Engenharia de estruturas semelhantes a capilares incorporadas em hidrogéis para cultura de células 3D

Nowadays, the biggest challenge in tissue engineering consists in developing structures and in the application of strategies to emulate the anatomical and cellular complexity and vascularization of native tissues to maintain cell viability and functionality. The presence of functional blood vessel networks is essential to ensure adequate nutrient flow and oxygen diffusion throughout the support structure, two key requirements for maintaining cell viability. This work aimed to develop a complex in vitro model that mimics the native vascular network. To this end, a multilayered membrane made of six bilayers of chitosan (CHI)/alginate (ALG) or CHI/ALG-RGD (tripeptide of Arginine (R)-Glycine (G)- Aspartic acid (D) responsible for the cellular adhesion to the extracellular matrix (ECM)) were produced via Layer-by-Layer (LbL) assembly technology on the ALG printed structures. The ALG structures coated with the multilayered membranes were embedded in xanthan gum, chemically modified with methacrylated groups in order to obtain a mechanically robust hydrogel structure after photocrosslinking by UV light exposure. The liquification of the ALG printed structures, coated with the CHI/ALG, CHI/ALG-RGD or without the multilayers membranes, with ethylenediaminetetraacetic acid (EDTA), led to the formation of microchannels in which human umbilical vein endothelial cells (HUVECs) were cultured for 24 hours. The obtained results demonstrate that the microchannels encompassing CHI/ALG-RGD multilayered membranes contributed to a larger cellular adhesion, demonstrating their potential to be applied in tissue engineering and regenerative medicine strategies.Atualmente, o maior desafio em engenharia de tecidos consiste no desenvolvimento de estruturas e aplicação de estratégias que visem mimetizar a complexidade anatómica e celular, assim como a vascularização de tecidos nativos, de forma a manter a viabilidade e funcionalidade das células. A presença de estruturas funcionais à base de vasos sanguíneos é essencial para garantir o fluxo adequado de nutrientes, assim como a difusão de oxigénio em toda a estrutura de suporte, dois requisitos essenciais para manter a viabilidade celular. Este trabalho teve como objetivo desenvolver um modelo complexo in vitro que mimetize a rede vascular nativa. Com esse intuito, membranas multicamadas compreendendo seis bicamadas de quitosana (CHI)/alginato (ALG) e CHI/ALG-RGD (tripéptido de Arginina (R)-Glicina (G)-Ácido aspártico (D) responsável pela adesão de células à matriz extracelular) foram produzidas, via tecnologia de deposição camada-a-camada (do inglês Layer-by-Layer assembly technology), em estruturas impressas de ALG. As fibras de ALG revestidas com os filmes multicamadas foram embebidas em goma xantana, quimicamente modificada com grupos metacrilatos, de modo a obter uma estrutura de hidrogel mecanicamente robusta após foto-reticulação por ação da luz UV. A liquefação das estruturas impressas de ALG, contendo as multicamadas de CHI/ALG ou CHi/ALG-RGD, com ácido etilenodiamino tetra-acético (EDTA), levou à formação de microcanais nos quais se cultivaram células endoteliais humanas, extraídas da veia umbilical durante 24 horas. Os resultados obtidos demonstraram que os microcanais compreendendo as membranas multicamadas à base de CHI/ALG-RGD contribuíram para uma maior adesão celular, demonstrando o seu potencial para estratégias de engenharia de tecidos e medicina regenerativa.Mestrado em Biotecnologi

Polymer- and Hybrid-Based Biomaterials for Interstitial, Connective, Vascular, Nerve, Visceral and Musculoskeletal Tissue Engineering

