Routes to advance vascularized bone tissue engineering constructs

Tese de Doutoramento em BioengenhariaOne of tissue engineering (TE) challenges concerns the vascularization of engineered constructs upon implantation into the defect. In fact, for the survival of the engineered tissue beyond the oxygen diffusion limit, the formation of new blood vessels is mandatory. Therefore, this thesis aimed at designing routes towards advanced vascularized bone analogs, based on the combination of cells, biomaterials and inorganic components. The major objectives of this thesis were 1) to identify a single cell source to obtain both endothelial (ECs) and osteoblast-like cells (OBs); 2) to identify the optimal conditions in which these cells synergistically communicate; 3) to trigger the osteogenic differentiation of stem/stromal cells by inorganic osteoinducers and 4) to design 3D hydrogel systems for the controlled spatial distribution of cells. The use of adipose tissue (AT) as a cell pool for TE purposes is highly appealing, since its stromal vascular fraction (SVF) contains stem/stromal-like cells (hASCs) that can be differentiated into specific lineages, enhancing their potential use in a clinical setting. Under this context, the SSEA-4+ cellular subset of SVF (SSEA-4+hASCs) was proven to hold enhanced differentiation potential into ECs- and OBs-like cells, the most relevant cell types for bone vascularization TE routes. Using immunomagnetic selection tools, SSEA-4+hASCs were successfully separated and differentiated towards both endothelial and osteogenic lineages. Furthermore, it was found that culturing these obtained ECs and pre-OBs at an initial ratio of 75:25 in a mixture of standard endothelial and osteogenic media, cells synergistically communicate to encourage the full differentiation of pre-OBs and the maintenance of ECs phenotype. Culturing SSEA-4+hASCs in presence of sNPs in basal condition lead to the deposition of a collagen-enriched matrix relevant for bone TE. When in combination with standard osteogenic factors, sNPs were able to significantly increase the osteogenic commitment of both hMSCs and SSEA-4+hASCs. Finally, to address the tri-dimensionality of the bone, hydrogels templates, based on kappa-carrageenan (κ-CA) and chitosan (CHT), were designed to accommodate SSEA-4+hASCs-derived ECs and OBs. The CHT coated κ-CA hydrogel microfibers, arranged in such a fashion to mimic the blood vessel network, were able to support the endothelial signature of entrapped ECs. These, upon assembly within a pre-OBs loaded matrix, are appealing to be templates to attain a 3D microvascular network. By decorating κ-CA with photocrosslinkable units, hydrogels with tunable mechanical properties and high recovery rates after deformation we obtained. The controlled spatial distribution of cells was achieved by patterning the hydrogels in well-defined geometries. In summary, the research work described in this thesis addressed new strategies within the TE field that might inspire the development of improved vascularized bone-engineered constructs. The use of SSEA-4+hASCs was proven to be an endearing choice of undifferentiated cells, while their combination with sNPs and κ-CA hydrogels displayed numerous advantages. Nonetheless, the unraveling of the real potential of these cells, alone or in combination with sNPs and/or κ-CA hydrogels, towards promoting vascularized bone formation yet requires in vivo confirmation.Um dos desafios da engenharia de tecidos consiste na vascularização após a implantação do implante no defeito. De facto, para a sobrevivência do substituto do tecido é essencial a difusão de oxigénio assim como a formação de novos vasos sanguíneos. Portanto, esta tese explora novas estratégias para o desenvolvimento de análogos de osso vascularizado, com base na combinação de células, biomateriais e componentes inorgânicos. Os objetivos principais desta tese foram: 1) identificar uma única fonte celular para obter tanto as células endoteliais (ECs), como as osteoblastos (OBs); 2) identificar as condições ideais em que estas células comunicam de uma forma sinérgica; 3) desencadear a diferenciação osteogénica das células estaminais através dos osteoindutores inorgânicos e 4) projetar sistemas de hidrogéis em 3D para controlar a distribuição espacial das células. O uso do tecido adiposo como uma fonte de células é altamente atraente para engenharia de tecidos. As células estaminais derivadas do tecido adiposo (hASCs) podem ser diferenciadas em linhagens específicas, melhorando assim o seu potencial para aplicações clínicas. Neste contexto, a população SSEA-4+, identificada na fração vascular do tecido adiposo (SSEA-4+hASCs), foi a que demonstrou melhor potencial de diferenciação em células endoteliais (ECs) e osteoblastos (OBs), as células mais envolvidas na vascularização óssea. Usando ferramentas de seleção imunomagnéticas, as SSEA-4+hASCs foram separadas e diferenciadas em ambas linhagens: endotelial e osteogénica. Além disso, verificou-se que a cultura de ECs e pré-OBs numa razão inicial de 75:25, num meio de cultura misto, levou a uma comunicação celular sinérgica, incentivando a diferenciação completa das pré-OBs e a manutenção do fenótipo endotelial das ECs. A cultura das SSEA-4+hASCs na presença de nanopartículas de silica (SNPs) num meio basal, levou à deposição de uma matriz enriquecida em colagénio, essencial na regeneração óssea. Em combinação com fatores osteogénicos, as SNPs foram capazes de significativamente aumentar o compromisso osteogénico de ambas as células mesenquimais humanas e SSEA-4+hASCs. Finalmente, para resolver a tridimensionalidade do osso, modelos 3D com base em hidrogéis de kappa-carragenina (κ-CA) e quitosano (CHT), foram desenvolvidos para acomodar as ECs e OBs. Microfibras de κ-CA revestidas com CHT, dispostas de tal forma que mimetizam a rede vascular, foram capazes de manter a assinatura endotelial das ECs. Após o arranjo dentro de uma matriz enriquecida em pré-OBs, espera-se que agissem como padrões para gerir uma rede microvascular funcional. Seguinte, a decoração da κ- CA com unidades foto-reticulaveis rendeu hidrogéis com propriedades mecânicas ajustáveis e altas taxas de recuperação após a deformação. Uma distribuição controlada de células foi obtido por patterning em geometrias bem definidas. Em resumo, o trabalho de investigação descrito nesta tese propõe novas estratégias dentro da engenharia de tecidos que podem inspirar o desenvolvimento de construções de osso vascularizado. O uso das SSEA-4+hASCs provou ser uma escolha cativante de células não diferenciadas, enquanto a combinação com SNPs e hidrogéis de κ-CA exibiu várias vantagens. No entanto, o desenrolar do verdadeiro potencial destas células, individualmente ou em combinação com SNPs e/ou hidrogéis de κ-CA, no sentido de promover a formação de tecido ósseo vascularizado, ainda requer confirmação in vivo.Foundation for Science and Technology (FTC) and MIT- doctoral grant (SFRH/BD/42968/2008)

