Rodrigo A. Rezende

Burr-like, laser-made 3D microscaffolds for tissue spheroid encagement.

The modeling, fabrication, cell loading, and mechanical and in vitro biological testing of biomimetic, interlockable, laser-made, concentric 3D scaffolds are presented. The scaffolds are made by multiphoton polymerization of an organic-inorganic zirconium silicate. Their mechanical properties are theoretically modeled using finite elements analysis and experimentally measured using a Microsquisher(®). They are subsequently loaded with preosteoblastic cells, which remain live after 24 and 72 h. The interlockable scaffolds have maintained their ability to fuse with tissue spheroids. This work represents a novel technological platform, enabling the rapid, laser-based, in situ 3D tissue biofabrication.

Design, physical prototyping and initial characterisation of ‘lockyballs’

Directed tissue self-assembly or bottom-up modular approach in tissue biofabrication is an attractive and potentially superior alternative to a classic top-down solid scaffold-based approach in tissue engineering. For example, rapidly emerging organ printing technology using self-assembling tissue spheroids as building blocks is enabling computer-aided robotic bioprinting of three-dimensional (3D) tissue constructs. However, achieving proper material properties while maintaining desirable geometry and shape of 3D bioprinted tissue engineered constructs using directed tissue self-assembly, is still a challenge. Proponents of directed tissue self-assembly see the solution of this problem in developing methods of accelerated tissue maturation and/or using sacrificial temporal supporting of removable hydrogels. In the meantime, there is a growing consensus that a third strategy based on the integration of a directed tissue self-assembly approach with a conventional solid scaffold-based approach could be a potential optimal solution. We hypothesise that tissue spheroids with ‘velcro®-like’ interlockable solid microscaffolds or simply ‘lockyballs’ could enable the rapid in vivo biofabrication of 3D tissue constructs at desirable material properties and high initial cell density. Recently, biocompatible and biodegradable photo-sensitive biomaterials could be fabricated at nanoscale resolution using two-photon polymerisation (2PP), a development rendering this technique with high potential to fabricate ‘velcro®-like’ interlockable microscaffolds. Here we report design studies, physical prototyping using 2PP and initial functional characterisation of interlockable solid microscaffolds or so-called ‘lockyballs’. 2PP was used as a novel enabling platform technology for rapid bottom-up modular tissue biofabrication of interlockable constructs. The principle of lockable tissue spheroids fabricated using the described lockyballs as solid microscaffolds is characterised by attractive new functionalities such as lockability and tunable material properties of the engineered constructs. It is reasonable to predict that these building blocks create the basis for a development of a clinical in vivo rapid biofabrication approach and form part of recent promising emerging bioprinting technologies.

Nanotechnological Strategies for Biofabrication of Human Organs

Nanotechnology is a rapidly emerging technology dealing with so-called nanomaterials which at least in one dimension have size smaller than 100 nm. One of the most potentially promising applications of nanotechnology is in the area of tissue engineering, including biofabrication of 3D human tissues and organs. This paper focused on demonstrating how nanomaterials with nanolevel size can contribute to development of 3D human tissues and organs which have macrolevel organization. Specific nanomaterials such as nanofibers and nanoparticles are discussed in the context of their application for biofabricating 3D human tissues and organs. Several examples of novel tissue and organ biofabrication technologies based on using novel nanomaterials are presented and their recent limitations are analyzed. A robotic device for fabrication of compliant composite electrospun vascular graft is described. The concept of self-assembling magnetic tissue spheroids as an intermediate structure between nano- and macrolevel organization and building blocks for biofabrication of complex 3D human tissues and organs is introduced. The design of in vivo robotic bioprinter based on this concept and magnetic levitation of tissue spheroids labeled with magnetic nanoparticles is presented. The challenges and future prospects of applying nanomaterials and nanotechnological strategies in organ biofabrication are outlined.

