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The surface properties of biomaterials interact directly with biological systems, influencing cellular responses, tissue integration, and biocompatibility. Surface topography plays a critical role in cardiac tissue engineering by affecting electrical conductivity, cardiomyocyte alignment, and contractile function. Current methods for controlling surface properties and topography in cardiac tissue engineering scaffolds are limited, expensive, and lack precision. This study introduces a low-cost, one-step degradation process to create scaffolds with well-defined micro-grooves from multilayered 3D printed poly(lactic acid)/thermoplastic polyurethane composites. The approach provides control over erosion rate and surface morphology, allowing easy tuning of scaffold topographical cues for tissue engineering applications. The findings reported in this study provide a library of easily tuneable scaffold topographical cues. A strong dependence of neonatal rat cardiomyocyte (NRCM) contact guidance with the multilayers' dimension and shape in partially degraded polylactic acid (PLA)/thermoplastic polyurethane (TPU) samples is observed. NRCMs cultured on samples with a layer thickness of 13 ± 2 µm and depth of 4.7 ± 0.2 µm demonstrate the most regular contractions. Hence, the proposed fabrication scheme can be used to produce a new generation of biomaterials with excellent controllability determined by multilayer thickness, printing parameters, and degradation treatment duration.
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Long-term stability and function are key challenges for optical nanosensors operating in complex biological environments. While much focus is rightly placed on issues related to specificity, sensitivity, reversibility, and response time, many nanosensors are not capable of transducing accurate results over prolonged time periods. Sensors could fail over time due to the degradation of scaffold material, degradation of signaling dyes and components, or a combination of both. It is critical to investigate how such degradative processes affect sensor output, as the consequences could be severe. Herein, we used fluorescent core-shell organosilica pH nanosensors as a model system, incubating them in a range of common aqueous solutions over time at different temperatures, and then searched for changes in fluorescence signal, particle size, and evidence of silica degradation. We found that these ratiometric nanosensors produced stable optical signals after aging for 30 days at 37 °C in standard saline buffers with and without 10% fetal bovine serum, and without any evidence of material degradation. Next, we evaluated their performance as real-time pH nanosensors in bacterial suspension cultures, observing a close agreement with a pH electrode for control nanosensors, yet observing obvious deviations in signal based on the aging conditions. The results show that while the organosilica scaffold does not degrade appreciably over time, careful selection of dyes and further systematic investigations into the effects of salt and protein levels are required to realize long-term stable nanosensors.
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Microgels are emerging as an outstanding platform for tissue regeneration because they overcome issues associated with conventional bulk/macroscopic hydrogels such as limited cell-cell contact and cell communication and low diffusion rates. Owing to the enhanced mass transfer and injectability via a minimally invasive procedure, these microgels are becoming a promising approach for bone regeneration applications. Nevertheless, there still remains a huge gap between the understanding of how the hydrogel matrix composition can influence cell response and overall tissue formation when switching from bulk formats to microgel format, which is often neglected or rarely studied. Here, we fabricated polyethylene glycol-based microgels and bulk hydrogels incorporating gelatin and hyaluronic acid (HA), either individually or together, and assessed the impact of both hydrogel composition and format upon the osteogenic differentiation of encapsulated human bone marrow-derived mesenchymal stem cells (hBMSCs). Osteogenesis was significantly greater in microgels than bulk hydrogels for both gelatin alone (Gel) and gelatin HA composite (Gel:HA) hydrogels, as determined by the expression of Runt-related transcription factor (Runx2) and alkaline phosphatase (ALP) genes and mineral deposition. Interestingly, Gel and Gel:HA hydrogels behaved differently between bulk and microgel format. In bulk format, overall osteogenic outcomes were better in Gel:HA hydrogels, but in microgel format, while the level of osteogenic gene expression was equivalent between both compositions, the degree of mineralization was reduced in Gel:HA microgels. Investigation into the affinity of hydroxyapatite for the different matrix compositions indicated that the decreased mineralization of Gel:HA microgels was likely due to a low affinity of hydroxyapatite to bind to HA and support mineral deposition, which has a greater impact on microgels than bulk hydrogels. Together, these findings suggest that both hydrogel composition and format can determine the success of tissue formation and that there is a complex interplay of these two factors on both cell behavior and matrix deposition. This has important implications for tissue engineering, showing that hydrogel composition and geometry must be evaluated together when optimizing conditions for cell differentiation and tissue formation.
