Frederico D. A. S. Pereira

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.

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.

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.

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.

Is characterizing the digital forensic facial reconstruction with hair necessary? A familiar assessors’ analysis

BACKGROUND In the international scientific literature, there are few studies that emphasize the presence or absence of hair in forensic facial reconstructions. There are neither Brazilian studies concerning digital facial reconstructions without hair, nor research comparing recognition tests between digital facial reconstructions with hair and without hair. The miscegenation of Brazilian people is considerable. Brazilian people, and, in particular, Brazilian women, even if considered as Caucasoid, may present the hair in very different ways: curly, wavy or straight, blonde, red, brown or black, long or short, etc. For this reason, it is difficult to find a correct type of hair for facial reconstruction (unless, in real cases, some hair is recovered with the skeletal remains). AIMS AND METHODS This study focuses on the performance of three different digital forensic facial reconstructions, without hair, of a Brazilian female subject (based on one international database and two Brazilian databases for soft facial-tissue thickness) and evaluates the digital forensic facial reconstructions comparing them to photographs of the target individual and nine other subjects, employing the recognition method. A total of 22 assessors participated in the recognition process; all of them were familiar with the 10 individuals who composed the face pool. RESULTS AND CONCLUSIONS The target subject was correctly recognized by 41% of the 22 examiners in the International Pattern, by 32% in the Brazilian Magnetic Resonance Pattern and by 32% in the Brazilian Fresh Cadavers Pattern. The facial reconstructions without hair were correctly recognized using the three databases of facial soft-tissue thickness. The observed results were higher than the results obtained using facial reconstructions with hair, from the same skull, which can indicate that it is better to not use hair, at least when there is no information concerning its characteristics.