Development of tissue engineered strategies combining stem cells and scaffolds aimed to regenerate bone and osteochondral interfaces

Tese de doutoramento em Engenharia de Tecidos, Medicina Regenerativa e Células EstaminaisBone is a specialized tissue characterized by its rigidity and hardness, yet light weighed to fulfill diverse functions as mineral storage, organ protection or body support and locomotion. Despite its extraordinary healing ability, bone response may be unsuccessful to repair severe damage caused by injury or degenerative diseases. Furthermore, when bone is affected, other tissues and interfaces might be quite distressed as well. Cartilage and bone interface of the joints (osteochondral interfaces) is particularly affected by traumatic injuries and aging diseases. The challenge lies in balancing the structural, functional and biological needs of bone and cartilage in a stable milieu. As currently used therapies do not provide the ideal treatment, the development of biological substitutes through tissue engineering (TE) approaches may provide the ultimate solutions to restore, maintain, or improve bone and osteochondral tissue function. Scaffolds play an imperative role in most TE strategies, where they are expected to guide cellular distribution and colonization, similarly to the natural occurring communications between cells and tissue, and to provide mechanical support during tissue regeneration. Nevertheless, in large damaged areas, scaffolding alone might be insufficient to promote a satisfactory healing response. Culturing stem cells onto the scaffolds has demonstrated to promote the regeneration of damaged tissues. Stem cells (SCs) can be found in almost every tissue, evidencing their role in repairing injuries. Bone marrow stem cells (BMSCs) are the most studied, and promising candidates for autologous TE approaches minimizing disease transmission risks but shown to be donor age-affected and had limited self-renewal capability. These limitations directed research into other stem cells sources, such as amniotic fluid (AF), that have shown to be an almost unlimited SCs source with high proliferative and osteogenic potential. Along with the almost endless ability to expand without telomere shortening, amniotic fluid stem cells (AFSCs) share with embryonic stem cells some markers and a high self renewal capacity. In this Thesis several potential approaches were considered aiming at bone and osteochondral TE, focusing on distinct scaffold design and composition, previously or newly developed, and distinct stem cells sources. Different animal models were used to evaluate the proposed strategies with scaffolds and/or cell-scaffold constructs. As a first approach, a multilayered scaffold was developed composed of tricalcium phosphate (TCP) granules entrapped in a polycaprolactone (PCL) nanofiber mesh, inspired from the natural organic-inorganic nanostructure of bone. A synergistic effect of PCL-TCP scaffolds and mechanical stimulation was observed in the osteogenic differentiation of BMSCs cultured onto these scaffolds, resulting in the production of a mineralized ECM, even in basal medium. Composite multilayered scaffolds showed an interesting behavior under dynamic conditions using cell culturing media without osteogenic supplements. Another approach consisted in the combination of wet-spinning technology and a calcium silicate solution to produce SPCL (starch and polycaprolactone blend) wet-spun fiber meshes with functionalized silanol groups (SPCL-Si). The purpose was to developed new bioactive materials linking the properties of classical bioactive ceramics, and the processability and degradability of an organic polymer. SPCL-Si scaffolds own intrinsic properties to sustain in vitro osteogenic features, and thus holding a great potential for bone engineering approaches. Additionally, this Thesis aimed at designing a new construct for the repair of osteochondral (OC) interfaces, proposing a novel bilayered scaffold, combining the well described agarose gels for cartilage and the promising SPCL scaffolds for bone, encapsulated/seeded with amniotic stem cells (AFSCs). An OC engineered system was successfully developed, where both osteo- or chondrogenic differentiated AFSCs maintained long term viability and phenotypic expression, even in basal medium after assembling of the bilayered construct. Another major original objective of this Thesis was to explore the potential of amniotic fluid stem cells (AFSCs), as compared to bone marrow stem cells (BMSCs), for bone TE applications. Besides their source, the environmental conditions are known to influence cell response, and thus, both AFSCs and BMSCs were seeded/cultured in either 2D or 3D (using SPCL scaffolds) conditions. AFSCs and BMSCs expressed different bone-related markers at different time points. This study demonstrated that the selection of a particular stem cell type may not be a simple and direct process and relies on the target TE strategy. Finally, non-critical sized defects were induced in goat femurs so as to understand the role of the scaffold material -SPCL- and the influence of culturing autologous mesenchymal cells with/without pre-culture in osteogenic medium. Neobone formation and cellular distribution was increased in cell seeded SPCL scaffolds (pre-differentiation condition), showing the relevance of implanted cells in the bone regeneration process, and suggesting the importance of the stage of osteogenic differentiation of seeded cells. In a similar approach, femoral critical sized defects were induced in nude rats and SPCL scaffolds were implanted with or without AFSCs under different stages of osteogenic differentiation. The bridging effect between the bone segments was more prominent in scaffolds with osteogenic committed cells, and large blood vessels were observed, especially in SPCL scaffolds seeded with undifferentiated cells or osteogenic-like cells. Both in vivo studies showed the potential of SPCL scaffolds as a tissue 3D support for the regeneration of bone and underlined the importance of stem cells and stem cells stage of differentiation for achieving enhanced bone tissue regeneration. In summary, the described scaffold design and composition show a great potential to be tailored to specific applications in bone tissue regeneration strategies. Nevertheless, SPCL meshes obtained from melt spun fibers are clearly one step ahead as scaffold structures, showing to provide the necessary support for bone and osteochondral TE strategies using different sources of stem cells. Most importantly, the work described in this Thesis clearly demonstrated that bone tissue engineering requires the presence of stem cells, and that their pre-differentiation into the osteoblastic phenotype facilitates bone regeneration