Role of extracellular matrix components and structure in new renal models in vitro

The extracellular matrix (ECM), a complex set of fibrillar proteins and proteoglycans, supports the renal parenchyma and provides biomechanical and biochemical cues critical for spatial-temporal patterning of cell development and acquisition of specialized functions. As in vitro models progress towards biomimicry, more attention is paid to reproducing ECM-mediated stimuli. ECM’s role in in vitro models of renal function and disease used to investigate kidney injury and regeneration is discussed. Availability, affordability, and lot-to-lot consistency are the main factors determining the selection of materials to recreate ECM in vitro. While simpler components can be synthesized in vitro, others must be isolated from animal or human tissues, either as single isolated components or as complex mixtures, such as Matrigel or decellularized formulations. Synthetic polymeric materials with dynamic and instructive capacities are also being explored for cell mechanical support to overcome the issues with natural products. ECM components can be used as simple 2D coatings or complex 3D scaffolds combining natural and synthetic materials. The goal is to recreate the biochemical signals provided by glycosaminoglycans and other signaling molecules, together with the stiffness, elasticity, segmentation, and dimensionality of the original kidney tissue, to support the specialized functions of glomerular, tubular, and vascular compartments. ECM mimicking also plays a central role in recent developments aiming to reproduce renal tissue in vitro or even in therapeutical strategies to regenerate renal function. Bioprinting of renal tubules, recellularization of kidney ECM scaffolds, and development of kidney organoids are examples. Future solutions will probably combine these technologies

The osteogenic differentiation of SSEA-4 sub-population of human adipose derived stem cells using silicate nanoplatelets