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Células-Tronco Mesenquimais , Osteogênese , Diferenciação Celular , Humanos , Hidrogéis , Engenharia TecidualRESUMO
Mesenchymal stem cells (MSCs) have attracted great interest in recent years for tissue engineering and regenerative medicine applications due to their ease of isolation and multipotent differentiation capacity. In the past, MSC research has focussed on the effects of soluble cues, such as growth factors and cytokines; however, there is now increasing interest in understanding how parameters such as substrate modulus, specific extracellular matrix (ECM) components and the ways in which these are presented to the cell can influence MSC properties. Here we use surfaces of self-assembled maleimide-functionalized polystyrene-block-poly(ethylene oxide) copolymers (PS-PEO-Ma) to investigate how the spatial arrangement of cell adhesion ligands affects MSC behaviour. By changing the ratio of PS-PEO-Ma in mixtures of block copolymer and polystyrene homopolymer, we can create surfaces with lateral spacing of the PEO-Ma domains ranging from 34 to 62 nm. Through subsequent binding of cysteine-GRGDS peptides to the maleimide-terminated end of the PEO chains in each of these domains, we are able to present tailored surfaces of controlled lateral spacing of RGD (arginine-glycine-aspartic acid) peptides to MSCs. We demonstrate that adhesion of MSCs to the RGD-functionalized block-copolymer surfaces is through specific attachment to the presented RGD motif and that this is mediated by α5, αV, ß1 and ß3 integrins. We show that as the lateral spacing of the peptides is increased, the ability of the MSCs to spread is diminished and that the morphology changes from well-spread cells with normal fibroblastic morphology and defined stress-fibres, to less-spread cells with numerous cell protrusions and few stress fibres. In addition, the ability of MSCs to form mature focal adhesions is reduced on substrates with increased lateral spacing. Finally, we investigate differentiation and use qRT-PCR determination of gene expression levels and a quantitative alkaline phosphatase assay to show that MSC osteogenesis is reduced on surfaces with increased lateral spacing while adipogenic differentiation is increased. We show here, for the first time, that the lateral spacing of adhesion peptides affects human MSC (hMSC) properties and might therefore be a useful parameter with which to modify hMSC behaviour in future tissue engineering strategies.
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Células-Tronco Mesenquimais/citologia , Oligopeptídeos/metabolismo , Adipogenia , Adesão Celular , Movimento Celular , Células Cultivadas , Citoesqueleto/ultraestrutura , Adesões Focais , Humanos , Cadeias alfa de Integrinas/metabolismo , Cadeias beta de Integrinas/metabolismo , Células-Tronco Mesenquimais/fisiologia , Células-Tronco Mesenquimais/ultraestrutura , Osteogênese , Polietilenoglicóis/química , Poliestirenos/químicaRESUMO
To encourage cell adhesion on biomaterial surfaces in a more facile, safe, and low-cost fashion, we have demonstrated a noncovalent approach to spatially conjugate ß-cyclodextrin (ß-CD) modified peptide sequences onto self-assembled adamantane-terminated polystyrene-b-poly(ethylene oxide) (PS-PEO-Ada) films through inclusion complexing interactions between ß-CDs and adamantane. By simply blending various ratios of unmodified PS-PEO with a newly synthesized PS-PEO-Ada, we produced PS polymer films that displayed well-organized adamantine-decorated cylindrical PEO domains with varying average interdomain spacings ranging from 29 to 47 nm. The presence of the adamantane moiety at the terminal end of the PEO chain permitted rapid, and importantly, oriented attachment of ß-CD functionalized peptides onto these surfaces. This one-step process not only converted these proven nonadherent PS-PEO surfaces into adherent surfaces, but also permitted precisely controlled presentation and surface distribution of the conjugated peptides. The utility of these surfaces as cell culture substrates was confirmed with human mesenchymal stem cells (hMSCs). We observed that with increasing PS-PEO-Ada content in the PEO cylindrical domains, these novel polymer films displayed improved cell attachment and spreading, with notable differences in hMSC morphology. We further confirmed that this novel PS-PEO-Ada surface provides a flexible platform for facile conjugation of mixtures of ß-CDs functionalized with different peptides, specifically RGD and IKVAV peptides. The cell adhesion and spreading assays on these surfaces indicated that the morphologies of hMSCs can be easily manipulated, while no significant changes in cell attachment were observed. The lock-and-key peptide conjugation technique presented in this work is applicable to any substrate that incorporates a moiety capable of forming inclusion complexes with α-, ß-, and γ-CDs, providing a facile and flexible method by which to construct peptide-conjugated biomaterial substrates for a multitude of applications in fields ranging from cell bioprocessing and regenerative medicine to cell-based assays.