In this review, materials based on polymers and hybrids possessing both organic and inorganic contents for repairing or facilitating cell growth in tissue engineering are discussed. Pure polymer based biomaterials are predominantly used to target soft tissues. Stipulated by possibilities of tuning the composition and concentration of their inorganic content, hybrid materials allow to mimic properties of various types of harder tissues. That leads to the concept of “one-matches-all” referring to materials possessing the same polymeric base, but different inorganic content to enable tissue growth and repair, proliferation of cells, and the formation of the ECM (extra cellular matrix). Furthermore, adding drug delivery carriers to coatings and scaffolds designed with such materials brings additional functionality by encapsulating active molecules, antibacterial agents, and growth factors. We discuss here materials and methods of their assembly from a general perspective together with their applications in various tissue engineering sub-areas: interstitial, connective, vascular, nervous, visceral and musculoskeletal tissues. The overall aims of this review are two-fold: (a) to describe the needs and opportunities in the field of bio-medicine, which should be useful for material scientists, and (b) to present capabilities and resources available in the area of materials, which should be of interest for biologists and medical doctors.</jats:p

Curvy surface conformal ultra-thin transfer printed Si optoelectronic penetrating microprobe arrays

Penetrating neural probe arrays are powerful bio-integrated devices for studying basic neuroscience and applied neurophysiology, underlying neurological disorders, and understanding and regulating animal and human behavior. This paper presents a penetrating microprobe array constructed in thin and flexible fashion, which can be seamlessly integrated with the soft curvy substances. The function of the microprobes is enabled by transfer printed ultra-thin Si optoelectronics. As a proof-of-concept device, microprobe array with Si photodetector arrays are demonstrated and their capability of mapping the photo intensity in space are illustrated. The design strategies of utilizing thin polyimide based microprobes and supporting substrate, and employing the heterogeneously integrated thin optoelectronics are keys to accomplish such a device. The experimental and theoretical investigations illustrate the materials, manufacturing, mechanical and optoelectronic aspects of the device. While this paper primarily focuses on the device platform development, the associated materials, manufacturing technologies, and device design strategy are applicable to more complex and multi-functionalities in penetrating probe array-based neural interfaces and can also find potential utilities in a wide range of bio-integrated systems

Electrothermal soft manipulator enabling safe transport and handling of thin cell/tissue sheets and bioelectronic devices

“Living” cell sheets or bioelectronic chips have great potentials to improve the quality of diagnostics and therapies. However, handling these thin and delicate materials remains a grand challenge because the external force applied for gripping and releasing can easily deform or damage the materials. This study presents a soft manipulator that can manipulate and transport cell/tissue sheets and ultrathin wearable biosensing devices seamlessly by recapitulating how a cephalopod’s suction cup works. The soft manipulator consists of an ultrafast thermo-responsive, microchanneled hydrogel layer with tissue-like softness and an electric heater layer. The electric current to the manipulator drives microchannels of the gel to shrink/expand and results in a pressure change through the microchannels. The manipulator can lift/detach an object within 10 s and can be used repeatedly over 50 times. This soft manipulator would be highly useful for safe and reliable assembly and implantation of therapeutic cell/tissue sheets and biosensing devices

Surface acoustic waves induced micropatterning of cells in gelatin methacryloyl (GelMA) hydrogels

Acoustic force patterning is an emerging technology that provides a platform to control the spatial location of cells in a rapid, accurate, yet contactless manner. However, very few studies have been reported on the usage of acoustic force patterning for the rapid arrangement of biological objects, such as cells, in a three-dimensional (3D) environment. In this study, we report on a bio-acoustic force patterning technique, which uses surface acoustic waves (SAWs) for the rapid arrangement of cells within an extracellular matrix-based hydrogel such as gelatin methacryloyl (GelMA). A proof-of-principle was achieved through both simulations and experiments based on the in-house fabricated piezoelectric SAW transducers, which enabled us to explore the effects of various parameters on the performance of the built construct. The SAWs were applied in a fashion that generated standing SAWs (SSAWs) on the substrate, the energy of which subsequently was transferred into the gel, creating a rapid, and contactless alignment of the cells (&lt;10 s, based on the experimental conditions). Following ultraviolet radiation induced photo-crosslinking of the cell encapsulated GelMA pre-polymer solution, the patterned cardiac cells readily spread after alignment in the GelMA hydrogel and demonstrated beating activity in 5–7 days. The described acoustic force assembly method can be utilized not only to control the spatial distribution of the cells inside a 3D construct, but can also preserve the viability and functionality of the patterned cells (e.g. beating rates of cardiac cells). This platform can be potentially employed in a diverse range of applications, whether it is for tissue engineering, in vitro cell studies, or creating 3D biomimetic tissue structures