How to surpass invitro stem cell differentiation, reducing cell manipulation, and lead the in situ regeneration process after transplantation, remains to be unraveled in bone tissue engineering (bTE). Recently, we showed that the combination of human bone marrow stromal cells with bioactive silicate nanoplatelets (sNPs) promotes the osteogenic differentiation without the use of standard osteogenic inductors. Even more, using SSEA-4(+) cell-subpopulations (SSEA-4(+)hASCs) residing within the adipose tissue, as a single-cellular source to obtain relevant cell types for bone regeneration, was also proposed. Herein, sNPs were used to promote the osteogenic differentiation of SSEA-4(+)hASCs. The interactions between SSEA-4(+)hASCs and sNPs, namely the internalization pathway and effect on cells osteogenic differentiation, were evaluated. SNPs below 100μg/mL showed high cytocompatibility and fast internalization via clathrin-mediated pathway. SNPs triggered an overexpression of osteogenic-related markers (RUNX2, osteopontin, osteocalcin) accompanied by increased alkaline phosphatase activity and deposition of a predominantly collagen-type I matrix. Consequently, a robust matrix mineralization was achieved, covering >90% of the culturing surface area. Overall, we demonstrated the high osteogenic differentiation potential of SSEA-4(+)hASCs, further enhanced by the addition of sNPs in a dose dependent manner. This strategy endorses the combination of an adipose-derived cell-subpopulation with inorganic compounds to achieve bone matrix-analogs with clinical relevance.Authors thank the Portuguese Foundation for Science and Technology (FCT) for the personal grant SFRH/BD/42968/2008 through the MIT-Portugal Program (SMM). The research leading to these results has received funding from the MIT/ECE/0047/2009 project and the European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement n degrees REGPOT-CT2012-316331-POLARIS and MIT/ECE/0047/2009 project

Role of extracellular matrix components and structure in new renal models in vitro

The extracellular matrix (ECM), a complex set of fibrillar proteins and proteoglycans, supports the renal parenchyma and provides biomechanical and biochemical cues critical for spatial-temporal patterning of cell development and acquisition of specialized functions. As in vitro models progress towards biomimicry, more attention is paid to reproducing ECM-mediated stimuli. ECM’s role in in vitro models of renal function and disease used to investigate kidney injury and regeneration is discussed. Availability, affordability, and lot-to-lot consistency are the main factors determining the selection of materials to recreate ECM in vitro. While simpler components can be synthesized in vitro, others must be isolated from animal or human tissues, either as single isolated components or as complex mixtures, such as Matrigel or decellularized formulations. Synthetic polymeric materials with dynamic and instructive capacities are also being explored for cell mechanical support to overcome the issues with natural products. ECM components can be used as simple 2D coatings or complex 3D scaffolds combining natural and synthetic materials. The goal is to recreate the biochemical signals provided by glycosaminoglycans and other signaling molecules, together with the stiffness, elasticity, segmentation, and dimensionality of the original kidney tissue, to support the specialized functions of glomerular, tubular, and vascular compartments. ECM mimicking also plays a central role in recent developments aiming to reproduce renal tissue in vitro or even in therapeutical strategies to regenerate renal function. Bioprinting of renal tubules, recellularization of kidney ECM scaffolds, and development of kidney organoids are examples. Future solutions will probably combine these technologies

Postbiotics and Kidney Disease

Chronic kidney disease (CKD) is projected to become the fifth global cause of death by 2040 as a result of key shortcomings in the current methods available to diagnose and treat kidney diseases. In this regard, the novel holobiont concept, used to describe an individual host and its microbial community, may pave the way towards a better understanding of kidney disease pathogenesis and progression. Microbiota-modulating or -derived interventions include probiotics, prebiotics, synbiotics and postbiotics. As of 2019, the concept of postbiotics was updated by the International Scientific Association of Probiotics and Prebiotics (ISAPP) to refer to preparations of inanimate microorganisms and/or their components that confer a health benefit to the host. By explicitly excluding purified metabolites without a cellular biomass, any literature making use of such term is potentially rendered obsolete. We now review the revised concept of postbiotics concerning their potential clinical applications and research in kidney disease, by discussing in detail several formulations that are undergoing preclinical development such as GABA-salt for diet-induced hypertension and kidney injury, sonicated Lactobacillus paracasei in high fat diet-induced kidney injury, GABA-salt, lacto-GABA-salt and postbiotic-GABA-salt in acute kidney injury, and O. formigenes lysates for hyperoxaluria. Furthermore, we provide a roadmap for postbiotics research in kidney disease to expedite clinical translation