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Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/fisiologia , Células-Tronco Mesenquimais/química , Células-Tronco Mesenquimais/fisiologia , Adesão Celular/fisiologia , Células Cultivadas , Humanos , Propriedades de SuperfícieRESUMO
Strategies incorporating mesenchymal stromal cells (MSC), hydrogels and osteoinductive signals offer promise for bone repair. Osteoinductive signals such as growth factors face challenges in clinical translation due to their high cost, low stability and immunogenicity leading to interest in microRNAs as a simple, inexpensive and powerful alternative. The selection of appropriate miRNA candidates and their efficient delivery must be optimised to make this a reality. This study evaluated pro-osteogenic miRNAs and used porous silicon nanoparticles modified with polyamidoamine dendrimers (PAMAM-pSiNP) to deliver these to MSC encapsulated within gelatin-PEG hydrogels. miR-29b-3p, miR-101-3p and miR-125b-5p are strongly pro-osteogenic and are shown to target FASN and ELOVL4 in the fatty acid biosynthesis pathway to modulate MSC osteogenesis. Hydrogel delivery of miRNA:PAMAM-pSiNP complexes enhanced transfection compared to 2D. The osteogenic potential of hBMSC in hydrogels with miR125b:PAMAM-pSiNP complexes is evaluated. Importantly, a dual-effect on osteogenesis occurred, with miRNAs increasing expression of alkaline phosphatase (ALP) and Runt-related transcription factor 2 (RUNX2) whilst the pSiNPs enhanced mineralisation, likely via degradation into silicic acid. Overall, this work presents insights into the role of miRNAs and fatty acid signalling in osteogenesis, providing future targets to improve bone formation and a promising system to enhance bone tissue engineering.
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Ácidos Graxos , Hidrogéis , Células-Tronco Mesenquimais , MicroRNAs , Nanopartículas , Osteogênese , Silício , MicroRNAs/genética , MicroRNAs/metabolismo , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Silício/química , Hidrogéis/química , Humanos , Nanopartículas/química , Porosidade , Ácidos Graxos/química , Dendrímeros/química , Ácido Graxo Sintase Tipo I/metabolismo , Ácido Graxo Sintase Tipo I/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
Oxygen plays a central role in aerobic metabolism, and while many approaches have been developed to measure oxygen concentration in biological environments over time, monitoring spatiotemporal changes in dissolved oxygen levels remains challenging. To address this, we developed a ratiometric core-shell organosilica nanosensor for continuous, real-time optical monitoring of oxygen levels in biological environments. The nanosensors demonstrate good steady state characteristics (KpSV = 0.40 L/mg, R2 = 0.95) and respond reversibly to changes in oxygen concentration in buffered solutions and report similar oxygen level changes in response to bacterial cell growth (Escherichia coli) in comparison to a commercial bulk optode-based sensing film. We further demonstrated that the oxygen nanosensors could be distributed within a growing culture of E. coli and used to record oxygen levels over time and in different locations within a static culture, opening the possibility of spatiotemporal monitoring in complex biological systems.
