Tissue engineering : key elements and some trends

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Outlook in tissue-engineered magnetic systems and biomagnetic control

The advancement of tissue engineering strategies has opened up new therapeutic avenues in the regeneration of many musculoskeletal tissues and cell niches. The burst of research in nanotechnology associated with tissue engineering brings inputs for the precise control of cells and cellular environments, that can play an important role in the development of these new therapies. Magnetic actuation, especially in combination with magnetic nanoparticles, may be a valuable tool in the interaction with living systems, such as stem cell guidance, retention, stimulation, and differentiation. Advances in the field of magnetic technology have also enabled the fabrication of increasingly complex systems such as cell sheets, organoids, or bioprinted scaffolds. Our Opinion article highlights this promising field of research and attempts to cover some of the most recent contributions to both tissue engineering and regenerative medicine. The advancement of tissue engineering strategies has opened up new therapeutic avenues in the regeneration of many musculoskeletal tissues and cell niches. The burst of research in nanotechnology associated with tissue engineering brings inputs for the precise control of cells and cellular environments, that can play an important role in the development of these new therapies. Magnetic actuation, especially in combination with magnetic nanoparticles, may be a valuable tool in the interaction with living systems, such as stem cell guidance, retention, stimula- tion, and differentiation. Advances in the field of magnetic technology have also enabled the fabrication of increasingly complex systems such as cell sheets, organoids, or bioprinted scaffolds. Our Opinion article highlights this promising field of research and attempts to cover some of the most recent con- tributions to both tissue engineering and regenerative medicine.Authors acknowledge the European Research Council COG MagTendon No. 772817, the H2020 Achilles Twinning project No. 810850, and the FCT e Fundação para a Ciência e a Tecnologia under the Scientific Employment Stimulus - Individual Call (CEEC Individual) - 2020.01157. CEECIND/CP1600/CT0024

Magnetic systems for regenerative medicine

[Excerpt] Over the last decade, magnetic-based systems have made remarkable breakthroughs in the field of tissue engineering and regenerative medicine. The ability for contactless manipulation of magnetic responsive biomaterials, or even living cells, has been leveraged to devise innovative concepts that are widening the available bioengineering design space that can be explored in this multidisciplinary field. From the fabrication of cellular constructs with bioinspired patterns and hierarchical structures up to the concepts of levitational bioassembly, magnetic systems are enabling to engineer 3D tissues that better recapitulate the complex biophysical and biological cues of their native counterparts. Moreover, the inherent magnetic responsiveness of this living systems is being explored as mechanical and electrical nanotransducers to further stimulate cell functions, not only in vitro but also in vivo. Remarkably, recent advances in the convergence of microfabrication technologies with magnetic materials is also opening prospects to further fabricate advanced living microrobots and microphysiological systems with new added functionalities. Due to their good track record of biological tolerance and biodegradability, iron oxide-based nanoparticles remain the first choice of (superpara)magnetic nanomaterials, but new variants and combinations of nanomaterial are being increasingly explored in this field. Altogether, magnetic systems are contributing in multiple ways to boost the regenerative potential of bioengineered constructs and may lead to the development of in vitro tissue/organ models with improved physiological relevance. [...

A bone tissue engineering strategy based on starch scaffolds and bone marrow cells cultured in a flow perfusion bioreactor

