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Triple-negative breast cancer (TNBC) is the most invasive type of breast cancer with high risk of brain metastasis. To better understand interactions between breast tumors with the brain extracellular matrix (ECM), a 3D cell culture model is implemented using a thiolated hyaluronic acid (HA-SH) based hydrogel. The latter is used as HA represents a major component of brain ECM. Melt-electrowritten (MEW) scaffolds of box- and triangular-shaped polycaprolactone (PCL) micro-fibers for hydrogel reinforcement are utilized. Two different molecular weight HA-SH materials (230 and 420 kDa) are used with elastic moduli of 148 ± 34 Pa (soft) and 1274 ± 440 Pa (stiff). Both hydrogels demonstrate similar porosities. The different molecular weight of HA-SH, however, significantly changes mechanical properties, e.g., stiffness, nonlinearity, and hysteresis. The breast tumor cell line MDA-MB-231 forms mainly multicellular aggregates in both HA-SH hydrogels but sustains high viability (75%). Supplementation of HA-SH hydrogels with ECM components does not affect gene expression but improves cell viability and impacts cellular distribution and morphology. The presence of other brain cell types further support numerous cell-cell interactions with tumor cells. In summary, the present 3D cell culture model represents a novel tool establishing a disease cell culture model in a systematic way.
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Breast cancer develops in close proximity to mammary adipose tissue and interactions with the local adipose environment have been shown to drive tumor progression. The specific role, however, of this complex tumor microenvironment in cancer cell migration still needs to be elucidated. Therefore, in this study, a 3D bioprinted breast cancer model was developed that allows for a comprehensive analysis of individual tumor cell migration parameters in dependence of adjacent adipose stroma. In this co-culture model, a breast cancer compartment with MDA-MB-231 breast cancer cells embedded in collagen is surrounded by an adipose tissue compartment consisting of adipose-derived stromal cell (ASC) or adipose spheroids in a printable bioink based on thiolated hyaluronic acid. Printing parameters were optimized for adipose spheroids to ensure viability and integrity of the fragile lipid-laden cells. Preservation of the adipogenic phenotype after printing was demonstrated by quantification of lipid content, expression of adipogenic marker genes, the presence of a coherent adipo-specific extracellular matrix, and cytokine secretion. The migration of tumor cells as a function of paracrine signaling of the surrounding adipose compartment was then analyzed using live-cell imaging. The presence of ASC or adipose spheroids substantially increased key migration parameters of MDA-MB-231 cells, namely motile fraction, persistence, invasion distance, and speed. These findings shed new light on the role of adipose tissue in cancer cell migration. They highlight the potential of our 3D printed breast cancer-stroma model to elucidate mechanisms of stroma-induced cancer cell migration and to serve as a screening platform for novel anti-cancer drugs targeting cancer cell dissemination.
Assuntos
Tecido Adiposo , Bioimpressão , Neoplasias da Mama , Movimento Celular , Impressão Tridimensional , Esferoides Celulares , Células Estromais , Humanos , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Esferoides Celulares/patologia , Esferoides Celulares/metabolismo , Movimento Celular/efeitos dos fármacos , Tecido Adiposo/citologia , Feminino , Linhagem Celular Tumoral , Células Estromais/patologia , Células Estromais/metabolismo , Células Estromais/citologia , Técnicas de Cocultura , Microambiente TumoralRESUMO
Articular cartilage is crucial for joint function but its avascularity limits intrinsic repair, leading to conditions like osteoarthritis (OA). Chondromodulin-I (Cnmd) has emerged as a key molecule in cartilage biology, with potential implications for OA therapy. Cnmd is primarily expressed in cartilage and plays an important role in chondrocyte proliferation, cartilage homeostasis, and the blocking of angiogenesis. In vivo and in vitro studies on Cnmd, also suggest an involvement in bone repair and in delaying OA progression. Its downregulation correlates with OA severity, indicating its potential as a therapeutic target. Further research is needed to fully understand the mode of action of Cnmd and its beneficial implications for managing OA. This comprehensive review aims to elucidate the molecular characteristics of Cnmd, from its expression pattern, role in cartilage maintenance, callus formation during bone repair and association with OA.
