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RATIONALE: The innate immune response contributes to cardiac injury in myocardial ischemia/reperfusion (MI/R). Neutrophils are an important early part of the innate immune response to MI/R. Adenosine, an endogenous purine, is a known innate immune modulator and inhibitor of neutrophil activation. However, its delivery to the heart is limited by its short half-life (<30 s) and off-target side effects. CD39 and CD73 are anti-inflammatory homeostatic enzymes that can generate adenosine from phosphorylated adenosine substrate such as ATP released from injured tissue. OBJECTIVE: We hypothesize that hydrogel-delivered CD39 and CD73 target the local early innate immune response, reduce neutrophil activation, and preserve cardiac function in MI/R injury. METHODS AND RESULTS: We engineered a poly(ethylene) glycol (PEG) hydrogel loaded with the adenosine-generating enzymes CD39 and CD73. We incubated the hydrogels with neutrophils in vitro and showed a reduction in hydrogen peroxide production using Amplex Red. We demonstrated availability of substrate for the enzymes in the myocardium in MI/R by LC/MS, and tested release kinetics from the hydrogel. On echocardiography, global longitudinal strain (GLS) was preserved in MI/R hearts treated with the loaded hydrogel. Delivery of purinergic enzymes via this synthetic hydrogel resulted in lower innate immune infiltration into the myocardium post-MI/R, decreased markers of macrophage and neutrophil activation (NETosis), and decreased leukocyte-platelet complexes in circulation. CONCLUSIONS: In a rat model of MI/R injury, CD39 and CD73 delivered via a hydrogel preserve cardiac function by modulating the innate immune response.
Assuntos
Isquemia Miocárdica , Traumatismo por Reperfusão Miocárdica , Ratos , Animais , Hidrogéis/uso terapêutico , Coração , Miocárdio , Adenosina , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Polietilenoglicóis/uso terapêuticoRESUMO
[Figure: see text].
Assuntos
5'-Nucleotidase/administração & dosagem , Adenosina/metabolismo , Portadores de Fármacos , Membro Posterior/irrigação sanguínea , Isquemia/tratamento farmacológico , Maleimidas/química , Doença Arterial Periférica/tratamento farmacológico , Polietilenoglicóis/química , 5'-Nucleotidase/química , 5'-Nucleotidase/metabolismo , Animais , Células Cultivadas , Preparações de Ação Retardada , Modelos Animais de Doenças , Composição de Medicamentos , Feminino , Humanos , Hidrogéis , Isquemia/enzimologia , Isquemia/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Ativação de Neutrófilo/efeitos dos fármacos , Doença Arterial Periférica/enzimologia , Doença Arterial Periférica/fisiopatologia , Fluxo Sanguíneo Regional , Regulação para CimaRESUMO
Exposure of aged mice to a young systemic milieu revealed remarkable rejuvenation effects on aged tissues, including skeletal muscle. Although some candidate factors have been identified, the exact identity and the underlying mechanisms of putative rejuvenating factors remain elusive, mainly due to the complexity of in vivo parabiosis. Here, we present an in vitro muscle parabiosis system that integrates young- and old-muscle stem cell vascular niche on a three-dimensional microfluidic platform designed to recapitulate key features of native muscle stem cell microenvironment. This innovative system enables mechanistic studies of cellular dynamics and molecular interactions within the muscle stem cell niche, especially in response to conditional extrinsic stimuli of local and systemic factors. We demonstrate that vascular endothelial growth factor (VEGF) signaling from endothelial cells and myotubes synergistically contribute to the rejuvenation of the aged muscle stem cell function. Moreover, with the adjustable on-chip system, we can mimic both blood transfusion and parabiosis and detect the time-varying effects of anti-geronic and pro-geronic factors in a single organ or multi-organ systems. Our unique approach presents a complementary in vitro model to supplement in vivo parabiosis for identifying potential anti-geronic factors responsible for revitalizing aging organs.