Bioengenharia de sistemas nano estruturados com base em superfícies inspiradas na natureza para regeneração de tecidos humanos

Modular tissue engineering aims to mimic the complexity of native tissue with well-defined 3D architectures and synergistic interactions of various cell lines by generating repeated functional modular units that will be assembled into a functional tissue. These modular blocks should exhibit specific microstructural characteristics to mimic the complex architecture of native tissues. For the direct production of modular units, patterned superhydrophobic-superhydrophilic (SH-SL) surfaces have emerged as promising platforms for scalable manufacturing of microscale modular units to develop functional tissues designed by the bottom-up approach. In this sense, and inspired by the Lotus effect, the present work aims at the production of freestanding (FS) stratificated micromembranes based on polyl- lysine (PLL) and alginate (ALG) biopolymers through the Layer-by-Layer (LbL) methodology. For this purpose, initially microscale SH-SL surfaces with different geometric shapes were developed. Subsequently, alginate hydrogels were formed in situ by the standing droplet method in the SL areas that served as a sacrificial template to the production of freestanding membranes by sequential deposition of electrolytes through electrostatic interactions. Regarding the deposition conditions of the polymers, in the zeta potential analysis, the charges of each compound were verified, while the quartz microbalance (QCM-D) showed the electrostatic interaction between PLL and ALG. ATR-FTIR analysis confirmed the presence of polymers in the resulting membrane. After detachment, the resulting membranes crosslinked with genipin (GnP) to improve mechanical properties to promote cell adhesion and proliferation. Biological assays with human umbilical vein endothelial cells (HUVECs) and human adipose stem cells (hASCs) showed that the crosslinked [PLL / ALG]100 membranes show cellular viability.A engenharia modular de tecidos visa mimetizar a complexidade do tecido nativo com arquiteturas 3D bem definidas e interações sinérgicas de várias linhas celulares através da geração de unidades modulares funcionais repetidas que serão montadas em um tecido funcional. Esses blocos modulares devem exibir características microestruturais específicas para imitar a arquitetura complexa de tecidos nativos. Para a produção direta de unidades modulares, as superfícies superhidrofóbicas-superhidrofílicas (SHSL) padronizadas surgiram como plataformas promissoras para uma fabricação escalável de unidades modulares à microescala para desenvolver tecidos funcionais projetados pela abordagem bottom-up. Neste sentido, e inspirado no efeito de Lotus, o presente trabalho visa a produção de micromembranas autónomas estratificadas baseadas nos biopolímeros poli-llisina (PLL) e alginato (ALG) através da metodologia Layer-by-Layer (LbL). Para este propósito, inicialmente foram desenvolvidas superfícies SH-SL padronizadas à microescala com diferentes formas geométricas. Posteriormente, hidrogéis de alginato foram formados in situ pelo método standing droplet nas áreas SL que serviram de template de sacrifício para a produção de membranas autónomas pela deposição sequencial dos polieletrólitos através de interações electrostáticas. Relativamente às condições de deposição dos polímeros, na análise do potencial zeta verificaram-se as cargas de cada composto, enquanto que a microbalança de quartzo (QCM-D) evidenciou a interação eletrostática entre a PLL e o ALG. A análise por ATR-FTIR, confirmou a presença dos polímeros na membrana resultante. Após o destaque, as membranas forma reticuladas com genipina (GnP) para melhorar as propriedades mecânicas a fim de promover a adesão e proliferação celular. Ensaios biológicos com human umbilical vein endotelial cells (HUVECs) e human adipose stem cells (hASCs) evidenciaram que as membranas de [PLL/ALG]100 reticuladas apresentam viabilidade celular.Mestrado em Materiais e Dispositivos Biomédico

