Tissue engineering is an emerging field aimed to combine biological, engineering and material methods to create a biomimetic three dimensional (3D) environment to control cells proliferation and functional tissue formation. In such an artificial structural environment, a scaffold, made from biomaterial(s), plays an essential role by providing a mechanical support and biological guidance platform. Hence, fabrication of tissue scaffolds is of a fundamental importance, yet a challenging task, in tissue engineering. This task becomes more challenging if living cells need to be encapsulated in the scaffolds so as to fabricate scaffolds with structures to mimic the native ones, mainly due to the issue of process-induced cell damage. This research aims to develop novel methods to model the process of fabricating cell-encapsulated scaffolds and process-induced cell damage. Particularly, this research focuses on the scaffold fabrication process based on the dispensing-based rapid prototyping technique - one of the most promising scaffold fabrication methods nowadays, by which a 3D scaffold is fabricated by laying down multiple, precisely formed layers in succession. In the dispensing-based scaffold fabrication process, the flow behavior of biomaterials solution can significantly affect the flow rate of material dispensed, thus the structure of scaffold fabricated. In this research, characterization of flow behavior of materials was studied; and models to represent the flow behaviour and its influence on the scaffold structure were developed. The resultant models were shown able to greatly improve the scaffold fabrication in terms of process parameter determination. If cells are encapsulated in hydrogel for scaffold fabrication, cell density can affect the mechanical properties of hydrogel scaffolds formed. In this research, the influence of cell density on mechanical properties of hydrogel scaffolds was investigated. Furthermore, finite element analysis (FEA) of mechanical properties of scaffolds with varying cell densities was performed.The results show that the local stress and strain energy on cells varies at different cell densities. The method developed may greatly facilitate hydrogel scaffolds design to minimize cell damage in scaffold and promote tissue regeneration. . In the cell-encapsulated scaffold fabrication process, cells inevitably suffer from mechanical forces and other process-induced hazards. In such a harsh environment, cells deform and may be injured, even damaged due to mechanical breakage of cell membrane. In this research, three primary physical variables: shear stress, exposure time, and temperature were examined and investigated with regard to their effects on cell damage. Cell damage laws through the development phenomenal models and computational fluidic dynamic (CFD) models were established; and their applications to the cell-encapsulated scaffold fabrication process were pursued. The results obtained show these models and modeling methods not only allow one to optimize process parameters to preserve cell viability but also provide a novel strategy to probe cell damage mechanism in microscopic view.