Toward organ printing: Design characteristics, virtual modelling and physical prototyping vascular segments of kidney arterial tree

Organ printing is defined as the layer by layer additive biofabrication of three-dimensional (3D) tissue and organ constructs using tissue spheroids as building blocks. Ultimately, successful bioprinting of human organ constructs is dependent on a ‘built in’ vascular tree to perfuse and maintain the viability of the organ constructs. Thus, the design of the vascular tree is a critically important step in practical implementation of organ printing technology. Bioprinting a vascular tree requires detailed knowledge of the morphometrical, morphological and biomechanical characteristics of the sequentially branched segments of the natural vascular tree as well as insight on post-printing tissue compaction and remodelling. Toward accomplishing this goal, we characterised the morphometrical, morphological and biomechanical characteristics of the initial segments of the natural kidney arterial vascular tree of the porcine kidney. Computer simulation was used to model compaction of tissue engineered tubular vascular segments with different wall thicknesses virtually biofabricated from closely packed and fused uniformly sized vascular tissue spheroids. The number of concentric layers of tissue spheroids required to bioprint tubular vascular segments with desirable wall thickness and diameter was theoretically estimated. Our results demonstrate that vascular segment compaction correlates well with reported experimental data. Finally, physical prototyping of linear and branched tubular constructs using silicon droplets as physical analogues of tissue spheroids was performed. Thus, virtual and physical prototyping provide important insights into the design parameters and demonstrate the principal feasibility of bioprinting a branched vascular tree using vascular tissue spheroids.

Additive Manufacturing and its future impact in logistics

Abstract This article presents an overview of the additive manufacturing (AM) area, as known as rapid prototyping and more recently as 3D printing, and its expected impact in logistics. The AM processes are classified in various categories and the most important processes are highlighted in this paper. Theoretically, any complex shape can be produced using AM that makes it very suitable for a fast and customized production. The applications for AM are expected to be present in the principal production chains and type of industry. It will impact as part production, systems or a complete product ranging from medical to aerospace industry. Therefore, this article discusses applications and new production paradigm that will affect in some extent the logistics the way it is today.

Delivery of Human Adipose Stem Cells Spheroids into Lockyballs

Adipose stem cells (ASCs) spheroids show enhanced regenerative effects compared to single cells. Also, spheroids have been recently introduced as building blocks in directed self-assembly strategy. Recent efforts aim to improve long-term cell retention and integration by the use of microencapsulation delivery systems that can rapidly integrate in the implantation site. Interlockable solid synthetic microscaffolds, so called lockyballs, were recently designed with hooks and loops to enhance cell retention and integration at the implantation site as well as to support spheroids aggregation after transplantation. Here we present an efficient methodology for human ASCs spheroids biofabrication and lockyballs cellularization using micro-molded non-adhesive agarose hydrogel. Lockyballs were produced using two-photon polymerization with an estimated mechanical strength. The Young’s modulus was calculated at level 0.1362 +/-0.009 MPa. Interlocking in vitro test demonstrates high level of loading induced interlockability of fabricated lockyballs. Diameter measurements and elongation coefficient calculation revealed that human ASCs spheroids biofabricated in resections of micro-molded non-adhesive hydrogel had a more regular size distribution and shape than spheroids biofabricated in hanging drops. Cellularization of lockyballs using human ASCs spheroids did not alter the level of cells viability (p › 0,999) and gene fold expression for SOX-9 and RUNX2 (p › 0,195). The biofabrication of ASCs spheroids into lockyballs represents an innovative strategy in regenerative medicine, which combines solid scaffold-based and directed self-assembly approaches, fostering opportunities for rapid in situ biofabrication of 3D building-blocks.