Actualmente o transplante de tecido do próprio doente ou de um dador continua a ser a técnica mais utilizada para tratar defeitos ósseos provocados por doenças ou acidentes. No entanto, esta prática apresenta sérias limitações devido à escassez de dadores, ao risco de transmissão de doenças e/ou de rejeição imunológica e também devido ao problema da lesão dos tecidos envolventes que normalmente ocorre no local de onde é removido o tecido para transplante. O elevado número de pessoas em todo o Mundo que são afectadas por estes problemas, bem como os consequentes custos sócio-económicos, são razões acrescidas para a necessidade de desenvolver terapias alternativas para tratar a perda ou mau funcionamento de tecido ósseo. A Engenharia de Tecidos é uma área científica em contínua expansão. Os desenvolvimentos conseguidos por esta área têm contribuído significativamente para diversos avanços no campo da Medicina Regenerativa. Esta ciência interdisciplinar combina os conhecimentos de diversas outras áreas, tão distintas como a Engenharia de Materiais e a Biologia, com o objectivo de desenvolver substitutos sintéticos para tecidos humanos. Para se atingir este objectivo utilizam-se, de uma forma genérica, combinações específicas de células e de um material de suporte tridimensional com propriedades adequadas, gerando um material híbrido cujas características podem ainda ser moduladas através do sistema de cultura usado. A presente tese é centrada no desenvolvimento de estratégias de engenharia de tecido ósseo baseadas na cultura in vitro de células previamente “semeadas” num suporte tridimensional (“scaffold”). Esta estratégia permite que as células adiram ao suporte, proliferem e segreguem matriz extracelular específica do tecido ósseo, até se obter um substituto artificial funcional com características do tecido original, que pode finalmente ser transplantado para tratar o defeito em causa. Para que uma estratégia deste tipo seja bem sucedida, pelo menos três componentes fundamentais devem ser cuidadosamente estudados: o material de suporte (scaffold), as células a usar e o sistema de cultura in vitro. Daí que os principais objectivos desta tese estejam relacionados com estes três aspectos, nomeadamente: • Desenvolvimento de scaffolds biodegradáveis a partir de polímeros à base de amido de milho que induzam a adesão e proliferação celular e que apresentem propriedades adequadas, tais como a porosidade e interconectividade entre poros, de forma a proporcionar um ambiente que favoreça o desenvolvimento in vitro de um material híbrido com características similares ao osso humano. • Estudo da utilização de células da medula óssea como uma potencial fonte de células para engenharia do tecido ósseo, uma vez que estas células podem ser facilmente recolhidas do próprio paciente a tratar por métodos não-invasivos (bioppsia) e em quantidades suficientes. Além disso, tratando-se de uma fonte de células autólogas (obtidas do próprio paciente) permitem evitar os riscos de transmissão de doenças contagiosas e/ou de rejeição pelo sistema imunológico. • Estudo da influência das condições de cultura in vitro geradas por um bioreactor de perfusão (em comparação com os métodos tradicionais de cultura em condições estáticas) no desenvolvimento dos materiais híbridos, compostos pelas células e scaffolds, assim como as interacções do ambiente proporcionado por este sistema de cultura com as diferentes estruturas/arquitecturas e porosidades dos scaffolds utilizados. Estes objectivos convergem para o objectivo geral desta tese que consistiu no desenvolvimento de uma terapia de engenharia do tecido ósseo alternativa ás existentes e com potencial para vir a ser posteriormente utilizada na prática clínica. Este objectivo foi avaliado através do estudo da funcionalidade dos materiais híbridos obtidos em diferentes condições de cultura in vitro (e utilizando diferentes scaffolds), partindo do principio que o sistema de perfusão poderia eventualmente superar as limitações de difusão típicas dos sistema de cultura estática e simultaneamente proporcionar estímulos mecânicos ás células, semelhantes aos encontrados em condições fisiológicas. O trabalhou desenvolvido permitiu propor várias metodologias de processamento que conduziram à obtenção de scaffolds com propriedades e estruturas porosas muito interessantes. De um modo geral, estes scaffolds permitem a adesão, proliferação e diferenciação das células de medula óssea, quando cultivadas em condições estáticas ou no bioreactor de perfusão. Foi demonstrado que a estrutura porosa dos scaffolds e specialmente a interconectividade entre poros, afecta a homogeneidade do tecido formado. A porosidade dos scaffolds influencia o desenvolvimento sequencial das células osteoblásticas e, em combinação com as condições de cultura usadas pode influenciar a funcionalidade dos “tecidos” formados in vitro. Os resultados obtidos salientam a importância do sistema de cultura utilizado em estratégias de engenharia de tecido ósseo como a que é proposta nesta tese. De facto, o bioreactor de perfusão contribui significativamente para melhorar a funcionalidade dos materiais híbridos/tecidos desenvolvidos in vitro, uma vez que combina factores biológicos e mecânicos que proporcionam um melhor desenvolvimento das células contidas no scaffolds, conduzindo á obtenção de um tecido mineralizado semelhante ao osso humano. Os resultados obtidos demonstram que a cultura de células de medula óssea em scaffolds biodegradáveis á base de amido de milho num bioreactor de perfusão, pode também constituir um modelo para o estudo de mecanismos biológicos associados ao processo de formação de tecido ósseo, o que por sua vez pode contribuir para a avaliação e melhoria de estrategias de engenharia de tecidos do osso.There is a very significant and well-known clinical need for the establishment of alternative therapies for the treatment of bone tissue loss or failure resulting from injury or disease, as the transplantation of tissues in these patients is severely limited by donor scarcity and is highly associated to the risk of immune rejection and disease transfer. The always evolving field of tissue engineering has brought a number of significant advances to regenerative medicine. This interdisciplinary science combines the knowledge and experience of many different fields, from materials science to biology, in order to develop tissue-like substitutes. This is generally achieved through a specific interplay between cells and scaffolds (and in some cases, growth factors), which can also be modulated by the culturing system used. This thesis focuses on bone tissue engineering approaches based on culturing cells-scaffold constructs in vitro, allowing the seeded cells to proliferate and secrete tissue specific extracellular matrix (ECM) until obtaining a functional tissue-like substitute that can be transplanted to the tissue defect to be treated. To achieve the success of such tissue engineering approach, there are at least three key issues that must be carefully studied: the scaffold material, the cells and the culturing environment. Therefore, the main objectives proposed for this thesis address these three aspects: - Development of appropriate starch-based scaffolds capable of inducing the attachment and proliferation of the seeded cells, and exhibiting adequate properties, such as porosity and pore interconnectivity, in order to provide an appropriate environment for the in vitro development of bone-like tissue. - Studying the use of bone marrow cells as a reliable cell source for bone tissue engineering application, as that can be readily available (in sufficient amounts) and obtained by simple procedures (biopsy) from the same patient, avoiding the risk risks of disease transmission and/or immune rejection. - Studying the influence of in vitro culturing conditions, namely flow perfusion, on the development of cell-scaffolds constructs, as well as the interactions of the environment provided by this culturing method with different scaffolds architectures and porosities. These objectives converge to the main goal of this thesis, which is the development of an improved bone tissue engineering therapy. This was assessed by the functionality of the tissue engineering constructs obtained under different in vitro culturing conditions (and from different scaffolds), in the light of using flow perfusion bioreactor having the potential to mitigate diffusion limitations typical of static culturing and simultaneously provide physiological-like stimulus to the seeded cells. This work allowed for the development of a range of processing methodologies leading to scaffolds with different properties and porous structures, also depending on the synthetic component of the starch-based polymeric blend. In general, these starch-based scaffolds allowed for the adhesion, proliferation and differentiation of marrow stromal cells towards the osteoblastic phenotype, under static and flow perfusion conditions. It was demonstrated that scaffold architecture and especially pore interconnectivity affect the homogeneity of the formed tissue. The scaffolds porosity influences the sequential development of osteoblastic cells and in combination with the culture conditions may affect the functionality of in vitro formed tissues. The work developed also emphasized the importance of the culturing system in bone tissue engineering approaches such as the one proposed in this thesis. Flow perfusion augments the functionality of scaffold/cell constructs grown in vitro as it combines both biological and mechanical factors that enhance cell differentiation and cell organization within the construct, towards the development of bone-like mineralized tissue. Additionally, this study also shows that flow perfusion bioreactor culture of marrow stromal cells combined with the use of appropriate starch based biodegradable scaffolds may also constitute a useful model to study bone formation and assess bone tissue engineering strategies in vitro.Fundação para a Ciência e a Tecnologia – POCTI

