Acrylic acid plasma coated 3D Scaffolds for Cartilage tissue engineering applications

Abstract The current generation of tissue engineered additive manufactured scaffolds for cartilage repair shows high potential for growing adult cartilage tissue. This study proposes two surface modification strategies based on non-thermal plasma technology for the modification of poly(ethylene oxide terephthalate/poly(butylene terephthalate) additive manufactured scaffolds to enhance their cell-material interactions. The first, plasma activation in a helium discharge, introduced non-specific polar functionalities. In the second approach, a carboxylic acid plasma polymer coating, using acrylic acid as precursor, was deposited throughout the scaffolds. Both surface modifications were characterized by significant changes in wettability, linked to the incorporation of new oxygen-containing functional groups. Their capacity for chondrogenesis was studied using ATDC5 chondroblasts as a model cell-line. The results demonstrate that the carboxylic acid-rich plasma coating had a positive effect on the generation of the glucoaminoglycans (GAG) matrix and stimulated the migration of cells throughout the scaffold. He plasma activation stimulated the formation of GAGs but did not stimulate the migration of chondroblasts throughout the scaffolds. Both plasma treatments spurred chondrogenesis by favoring GAG deposition. This leads to the overall conclusion that acrylic acid based plasma coatings exhibit potential as a surface modification technique for cartilage tissue engineering applications

Dimensionality changes actin network through lamin A/C and zyxin

Mechanosensing proteins have mainly been investigated in 2D culture platforms, while understanding their regulation in 3D enviroments is critical for tissue engineering. Among mechanosensing proteins, the actin cytoskeleton plays a key role in human mesenchymal stromal cells (hMSCs) activity, but its regulation in 3D tissue engineered scaffolds remains poorly studied. Here, we show that human mesenchymal stromal cells (hMSCs) cultured on 3D electrospun scaffolds made of a stiff material do not form actin stress fibers, contrary to hMSCs on 2D films of the same material. On 3D electrospun and additive manufactured scaffolds, hMSCs also displayed fewer focal adhesions, lower lamin A and C expression and less YAP1 nuclear localization and myosin light chain phosphorylation. Together, this strongly suggests that dimensionality prevents the build-up of cellular tension, even on stiff materials. Knock down of either lamin A and C or zyxin resulted in fewer stress fibers in the cell center. Zyxin knock down reduced lamin A and C expression, but not vice versa, showing that this signal chain starts from the outside of the cell. Lineage commitment was not affected by the lack of these important osteogenic proteins in 3D, as all cells committed to osteogenesis in bi-potential medium. Our study demonstrates that dimensionality changes the actin cytoskeleton through lamin A and C and zyxin, and highlights the difference in the regulation of lineage commitment in 3D enviroments. Together, these results can have important implications for future scaffold design for both stiff- and soft tissue engineering constructs

Mechanosensitive regulation of stanniocalcin-1 by zyxin and actin-myosin in human mesenchymal stromal cells

Stanniocalcin-1 (STC1) secreted by mesenchymal stromal cells (MSCs) has anti-inflammatory functions, reduces apoptosis, and aids in angiogenesis, both in vitro and in vivo. However, little is known about the molecular mechanisms of its regulation. Here, we show that STC1 secretion is increased only under specific cell-stress conditions. We find that this is due to a change in actin stress fibers and actin-myosin tension. Abolishment of stress fibers by blebbistatin and knockdown of the focal adhesion protein zyxin leads to an increase in STC1 secretion. To also study this connection in 3D, where few focal adhesions and actin stress fibers are present, STC1 expression was analyzed in 3D alginate hydrogels and 3D electrospun scaffolds. Indeed, STC1 secretion was increased in these low cellular tension 3D environments. Together, our data show that STC1 does not directly respond to cell stress, but that it is regulated through mechanotransduction. This research takes a step forward in the fundamental understanding of STC1 regulation and can have implications for cell-based regenerative medicine, where cell survival, anti-inflammatory factors and angiogenesis are critical