Estratégias biomiméticas usando a técnica camada-a-camada para aplicações biomédicas e engenharia de tecidos

The development of a suitable coating or material, which physico-chemical, mechanical or biological properties, that can be tailored according the features of the target tissue, has been gaining increased importance in biomedical and tissue engineering and regenerative medicine (TERM) fields. Biomimetic strategies have contributed significantly for the progress of biomedical field during the last years. This is possible to be achieved at different levels: imitating Nature form or function and mimicking natural processes and systems are the most used biomimetic approaches. In this thesis, Layer-by-Layer (LbL) methodology was used as a hierarchical biomimetic tool to modify surfaces and to produce freestanding membranes based on polyelectrolyte multilayers (PEMs). The possibility to functionalize or engineer biomaterials combined with the ability to incorporate a wide range of building blocks, makes LbL a powerful processing technique in the biomedical field. Synthetic polymers have been used to construct PEMs for biomedical and TERM applications; however, they lack often on adhesive cues for cell attachment and tissue growth. To overcome such issue, biomimetic synthetic polymers have been developed. Elastin-like polypeptides (ELPs) are a class of nature-inspired polymers, nonimmunogenic, genetically encodable and biocompatible. These materials are based on the repetition of short peptides considered to be building blocks in natural elastin and can include specific bioactive sequences, as the tripeptide Arginine-Glycine-Aspartame (RGD) known by promoting cell adhesion. For the first work of this thesis, ELPs were functionalized with azide and alkyne groups to introduce the reactivity required to carry out the 1,3-dipolar cycloaddition under mild biocompatible conditions, with no toxic by-products and in short reaction times. This reaction was done by means of a LbL assembly, driven by covalent interactions instead of being driven by electrostatic interactions, obtaining a bioactive and biomimetic multilayer coating. Moreover, these polymers are characterized by a critical temperature, known as the transition temperature in aqueous solution (Tt), which is related with a conformational reorganization. Thus, below Tt the polymer chains were soluble in water and above Tt they formed nano- and micro-aggregates becoming insoluble in a reversible process, making these coatings stimuli-responsive. In the following chapters, several polysaccharides as chitosan (CHT), alginate (ALG), hyaluronic acid (HA) or chondroitin sulfate (CS) were used to produce freestanding structured membranes through LbL processes, mainly driven by electrostatic interactions. The use of PEMs containing biopolymers are particularly appealing to coat and develop multilayered structures with biochemical functionalities, biocompatibility, and to mimic the interactions observed in native extracellular matrix (ECM). CHI/CS multilayers were used throughout the thesis, revealing some unique properties, when compared with other polysaccharide-based multilayers, such as their elasticity and degradation rate. However, natural origin polymer-based multilayers present low stiffness and higher hydration rates, which hinder cell adhesion. To overcome this, the CHT/CS multilayers were crosslinked with genipin. This is also a natural product, that is extracted from gardenia fruits and presents the ability to improve the mechanical properties, while preserves the biocompatibility and even enhances the cell adhesive properties. The ability to tailor the multilayers properties can be applied during their assembly or postassembly. Upon adjusting cross-linking parameters (e.g., cross-linker concentration and reaction time) the morphology, thickness, water uptake, rate of biodegradation, mechanical properties and cell adhesive properties can be tuned. Studies of shape-memory of these multilayered films, presented promising results regarding their use in biomedical applications. The mechanical properties of the multilayers can be further improved combining covalent and ionic crosslinking, which gives rise to a full interpenetrating polymer network. More interesting, it was possible to create a well-organized patterned topography at the surface of the freestanding multilayered membrane, just by using a different underlying substrate. This strategy envisaged to mimic the topography of the ECM of some tissues, as bone, skin or nerves, creating