Submitted by Maria Anália Conceição (marianaliaconceicao@gmail.com) on 2016-06-27T19:41:41Z No. of bitstreams: 1 Publico-NOVO-21.pdf: 2987989 bytes, checksum: fedc0b7588511ac9f1b74f18e86aed33 (MD5) / Approved for entry into archive by Maria Anália Conceição (marianaliaconceicao@gmail.com) on 2016-06-27T19:42:44Z (GMT) No. of bitstreams: 1 Publico-NOVO-21.pdf: 2987989 bytes, checksum: fedc0b7588511ac9f1b74f18e86aed33 (MD5) / Made available in DSpace on 2016-06-27T19:42:44Z (GMT). No. of bitstreams: 1 Publico-NOVO-21.pdf: 2987989 bytes, checksum: fedc0b7588511ac9f1b74f18e86aed33 (MD5) Previous issue date: 2015 / Agência Brasileira de Cooperação (ABC) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Rede Ibero-Americana de Biofabricação / Introdução: A Engenharia Tecidual (ET) é uma ciência multidisciplinar que visa produzir órgãos e partes humanas substitutas acometidas por lesões traumáticas, doenças degenerativas ou agenesias. Uma das suas etapas é a produção de arcabouços biocompatíveis para aplicação na Medicina Regenerativa. Estas estruturas são conhecidas como scaffolds, que apresentam macrogeometria semelhante ao tecido original, em textura e porosidade e direcionam o comportamento das células que serão semeadas. A recuperação da integridade anatômica e funcional de tecidos lesados garante a sobrevivência dos seres vivos e o tratamento de perdas extensas é desafiador. Objetivo: Avaliar a eficiência para desmineralizar e descelularizar dentes viabilizando-os como scaffolds naturais. Métodos: As amostras foram submetidas a um tratamento com soluções desmineralizadoras/descelularizadoras. Foram usadas 5 soluções: G1-Formol 10% controle, EDTA 28% para desmineralização nos quatro grupos; G2- hipoclorito de sódio 2,5%; e G3-peróxido de hidrogênio 9%; G4- hipoclorito de sódio 2,5% associado com detergente enzimático; G5- detergente enzimático associado a peróxido de hidrogênio 9%. A evolução da desmineralização e descelularização foi acompanhada durante 12 semanas, por meio de pesagem, técnicas analíticas MEV (Microscopia eletrônica de Varredura), fotografia e radiografia. As amostras foram pesadas a cada sete dias para controle da perda de mineral. Os resultados receberam análise estatística de variância de Friedman, Kruskal-Wallis, Resumo xix Teste do Quiquadrado e Teste exato de Fisher. Foi fixado em 0,05 ou 5% o nível de rejeição da hipótese de nulidade. Conclusão: O grupo 5 mostrouse microscopicamente a melhor solução, mesmo mantendo em 30% das amostras resíduos biológicos. / Tissue Engineering (TE) is a multidisciplinary science that aims to produce replacement organs and parts affected by trauma, degenerative diseases or agenesis. One of its goals is to produce biocompatible scaffolds for application in regenerative medicine. These structures are known as scaffolds, presenting three-dimensional shape similar to the original tissue in texture and porosity and directing the behavior of cells to be seeded. The recovery of anatomical and functional integrity of damaged tissues ensures the survival of living beings and the treatment of extensive losses is challenging. Objective: Get decelullarized and demineralized teeth to make them feasible as natural scaffolds. Methods: The samples will be subjected to a treatment with demineralizing/decelularizing solutions. 4 different solutions were used (14% EDTA, 2.5% sodium hypochlorite, hydrogen peroxide and 9% and one group of control (10% formaldehyde). The evolution of demineralization/decellularization was monitored for 90 days through the use of electron scanning microscopy, X-ray and photography. Samples’ weights were measured each seven days to control the mineral loss. Results were subjected to statistical analysis of variance, KruskalWallis test Wilcoxan and Chi-square Test. Was fixed at 0,05 or 5% rejection level of the null hypothesis. / FAPESP: 07/58856-7 / FAPESP: 07/59488-1 / FAPESP: 07/51227-4 / CNPq: 573661/2008-1 / FAPESP: 08/57860-3

This thesis investigates new processing methods and bone tissue engineering applications of cross-linked cellulose nanocrystal (CNC) aerogels. Aerogels are highly porous, low-density materials that have been praised for their high surface area and interconnected pores, but criticized for their brittleness. This prompted a search for new aerogel “building blocks” to produce more flexible materials; CNCs meet this need and chemically cross-linked CNC aerogels have good compressive strength and shape recovery properties in air and liquid environments. CNCs are high aspect ratio, non-toxic and renewably-sourced nanoparticles. Literature has demonstrated CNC aerogel production using cryo-templating with controlled drying. In this work, we produce aerogels using a new scalable process called pressurized gas expansion (PGX) and compare them to conventional cryo-templated aerogels. PGX aerogels were found to have more expanded fibrillar morphology, a range of mesopore sizes and smaller macropores, in contrast to cryo-templated aerogels that had a sheet-like morphology surrounding larger macropores. Additionally, PGX aerogels had higher specific surface area and porosity, but lower compressive strength due to a lower cross-link density. While neither CNC aerogel type dispersed in water, PGX aerogels partially shrank whereas cryo-templated aerogels did not; this is attributed to their morphological differences. This work shows that new aerogel processing methods can introduce new properties and thus broaden the potential applications of CNC aerogels. One specific biomedical application was evaluated for CNC aerogels – their use as bone tissue scaffolds. Cryo-templated aerogels comprised of CNCs with different surface chemistries, either sulfate or phosphate groups, were found to have attractive chemical, physical and mechanical properties for bone tissue engineering. This work shows that both types of CNC aerogels can facilitate cell proliferation, favorable differentiation, and can nucleate uniform hydroxyapatite growth. These positive in vitro results and the bimodal pore morphology of CNC aerogels make them promising bone scaffolds for in vivo studies. / Thesis / Master of Applied Science (MASc) / Aerogels are light, porous, sponge-like materials that are essentially 99% air by volume. In this work, the aerogels are made from non-toxic plant-based nanoparticles called cellulose nanocrystals (CNCs). This thesis investigates: 1) new ways to control CNC aerogel properties and pore size through different processing methods and 2) the use of CNC aerogels to aid in the repair of damaged bones. High-resolution microscopy and nano-characterization tools show that CNC aerogels have tunable properties, which may extend their possible applications. The internal structure, sponge-like mechanical properties and biocompatibility of CNC aerogels allowed them to be successfully utilized to support bone cells and grow bone-like mineral.