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Cartilagem Articular , Peptídeos e Proteínas de Sinalização Intercelular , Osteoartrite , Animais , Humanos , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Condrócitos/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Membrana/metabolismo , Osteoartrite/metabolismo , Osteoartrite/patologia , AdultoRESUMO
Three-dimensional (3D) cell culture techniques have become a valuable tool to mimic the complex interactions of cells with each other and their surrounding extracellular matrix as they occur in vivo. In this respect, 3D spheroids are widely acknowledged as self-assembled cellular aggregates that can be generated from a variety of cell types without the need for exogenous material while being highly reproducible, easy to handle, and cost-effective. Furthermore, due to their capacity to be developed into microtissues, spheroids represent potential building blocks for various tissue engineering applications, including 3D bioprinting approaches for tissue model development. Adipose-derived stromal/stem cells (ASCs), due to their ease of isolation, multipotent nature, and secretory capacity, represent an attractive cell source employed in numerous tissue engineering studies and other cell-based therapy approaches. In this chapter, we describe two procedures for robust spheroid generation, namely the liquid overlay technique, either using agarose-coated 96-well plates or employing agarose-cast micromolds. Furthermore, we show, in principle, the generation of ASC spheroids with subsequent adipogenic differentiation and the spheroid generation using adipogenically differentiated ASCs, as well as the morphological characterization of generated spheroids.
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Adipócitos , Esferoides Celulares , Sefarose , Diferenciação Celular , Engenharia Tecidual/métodos , Tecido AdiposoRESUMO
Volumetric bioprinting (VBP) is a light-based 3D printing platform, which recently prompted a paradigm shift for additive manufacturing (AM) techniques considering its capability to enable the fabrication of complex cell-laden geometries in tens of seconds with high spatiotemporal control and pattern accuracy. A flexible allyl-modified gelatin (gelAGE)-based photoclick resin is developed in this study to fabricate matrices with exceptionally soft polymer networks (0.2-1.0 kPa). The gelAGE-based resin formulations are designed to exploit the fast thiol-ene crosslinking in combination with a four-arm thiolated polyethylene glycol (PEG4SH) in the presence of a photoinitiator. The flexibility of the gelAGE biomaterial platform allows one to tailor its concentration spanning from 2.75% to 6% and to vary the allyl to thiol ratio without hampering the photocrosslinking efficiency. The thiol-ene crosslinking enables the production of viable cell-material constructs with a high throughput in tens of seconds. The suitability of the gelAGE-based resins is demonstrated by adipogenic differentiation of adipose-derived stromal cells (ASC) after VBP and by the printing of more fragile adipocytes as a proof-of-concept. Taken together, this study introduces a soft photoclick resin which paves the way for volumetric printing applications toward soft tissue engineering.
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Bioimpressão , Engenharia Tecidual , Engenharia Tecidual/métodos , Gelatina , Bioimpressão/métodos , Hidrogéis , Impressão Tridimensional , Compostos de Sulfidrila , Alicerces TeciduaisRESUMO
The tumor microenvironment (TME) in breast cancer is determined by the complex crosstalk of cancer cells with adipose tissue-inherent cells such as adipose-derived stromal cells (ASCs) and adipocytes resulting from the local invasion of tumor cells in the mammary fat pad. This leads to heterotypic cellular contacts between these cell types. To adequately mimic the specific cell-to-cell interaction in an in vivo-like 3D environment, we developed a direct co-culture spheroid model using ASCs or differentiated adipocytes in combination with MDA-MB-231 or MCF-7 breast carcinoma cells. Co-spheroids were generated in a well-defined and reproducible manner in a high-throughput process. We compared the expression of the tumor-promoting chemokine CCL5 and its cognate receptors in these co-spheroids to indirect and direct standard 2D co-cultures. A marked up-regulation of CCL5 and in particular the receptor CCR1 with strict dependence on cell-cell contacts and culture dimensionality was evident. Furthermore, the impact of direct contacts between ASCs and tumor cells and the involvement of CCR1 in promoting tumor cell migration were demonstrated. Overall, these results show the importance of direct 3D co-culture models to better represent the complex tumor-stroma interaction in a tissue-like context. The unveiling of tumor-specific markers that are up-regulated upon direct cell-cell contact with neighboring stromal cells, as demonstrated in the 3D co-culture spheroids, may represent a promising strategy to find new targets for the diagnosis and treatment of invasive breast cancer.