grooves on the material’s surface at nanoscale. Using this approach, it was possible to control some cellular functions and behavior as alignment and differentiation. Further in this thesis and inspired by the composition of the adhesive proteins in mussels, freestanding multilayered membranes containing dopamine-modified hyaluronic acid (HA-DN) were produced. The presence of DN along with the thickness of the membranes presented better lap-shear adhesion strength than the control membranes (hyaluronic acid and alginate films – two polysaccharides often regarded as good natural adhesives – were assembled together). Moreover, in vitro tests showed an enhanced cell adhesion for the membranes containing HA-DN and ability to use such kind of membranes for different biomedical and TERM applications, particularly for bone regeneration and skin wound healing. Combining different biomimetic concepts, it was also possible to recreate the complex environment of osteoarthritic articular cartilage by preparing human circular discs of superficially damaged articular cartilage from human samples. Herein, the adhesive freestanding multilayered membranes were used as a vehicle to deliver human adipose stem cells (hASCs) to help to repair the damaged cartilage. hASCs temporarily adhered to the adhesive LbL-based membranes, and were transported to the cartilage discs, creating a bridge of cells between the membranes and the surface of the cartilage. The cells started to migrate into the defects of the cartilage, proliferating and secreting factors capable of repairing the cartilage. Overall, the developed work in this thesis shows that LbL is a very versatile technique that provides the means to develop a wide range of solutions to be useful in biomedical and TERM applications.O desenvolvimento de um revestimento ou material cujas propriedades físicoquímica, mecânicas ou biológicas podem ser modificadas de acordo com as propriedades do tecido alvo, tem ganho cada vez mais importância, nomeadamente para fins biomédicos e de engenharia de tecidos e medicina regenerativa. Durante os últimos anos, diferentes estratégias biomiméticas têm contribuído significativamente para o progresso destas áreas. Estas são possíveis de implementar a diferentes níveis: imitar formas e funções existentes na natureza ou mimetizar processos e sistemas naturais. Na presente tese, a técnica camada-a-camada (LbL) foi usada como uma ferramenta biomimética para modificar superfícies ou produzir membranas com base em múltiplas camadas de polieletrólitos. A crescente utilização desta técnica, concretamente na área biomédica, prende-se com a possibilidade de funcionalizar ou produzir biomateriais aliada à capacidade de incorporar uma gama alargada de blocos de construção. Aqui, diferentes polímeros sintéticos e naturais têm sido usados para construir estruturas multicamada; no entanto, a generalidade dos polímeros sintéticos não apresenta naturalmente locais de ligação e adesão celular. Para contornar este obstáculo, algumas modificações químicas aos polímeros sintéticos têm sido sugeridas e novos compostos têm sido desenvolvidos, inspirados na composição de sistemas naturais. Por exemplo, polipéptidos tipo-elastina (ELPs) são uma classe de polímeros inspirados na natureza, que apresentam propriedades não-imunogénicas e biocompatíveis, podendo ser geneticamente programados conforme desejado. A sua composição baseia-se na repetição de pequenos péptidos também presentes na elastina humana, com a possibilidade também de incorporar outras sequências bioativas especificas, como o tripéptido Arginina-GlicinaÁcido Aspártico (RGD), reconhecido por promover a adesão celular. Para esta tese foram produzidos ELPs, que mais tarde foram funcionalizados com grupos azida e alquino para introduzir a reatividade necessária para uma reação 1,3-dipolar de ciclo-adição se realizar em condições biocompatíveis, sem produtos tóxicos resultantes e em curtos tempos de reação. Esta reação foi realizada sob a técnica LbL, mas conduzida por interações covalentes ao invés de electroestáticas, para atuar como revestimento biomédico. Estes polímeros são ainda reconhecidos pela sua temperatura de transição (Tt) em solução aquosa, relacionada com uma reorganização conformacional da cadeia polimérica. Abaixo da Tt as suas cadeias poliméricas são solúveis, mas acima de Tt formam-se micro-agregados; este é um processo reversível que confere propriedades responsivas aos revestimentos. Nos seguintes capítulos, diferentes polissacarídeos como quitosano (CHT), alginato (ALG), sulfato de condroitina (CS) ou ácido hialurónico (HA), foram usados para produzir membranas multicamadas conduzidas maioritariamente via interações electroestáticas. Esta abordagem tem ganho cada vez mais importância para desenvolver materiais com funcionalidade bioquímica, biocompatibilidade e para mimetizar algumas interações observadas na matriz extracelular (ECM). Ao longo desta tese foram usadas membranas multicamada de CHT/CS; estes materiais revelaram algumas propriedades muito particulares, quando comparadas com outros sistemas de multicamada, como a sua elasticidade e taxas de degradação mais rápidas. No entanto, a baixa rigidez e maiores taxas de hidratação, que muitas vezes impedem a adesão celular, surgem frequentemente associados a sistemas multicamada compostos somente por polissacarídeos. Para contornar este obstáculo, as membranas multicamada de CHT/CS foram reticuladas com genipina. De notar que este composto é de origem natural, sendo extraído da fruta da gardénia; a pós-modificação das membranas com genipina resultou na melhoria das propriedades mecânicas e biocompatibilidade, e ainda, no aumentando das propriedades bio-adesivas. Na realidade, a possibilidade de modular as propriedades destes sistemas multicamada por reticulação química pode ser conseguida logo durante a adsorção de cada camada ou no fim do processo. Características dos biomateriais como a morfologia, espessura, taxas de adsorção de água ou biodegradação, propriedades mecânicas e biológicas podem ser moduladas ajustando certos parâmetros de reticulação (por exemplo, agente de reticulação, concentração ou tempo de reação). Para além do mais, estudos de memória de forma destas membranas multicamada mostraram resultados promissores, considerando o seu uso para fins biomédicos. As propriedades mecânicas destes sistemas foram melhoradas combinando as ligações electroestáticas já existentes com ligações covalentes conferidas pela reticulação química, dando origem a uma rede polimérica multicamada, mas interpenetrada. Na continuação deste trabalho foi possível criar uma topografia com padrão bem organizado na superfície das membranas, alterando somente o material onde efetuamos a deposição das multicamadas. Esta estratégia visou mimetizar a topografia da ECM de diferentes tecidos, como o osso, a pele ou os nervos, criando canais alinhados na superfície do material. Usando este tipo de materiais multicamada padronizados foi possível modular funções e comportamentos celulares como o alinhamento ou a diferenciação. Em seguida, inspirados pela composição das proteínas que conferem adesividade aos mexilhões, foram produzidas membranas multicamada contendo HA modificado com dopamina (DN). A presença de DN ao longo da espessura das membranas multicamada parece ter contribuído para uma melhor e maior força de adesão, quando comparadas com as membranas controlo (membranas multicamada CHT/HA e CHT/ALG). Para além do mais, os testes in vitro resultaram em uma significante melhoria da adesão celular às membranas contendo DN. Esta estratégia mostrou ser promissora para diferentes aplicações biomédicas e de engenharia de tecidos, particularmente para a regeneração de tecido ósseo e a cicatrização de feridas da pele. Combinando diferentes estratégias e conceitos biomiméticos, foi também possível recriar um sistema complexo associado à cartilagem articular e concretamente a doenças como a osteoartrite. Assim sendo, na última parte desta tese, estas membranas multicamada com propriedades adesivas foram utilizadas como veículo para transportar células estaminais humanas do tecido adiposo (hASCs) para o local onde a cartilagem se encontra danificada. A presença deste tipo de células tem sido utilizada como tratamento para cartilagem danificada. Aqui, hASCs aderiram temporariamente às membranas multicamada, e foram assim transportadas diretamente para discos de cartilagem humana danificada, permitindo a criação de uma ponte celular entre as membranas e a superfície da cartilagem. Desta forma, estas células começaram a proliferar na superfície da cartilagem começando a migrar para os defeitos (em profundidade), segregando fatores capazes de ajudar na reparação da cartilagem. No geral, o trabalho desenvolvido para a presente tese mostra a grande versatilidade da técnica LbL, que proporciona os meios necessários para desenvolver uma gama alargada de materiais, estratégias e soluções muito necessárias e promissoras para aplicações biomédicas e de engenharia de tecidos e medicina regenerativa.Programa Doutoral em Químic

