM) applications. Based on similar methodologies applied in past years' reviews, we identified the most high-impact publications and reviewed the major concepts, findings, and research outcomes in the context of advancement beyond the state-of-the-art in the field. We first aim to clarify the confusion in terminology and concepts in biofabrication to therefore introduce the striking evolutions in three-dimensional and four-dimensional bioprinting of tissues. We conclude with a short discussion on the future outlooks for innovation that biofabrication could bring to TERM research

Alignment of Cells and Extracellular Matrix Within Tissue- Engineered Substitutes

Most of the cells in our body are in direct contact with extracellular matrix (ECM) compo‐ nents which constitute a complex network of nano-scale proteins and glycosaminoglycans. Those cells constantly remodel the ECM by different processes. They build it by secreting dif‐ ferent proteins such as collagen, proteoglycans, laminins or degrade it by producing factors such as matrix metalloproteinase (MMP). Cells interact with the ECM via specific receptors, the integrins [1]. They also organize this matrix, guided by different stimuli, to generate pat‐ terns, essential for tissue and organ functions. Reciprocally, cells are guided by the ECM, they modify their morphology and phenotype depending on the protein types and organization via bidirectional integrin signaling [2-4]. In the growing field of tissue engineering [5], control of these aspects are of the utmost importance to create constructs that closely mimic native tis‐ sues. To do so, we must take into account the composition of the scaffold (synthetic, natural, biodegradable or not), its organization and the dimension of the structure. The particular alignment patterns of ECM and cells observed in tissues and organs such as the corneal stroma, vascular smooth muscle cells (SMCs), tendons, bones and skeletal mus‐ cles are crucial for organ function. SMCs express contraction proteins such as alpha-smoothmuscle (SM)-actin, desmin and myosin [6] that are essential for cell contraction [6]. To result in vessel contraction, the cells and ECM need to be organized in such a way that most cells are elongated in the same axis. For tubular vascular constructs, it is suitable that SMCs align in the circumferential direction, as they do in vivo [7, 8]. Another striking example of align‐ ment is skeletal muscle cells that form long polynuclear cells, all elongated in the same axis. Each cell generates a weak and short contraction pulse but collectively, it results in a strong, long and sustained contraction of the muscle and, in term, a displacement of the member. In the corneal stroma, the particular arrangement of the corneal fibroblasts (keratocytes) and ECM is essential to keep the transparency of this tissue [9-13]. Tendons also present a pecu‐ liar matrix alignment relative to the muscle axis. It gives a substantial resistance and excep‐ tional mechanical properties to the tissue in that axis [14, 15]. Intervertebral discs [16], cartilage [17], dental enamel [18], and basement membrane of epithelium are other examples of tissues/organs that present peculiar cell and matrix organization. By reproducing and controlling those alignment patterns within tissue-engineered substitutes, a more physiolog‐ ical representation of human tissues could be achieved. Taking into account the importance of cell microenvironment on the functionality of tissue engineered organ substitutes, one can assume the importance of being able to customise the 3D structure of the biomaterial or scaffold supporting cell growth. To do so, some methods have been developed and most of them rely on topographic or contact guidance. This is the phenomenon by which cells elongate and migrate in the same axis as the ECM. Topographic guidance was so termed by Curtis and Clark [19] to include cell shape, orientation and movement in the concept of contact guidance described by Harrison [20] and implemented by Weiss [21, 22]. Therefore, if one can achieve ECM alignment, cells will follow the same pattern. Inversely, if cells are aligned on a patterned culture plate, the end result would be aligned ECM deposition [23]. The specific property of tissues or materials that present a variation in their mechanical and structural properties in different axis is called anisotropy. This property can be evaluated ei‐ ther by birefringence measurements [24, 25], mechanical testing in different axis [26], immu‐ nological staining of collagen or actin filaments [23] or direct visualisation of collagen fibrils using their self-fluorescence around 488 nm [27, 28]. Several techniques have been recently developed to mimic the specific alignment of cells within tissues to produce more physiologically relevant constructs. In this chapter, we will describe five different techniques, collagen gel compaction, electromagnetic field, electro‐ spinning of nanofibers, mechanical stimulation and microstructured culture plates

Bio-nanoparticles and bio-microfibers for improved gene transfer into glioma cells

Ph.DDOCTOR OF PHILOSOPH

In vitro Tissue Engineering of Liver and Primary Lymphoid Tissues with Inverted Colloidal Crystal Scaffolds for Drug Testing Application.