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Escherichia coli , Oxigênio , Oxigênio/metabolismo , Oxigênio/análise , Escherichia coli/metabolismo , Escherichia coli/isolamento & purificação , Técnicas Biossensoriais/métodos , Nanotecnologia , Compostos de Organossilício/químicaRESUMO
Idiopathic pulmonary fibrosis (IPF) is characterised by lung scarring and stiffening, for which there is no effective cure. Based on the immunomodulatory and anti-fibrotic effects of induced pluripotent stem cell (iPSC) and mesenchymoangioblast-derived mesenchymal stem cells (iPSCs-MSCs), this study evaluated the therapeutic effects of iPSCs-MSCs in a bleomycin (BLM)-induced model of pulmonary fibrosis. Adult male C57BL/6 mice received a double administration of BLM (0.15â¯mg/day) 7-days apart and were then maintained for a further 28-days (until day-35), whilst control mice were administered saline 7-days apart and maintained for the same time-period. Sub-groups of BLM-injured mice were intravenously-injected with 1×106 iPSC-MSCs on day-21 alone or on day-21 and day-28 and left until day-35 post-injury. Measures of lung inflammation, fibrosis and compliance were then evaluated. BLM-injured mice presented with lung inflammation characterised by increased immune cell infiltration and increased pro-inflammatory cytokine expression, epithelial damage, lung transforming growth factor (TGF)-ß1 activity, myofibroblast differentiation, interstitial collagen fibre deposition and topology (fibrosis), in conjunction with reduced matrix metalloproteinase (MMP)-to-tissue inhibitor of metalloproteinase (TIMP) ratios and dynamic lung compliance. All these measures were ameliorated by a single or once-weekly intravenous-administration of iPSC-MSCs, with the latter reducing dendritic cell infiltration and lung epithelial damage, whilst promoting anti-inflammatory interleukin (IL)-10 levels to a greater extent. Proteomic profiling of the conditioned media of cultured iPSC-MSCs that were stimulated with TNF-α and IFN-γ, revealed that these stem cells secreted protein levels of immunosuppressive factors that contributed to the anti-fibrotic and therapeutic potential of iPSCs-MSCs as a novel treatment option for IPF.
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Bleomicina , Células-Tronco Pluripotentes Induzidas , Pulmão , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Camundongos Endogâmicos C57BL , Fibrose Pulmonar , Animais , Masculino , Células-Tronco Mesenquimais/metabolismo , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/patologia , Fibrose Pulmonar/terapia , Pulmão/patologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Transplante de Células-Tronco Mesenquimais/métodos , Camundongos , Diferenciação Celular/efeitos dos fármacos , Citocinas/metabolismo , Modelos Animais de Doenças , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Background: High post-surgical failure rates following tendon injury generate high medical costs and poor patient recovery. Cell-based tendon tissue engineering has the potential to produce fully functional replacement tissue and provide new strategies to restore tendon function and healing. In this endeavour, the application of mesenchymal stromal cells (MSCs) encapsulated in biomaterial scaffolds has shown great promise. However, a consensus on optimal promotion of tenogenic differentiation of MSCs has yet to be reached, although growth factors and mechanical cues are generally acknowledged as important factors. Methods: In this study, we prepared a hydrogel cell culture system consisting of methacrylated poly(d,l-lactic acid-ethylene glycol-d,l-lactic acid) (P(LA-EG-LA)) and gelatin methacrylate (GelMA) to encapsulate human bone marrow-derived MSCs (hBMSCs). We further systematically investigated the influence of static and intermittent cyclic uniaxial strain mechanical stimulation, in combination with transforming growth factor-ß3 (TGF-ß3) supplementation, on tenogenic differentiation of hBMSCs. Results: Increased TGF-ß3 concentration upregulated the tenogenic genes Scleraxis (SCX) and collagen type I (COL1A1) but showed no effects on tenascin-c (TNC) and collagen type III (COL3A1) expression. Mechanical stimulation had no observable effect on gene expression, but intermittent cyclic uniaxial strain stimulation improved matrix deposition. Together, these data provide new insights into how TGF-ß3 and mechanical stimulation regulate MSC tenogenesis, with TGF-ß3 promoting the expression of key tenogenic genes whilst mechanical stimulation aided matrix deposition in the engineered tissue. Furthermore, intermittent cyclic uniaxial strain at 3% elongation and 0.33 âHz for 1 âh/day showed improved matrix effects compared to static strain. Conclusion: Together, the most promising result for tenogenic differentiation of hBMSCs was identified as treatment with 5 âng/ml TGF-ß3 under intermittent cyclic uniaxial strain (3% strain; 0.33 âHz; 1 âh/day). This knowledge is of importance for the development of an improved protocol for tenogenic differentiation of MSCs and thereby for tendon tissue engineering. The translational potential of this article: Tissue-engineered strategies for tendon repair require a consensus on the differentiation of mesenchymal stromal cells to tenocytes, which is currently lacking. This article provides a systematic investigation of two main tenogenic differentiation conditions to further development of a tenogenic differentiation protocol.