Assembly of living building blocks to engineer complex tissues

The great demand for tissue and organ grafts, compounded by an aging demographic and a shortage of available donors, has driven the development of bioengineering approaches that can generate biomimetic tissues in vitro. Despite the considerable progress in conventional scaffold‐based tissue engineering, the recreation of physiological complexity has remained a challenge. Bottom‐up tissue engineering strategies have opened up a new avenue for the modular assembly of living building blocks into customized tissue architectures. This Progress Report overviews the recent progress and trends in the fabrication and assembly of living building blocks, with a key highlight on emerging bioprinting technologies that can be used for modular assembly and complexity in tissue engineering. By summarizing the work to date, providing new classifications of different living building blocks, highlighting state‐of‐the‐art research and trends, and offering personal perspectives on future opportunities, this Progress Report aims to aid and inspire other researchers working in the field of modular tissue engineering

Scaffolding strategies for tissue engineering and regenerative medicine applications

During the past two decades, tissue engineering and the regenerative medicine field have invested in the regeneration and reconstruction of pathologically altered tissues, such as cartilage, bone, skin, heart valves, nerves and tendons, and many others. The 3D structured scaffolds and hydrogels alone or combined with bioactive molecules or genes and cells are able to guide the development of functional engineered tissues, and provide mechanical support during in vivo implantation. Naturally derived and synthetic polymers, bioresorbable inorganic materials, and respective hybrids, and decellularized tissue have been considered as scaffolding biomaterials, owing to their boosted structural, mechanical, and biological properties. A diversity of biomaterials, current treatment strategies, and emergent technologies used for 3D scaffolds and hydrogel processing, and the tissue-specific considerations for scaffolding for Tissue engineering (TE) purposes are herein highlighted and discussed in depth. The newest procedures focusing on the 3D behavior and multi-cellular interactions of native tissues for further use for in vitro model processing are also outlined. Completed and ongoing preclinical research trials for TE applications using scaffolds and hydrogels, challenges, and future prospects of research in the regenerative medicine field are also presented.This research was funded by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) (NORTE-01-0145-FEDER-000023) and by the Portuguese Foundation for Science and Technology ((M-ERA-NET/0022/2016), Transitional Rule DL 57/2016 (CTTI-57/18-I3BS(5)), and (IF/01285/2015))

Designing a Scaffold-Free Bio-Orthogonal Click Chemistry Method of Cell Assembly for Application in Tissue Engineering

Tissue engineering is a growing field of science that relies on the use of material chemistry, engineering, genetics, and cell biology to produce functional tissues for use in transplantation, drug testing and disease modelling. Presently, there is an urgent need for a technology which would enable assembly of cells into 3-dimensional multilayered tissues. Current cell-assembly technologies rely on biodegradable polymer scaffolds to assemble cells into 3D structures and to support the cell mass of the growing tissue. The presence of these materials in tissues, however, lowers the cell density and the process of scaffold biodegradation results in accumulation of monomer byproducts within the tissue. To overcome these issues we developed a scaffold free method of cell-assembly based on bio-orthogonal ligation reactions between oxyamine and ketone groups to form a stable oxime bond. The reaction is quick, specific and occurs under physiological conditions without a catalyst. To deliver the bio-orthogonal functionalities onto cell surfaces, ketone- and oxyamine- functionalized lipids were incorporated into liposomes which were subsequently fused with cell membranes. The surface engineered cells were assembled into three-dimensional tissues. Using this approach, we were able to produce functional cardiac and liver tissues with variable thicknesses and cell orientations for drug testing as well as the complex 3D co-cultures of stem cells to study stem cell differentiation. The rapid bio-orthogonal cell ligation process also enables assembly of cells into co-culture spheroids in flow, inside a microchannel. The introduction of a bi-functional oxyamine crosslinker molecule allowed for the rapid crosslinking of ketone-functionalized cells into 3D tissues. This bio-orthogonal click chemistry technology can be used with different cell types to produce customized tissues for applications in drug development and regenerative medicine