3D-printed PCL scaffolds for the cultivation of mesenchymal stem cells

Introduction Tissue engineering is a field which is currently under a great deal of investigation for the development and/or restoration of tissue and organs, through the combination of cell therapy with biomaterials. Rapid prototyping or additive manufacturing is a versatile technology which makes possible the fabrication of three dimensional (3D) structures from a wide range of materials with complex geometry and accuracy, such as scaffolds. Aim The aim of this study has been to investigate the interaction between mesenchymal stem cells with poly (ε-caprolactone) (PCL) biomaterials used for obtaining scaffolds through additive manufacturing. Materials and Methods Scanning electron microscopy, confocal microscopy and biological assays were performed to analyse the successful interaction between the cells and the biomaterials. Results As a result, the number of viable cells attached to the scaffolds was lower when compared to the control group; however, it was possible to observe cells in the scaffolds since day 1 of analysis, with regions of confluence after 21 days of seeding. Conclusions To conclude, these biomaterials are interesting if used as medical artifacts, principally in tissue with prolonged regeneration time and which requires 3D supports with good mechanical properties.

Starch and chitosan oligosaccharides as interpenetrating phases in poly(N-isopropylacrylamide) injectable gels

Thermosensitive interpenetrating gels were prepared by physically blending poly(N-isopropylacrylamide) (PNIPA) as the matrix and the following polysaccharides as interpenetrating phases: chitosan oligosaccharides (identified as QNAD and QNED) and soluble starch (STARCH). The molecular weight of the dispersed phase, the free water/bound water ratio and the thermosensitivity (transition temperature: LCST) of the gels were determined. It was found that these gels are pseudoplastic and that their viscosity depends on the molecular weight of the dispersed phase. LCST transition occurred around 35-37°C. The morphology of the porosity of the freeze-dried samples was studied by Scanning Electron Microscopy (SEM). An in vitro test of cell hemolysis on blood agar showed that these gels are noncytotoxic. According to the results obtained, these interpenetrating gels show characteristics of an injectable material, and have a transition LCST at body temperature, which reinforces their potential to be used in the surgical field and as scaffolds for tissue engineering.

Virtual Biofabrication Line

Abstract Virtual manufacturing (VM) is one of the most important tools in the development of manufacturing technology and it is now a standard practice in many industries. Virtual refineries have been successfully used in the design and optimization of refinery process in the oil industry. It is becoming obvious that practical implementation of organ biofabrication at industrial scale needs more than just one robotic bioprinter. Future organ biofabrication line must include series of well integrated robotic biofabrication tools such as cell sorters, tissue spheroid biofabricators, robotic bioprinters and perfusion bioreactors. Here we report a first attempt of the design and computer simulation of an organ biofabrication line. Virtual biofabrication line could be used for designing of economically effective organization of organ biofabrication process at industrial scale, analysis and optimization logistics of bioprocessing as well as for student education and personal training.

In vitro biocompatibility study of biodegradable polyester scaffolds constructed using Fused Deposition Modeling (FDM)

Abstract The growing interest in tissue engineering has stimulated the research of biomaterials that can be used as cellular supports and/or scaffolds to subsequently stimulate and/or regenerate tissues. Based on this premise, biodegradable polyesters: amorphous Poly (Lactic-acid) (PLA) and semi-crystalline Poly(e-caprolactone) (PCL), were used for manufacturing 3D scaffolds. These structures were designed using a a free software called Rhinoceros ® version 4.0. The software parameters considered for the design of these structures were: the distance between adjacent filaments, number of layers and the filaments orientation between layers. Through this information, and using PLA and PCL filaments (with diameters 2mm ⩽ o ⩽ 3 mm, obtained by extrusion), scaffolds were fabricated using Fused Deposition Modeling (FDM), a rapid prototyping technology. The Morphology of all structures was observed by Scanning Electron Microscopy (SEM). To assess biocompatibility, human fibroblasts were seeded on these scaffolds, and cultured for 4 and 8 days. The biocompatibility was assessed by a metabolic activity assay based on MTT, where an increase in metabolic activity is interpreted as cell proliferation. The results led to appreciate the interaction of fibroblast cultures with these materials, with a noticeable increase in the cellular metabolism indicative of the material´s cytocompatibility and its capacity to support proliferation, making them strong candidates for tissue engineering.