Effective early stage drug toxicity testing is imperative to minimize failures in the late clinical stages of the drug development process. 2D cell cultures have been dominantly used, but they cannot adequately estimate actual toxic effects of drug molecules due to the limited capability in restoring original cellular behaviors in 3D tissues. As a potential solution to improve the predictive power of in vitro screening procedures, this dissertation explored a new opportunity of in vitro tissue engineering as a part of the drug development process. Besides the biological significance in functional tissue formation, scaffolds should be transparent and support standardized tissue growth. Inverted colloidal crystal (ICC) hydrogel scaffolds having standardized 3D structure and materials as well as retaining a high analytical capability were developed for this purpose. Uniform size spherical pore arrays prepared with cell repulsive polyacrylamide promoted homogenous HepG2 liver tissue spheroid formation, while the transparent hydrogel matrix allowed convenient characterization of cellular processes. The standardized spheroid culture model was successfully applied to the in vitro toxicity testing of CdTe and Au nanoparticles. Significantly reduced toxic effects were observed compared to the conventional 2D culture attributed by tissue-like morphology and cell phenotypic change in the spheroid culture. In addition, ICC scaffolds combined with a LBL surface modification technique served as a platform for engineering primary lymphoid tissue, i.e. bone marrow and thymus. Under dynamic culture condition, hematopoietic stem cells (HSCs) could travel deep into the scaffold via interconnecting channels, while they were temporarily entrapped due to limited channel size and number. As a result, HSCs extensively interacted with stromal cells growing along the LBL coated pore surface. Such intimate cell-cell and cell-matrix interaction is the key process in HSCs survival and differentiation that was substantiated by ex vivo expansion and B-/T-cell differentiation of HSCs. Overall this thesis introduces a promising application of in vitro tissue engineering as a practical and valuable early stage toxicity testing tool. ICC scaffolds exhibited unique advantage in preparation of spheroid culture model and lymphoid tissue engineering. Standardized in vitro tissue models substantiate the capability to extend current cellular level cytotoxicity to the tissue level.Ph.D.Biomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/64654/1/jungwoo_1.pd

A prototype for 3D electrohydrodynamic printing

Electrohydrodynamic direct writing is a flexible cost effective alternative technique that is capable of producing a very fine jet of liquid in the presence of an external electric field. This jet can then be used to pattern surfaces in an ordered and controlled fashion and offers a robust route to low cost large area micro and nano-manufacturing. Unlike other types of direct writing techniques, the liquid in electrohydrodynamic printing is subjected to both pushing and pulling forces. The pushing force is brought about by the constant flow rate that is maintained via high precision mechanical pumps while a pulling force is applied through a potential difference that is applied between the nozzle and the ground electrode and as a result a fine jet can be generated to pattern surfaces. The impracticality of use and the cost of building micrometre and sub-micrometre sized nozzles to print narrow line widths warrant an investigation into alternative means of dispensing printing inks using nozzles that are cheap to produce, easy to handle and consistent in delivery. The enormous capillary pressures that would have to be overcome in order to print highly viscous materials with micrometre and sub-micrometre sized nozzles may also limit the types of feed that could be used in printing narrow line widths. Thus, the initial work described is focused on improving print head design in an attempt to electrohydrodynamic print pattern narrow line widths using silk fibroin. This is followed by work where we attempt to design and construct of a new electrohydrodynamic printing machine with the sole purpose of expediting research in electrohydrodynamic printing in a flexible, feasible and user friendly manner. To achieve this, replicating rapid prototype technology is merged with conventional electrohydrodynamic printing phenomena to produce a EHD printing machine capable of print depositing narrow line widths. In order to validate the device the work also describes an attempt to print a fully formed human ear out of polycaprolactone. Finally, we investigate an approach to the electohydrodynamic printing of nasal septal scaffolds using the microfabrication system that was developed and optimized in our laboratory. In these initial stages we were successful in showing the degree of control and flexibility we possess when manufacturing constructs out of a biodegradable polymer ( polycaprolactone) from the micro to macro scale through manipulation of just one process parameter (concentration). This work also features characterization of scaffold mechanical properties using a recently invented Atomic force microscopy technique called PeakForce QNM (Quantitative Nanomechanical Property Mapping)