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In living tissues, cells express their functions following complex signals from their surrounding microenvironment. Capturing both hierarchical architectures at the micro- and macroscale, and anisotropic cell patterning remains a major challenge in bioprinting, and a bottleneck toward creating physiologically-relevant models. Addressing this limitation, a novel technique is introduced, termed Embedded Extrusion-Volumetric Printing (EmVP), converging extrusion-bioprinting and layer-less, ultra-fast volumetric bioprinting, allowing spatially pattern multiple inks/cell types. Light-responsive microgels are developed for the first time as bioresins (µResins) for light-based volumetric bioprinting, providing a microporous environment permissive for cell homing and self-organization. Tuning the mechanical and optical properties of gelatin-based microparticles enables their use as support bath for suspended extrusion printing, in which features containing high cell densities can be easily introduced. µResins can be sculpted within seconds with tomographic light projections into centimeter-scale, granular hydrogel-based, convoluted constructs. Interstitial microvoids enhanced differentiation of multiple stem/progenitor cells (vascular, mesenchymal, neural), otherwise not possible with conventional bulk hydrogels. As proof-of-concept, EmVP is applied to create complex synthetic biology-inspired intercellular communication models, where adipocyte differentiation is regulated by optogenetic-engineered pancreatic cells. Overall, EmVP offers new avenues for producing regenerative grafts with biological functionality, and for developing engineered living systems and (metabolic) disease models.
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Bioimpressão , Microgéis , Engenharia Tecidual/métodos , Hidrogéis , Bioimpressão/métodos , Impressão Tridimensional , Alicerces TeciduaisRESUMO
PURPOSE: Hypertrophic cartilage is an important characteristic of osteoarthritis and can often be found in patients suffering from osteoarthritis. Although the exact pathomechanism remains poorly understood, hypertrophic de-differentiation of chondrocytes also poses a major challenge in the cell-based repair of hyaline cartilage using mesenchymal stromal cells (MSCs). While different members of the transforming growth factor beta (TGF-ß) family have been shown to promote chondrogenesis in MSCs, the transition into a hypertrophic phenotype remains a problem. To further examine this topic we compared the effects of the transcription growth and differentiation factor 5 (GDF-5) and the mutant R57A on in vitro chondrogenesis in MSCs. METHODS: Bone marrow-derived MSCs (BMSCs) were placed in pellet culture and in-cubated in chondrogenic differentiation medium containing R57A, GDF-5 and TGF-ß1 for 21 days. Chondrogenesis was examined histologically, immunohistochemically, through biochemical assays and by RT-qPCR regarding the expression of chondrogenic marker genes. RESULTS: Treatment of BMSCs with R57A led to a dose dependent induction of chondrogenesis in BMSCs. Biochemical assays also showed an elevated glycosaminoglycan (GAG) content and expression of chondrogenic marker genes in corresponding pellets. While treatment with R57A led to superior chondrogenic differentiation compared to treatment with the GDF-5 wild type and similar levels compared to incubation with TGF-ß1, levels of chondrogenic hypertrophy were lower after induction with R57A and the GDF-5 wild type. CONCLUSIONS: R57A is a stronger inducer of chondrogenesis in BMSCs than the GDF-5 wild type while leading to lower levels of chondrogenic hypertrophy in comparison with TGF-ß1.