Unlike traditional stochastic scaffold fabrication techniques, additive manufacturing (AM) can be used to create tissue-specific three-dimensional scaffolds with controlled porosity and pore geometry (meso-structure). However, due to the relatively few biocompatible materials available for processing in AM machines, direct fabrication of tissue scaffolds is limited. To alleviate material limitations and improve feature resolution, a new indirect scaffold fabrication method is developed. A four step fabrication process is explored: Fused Deposition Modeling (FDM) is used to fabricate scaffold patterns of varied pore size and geometry. Next, scaffold patterns are surface treated, and then mineralized via simulated body fluid (SBF); forming a bone-like ceramic throughout the scaffold pattern. Finally, mineralized patterns are heat treated to pyrolyze the pattern and sinter the minerals. Two scaffold meso-structures are tested: "tube" and "backfill." Two pattern materials are tested [acrylonitrile butadiene styrene (ABS) and investment cast wax (ICW)] to determine which material is the most appropriate for mineralization and sintering. Mineralization is improved through plasma surface treatment and dynamic flow conditions. Appropriate burnout and sintering temperatures to remove pattern material are determined experimentally. While the "tube scaffolds" were found to fail structurally, "backfill scaffolds" were successfully created using the new fabrication process. The "backfill scaffold" meso-structure had wall thicknesses of 470 – 530 µm and internal channel diameters of 280 – 340 µm, which is in the range of appropriate pore size for bone tissue engineering. "Backfill scaffolds" alleviated material limitations, and had improved feature resolution compared to current indirect scaffold fabrication processes. / Master of Science

This dissertation presents the application of MEMS-based approaches for the construction of engineered tissue substitutes. MEMS technology can offer the physical scale, resolution, and organization necessary for mimicking native tissue architecture. Micromachined nozzles and templates were explored for the fabrication of acellular, biomimetic collagenous fibrous scaffolds, microvascular tissue structures, and the combination of these structures with cell-based therapeutics. The influence of the microstructure of the tissue constructs on their macro-scale characteristics was investigated.

The tool-less additive manufacturing (AM) or 3D printing processes (3DP) use incremental consolidation of feed-stock materials to construct part. The layer by layer AM processes can achieve spatial material distribution and desired microstructure pattern with high resolution. This unique characteristics of AM can bring custom-made form and tailored functionality within the same object. However, incorporating form and functionality has their own challenge in both design and manufacturing domain. This research focuses on designing manufacturable topology by marrying form and functionality in additively manufactured part using infill structure. To realize the goal, this thesis presents a systematic design framework that focuses on reducing the gap between design and manufacturing of complex architecture. The objective is to develop a design methodology of lattice infill and thin shell structure suitable for additive manufacturing processes. Particularly, custom algorithmic approaches have been developed to adapt the existing porous structural patterns for both interior and exterior of objects considering application specific functionality requirements. The object segmentation and shell perforation methodology proposed in this work ensures manufacturability of large scale thin shell or hollowed objects and incorporates tailored part functionality. Furthermore, a computational design framework developed for tissue scaffold structures incorporates the actual structural heterogeneity of natural bones obtained from their medical images to facilitate the tissue regeneration process. The manufacturability is considered in the design process and the performances are measured after their fabrication. Thus, the present thesis demonstrates how the form of porous structures can be adapted to mingle with functionality requirements of the application as well as fabrication constraints. Also, this work bridges the design framework (virtual) and the manufacturing platform (realization) through intelligent data management which facilitates smooth transition of information between the two ends. / National Science Foundation #OIA-1355466 / National Science Foundation-DMR- MRI #1625704 / National Institute of Health - COBRE: CDTSPC; Grant # P20GM109024 / US-DOT # 693JK31850009CAAP / Dept. of Commerce Research-ND, Award # 17-08-G-191 / CSMS, NDEPSCoR / NDSU Grand Challenge and Development Foundation