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Microgels are microscale particles of hydrogel that can be laden with cells and used to create macroporous tissue constructs. Their ability to support cell-ECM and cell-cell interactions, along with the high levels of nutrient and metabolite exchange facilitated by their high surface area-to-volume ratio, means that they are attracting increasing attention for a variety of tissue regeneration applications. Here, we present methods for fabricating and modifying the structure of microfluidic devices using commonly available laboratory consumables including pipet tips and PTFE and silicon tubing to produce microgels. Different microfluidic devices realized the controlled generation of a wide size range (130-800 µm) of microgels for cell encapsulation. Subsequently, we describe the process of encapsulating mesenchymal stromal cells in microgels formed by photo-cross-linking of gelatin-norbornene and PEG dithiol. The introduced pipet-based chip offers simplicity, tunability, and versatility, making it easily assembled in most laboratories to effectively produce cell-laden microgels for various applications in tissue engineering.
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Microgéis , Encapsulamento de Células , Gelatina/química , Engenharia Tecidual/métodos , Hidrogéis/químicaRESUMO
Mesenchymal stromal cells (MSCs) have significant therapeutic potential due to their ability to differentiate into musculoskeletal lineages suitable for tissue-engineering, as well as the immunomodulatory and pro-regenerative effects of the paracrine factors that these cells secrete. Cues from the extracellular environment, including physical stimuli such as substrate stiffness, are strong drivers of MSC differentiation, but their effects upon MSC paracrine activity are not well understood. This study, therefore sought to determine the impact of substrate stiffness on the paracrine activity of MSCs, analysing both effects on MSC fate and their effect on T-cell and macrophage activity and angiogenesis. The data show that conditioned medium (CM) from MSCs cultured on 0.2 kPa (soft) and 100 kPa (stiff) polyacrylamide hydrogels have differing effects on MSC proliferation and differentiation, with stiff CM promoting proliferation whilst soft CM promoted differentiation. There were also differences in the effects upon macrophage phagocytosis and angiogenesis, with the most beneficial effects from soft CM. Analysis of the media composition identified differences in the levels of proteins including IL-6, OPG, and TIMP-2. Using recombinant proteins and blocking antibodies, we confirmed a role for OPG in modulating MSC proliferation with a complex combination of factors involved in the regulation of MSC differentiation. Together the data confirm that the physical microenvironment has an important influence on the MSC secretome and that this can alter the differentiation and regenerative potential of the cells. These findings can be used to tailor the culture environment for manufacturing potent MSCs for specific clinical applications or to inform the design of biomaterials that enable the retention of MSC activity after delivery into the body. STATEMENT OF SIGNIFICANCE: ⢠MSCs cultured on 100 kPa matrices produce a secretome that boosts MSC proliferation ⢠MSCs cultured on 0.2 kPa matrices produce a secretome that promotes MSC osteogenesis and adipogenesis, as well as angiogenesis and macrophage phagocytosis ⢠IL-6 secretion is elevated in MSCs on 0.2 kPa substrates ⢠OPG, TIMP-2, MCP-1, and sTNFR1 secretion are elevated in MSCs on 100 kPa substrates.