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Cirurgiões , Cirurgia Plástica , Humanos , Estados Unidos , Segurança do Paciente , Obesidade , Tecido Adiposo/transplante , EstéticaRESUMO
3D neuronal cultures attempt to better replicate the in vivo environment to study neurological/neurodegenerative diseases compared to 2D models. A challenge to establish 3D neuron culture models is the low elastic modulus (30-500 Pa) of the native brain. Here, an ultra-soft matrix based on thiolated hyaluronic acid (HA-SH) reinforced with a microfiber frame is formulated and used. Hyaluronic acid represents an essential component of the brain extracellular matrix (ECM). Box-shaped frames with a microfiber spacing of 200 µm composed of 10-layers of poly(É-caprolactone) (PCL) microfibers (9.7 ± 0.2 µm) made via melt electrowriting (MEW) are used to reinforce the HA-SH matrix which has an elastic modulus of 95 Pa. The neuronal viability is low in pure HA-SH matrix, however, when astrocytes are pre-seeded below this reinforced construct, they significantly support neuronal survival, network formation quantified by neurite length, and neuronal firing shown by Ca2+ imaging. The astrocyte-seeded HA-SH matrix is able to match the neuronal viability to the level of Matrigel, a gold standard matrix for neuronal culture for over two decades. Thus, this 3D MEW frame reinforced HA-SH composite with neurons and astrocytes constitutes a reliable and reproducible system to further study brain diseases.
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Matriz Extracelular , Ácido Hialurônico , Neuritos , Neurônios , Sobrevivência CelularRESUMO
In 3D bioprinting for cartilage regeneration, bioinks that support chondrogenic development are of key importance. Growth factors covalently bound in non-printable hydrogels have been shown to effectively promote chondrogenesis. However, studies that investigate the functionality of tethered growth factors within 3D printable bioinks are still lacking. Therefore, in this study, we established a dual-stage crosslinked hyaluronic acid-based bioink that enabled covalent tethering of transforming growth factor-beta 1 (TGF-ß1). Bone marrow-derived mesenchymal stromal cells (MSCs) were cultured over three weeks in vitro, and chondrogenic differentiation of MSCs within bioink constructs with tethered TGF-ß1 was markedly enhanced, as compared to constructs with non-covalently incorporated TGF-ß1. This was substantiated with regard to early TGF-ß1 signaling, chondrogenic gene expression, qualitative and quantitative ECM deposition and distribution, and resulting construct stiffness. Furthermore, it was successfully demonstrated, in a comparative analysis of cast and printed bioinks, that covalently tethered TGF-ß1 maintained its functionality after 3D printing. Taken together, the presented ink composition enabled the generation of high-quality cartilaginous tissues without the need for continuous exogenous growth factor supply and, thus, bears great potential for future investigation towards cartilage regeneration. Furthermore, growth factor tethering within bioinks, potentially leading to superior tissue development, may also be explored for other biofabrication applications.
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Bioimpressão/métodos , Cartilagem Articular/citologia , Ácido Hialurônico/química , Células-Tronco Mesenquimais/citologia , Fator de Crescimento Transformador beta1/farmacologia , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/metabolismo , Diferenciação Celular , Células Cultivadas , Matriz Extracelular/metabolismo , Humanos , Hidrogéis , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Impressão Tridimensional , Engenharia Tecidual/métodos , Alicerces Teciduais , Fator de Crescimento Transformador beta1/químicaRESUMO
3D bioprinting often involves application of highly concentrated polymeric bioinks to enable fabrication of stable cell-hydrogel constructs, although poor cell survival, compromised stem cell differentiation, and an inhomogeneous distribution of newly produced extracellular matrix (ECM) are frequently observed. Therefore, this study presents a bioink platform using a new versatile dual-stage crosslinking approach based on thiolated hyaluronic acid (HA-SH), which not only provides stand-alone 3D printability but also facilitates effective chondrogenic differentiation of mesenchymal stromal cells. A range of HA-SH with different molecular weights is synthesized and crosslinked with acrylated (PEG-diacryl) and allylated (PEG-diallyl) polyethylene glycol in a two-step reaction scheme. The initial Michael addition is used to achieve ink printability, followed by UV-mediated thiol-ene reaction to stabilize the printed bioink for long-term cell culture. Bioinks with high molecular weight HA-SH (>200 kDa) require comparably low polymer content to facilitate bioprinting. This leads to superior quality of cartilaginous constructs which possess a coherent ECM and a strongly increased stiffness of long-term cultured constructs. The dual-stage system may serve as an example to design platforms using two independent crosslinking reactions at one functional group, which allows adjusting printability as well as material and biological properties of bioinks.