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Treatment strategies to address pathologies of fibrocartilaginous tissue are in part limited by an incomplete understanding of structure-function relationships in these load-bearing tissues. There is therefore a pressing need to develop micro-engineered tissue platforms that can recreate the highly inhomogeneous tissue microstructures that are known to influence mechanotransductive processes in normal and diseased tissue. Here, we report the quantification of proteoglycan-rich microdomains in developing, ageing and diseased fibrocartilaginous tissues, and the impact of these microdomains on endogenous cell responses to physiologic deformation within a native-tissue context. We also developed a method to generate heterogeneous tissue-engineered constructs (hetTECs) with non-fibrous proteoglycan-rich microdomains engineered into the fibrous structure, and show that these hetTECs match the microstructural, micromechanical and mechanobiological benchmarks of native tissue. Our tissue-engineered platform should facilitate the study of the mechanobiology of developing, homeostatic, degenerating and regenerating fibrous tissues.
Assuntos
Sinalização do Cálcio , Condrócitos/metabolismo , Fibrocartilagem/metabolismo , Mecanotransdução Celular , Proteoglicanas/metabolismo , Estresse Mecânico , Adulto , Idoso , Animais , Bovinos , Células Cultivadas , Feminino , Fibrocartilagem/citologia , Humanos , Masculino , Células-Tronco Mesenquimais/metabolismo , Pessoa de Meia-Idade , Engenharia Tecidual , Suporte de CargaRESUMO
Mechanical cues play important roles in directing the lineage commitment of mesenchymal stem cells (MSCs). In this study, we explored the molecular mechanisms by which dynamic tensile loading (DL) regulates chromatin organization in this cell type. Our previous findings indicated that the application of DL elicited a rapid increase in chromatin condensation through purinergic signaling mediated by ATP. Here, we show that the rate and degree of condensation depends on the frequency and duration of mechanical loading, and that ATP release requires actomyosin-based cellular contractility. Increases in baseline cellular contractility via the addition of an activator of G-protein coupled receptors (lysophosphatidic acid) induced rapid ATP release, resulting in chromatin condensation independent of loading. Conversely, inhibition of contractility through pretreatment with either a RhoA/Rock inhibitor (Y27632) or MLCK inhibitor (ML7) abrogated ATP release in response to DL, blocking load-induced chromatin condensation. With loading, ATP release occurred very rapidly (within the first 10-20 s), whereas changes in chromatin occurred at a later time point (â¼10 min), suggesting a downstream biochemical pathway mediating this process. When cells were pretreated with blockers of the transforming growth factor (TGF) superfamily, purinergic signaling in response to DL was also eliminated. Further analysis showed that this pretreatment decreased contractility, implicating activity in the TGF pathway in the establishment of the baseline contractile state of MSCs (in the absence of exogenous ligands). These data indicate that chromatin condensation in response to DL is regulated through the interplay between purinergic and RhoA/Rock signaling, and that ligandless activity in the TGF/bone morphogenetic proteins signaling pathway contributes to the establishment of baseline contractility in MSCs.
Assuntos
Cromatina/química , Cromatina/metabolismo , Fenômenos Mecânicos , Células-Tronco Mesenquimais/citologia , Trifosfato de Adenosina/metabolismo , Animais , Fenômenos Biomecânicos , Proteínas Morfogenéticas Ósseas/metabolismo , Bovinos , Espaço Extracelular/metabolismo , Células-Tronco Mesenquimais/metabolismo , Transdução de Sinais , Resistência à Tração , Fator de Crescimento Transformador beta/metabolismoRESUMO
Significance: Volumetric muscle loss (VML) is caused by the loss of significant amounts of skeletal muscle tissue. VML cannot be repaired by intrinsic regenerative processes, resulting in permanent loss of muscle function and disability. Current rehabilitative-focused treatment strategies lack efficacy and do not restore muscle function, indicating the need for the development of effective regenerative strategies. Recent Advances: Recent developments implicate biomaterial-based approaches for promoting muscle repair and functional restoration post-VML. Specifically, bioscaffolds transplanted in the injury site have been utilized to mimic endogenous cues of the ablated tissue to promote myogenic pathways, increase neo-myofiber synthesis, and ultimately restore contractile function to the injured unit. Critical Issues: Despite the development and preclinical testing of various biomaterial-based regenerative strategies, effective therapies for patients are not available. The unique challenges posed for biomaterial-based treatments of VML injuries, including its scalability and clinical applicability beyond small-animal models, impede progress. Furthermore, production of tissue-engineered constructs is technically demanding, with reproducibility issues at scale and complexities in achieving vascularization and innervation of large constructs. Future Directions: Biomaterial-based regenerative strategies designed to comprehensively address the pathophysiology of VML are needed. Considerations for clinical translation, including scalability and regulatory compliance, should also be considered when developing such strategies. In addition, an integrated approach that combines regenerative and rehabilitative strategies is essential for ensuring functional improvement.