Localization and regulation of trpv4 channels in CILIATED epithelia

La neteja del moc i dels patògens dels pulmons, i el transport de gàmets i embrions en els òrgans reproductius de les femelles són funcions clau en els epitelis ciliats, tals com aquells que es troben presents en les vies respiratòries i l'oviducte. La taxa de transport mucociliar és funció de la freqüència de batut ciliar (CBF) i aquesta freqüència és augmentada per increments en la concentració de Ca2+ intracelul·lar. El canal catiònic "transient potential vanilloid 4" (TRPV4) intervé en l'entrada de Ca2+ en resposta a estímuls mecànics i osmòtics. L'expressió del TRPV4 en l'epiteli ciliat de les vies respiratòries i de l'oviducte és confirmada mitjançant la localització per immunofluorescència del canal iònic a la membrana apical de l'epiteli ciliat i polaritzat, allà on la senyalització de Ca2+ és requerida per la regulació de la CBF. Cèl·lules ciliades de la tràquea de ratolins TRPV4-/- no expressen el canal TRPV4, no responen a l'activador específic del TRPV4, el 4α-phorbol 12,13-didecanoate (4α-PDD) i presenten respostes de Ca2+ reduïdes a temperatures mitjanes (~25ºC- 8ºC), un altre estímul dels canals TRPV4. L'activació dels canals TRPV4 per solucions altament viscoses i per hypotonicitat depèn de l'activació de la via de la fosfolipasa A2(PLA2)i la subseqüent producció de àcid epoxieicosatrienoic (EET). En condicions de baixa activació de la PLA2, estímuls mecànics i hipotònics alliberen ATP per a l'activació de la via de la fosfolipasa C (PLC)-inositol trifosfat (IP3) per contribuir a l'activació dels canals TRPV4. Descrivim que el metabòlit IP3 sense ser un agonista per ell mateix, sensibilitza el TRPV4 per a l'activació de EET, essent aquest un mecanisme general. L'acoblament funcional entre els canals TRPV4 de la membrana plasmàtica i els receptors de IP3 (IP3R) és necessari tant per iniciar com mantenir la senyalització oscil·latòria del Ca2+ desencadenada per estímuls viscosos i hipotònics. Un dels principals activadors de la CBF, la adenosina-5'-trifosfat (ATP), desencadena una resposta cel·lular mediada per Ca2+ en la que es desencadena tant l'alliberament de Ca2+ des dels dipòsits intracel·lulars com l'entrada de Ca2+. És destacable la contribució de el TRPV4 en l'augment de la CBF mediada per ATP. És més, el nostre treball implica als canals TRPV4 exclusivament en l'entrada de Ca2+ activada per receptor (ROCE). Tot plegat, aquesta tesi doctoral mostra el paper dels canals TRPV4 en l'acoblament d'estímuls fisiològics tipus mecànic, osmòtic i químic a la regulació de la CBF en l'epiteli ciliat destinat al transport mucociliar.Clearance of mucus and pathogenic agents from lungs and the transport of gametes and embryos in the female reproductive organs are key functions of ciliated epithelia such as those present in the airways and the oviduct. The rate of mucociliary transport is a function of ciliary beat frequency (CBF) and this, in turn, is increased by increases in intracellular calcium. Transient potential vanilloid 4 (TRPV4)cation channel mediates Ca2+ influx in response to mechanical and osmotic stimuli. TRPV4 expression in ciliated epithelia from airways and oviduct is confirmed by immunofluorescence localization of the channel at the apical membrane of the polarized ciliated epithelia, where the Ca2+ signalling is required for CBF regulation. Ciliated tracheal cells from TRPV4-/-mice show no TRPV4 expression, neither increases in intracellular Ca2+ and CBF in response to the TRPV4-specific activator 4α- phorbol 12,13- idecanoate (4α-PDD), and reduced responses to mild temperatures (~25ºC - 38ºC), another TRPV4-activating stimulus. TRPV4 gating by high viscous loads and hypotonicity depends on phospholipase A2 (PLA2) pathway activation and subsequent production of epoxyeicosatrienoic acid (EET). Under conditions of low PLA2 activation, mechanical and hypotonic stimuli use extracellular ATP release-mediated activation of phospholipase C (PLC)-inositol triphosphate(IP3)signalling to support TRPV4 gating. We describe that IP3, without being an agonist itself, sensitizes TRPV4 to EET activation. Besides, the functional coupling between plasma membrane TRPV4 channels and IP3 receptors (IP3R) is required to initiate and maintain the cellular oscillatory Ca2+ signal triggered by high viscous loads and hypotonic stimuli. One of the main CBF activators, adenosine-5'-triphosphate (ATP), triggers both Ca2+ release from intracellular Ca2+ stores and Ca2+ entry. Interestingly, TRPV4 contributes to ATP-induced increase in CBF. Furthermore, our work implicates TRPV4 channel exclusively in receptor-operated Ca2+ entry. Collectively, this PhD thesis shows the role of TRPV4 channels coupling physiologically relevant mechanical, hypotonic and chemical stimuli to CBF regulation in motile ciliary epithelia