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Bioimpressão , Células-Tronco Mesenquimais , Diferenciação Celular , Ácido Hialurônico/farmacologia , Impressão Tridimensional , Engenharia Tecidual , Alicerces TeciduaisRESUMO
Hyaluronic acid (HA)-based hydrogels are very commonly applied as cell carriers for different approaches in regenerative medicine. HA itself is a well-studied biomolecule that originates from the physiological extracellular matrix (ECM) of mammalians and, due to its acidic polysaccharide structure, offers many different possibilities for suitable chemical modifications which are necessary to control, for example, network formation. Most of these chemical modifications are performed using the free acid function of the polymer and, additionally, lead to an undesirable breakdown of the biopolymer's backbone. An alternative modification of the vicinal diol of the glucuronic acid is oxidation with sodium periodate to generate dialdehydes via a ring opening mechanism that can subsequently be further modified or crosslinked via Schiff base chemistry. Since this oxidation causes a structural destruction of the polysaccharide backbone, it was our intention to study a novel synthesis protocol frequently applied to selectively oxidize the C6 hydroxyl group of saccharides. On the basis of this TEMPO/TCC oxidation, we studied an alternative hydrogel platform based on oxidized HA crosslinked using adipic acid dihydrazide as the crosslinker.
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Óxidos N-Cíclicos/química , Ácido Hialurônico/química , Hidrogéis/química , Células-Tronco Mesenquimais/citologia , Adipatos/química , Diferenciação Celular , Sobrevivência Celular , Condrogênese , Reagentes de Ligações Cruzadas/química , Módulo de Elasticidade , Humanos , Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/metabolismo , Oxirredução , Bases de Schiff/química , Ressonância de Plasmônio de SuperfícieRESUMO
Biofabrication, including printing technologies, has emerged as a powerful approach to the design of disease models, such as in cancer research. In breast cancer, adipose tissue has been acknowledged as an important part of the tumor microenvironment favoring tumor progression. Therefore, in this study, a 3D-printed breast cancer model for facilitating investigations into cancer cell-adipocyte interaction was developed. First, we focused on the printability of human adipose-derived stromal cell (ASC) spheroids in an extrusion-based bioprinting setup and the adipogenic differentiation within printed spheroids into adipose microtissues. The printing process was optimized in terms of spheroid viability and homogeneous spheroid distribution in a hyaluronic acid-based bioink. Adipogenic differentiation after printing was demonstrated by lipid accumulation, expression of adipogenic marker genes, and an adipogenic ECM profile. Subsequently, a breast cancer cell (MDA-MB-231) compartment was printed onto the adipose tissue constructs. After nine days of co-culture, we observed a cancer cell-induced reduction of the lipid content and a remodeling of the ECM within the adipose tissues, with increased fibronectin, collagen I and collagen VI expression. Together, our data demonstrate that 3D-printed breast cancer-adipose tissue models can recapitulate important aspects of the complex cell-cell and cell-matrix interplay within the tumor-stroma microenvironment.