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Transplante de Células-Tronco Mesenquimais , Inibidor Tecidual de Metaloproteinase-2 , Inibidor Tecidual de Metaloproteinase-2/farmacologia , Interleucina-6 , Diferenciação Celular , Engenharia Tecidual , Meios de Cultivo Condicionados/farmacologiaRESUMO
Tendons are integral to our daily lives by allowing movement and locomotion but are frequently injured, leading to patient discomfort and impaired mobility. Current clinical procedures are unable to fully restore the native structure of the tendon, resulting in loss of full functionality, and the weakened tissue following repair often re-ruptures. Tendon tissue engineering, involving the combination of cells with biomaterial scaffolds to form new tendon tissue, holds promise to improve patient outcomes. A key requirement for efficacy in promoting tendon tissue formation is the optimal differentiation of the starting cell populations, most commonly adult tissue-derived mesenchymal stem/stromal cells (MSCs), into tenocytes, the predominant cellular component of tendon tissue. Currently, a lack of consensus on the protocols for effective tenogenic differentiation is hampering progress in tendon tissue engineering. In this review, we discuss the current state of knowledge regarding human stem cell differentiation towards tenocytes and tendon tissue formation. Tendon development and healing mechanisms are described, followed by a comprehensive overview of the current protocols for tenogenic differentiation, including the effects of biochemical and biophysical cues, and their combination, on tenogenesis. Lastly, a synthesis of the key features of these protocols is used to design future approaches. The holistic evaluation of current knowledge should facilitate and expedite the development of efficacious stem cell tenogenic differentiation protocols with future impact in tendon tissue engineering. STATEMENT OF SIGNIFICANCE: The lack of a widely-adopted tenogenic differentiation protocol has been a major hurdle in the tendon tissue engineering field. Building on current knowledge on tendon development and tendon healing, this review surveys peer-reviewed protocols to present a holistic evaluation and propose a pathway to facilitate and expedite the development of a consensus protocol for stem cell tenogenic differentiation and tendon tissue engineering.
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Células-Tronco Mesenquimais/citologia , Traumatismos dos Tendões/terapia , Tendões/fisiologia , Engenharia Tecidual , Adulto , Diferenciação Celular , Humanos , Células-Tronco Mesenquimais/metabolismo , Células-Tronco , Traumatismos dos Tendões/patologia , Tendões/citologia , Engenharia Tecidual/métodos , Engenharia Tecidual/tendênciasRESUMO
The ability to create complex three-dimensional cellular models that can effectively replicate the structure and function of human organs and tissues in vitro has the potential to revolutionize medicine. Such models could facilitate the interrogation of developmental and disease processes underpinning fundamental discovery science, vastly accelerate drug development and screening, or even be used to create tissues for implantation into the body. Realization of this potential, however, requires the recreation of complex biochemical, biophysical, and cellular patterns of 3D tissues and remains a key challenge in the field. Recent advances are being driven by improved knowledge of tissue morphogenesis and architecture and technological developments in bioengineering and materials science that can create the multidimensional and dynamic systems required to produce complex tissue microenvironments. In this article, we discuss challenges for in vitro models of tissues and organs and summarize the current state-of-the art in biomaterials and bioengineered systems that aim to address these challenges. This includes both top-down technologies, such as 3D photopatterning, magnetism, acoustic forces, and cell origami, as well as bottom-up patterning using 3D bioprinting, microfluidics, cell sheet technology, or composite scaffolds. We illustrate the varying ways that these can be applied to suit the needs of different tissues and applications by focussing on specific examples of patterning the bone-tendon interface, kidney organoids, and brain cancer models. Finally, we discuss the challenges and future prospects in applying materials science and bioengineering to develop high-quality 3D tissue structures for in vitro studies.
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Materiais Biocompatíveis , Bioimpressão , Humanos , Materiais Biocompatíveis/farmacologia , Materiais Biocompatíveis/química , Organoides , Impressão Tridimensional , Engenharia Tecidual/métodos , Células-TroncoRESUMO
Cells sense and respond to a variety of physical cues from their surrounding microenvironment, and these are interpreted through mechanotransductive processes to inform their behavior. These mechanisms have particular relevance to stem cells, where control of stem cell proliferation, potency, and differentiation is key to their successful application in regenerative medicine. It is increasingly recognized that surface micro- and nanotopographies influence stem cell behavior and may represent a powerful tool with which to direct the morphology and fate of stem cells. Current progress toward this goal has been driven by combined advances in fabrication technologies and cell biology. Here, the capacity to generate precisely defined micro- and nanoscale topographies has facilitated the studies that provide knowledge of the mechanotransducive processes that govern the cellular response as well as knowledge of the specific features that can drive cells toward a defined differentiation outcome. However, the path forward is not fully defined, and the "bumpy road" that lays ahead must be crossed before the full potential of these approaches can be fully exploited. This review focuses on the challenges and opportunities in applying micro- and nanotopographies to dictate stem cell fate for regenerative medicine. Here, key techniques used to produce topographic features are reviewed, such as photolithography, block copolymer lithography, electron beam lithography, nanoimprint lithography, soft lithography, scanning probe lithography, colloidal lithography, electrospinning, and surface roughening, alongside their advantages and disadvantages. The biological impacts of surface topographies are then discussed, including the current understanding of the mechanotransductive mechanisms by which these cues are interpreted by the cells, as well as the specific effects of surface topographies on cell differentiation and fate. Finally, considerations in translating these technologies and their future prospects are evaluated.