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)

Internalization of magnetic iron oxide nanoparticles for stem cell functionalization

Cell monitoring and cell localization can be potentially achieved by the internalization of magnetic nanoparticles (MNPs) in the cells. This might allow for the investigation of migratory patterns through tracking studies, the targeting of particle-labelled cells to desired locations via the application of an external magnetic field and, finally, for activation stem cells to initiate desired cellular responses as inducing the differentiation process [1]. This study focus on determining the effect of magnetic stimulation in human adipose stem cells (hASCs) differentiation towards tendon cells. Firstly, the MNPs uptake by the cell and magnitude/frequency of the external magnetic field was verified. Afterwards, initial studies on understanding the endocytic mechanisms involved in the uptake of MNPs were performed. For this, cells were treated with pharmacological inhibitors before exposure to fluorescent labeled MNPs

Induction of tenogenic differentiation in human adipose stem cells by manipulating culture medium supplements

The limited ability of tendon to self-repair and the limitation of treatment regimes have hastened the motivation to develop cell-based strategies for tendon repair. Growth factors (GFs) such as EGF, FGF, PDGF and TGF-b participate in tendon formation, ECM synthesis or healing, and may assist tenogenic differentiation. Thus, this work aims to establish culturing conditions that induce tenogenic differentiation of human adipose stem cells (hASCs) using these GFs. Materials&Methods: hASCs were isolated and expanded in a-MEM basic medium. Both freshly isolated and cryopreserved hASCs (P3) were further cultured with different supplements namely basic medium with glutamine (2 mM) and ascorbic acid (0.2 mM) plus i) EGF (10 ng/ ml), ii) FGF (10 ng/ml), iii) PDGF (10 ng/ml) or iv) TGF-b (10 ng/ ml) for 7, 14, 21 or 28 days. hASCs differentiation into tenocytes was assessed by real time PCR analysis of the expression of collagen type I and type III, decorin, and scleraxis. Results: In i) and ii) media, hASCs showed a tenocyte-like aligned distribution after 14 days, while in iii) and iv) it was only observed by 21 days. Overall, hASCs showed a higher gene expression in media i) and ii)

Magnetic micellar nanovehicles: prospects of multifunctional hybrid systems for precision theranostics

Hybrid nanoarchitectures such as magnetic polymeric micelles (MPMs) are among the most promising nanotechnology-enabled materials for biomedical applications combining the benefits of polymeric micelles and magnetic nanoparticles within a single bioinstructive system. MPMs are formed by the self-assembly of polymer amphiphiles above the critical micelle concentration, generating a colloidal structure with a hydrophobic core and a hydrophilic shell incorporating magnetic particles (MNPs) in one of the segments. MPMs have been investigated most prominently as contrast agents for magnetic resonance imaging (MRI), as heat generators in hyperthermia treatments, and as magnetic-susceptible nanocarriers for the delivery and release of therapeutic agents. The versatility of MPMs constitutes a powerful route to ultrasensitive, precise, and multifunctional diagnostic and therapeutic vehicles for the treatment of a wide range of pathologies. Although MPMs have been significantly explored for MRI and cancer therapy, MPMs are multipurpose functional units, widening their applicability into less expected fields of research such as bioengineering and regenerative medicine. Herein, we aim to review published reports of the last five years about MPMs concerning their structure and fabrication methods as well as their current and foreseen expectations for advanced biomedical applications.This research was funded by European Research Council, Consolidator Grant, grant number 772817. European Union’s Horizon 2020 Research and Innovation programme, grant number 810850. Fundação para a Ciência e a Tecnologia, grant number SFRD/BD/144816/2019

Normalização de textos escritos no ensino superior

A escrita no ensino superior particulariza- se por estar associada ao uso e ao domínio de diversas normas de apresentação e de organização, conforme as Normas Brasileiras de Referência (NBRs), fornecidas pela ABNT. Conforme essa prática, objetivamos, de forma geral: Identificar, em diferentes áreas do conhecimento, o papel da ABNT e de diferentes NBRs na produção escrita do ensino superior. E, de modo específico: 1) Analisar documentos que atestem a adoção das NBRs como orientação e normalização da escrita acadêmica; 2) Descrever o que alunos de graduação revelam conhecer sobre as NBRs que orientam a produção e a organização de textos acadêmicos. Esta pesquisa insere-se no paradigma qualitativo e interpretativo de investigação (TOZZONI-REIS, 2010), na análise dos planos de curso das disciplinas que direta ou indiretamente adotam as NBRs para normalizar a apresentação dos gêneros escritos, dos cursos de graduação da Universidade Federal de Campina Grande (UFCG-PB). Enquanto resultado, os dados apontam que os planos de cursos não sinalizam de forma explícita e recorrente a prática de normalização de textos acadêmicos, o que pode impliicar em exaustivas tentativas de adequação do que se escreve ao conjunto de normas requisitadas pela própria disciplina ou instituição