Organs-on-chip technology: a tool to tackle genetic kidney diseases

Chronic kidney disease (CKD) is a major healthcare burden that takes a toll on the quality of life of many patients. Emerging evidence indicates that a substantial proportion of these patients carry a genetic defect that contributes to their disease. Any effort to reduce the percentage of patients with a diagnosis of nephropathy heading towards kidney replacement therapies should therefore be encouraged. Besides early genetic screenings and registries, in vitro systems that mimic the complexity and pathophysiological aspects of the disease could advance the screening for targeted and personalized therapies. In this regard, the use of patient-derived cell lines, as well as the generation of disease-specific cell lines via gene editing and stem cell technologies, have significantly improved our understanding of the molecular mechanisms underlying inherited kidney diseases. Furthermore, organs-on-chip technology holds great potential as it can emulate tissue and organ functions that are not found in other, more simple, in vitro models. The personalized nature of the chips, together with physiologically relevant read-outs, provide new opportunities for patient-specific assessment, as well as personalized strategies for treatment. In this review, we summarize the major kidney-on-chip (KOC) configurations and present the most recent studies on the in vitro representation of genetic kidney diseases using KOC-driven strategies

Portable, wearable and implantable artificial kidney systems: needs, opportunities and challenges

Haemodialysis is life sustaining but expensive, provides limited removal of uraemic solutes, is associated with poor patient quality of life and has a large carbon footprint. Innovative dialysis technologies such as portable, wearable and implantable artificial kidney systems are being developed with the aim of addressing these issues and improving patient care. An important challenge for these technologies is the need for continuous regeneration of a small volume of dialysate. Dialysate recycling systems based on sorbents have great potential for such regeneration. Novel dialysis membranes composed of polymeric or inorganic materials are being developed to improve the removal of a broad range of uraemic toxins, with low levels of membrane fouling compared with currently available synthetic membranes. To achieve more complete therapy and provide important biological functions, these novel membranes could be combined with bioartificial kidneys, which consist of artificial membranes combined with kidney cells. Implementation of these systems will require robust cell sourcing; cell culture facilities annexed to dialysis centres; large-scale, low-cost production; and quality control measures. These challenges are not trivial, and global initiatives involving all relevant stakeholders, including academics, industrialists, medical professionals and patients with kidney disease, are required to achieve important technological breakthroughs

Fabrication of endothelial cell-laden carrageenan microfibers for microvascularized bone tissue engineering applications

ecent achievements in the area of tissue engineering (TE) have enabled the development of three-dimensional (3D) cell-laden hydrogels as in vitro platforms that closely mimic the 3D scenario found in native tissues. These platforms are extensively used to evaluate cellular behavior, cell-cell interactions, and tissue-like formation in highly defined settings. In this study, we propose a scalable and flexible 3D system based on microsized hydrogel fibers that might be used as building blocks for the establishment of 3D hydrogel constructs for vascularized bone TE applications. For this purpose, chitosan (CHT) coated κ-carrageenan (κ-CA) microfibers were developed using a two-step procedure involving ionotropic gelation (for the fiber formation) of κ-CA and its polyelectrolyte complexation with CHT (for the enhancement of fiber stability). The performance of the obtained fibers was assessed regarding their swelling and stability profiles, as well as their ability to carry and, subsequently, promote the outward release of microvascular-like endothelial cells (ECs), without compromising their viability and phenotype. Finally, the possibility of assembling and integrating these cell-laden fibers within a 3D hydrogel matrix containing osteoblast-like cells was evaluated. Overall, the obtained results demonstrate the suitability of the microsized κ-CA fibers to carry and deliver phenotypically apt microvascular-like ECs. Furthermore, it is shown that it is possible to assemble these cell-laden microsized fibers into 3D heterotypic hydrogels constructs. This in vitro 3D platform provides a versatile approach to investigate the interactions between multiple cell types in controlled settings, which may open up novel 3D in vitro culture techniques to better mimic the complexity of tissues.Authors thank the Portuguese Foundation for Science and Technology (FCT) for the personal grants SFRH/BD/42968/2008 through the MIT-Portugal Program (SMM) and SFRH/BD/64070/2009 (EGP). The research leading to these results has received funding from the European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement no REGPOT-CT2012-316331-POLARIS and MIT/ECE/0047/2009 project