3D Culture Modeling of Metastatic Breast Cancer Cells in Additive Manufactured Scaffolds

Cancer biology research is increasingly moving toward innovative in vitro 3D culture models, as conventional and current 2D cell cultures fail to resemble in vivo cancer biology. In the current study, porous 3D scaffolds, designed with two different porosities along with 2D tissue culture polystyrene (TCP) plates were used with a model breast cancer human cell line. The 3D engineered system was evaluated for the optimal seeding method (dynamic versus static), adhesion, and proliferation rate of MDA-MB-231 breast cancer cells. The expression profiles of proliferation-, stemness-, and dormancy-associated cancer markers, namely, ki67, lamin A/C, SOX2, Oct3/4, stanniocalcin 1 (STC1), and stanniocalcin 2 (STC2), were evaluated in the 3D cultured cells and compared to the respective profiles of the cells cultured in the conventional 2D TCP. Our data suggested that static seeding was the optimal seeding method with porosity-dependent efficiency. Moreover, cells cultured in 3D scaffolds displayed a more dormant phenotype in comparison to 2D, which was manifested by the lower proliferation rate, reduced ki67 expression, increased lamin A/C expression, and overexpression of STCs. The possible relationship between the cell affinity to different extracellular matrix (ECM) proteins and the RANK expression levels was also addressed after deriving collagen type I (COL-I) and fibronectin (FN) MDA-MB-231 filial cell lines with enhanced capacity to attach to the respective ECM proteins. The new derivatives exhibited a more mesenchymal like phenotype and higher RANK levels in relation to the parental cells, suggesting a relationship between ECM cell affinity and RANK expression. Therefore, the present 3D cell culture model shows that cancer cells on printed scaffolds can work as better representatives in cancer biology and drug screening related studies

3D Culture Modeling of Metastatic Breast Cancer Cells in Additive Manufactured Scaffolds

Cancer biology research is increasingly moving toward innovative in vitro 3D culture models, as conventional and current 2D cell cultures fail to resemble in vivo cancer biology. In the current study, porous 3D scaffolds, designed with two different porosities along with 2D tissue culture polystyrene (TCP) plates were used with a model breast cancer human cell line. The 3D engineered system was evaluated for the optimal seeding method (dynamic versus static), adhesion, and proliferation rate of MDA-MB-231 breast cancer cells. The expression profiles of proliferation-, stemness-, and dormancy-associated cancer markers, namely, ki67, lamin A/C, SOX2, Oct3/4, stanniocalcin 1 (STC1), and stanniocalcin 2 (STC2), were evaluated in the 3D cultured cells and compared to the respective profiles of the cells cultured in the conventional 2D TCP. Our data suggested that static seeding was the optimal seeding method with porosity-dependent efficiency. Moreover, cells cultured in 3D scaffolds displayed a more dormant phenotype in comparison to 2D, which was manifested by the lower proliferation rate, reduced ki67 expression, increased lamin A/C expression, and overexpression of STCs. The possible relationship between the cell affinity to different extracellular matrix (ECM) proteins and the RANK expression levels was also addressed after deriving collagen type I (COL-I) and fibronectin (FN) MDA-MB-231 filial cell lines with enhanced capacity to attach to the respective ECM proteins. The new derivatives exhibited a more mesenchymal like phenotype and higher RANK levels in relation to the parental cells, suggesting a relationship between ECM cell affinity and RANK expression. Therefore, the present 3D cell culture model shows that cancer cells on printed scaffolds can work as better representatives in cancer biology and drug screening related studies

Gradients in pore size enhance the osteogenic differentiation of human mesenchymal stromal cells in three-dimensional scaffolds

Small fractures in bone tissue can heal by themselves, but in case of larger defects current therapies are not completely successful due to several drawbacks. A possible strategy relies on the combination of additive manufactured polymeric scaffolds and human mesenchymal stromal cells (hMSCs). The architecture of bone tissue is characterized by a structural gradient. Long bones display a structural gradient in the radial direction, while flat bones in the axial direction. Such gradient presents a variation in bone density from the cancellous bone to the cortical bone. Therefore, scaffolds presenting a gradient in porosity could be ideal candidates to improve bone tissue regeneration. In this study, we present a construct with a discrete gradient in pore size and characterize its ability to further support the osteogenic differentiation of hMSCs. Furthermore, we studied the behaviour of hMSCs within the different compartments of the gradient scaffolds, showing a correlation between osteogenic differentiation and ECM mineralization, and pore dimensions. Alkaline phosphatase activity and calcium content increased with increasing pore dimensions. Our results indicate that designing structural porosity gradients may be an appealing strategy to support gradual osteogenic differentiation of adult stem cells