Multifunctional and liquified capsules for tissue regeneration

Tese de Doutoramento em Engenharia de Tecidos, Medicina Regenerativa e Células EstaminaisCell encapsulation systems, in which cells and/or biomaterials and molecules are physically isolated from the surrounding environment, are being increasingly applied as multifunctional strategies in Tissue Engineering and Regenerative Medicine. In the present thesis it is proposed a rather unique combination of functional biomaterials and different cell types for the groundbreaking advance of liquified cell encapsulation systems. The proposed system aims to transfigure the concept of conventional three-dimensional scaffolds, typically associated on the use of porous structures or hydrogels to support cells, by using an alternative and hierarchical methodology. Capsules are composed by different components: (i) a permselective multilayered membrane composed by various polyelectrolytes, namely alginate, chitosan, and poly(L-lysine), and also electrostatically bounded magnetic-nanoparticles which confer magnetic-responsive ability to the system; (ii) poly(L-lactic acid) microparticles surface functionalized by combining plasma treatment with different coating materials; and (iii) different cell types, namely L929, stem and endothelial cells. The membrane wraps the liquefied core of the capsules, ensuring permeability to essential molecules for cell survival, and enhancing direct contact between the encapsulated materials. The microparticles confer cell adhesion sites, and also influence biological processes of the encapsulated cells due to its chemically modified surface. Multilayered and liquified capsules encapsulating microparticles were first validated as successful cell encapsulation systems for tissue regeneration. Different parameters of the production process were optimized, such as the number, type and concentration of multilayers, and the required time de-crosslinker concentration to liquefy the alginate core. Capsules were further successfully proposed as bioencapsulation systems for the regeneration of specific tissues, namely cartilage and bone. Ultimately, the biological outcome of capsules was tested in vivo, demonstrating the biotolerability of the developed system. It is expected that the proposed capsules will have a strong impact and open new prospects in cell encapsulation systems for tissue regeneration.Sistemas de encapsulamento celular, nos quais células e/ou biomateriais e moléculas estão fisicamente isolados do exterior, estão a ser cada vez mais propostos como estratégias multifuncionais para Engenharia de Tecidos e Medicina Regenerativa. Na presente tese é proposta a combinação de biomateriais funcionais e vários tipos de células para o progresso de sistemas liquefeitos de encapsulamento celular. O sistema proposto pretende transfigurar o conceito convencional de scaffolds tridimensionais, tipicamente associados a estruturas porosas ou hidrogéis para suporte celular, propondo uma metodologia hierárquica. As cápsulas são constituídas por diferentes componentes: (i) uma membrana com permeabilidade seletiva composta por vários polieletrólitos, nomeadamente alginato, quitosano e poli(L-lisina), e ainda nanopartículas magnéticas electrostaticamente acopladas à membrana que conferem ao sistema capacidade de resposta magnética; (ii) micropartículas de poli(L-ácido láctico) com a superfície funcionalizada por tratamento de plasma combinado com diferentes materiais de revestimento; (iii) diferentes tipos de células, nomeadamente L929, estaminais e endoteliais. A membrana envolve o núcleo liquefeito das cápsulas, assegurando a permeabilidade de moléculas essenciais para a viabilidade celular, e ainda maximiza o contacto direto entre os diferentes componentes encapsulados. As micropartículas oferecem locais de adesão celular, e ainda influenciam os processos biológicos das células encapsuladas devido à superfície quimicamente modificada. Cápsulas com multicamadas e liquefeitas com micropartículas encapsuladas foram primeiramente validadas com sucesso como sistemas de encapsulamento celular para regeneração de tecidos. Diferentes parâmetros do processo de produção foram optimizados, tais como o número, tipo e concentração das multicamadas, e o tempo necessário e concentração do des-reticulador para liquefazer o interior de alginato. As cápsulas foram ainda propostas com sucesso como sistemas de bio-encapsulamento para a regeneração de tecidos específicos, nomeadamente cartilagem e osso. Em última análise, a resposta biológica das cápsulas foi testada in vivo, demonstrando a biotolerância do sistema desenvolvido. Espera-se que as cápsulas propostas tenham um impacto considerável e que abram novas perspectivas nos sistemas de encapsulamento celular para regeneração de tecidos