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Therapeutic benefits of mesenchymal stem cells (MSCs) are now widely believed to come from their paracrine signalling, i.e. secreted factors such as cytokines, chemokines, and extracellular vesicles (EVs). Cell-free therapy using EVs is an active and emerging field in regenerative medicine. Typical 2D cultures on tissue culture plastic is far removed from the physiological environment of MSCs. The application of 3D cell culture allows MSCs to adapt to their cellular environment which, in turn, influences their paracrine signalling activity. In this study we evaluated the impact of 3D MSCs culture on EVs secretion, cargo proteome composition, and functional assessment in immunomodulatory, anti-inflammatory and anti-fibrotic properties. MSC-EVs from 2D and 3D cultures expressed classical EV markers CD81, CD63, and CD9 with particle diameter of <100 nm. There were distinct changes in immunomodulatory potencies where 3D cultures exhibited reduced indoleamine 2,3-dioxygenase (IDO) activity and significantly reduced macrophage phagocytosis. Administration of 2D and 3D EVs following double dose bleomycin challenge in aged mice showed a marked increase of bodyweight loss in 3D group throughout days 7-28. Histopathological observations of lung tissues in 3D group showed increased collagen deposition, myofibroblast differentiation and leukocytes infiltrations. Assessment of lung mechanics showed 3D group did not improve lung function and instead exhibited increased resistance and tissue damping. Proteome profiling of MSC-EV composition revealed molecular enrichment of EV markers (compared to parental cells) and differential proteome between EVs from 2D and 3D culture condition associated with immune-based and fibrosis/extracellular matrix/membrane organization associated function. This study provides insight into distinct variation in EV protein composition dependent on the cellular microenvironment of the parental cells, which could have implications in their therapeutic effect and potency. Overall, this work suggests that EVs produced from 3D MSC cultures did not enhance typical MSC-EV properties expected from 2D cultures (immunomodulation, anti-fibrotic, anti-inflammatory). The outcome highlights critical differences between MSC-EVs obtained from different culture microenvironments, which should be considered when scaling up MSC culture for clinical manufacturing.
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Reactive oxygen species (ROS) and dissolved oxygen play key roles across many biological processes, and fluorescent stains and dyes are the primary tools used to quantify these species in vitro. However, spatio-temporal monitoring of ROS and dissolved oxygen in biological systems are challenging due to issues including poor photostability, lack of reversibility, and rapid off-site diffusion. In particular, ROS monitoring is hindered by the short lifetime of ROS molecules and their low abundance. The combination of nanomaterials and fluorescent detection has led to new opportunities for development of imaging probes, sensors, and theranostic products, because the scaffolds lead to improved optical properties, tuneable interactions with cells and media, and ratiometric sensing robust to environmental drift. In this review, we aim to critically assess and highlight recent development in nanosensors and nanomaterials used for the detection of oxygen and ROS in biological systems, and their future potential use as diagnosis tools.
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Injectable hydrogels have been employed extensively as versatile materials for cartilage regeneration due to their excellent biocompatibility, tunable structure, and ability to accommodate bioactive factors, as well as their ability to be locally delivered via minimally invasive injection to fill irregular defects. More recently, in vitro and in vivo studies have revealed that processing these materials to produce cell-laden microgels can enhance cell-cell and cell-matrix interactions and boost nutrient and metabolite exchange. Moreover, these studies have demonstrated gene expression profiles and matrix regeneration that are superior compared to conventional injectable bulk hydrogels. As cell-laden microgels and their application in cartilage repair are moving closer to clinical translation, this review aims to present an overview of the recent developments in this field. Here we focus on the currently used biomaterials and crosslinking strategies, the innovative fabrication techniques being used for the production of microgels, the cell sources used, the signals used for induction of chondrogenic differentiation and the resultant biological responses, and the ability to create three-dimensional, functional cartilage tissues. In addition, this review also covers the current clinical approaches for repairing cartilage as well as specific challenges faced when attempting the regeneration of damaged cartilage tissue. New findings related to the macroporous nature of the structures formed by the assembled microgel building blocks and the novel use of microgels in 3D printing for cartilage tissue engineering are also highlighted. Finally, we outline the challenges and future opportunities for employing cell-laden microgels in clinical applications.