Mechanosensitive regulation of stanniocalcin-1 by zyxin and actin-myosin in human mesenchymal stromal cells

Stanniocalcin-1 (STC1) secreted by mesenchymal stromal cells (MSCs) has anti-inflammatory functions, reduces apoptosis, and aids in angiogenesis, both in vitro and in vivo. However, little is known about the molecular mechanisms of its regulation. Here, we show that STC1 secretion is increased only under specific cell-stress conditions. We find that this is due to a change in actin stress fibers and actin-myosin tension. Abolishment of stress fibers by blebbistatin and knockdown of the focal adhesion protein zyxin leads to an increase in STC1 secretion. To also study this connection in 3D, where few focal adhesions and actin stress fibers are present, STC1 expression was analyzed in 3D alginate hydrogels and 3D electrospun scaffolds. Indeed, STC1 secretion was increased in these low cellular tension 3D environments. Together, our data show that STC1 does not directly respond to cell stress, but that it is regulated through mechanotransduction. This research takes a step forward in the fundamental understanding of STC1 regulation and can have implications for cell-based regenerative medicine, where cell survival, anti-inflammatory factors and angiogenesis are critical

Probing the pH Microenvironment of Mesenchymal Stromal Cell Cultures on Additive-Manufactured Scaffolds

Despite numerous advances in the field of tissue engineering and regenerative medicine, monitoring the formation of tissue regeneration and its metabolic variations during culture is still a challenge and mostly limited to bulk volumetric assays. Here, a simple method of adding capsules based optical sensors in cell seeded 3D scaffolds is presented and the potential of these sensors to monitor the pH changes in space and time during cell growth is demonstrated. It is shown that the pH decreased over time in the 3D scaffolds, with a more prominent decrease at the edges of the scaffolds. Moreover, the pH change is higher in 3D scaffolds compared to monolayered 2D cell cultures. The results suggest that this system, composed by capsules based optical sensors and 3D scaffolds with predefined geometry and pore architecture network, can be a suitable platform for monitoring pH variations during 3D cell growth and tissue formation. This is particularly relevant for the investigation of 3D cellular microenvironment alterations occurring both during physiological processes, such as tissue regeneration, and pathological processes, such as cancer evolution.Comment: 8 pages, 5 figures. SMALL, 202

Influencing chondrogenic differentiation of human mesenchymal stromal cells in scaffolds displaying a structural gradient in pore size

Articular cartilage lesions have a limited ability to heal by themselves. Yet, golden standard treatments for cartilage repair such as drilling, microfracture and mosaicplasty provide further damage and an unstable solution that degenerates into fibrocartilage in time. Articular cartilage presents a number of gradients in cell number and size along with structural gradients in extra cellular matrix (ECM) composition. Therefore, creating scaffolds that display a structural gradient can be an appealing strategy for cartilage tissue regeneration treatments. In the present study, a scaffold with an in-built discrete gradient in pore size was produced by additive manufacturing. Human mesenchymal stromal cells (hMSCs) were seeded within the gradient scaffolds and their proliferation, differentiation and ECM deposition was evaluated with respect to 2 non-gradient scaffolds. Glycosaminoglycan (GAG) deposition was significantly higher in gradient scaffolds and non-gradient scaffolds with the smallest pore size compared to non-gradient scaffolds with the largest pore size. A gradual increase of chondrogenic markers was observed within the gradient structures with decreasing pore size, which was also accompanied by an increasingly compact ECM formation. Therefore, scaffolds displaying a structural gradient in pore size seem to be a promising strategy to aid in the process of hMSC chondrogenic differentiation and could be considered for improved cartilage tissue regeneration applications. Statement of Significance We present the development of a novel hierarchical scaffold obtained by additive manufacturing. Structural hierarchy is obtained by changing pore size within the pore network characterizing the fabricated scaffolds and proves to be a functional element in the scaffold to influence adult stem cell differentiation in the chondrogenic lineage. Specifically, in regions of the scaffolds presenting smaller pores an increasing differentiation of stem cells toward the chondrogenic differentiation is displayed. Taking inspiration from the zonal organization of articular cartilage tissue, pore size gradients could, therefore, be considered as a new and important element in designing 3D scaffolds for regenerative medicine applications, in particular for all those tissues where gradient physical properties are present