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Tecido Adiposo/citologia , Bioimpressão , Neoplasias da Mama/patologia , Diferenciação Celular , Modelos Biológicos , Esferoides Celulares/citologia , Adipogenia , Sobrevivência Celular , Matriz Extracelular/metabolismo , Feminino , Humanos , Ácido Hialurônico/química , Hidrogéis/química , Impressão Tridimensional , Células Estromais/citologiaRESUMO
Identification of articular cartilage progenitor cells (ACPCs) has opened up new opportunities for cartilage repair. These cells may be used as alternatives for or in combination with mesenchymal stromal cells (MSCs) in cartilage engineering. However, their potential needs to be further investigated, since only a few studies have compared ACPCs and MSCs when cultured in hydrogels. Therefore, in this study, we compared chondrogenic differentiation of equine ACPCs and MSCs in agarose constructs as monocultures and as zonally layered co-cultures under both normoxic and hypoxic conditions. ACPCs and MSCs exhibited distinctly differential production of the cartilaginous extracellular matrix (ECM). For ACPC constructs, markedly higher glycosaminoglycan (GAG) contents were determined by histological and quantitative biochemical evaluation, both in normoxia and hypoxia. Differential GAG production was also reflected in layered co-culture constructs. For both cell types, similar staining for type II collagen was detected. However, distinctly weaker staining for undesired type I collagen was observed in the ACPC constructs. For ACPCs, only very low alkaline phosphatase (ALP) activity, a marker of terminal differentiation, was determined, in stark contrast to what was found for MSCs. This study underscores the potential of ACPCs as a promising cell source for cartilage engineering.
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Cartilagem Articular/citologia , Condrogênese , Células-Tronco Mesenquimais/citologia , Células-Tronco/citologia , Engenharia Tecidual , Animais , Diferenciação Celular , Células Cultivadas , CavalosRESUMO
Adipose-derived stromal/stem cells (ASCs) have been shown to exert regenerative functions, which are mainly attributed to the secretion of trophic factors. Upon transplantation, ASCs are facing an ischemic environment characterized by oxygen and nutrient deprivation. However, current knowledge on the secretion capacity of ASCs under such conditions is limited. Thus, the present study focused on the secretory function of ASCs under glucose and oxygen deprivation as major components of ischemia. After exposure to glucose/oxygen deprivation, ASCs maintained distinct viability, but the metabolic activity was greatly reduced by glucose limitation. ASCs were able to secrete a broad panel of factors under glucose/oxygen deprivation as revealed by a cytokine antibody array. Quantification of selected factors by ELISA demonstrated that glucose deprivation in combination with hypoxia led to markedly higher secretion levels of the angiogenic and anti-apoptotic factors IL-6, VEGF, and stanniocalcin-1 as compared to the hypoxic condition alone. A conditioned medium of glucose/oxygen-deprived ASCs promoted the viability and tube formation of endothelial cells, and the proliferation and migration of fibroblasts. These findings indicate that ASCs are stimulated by ischemia-like stress conditions to secrete trophic factors and would be able to exert their beneficial function in an ischemic environment.
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Tecido Adiposo/metabolismo , Isquemia/metabolismo , Células-Tronco/metabolismo , Células Estromais/metabolismo , HumanosRESUMO
Bioprinting offers the opportunity to fabricate precise 3D tumor models to study tumor pathophysiology and progression. However, the choice of the bioink used is important. In this study, cell behavior was studied in three mechanically and biologically different hydrogels (alginate, alginate dialdehyde crosslinked with gelatin (ADA-GEL), and thiol-modified hyaluronan (HA-SH crosslinked with PEGDA)) with cells from breast cancer (MDA-MB-231 and MCF-7) and melanoma (Mel Im and MV3), by analyzing survival, growth, and the amount of metabolically active, living cells via WST-8 labeling. Material characteristics were analyzed by dynamic mechanical analysis. Cell lines revealed significantly increased cell numbers in low-percentage alginate and HA-SH from day 1 to 14, while only Mel Im also revealed an increase in ADA-GEL. MCF-7 showed a preference for 1% alginate. Melanoma cells tended to proliferate better in ADA-GEL and HA-SH than mammary carcinoma cells. In 1% alginate, breast cancer cells showed equally good proliferation compared to melanoma cell lines. A smaller area was colonized in high-percentage alginate-based hydrogels. Moreover, 3% alginate was the stiffest material, and 2.5% ADA-GEL was the softest material. The other hydrogels were in the same range in between. Therefore, cellular responses were not only stiffness-dependent. With 1% alginate and HA-SH, we identified matrices that enable proliferation of all tested tumor cell lines while maintaining expected tumor heterogeneity. By adapting hydrogels, differences could be accentuated. This opens up the possibility of understanding and analyzing tumor heterogeneity by biofabrication.