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Microgéis , Cartilagem , Condrogênese , Hidrogéis , Regeneração , Engenharia TecidualRESUMO
Cells are able to perceive complex mechanical cues from their microenvironment, which in turn influences their development. Although the understanding of these intricate mechanotransductive signals is evolving, the precise roles of substrate microtopography in directing cell fate is still poorly understood. Here, UV nanoimprint lithography is used to generate micropillar arrays ranging from 1 to 10 µm in height, width, and spacing to investigate the impact of microtopography on mechanotransduction. Using mesenchymal stem cells (MSCs) as a model, stark pattern-specific changes in nuclear architecture, lamin A/C accumulation, chromatin positioning, and DNA methyltransferase expression, are demonstrated. MSC osteogenesis is also enhanced specifically on micropillars with 5 µm width/spacing and 5 µm height. Intriguingly, the highest degree of osteogenesis correlates with patterns that stimulated maximal nuclear deformation which is shown to be dependent on myosin-II-generated tension. The outcomes determine new insights into nuclear mechanotransduction by demonstrating that force transmission across the nuclear envelope can be modulated by substrate topography, and that this can alter chromatin organisation and impact upon cell fate. These findings have potential to inform the development of microstructured cell culture substrates that can direct cell mechanotransduction and fate for therapeutic applications in both research and clinical sectors.
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The encapsulation of cells into microgels is attractive for applications in tissue regeneration. While cells are protected against shear stress during injection, the assembly of microgels after injection into a tissue defect also forms a macroporous scaffold that allows effective nutrient transport throughout the construct. However, in most of current strategies that form microgel-based macroporous scaffold or higher-order structures, cells are seeded during or post the assembly process and not microencapsulated in situ. The objective of this study is to investigate the chondrogenic phenotype of microencapsulated fetal chondrocytes in a biocompatible, assembled microgel system vs. bulk gels and to test the stability of the constructs in vivo. Here, we demonstrate that cell microencapsulation leads to increased expression of cartilage-specific genes in a TGF-ß1-dependent manner. This correlates, as shown by histological staining, with the ability of microencapsulated cells to deposit cartilaginous matrix after migrating to the surface of the microgels, while keeping a macroscopic granular morphology. Implantation of precultured scaffolds in a subcutaneous mouse model results in vessel infiltration in bulk gels but not in assembled microgels, suggesting a higher stability of the matrix produced by the cells in the assembled microgel constructs. The cells are able to remodel the microgels as demonstrated by the gradual disappearance of the granular structure in vivo. The biocompatible microencapsulation and microgel assembly system presented in this article therefore hold great promise as an injectable system for cartilage repair.
Assuntos
Cartilagem/metabolismo , Encapsulamento de Células/métodos , Condrócitos/citologia , Condrogênese , Animais , Linhagem Celular , Condrócitos/metabolismo , Humanos , Camundongos , Modelos Animais , Transdução de Sinais , Alicerces Teciduais , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Over the last decade, the acceleration in the clinical use of mesenchymal stromal cells (MSCs) has been nothing short of spectacular. Perhaps most surprising is how little we know about the "MSC product." Although MSCs are being delivered to patients at an alarming rate, the regulatory requirements for MSC therapies (for example in terms of quality assurance and quality control) are nowhere near the expectations of traditional pharmaceuticals. That said, the standards that define a chemical compound or purified recombinant protein cannot be applied with the same stringency to a cell-based therapy. Biological processes are dynamic, adaptive and variable. Heterogeneity will always exist or emerge within even the most rigorously sorted clonal cell populations. With MSCs, perhaps more so than any other therapeutic cell, heterogeneity pervades at multiple levels, from the sample source to the single cell. The research and clinical communities collectively need to recognize and take steps to address this troublesome truth, to ensure that the promise of MSC-based therapies is fulfilled.