Inter-bonded fibrous matrices for 3D tissue engineering scaffolds

The thesis established a stable three-dimensional fibrous tissue scaffold that has controlled pore structure and inter-bonded fibrous structure, and also examined the effects of the 3D fibrous matrices and functional surfaces including nano-scale topography, bioactive CaP coating and antibacterial treatment on the cell growth behavior for tissue engineering application

3D Bioprinting Tissue Scaffolds with Living Cells for Tissue Engineering Applications

In tissue engineering, tissue scaffolds are used as temporary supports to promote regeneration of dysfunctional tissues. Of the available strategies, scaffolds produced from hydrogels and living cells show the great potential for their enhanced biological properties. To produce such scaffolds, three-dimensional (3D) bioprinting has evolved and is showing promise as a fabrication technique. However, its applications for fabricating customized hydrogel scaffolds containing living cells is still in its infancy. The major challenge with this approach is to print scaffolds while preserving cell viability and functionality as well as ensuring the structural integrity of the scaffold. To overcome this challenge, the present thesis aims to investigate the influences of hydrogel properties and the bioprinting process on cell viability and functionality, while also ensuing structural integrity, and on this basis, to develop bioprinting processes to produce tissue scaffolds with living cells for potential tissue engineering applications. This thesis first examined the influence of the mechanical properties of hydrogel on cell viability and functionality, utilizing alginate hydrogels and Schwann cells (the major glial cells of peripheral nervous system). Due to its poor cell adhesion, the alginate hydrogel was modified in this study with cell-adhesion supplements, including fibronectin, poly-l-lysine (PLL), and RGD (Arg-Gly-Asp) peptides. The RGD-modified alginate substrates were prepared with varying alginate concentrations in order to alter the mechanical properties of hydrogels, which were then seeded and encapsulated with Schwann cells. Cell viability and functionality, including proliferation, morphology, and expression of the extracellular matrix protein, were examined and correlated to the hydrogel mechanical properties. The results demonstrate that the viability and functionality of Schwann cells within alginate-based hydrogel vary with hydrogel mechanical properties, thus highlighting the importance of regulating the mechanical properties of hydrogel for improved cell viability and functionality in scaffold bioprinting. During the bioprinting process, cells are subject to process-induced forces, such as shear and extensional stresses, which can result in cell damage and therefore loss of cell function and even cell death. A method was developed to study the cell damage introduced by the shear and extensional stresses in the bioprinting process. A plate-and-cone rheometer was adopted to examine the effect of shear stress on cell damage. In these experiments, the relationship of cell damage to the shear stress was examined and quantified, which was then applied to identify the cell damage attributed to shear stress in bioprinting. On this basis, the damage to cells caused by extensional stress was inferred from the difference between the total cell damage occurring during the bioprinting process and the cell damage attributed to shear stress. This developed method allowed a relationship to be established between cell damage and both shear and extensional stresses during bioprinting. The experiments on this method provide insight into both the cell damage that occurs during bioprinting and the effect on cell viability and proliferative ability thereafter, which can be used to optimize the bioprinting process so as to preserve cell functionality. Based on the previous investigations, bioprinting processes were developed to fabricate tissue scaffolds containing Schwann cells for potential applications in nerve tissue engineering. Composite hydrogels consisting of alginate, fibrin, hyaluronic acid, and RGD peptide were prepared, and their hydrogel microstructures, mechanical stiffness after gelation, and capability to support the Schwann cell spreading were examined for identifying appropriate composite hydrogel for bioprinting processes. The flow behavior of composite hydrogel solutions and bioprinting process parameters (e.g., dispensing pressure, dispensing head speed, crosslinking process) were then examined with regard to their influence on the structure of the printed scaffolds and on this basis, bioprinting process were developed to fabricate scaffolds with Schwann cells. The functionality of Schwann cells within the printed scaffolds were assessed in terms of cell viability, proliferation, morphology, orientation, and protein expression, demonstrating that the printed scaffolds have potential for nerve tissue engineering applications. This thesis presents a comprehensive study on the bioprinting of scaffolds with living cells. The method developed and the study results will pave the way to fabricate scaffolds with living cells for more tissue engineering applications