Amphiphilic beads as depots for sustained drug release integrated into fibrillar scaffolds

Native extracellular matrix (ECM) is a complex fibrous structure loaded with bioactive cues that affects the surrounding cells. A promising strategy to mimicking native tissue architecture for tissue engineering applications is to engineer fibrous scaffolds using electrospinning. By loading appropriate bioactive cues within these fibrous scaffolds, various cellular functions such as cell adhesion, proliferation and differentiation can be regulated. Here, we report on the encapsulation and sustained release of a model hydrophobic drug (dexamethasone (Dex)) within beaded fibrillar scaffold of poly(ethylene oxide terephthalate)-poly(butylene terephthalate) (PEOT/PBT), a polyether-ester multiblock copolymer to direct differentiation of human mesenchymal stem cells (hMSCs). The amphiphilic beads act as depots for sustained drug release that is integrated into the fibrillar scaffolds. The entrapment of Dex within the beaded structure results in sustained release of the drug over the period of 28days. This is mainly attributed to the diffusion driven release of Dex from the amphiphilic electrospun scaffolds. In vitro results indicate that hMSCs cultured on Dex containing beaded fibrillar scaffolds exhibit an increase in osteogenic differentiation potential, as evidenced by increased alkaline phosphatase (ALP) activity, compared to the direct infusion of Dex in the culture medium. The formation of a mineralized matrix is also significantly enhanced due to the controlled Dex release from the fibrous scaffolds. This approach can be used to engineer scaffolds with appropriate chemical cues to direct tissue regenerationAKG, SMM, LM and AK conceived the idea and designed the experiments. AKG and SMM fabricated electrospun scaffolds and performed the structural (SEM, FTIR), mechanical, and in vitro studies. AAK and AKGperformedDex release study. AKGand AP performed thermal analysis. AKG analyzed experimental data. AKG, SMM, LMand AK wrote the manuscript. ADL and CvB provided the polymers and corrected the manuscript. AKK, AP, MG and RLR revised the paper. All authors discussed the results and commented on the manuscript. Authors would like to thank Shilpaa Mukundan, Poornima Kulkarni and Dr. Arghya Paul for help with image analysis, drug release modeling and technical discussion respectively. AKG would like to thank Prof. Robert Langer for access to equipment and acknowledge financial support from MIT Portugal Program (MPP-09Call-Langer-47). SMMthanks the Portuguese Foundation for Science and Technology (FCT) for the personal grant SFRH/BD/42968/2008 (MIT-Portugal Program). This research was funded by the office of Naval Research Young National Investigator Award (AK), the Presidential Early Career Award for Scientists and Engineers (PECASE) (AK), the NIH (EB009196; DE019024; EB007249; HL099073; AR057837), the National Science Foundation CAREER award (DMR 0847287; AK), and the Dutch Technology Foundation (STW # 11135; LM, CvB, and AD)

THE ECONOMIC CRISIS AND THE EVOLUTION OF THE ECONOMIC AND FINANCIAL ACTIVITY OF THE SME

The main effects of the economic crisis on the SMEs are a more difficult financing (the low accessibility of credits "smothers" viable businesses); lower exports; lower domestic demand; financial blockage created by the state, on the one hand, by the duties towards the private sector, and by other companies experiencing financial difficulties, on the other hand; decrease of investments; the psychological effect ("crisis syndrome" which acts at the psychological level by inhibiting the investments run by SMEs). The crisis must not be seen as a catastrophe, rather as a broad movement of global change, whose effects are not just economic, but also social and political. It is increasingly obvious that this is more than just an economic crisis: it is the signal for a major change in the system.SMEs, economic crisis, economic activity, obstacles to then SMEs