Tenomodulin subpopulation of human adipose stem cells as a promising source of tendon progenitor cells

Cell based strategies envision promising insights for tendon therapies due to the naturally limited cellularity and regeneration of these tissues. Human stem cells from the stromal vascular fraction of adipose tissue (hASCs) have shown their tenogenic potential for tendon strategies[1]. However, the heterogeneous populations within the hASCs pool may hinder their use in specific applications. Tenomodulin (TNMD) has been recognized as an important marker of tendon progenitor cells[2], thus herein we hypothesized that TNMD positive (TNMD+) cells are more prone to differentiate into tendon-like cells.Funds from (FCT/MCTES) and (FSE/POCH), PD/59/2013.info:eu-repo/semantics/publishedVersio

The effect of magnetic stimulation on the osteogenic and chondrogenic differentiation of human stem cells derived from the adipose tissue (hASCs)

The use of magnetic nanoparticles (MNPs) towards the musculoskeletal tissues has been the focus of many studies, regarding MNPs ability to promote and direct cellular stimulation and orient tissue responses. This is thought to be mainly achieved by mechano-responsive pathways, which can induce changes in cell behavior, including the processes of proliferation and differentiation, in response to external mechanical stimuli. Thus, the application of MNP-based strategies in tissue engineering may hold potential to propose novel solutions for cell therapy on bone and cartilage strategies to accomplish tissue regeneration. The present work aims at studying the influence of MNPs on the osteogenic and chondrogenic differentiation of human adipose derived stem cells (hASCs). MNPs were incorporated in hASCs and cultured in medium supplemented for osteogenic and chondrogenic differentiation. Cultures were maintained up to 28 days with/without an external magnetic stimulus provided by a magnetic bioreactor, to determine if the MNPs alone could affect the osteogenic or chondrogenic phenotype of the hASCs. Results indicate that the incorporation of MNPs does not negatively affect the viability nor the proliferation of hASCs. Furthermore, Alizarin Red staining evidences an enhancement in extracellular (ECM) mineralization under the influence of an external magnetic field. Although not as evident as for osteogenic differentiation, Toluidine blue and Safranin-O stainings also suggest the presence of a cartilage-like ECM with glycosaminoglycans and proteoglycans under the magnetic stimulus provided. Thus, MNPs incorporated in hASCs under the influence of an external magnetic field have the potential to induce differentiation towards the osteogenic and chondrogenic lineages.This research was supported by Project PEst-C/SAU/LA0026/2013/; NORTE-01-0124-FEDER-000020 under ON.2SR&TD Integrated Program; 7th Framework Programme (FP7/2007-2013) under grant agreement no. REGPOT-CT2012-316331-POLARIS

Pulsed electromagnetic field modulates tendon cells response in il‐1β‐conditioned environment

Strategies aiming at controlling and modulating inflammatory cues may offer therapeutic solutions for improving tendon regeneration. This study aims to investigate the modulatory effect of pulsed electromagnetic field (PEMF) on the inflammatory profile of human tendonâ derived cells (hTDCs) after supplementation with interleukinâ 1β (ILâ 1β). ILâ 1β was used to artificially induce in-flammatory cues associated with injured tendon environments. The PEMF effect was investigated varying the frequency (5 or 17 Hz), intensity (1.5, 4, or 5 mT), and dutyâ cycle (10% or 50%) parameters to which ILâ 1βâ treated hTDCs were exposed to. A PEMF actuation with 4 mT, 5 Hz and a 50% duty cycle decreased the production of ILâ 6 and tumor necrosis factorâ α (TNFâ α), as well as the expression of TNFα, ILâ 6, ILâ 8, COXâ 2, MMPâ 1, MMPâ 2, and MMPâ 3, while ILâ 4, ILâ 10, and TIMPâ 1 expression increased. These results suggest that PEMF stimulation can modulate hTDCs response in an inflammatory environment holding therapeutic potential for tendon regenerative strategies.The authors thank Hospital da Prelada (Porto, Portugal) for providing tendon tissue and acknowledge the financial support from Fundação para a Ciência e Tecnologia (FCT) for the doctoral grant PD/BD/128089/2016 and for MagTT project PTDC/CTM‐CTM/29930/2017 (POCI‐01‐0145‐FEDER‐29930), the project NORTE‐01‐0145‐FEDER‐000021 supported by Norte Portugal Regional Operational Programme (NORTE 2020), HORIZON 2020 under the TEAMING GRANT agreement No 739572—The Discoveries CTR and MagTendon No. 772817