UV-curing is one of the most commonly used methods for producing hydrogels for soft and hard tissue scaffolds. Spontaneous curing is an alternative method which possesses some advantages in comparison to the conventional UV-curing methods; for example, in situ crosslinking and excluding initiators. The main objective of this study was to investigate promising materials for producing UV and spontaneously cured hydrogels, and subsequently to perform a comparison between the produced hydrogels with regard to their different mechanical and physical properties.Seventeen different hydrogels including five UV-cured and twelve spontaneously cured hydrogels were produced by applying thiol-ene chemistry and by varying precursor materials. Hydrogel systems including di- and tetra- functional PEGs of different lengths (2 kDa and 6 kDa) and two different thiol-crosslinkers (ETTMP 1300 Da and DTT) were subsequently characterized and evaluated. The evaluation tests applied in this study were Raman spectroscopy, weight and volumetric swelling test, leaching test, tensile test, and rheology test. Between all the systems, tetra-acrylated PEG (6 kDa) BisMPA was found to be the most promising system. The pH level of the applied solvent (PBS) for spontaneously cured hydrogels was varied from the physiologically relevant level of 7.4 to 7.0 and 7.8 in order to investigate the dependency of physical and mechanical properties of the hydrogels to this parameter.Spontaneous curing of tetra-acrylated PEG (6 kDa) BisMPA with ETTMP 1300 Da as the thiol-crosslinker, was accomplished within 3½ min in PBS with a pH level of 7.4; and it came out to be the fastest spontaneously cured system between all the tested hydrogels. Increasing the PBS pH level resulted in a faster curing process (accomplished in 1½ min). Spontaneously cured hydrogels generally showed decreased mechanical properties, but improved swelling behavior compared to UV-cured hydrogels. Nevertheless, the discussed system still possessed 50% of the elastic modulus in the tensile test in comparison to the UV-cured state; and showed the highest elastic modulus in comparison to other spontaneously cured systems. The storage modulus of the mentioned hydrogel in the spontaneously cured state was very close to the same parameter in the UV-cured hydrogel based on the same precursors. It also possessed the highest storage modulus between all the spontaneously cured hydrogels. Although the obtained swelling properties of this system were not the highest between all the tested hydrogels, these parameters were still in an acceptable range as for a hydrogel proposed for tissue scaffold application (swelling ratio: 9.72, water content: 89.71%, volumetric swelling ratio: 9.05). Furthermore, the system had the lowest weight loss ratio between all the acrylate-based hydrogels (including both UV and spontaneously cured systems), which along with the Raman spectroscopy results shows the high crosslinking efficiency of the system.

This article describes the development of a component-based technology robot system that can be rapidly configured to perform a specific manufacturing task. The system is conceived with standard and inter-operable components including actuator modules, rigid link connectors and tools that can be assembled into robots with arbitrary geometry and degrees of freedom. The reconfigurable "plug-and-play" robot kinematic and dynamic modeling algorithms are developed. These algorithms are the basis for the control and simulation of reconfigurable robots. The concept of robot configuration optimization is introduced for the effective use of the rapidly reconfigurable robots. Control and communications of the workcell components are facilitated by a workcell-wide TCP/IP network and device level CAN-bus networks. An object-oriented simulation and visualization software for the reconfigurable robot is developed based on Windows NT. Prototypes of the robot systems configured to perform 3D contour following task and the positioning task are constructed and demonstrated. Applications of such systems for biomedical tissue scaffold fabrication are considered. / Singapore-MIT Alliance (SMA)

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