Adipose tissue-derived stem cells and their application in bone and cartilage tissue engineering

The adipose tissue was considered a reserve of energy until the ’80s, when it was found that this tissue was involved in the metabolism of sex steroids such as estrogens. From then on, the importance attributed to this tissue radically changed as it was then considered an active organ, involved in important functions of the human body. In 2001, for the first time, the existence of stem cells within this tissue was reported, and since then, this tissue has been gaining an increased importance as a stem cell source for a wide range of potential applications in cell therapies and=or tissue engineering and regenerative medicine strategies, mainly due to its wide availability and easy access. This manuscript provides an overview on adipose stem cells (i.e., adipose tissue–derived stem cells, ASCs) considering the tissue of origin, the niche of the ASCs, and their phenotype in all aspects. In this paper it is also discussed the markers that have been used for the characterization of these cells, their differentiation properties, and their immunological reactivity, reporting studies from 2001 until this date. The ASCs are also compared with bone marrow stem cells (BMSCs), until now considered as the gold standard source of stem cells, underlining the common characteristics and the differences between the stem cells obtained from these two sources, as well as the advantages and disadvantages of their potential use in different applications. Finally, this review will also focus on the potential application of ASCs in tissue engineering applications, particularly in the regeneration of bone and cartilage, commenting on the progress of this approach and future trends of the field.T. Rada thanks the European Marie Curie EST Project (Alea Jacta Est) for the Ph. D. fellowship. The authors acknowledge the Portuguese Foundation for Science and Technology (FCT) for the partial financial support through funds from POCTI and/or FEDER programs and to the European Union funded STREP Project HIPPOCRATES (NMP3-CT-2003-505758) and the European NoE EXPER-TISSUES (NMP3-CT-2004-500283)

Current strategies for osteochondral regeneration : from stem cells to pre-clinical approaches