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In 3D bioprinting, bioinks with high concentrations of polymeric materials are frequently used to enable fabrication of 3D cell-hydrogel constructs with sufficient stability. However, this is often associated with restricted cell bioactivity and an inhomogeneous distribution of newly produced extracellular matrix (ECM). Therefore, this study investigates bioink compositions based on hyaluronic acid (HA), an attractive material for cartilage regeneration, which allow for reduction of polymer content. Thiolated HA and allyl-modified poly(glycidol) in varying concentrations are UV-crosslinked. To adapt bioinks to poly(ε-caprolactone) (PCL)-supported 3D bioprinting, the gels are further supplemented with 1 wt% unmodified high molecular weight HA (hmHA) and chondrogenic differentiation of incorporated human mesenchymal stromal cells is assessed. Strikingly, addition of hmHA to gels with a low polymer content (3 wt%) results in distinct increase of construct quality with a homogeneous ECM distribution throughout the constructs, independent of the printing process. Improved ECM distribution in those constructs is associated with increased construct stiffness after chondrogenic differentiation, as compared to higher concentrated constructs (10 wt%), which only show pericellular matrix deposition. The study contributes to effective bioink development, demonstrating dual function of a supplement enabling PCL-supported bioprinting and at the same time improving biological properties of the resulting constructs.
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Bioimpressão , Cartilagem , Matriz Extracelular , Humanos , Ácido Hialurônico , Impressão Tridimensional , Engenharia TecidualRESUMO
When aiming at cell-based therapies in osteoarthritis (OA), proinflammatory conditions mediated by cytokines such as IL-1ß need to be considered. In recent studies, the phytoalexin resveratrol (RSV) has exhibited potent anti-inflammatory properties. However, long-term effects on 3D cartilaginous constructs under inflammatory conditions with regard to tissue quality, especially extracellular matrix (ECM) composition, have remained unexplored. Therefore, we employed long-term model cultures for cell-based therapies in an in vitro OA environment and evaluated effects of RSV. Pellet constructs made from expanded porcine articular chondrocytes were cultured with either IL-1ß (1-10 ng/ml) or RSV (50 µM) alone, or a cotreatment with both agents. Treatments were applied for 14 days, either directly after pellet formation or after a preculture period of 7 days. Culture with IL-1ß (10 ng/ml) decreased pellet size and DNA amount and severely compromised glycosaminoglycan (GAG) and collagen content. Cotreatment with RSV distinctly counteracted the proinflammatory catabolism and led to partial rescue of the ECM composition in both culture systems, with especially strong effects on GAG. Marked MMP13 expression was detected in IL-1ß-treated pellets, but none upon RSV cotreatment. Expression of collagen type I was increased upon IL-1ß treatment and still observed when adding RSV, whereas collagen type X, indicating hypertrophy, was detected exclusively in pellets treated with RSV alone. In conclusion, RSV can counteract IL-1ß-mediated degradation and distinctly improve cartilaginous ECM deposition in 3D long-term inflammatory cultures. Nevertheless, potential hypertrophic effects should be taken into account when considering RSV as cotreatment for articular cartilage repair techniques.