Damaged cartilage tissue has no functional replacement alternatives and current therapies for bone injury treatment are far from being the ideal solutions emphasizing an urgent need for alternative therapeutic approaches for osteochondral regeneration. The tissue engineering field provides new possibilities for therapeutics and regeneration in rheumatology and orthopaedics, holding the potential for improving the quality of life of millions of patients by exploring new strategies towards the development of biological substitutes to maintain, repair and improve osteochondral tissue function. Numerous studies have focused on the development of distinct tissue engineering strategies that could result in promising solutions for this delicate interface. In order to outperform currently used methods, novel tissue engineering approaches propose, for example, the design of multi-layered scaffolds, the use of stem cells, bioreactors or the combination of clinical techniques.MT Rodrigues thanks the Portuguese Foundation for Science and Technology (FCT) for providing a PhD scholarship (SFRH/BD/30745/2006)

Human tendon-derived cell sheets created by magnetic force-based tissue engineering hold tenogenic and immunomodulatory potential

Cell sheet technology and magnetic based tissue engineering hold the potential to become instrumen- tal in developing magnetically responsive living tissues analogues that can be potentially used both for modeling and therapeutical purposes. Cell sheet constructions more closely recreate physiological niches, through the preservation of contiguous cells and cell-ECM interactions, which assist the cellular guidance in regenerative processes. We herein propose to use magnetically assisted cell sheets (magCSs) constructed with human tendon- derived cells (hTDCs) and magnetic nanoparticles to study inflammation activity upon magCSs exposure to IL-1 β, anticipating its added value for tendon disease modeling. Our results show that IL-1 βinduces an inflammatory profile in magCSs, supporting its in vitro use to en- lighten inflammation mediated events in tendon cells. Moreover, the response of magCSs to IL-1 βis mod- ulated by pulsed electromagnetic field (PEMF) stimulation, favoring the expression of anti-inflammatory genes, which seems to be associated to MAPK(ERK1/2) pathway. The anti-inflammatory response to PEMF together with the immunomodulatory potential of magCSs opens new perspectives for their applicability on tendon regeneration that goes beyond advanced cell based modeling.This research was funded by the ERC CoG MagTendon (No. 772817), Fundação para a Ciência e Tecnologia (FCT) for the doctoral grant PD/BD/128089/2016 of A. Vinhas and the project MagTT PTDC/CTM-CTM/29930/2017 (POCI-01-0145-FEDER-29930), project Norte-01-0145-FEDER-02219015 supported by Norte Portugal Regional Operational Programme (NORTE 2020) and EC Twinning project Achilles (No. 810850)

Isolation of adipose stem cells (ASCs) subpopulations with distinct differentiation potential

[Excerpt] ASCs are becoming the elected cells for TE applications because ASCs have been easily isolated and have shown good differentiation potential. The aim of this work was to isolate the ASCs using immunomagnetic beads coated with different antibodies (Ab) markers and to test the differentiation potential of the different subpopulations isolated. [...]Marie Curie Actions Alea Jacta Est, Project HIPPOCRATES, NoE EXPERTISSUESinfo:eu-repo/semantics/publishedVersio

Influence of the porosity of starch-based fiber mesh scaffolds on the proliferation and osteogenic differentiation of bone marrow stromal cells cultured in a flow perfusion bioreactor

This study investigates the influence of the porosity of fiber mesh scaffolds obtained from a blend of starch and poly(!-caprolactone) on the proliferation and osteogenic differentiation of marrow stromal cells cultured under static and flow perfusion conditions. For this purpose, biodegradable scaffolds were fabricated by a fiber bonding method into mesh structures with two different porosities– 50 and 75%. These scaffolds were then seeded with marrow stromal cells harvested from Wistar rats and cultured in a flow perfusion bioreactor or in 6-well plates for up to 15 days. Scaffolds of 75% porosity demonstrated significantly enhanced cell proliferation under both static and flow perfusion culture conditions. The expression of alkaline phosphatase activity was higher in flow cultures, but only for cells cultured onto the higher porosity scaffolds. Calcium deposition patterns were similar for both scaffolds, showing a significant enhancement of calcium deposition on cellscaffold constructs cultured under flow perfusion, as compared to static cultures. Calcium deposition was higher in scaffolds of 75% porosity, but this difference was not statistically significant. Observation by scanning electron microscopy showed the formation of pore-like structures within the extracellular matrix deposited on the higher porosity scaffolds. Fourier transformed infrared spectroscopy with attenuated total reflectance and thin-film X-ray diffraction analysis of the cell-scaffold constructs after 15 days of culture in a flow perfusion bioreactor revealed the presence of a mineralized matrix similar to bone. These findings indicate that starch-based scaffolds, in conjunction with fluid flow bioreactor culture, minimize diffusion constraints and provide mechanical stimulation to the marrow stromal cells, leading to enhancement of differentiation toward development of bone-like mineralized tissue. These results also demonstrate that the scaffold structure, namely, the porosity, influences the sequential development of osteoblastic cells and, in combination