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Mechanical deformation applied at the joint or tissue level is transmitted through the macroscale extracellular matrix to the microscale local matrix, where it is transduced to cells within these tissues and modulates tissue growth, maintenance, and repair. The objective of this study was to investigate how applied tissue strain is transferred through the local matrix to the cell and nucleus in meniscus, tendon, and the annulus fibrosus, as well as in stem cell-seeded scaffolds engineered to reproduce the organized microstructure of these native tissues. To carry out this study, we developed a custom confocal microscope-mounted tensile testing device and simultaneously monitored strain across multiple length scales. Results showed that mean strain was heterogeneous and significantly attenuated, but coordinated, at the local matrix level in native tissues (35-70% strain attenuation). Conversely, freshly seeded scaffolds exhibited very direct and uniform strain transfer from the tissue to the local matrix level (15-25% strain attenuation). In addition, strain transfer from local matrix to cells and nuclei was dependent on fiber orientation and tissue type. Histological analysis suggested that different domains exist within these fibrous tissues, with most of the tissue being fibrous, characterized by an aligned collagen structure and elongated cells, and other regions being proteoglycan (PG)-rich, characterized by a dense accumulation of PGs and rounder cells. In meniscus, the observed heterogeneity in strain transfer correlated strongly with cellular morphology, where rounder cells located in PG-rich microdomains were shielded from deformation, while elongated cells in fibrous microdomains deformed readily. Collectively, these findings suggest that different tissues utilize distinct strain-attenuating mechanisms according to their unique structure and cellular phenotype, and these differences likely alter the local biologic response of such tissues and constructs in response to mechanical perturbation.
Assuntos
Cartilagem/citologia , Resistência à Tração , Animais , Cartilagem/metabolismo , Bovinos , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Matriz Extracelular/ultraestrutura , Microscopia Confocal/instrumentação , Especificidade de Órgãos , Proteoglicanas/metabolismo , Estresse MecânicoRESUMO
Intramuscular fatty infiltration in muscle injuries and diseases, caused by aberrant adipogenesis of fibro-adipogenic progenitors, negatively impacts function. Intramuscular delivery of wingless-type MMTV integration site family 7a (WNT7A) offers a promising strategy to stimulate muscle regeneration, but its effects on adipogenic conversion of fibro-adipogenic progenitors remain unknown. Here, we show that WNT7A decreases adipogenesis of fibro-adipogenic progenitors (FAPs) by inducing nuclear localization of Yes-associated protein (YAP) through Rho in a ß-CATENIN-independent manner and by promoting nuclear retention of YAP and transcriptional co-activator with PDZ-binding motif (TAZ) in differentiating FAPs. Furthermore, intramuscular injection of WNT7A in vivo effectively suppresses fatty infiltration in mice following glycerol-induced injury. Our results collectively suggest WNT7A as a potential protein-based therapeutic for diminishing adipogenesis of FAPs and intramuscular fatty infiltration in pathological muscle injuries or diseases.
Assuntos
Adipogenia , Células-Tronco Mesenquimais , Proteínas Wnt , Animais , Camundongos , Diferenciação Celular , Músculo Esquelético/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Wnt/metabolismo , Via de Sinalização WntRESUMO
Duchenne muscular dystrophy (DMD) causes patients to suffer from ambulatory disability and cardiorespiratory failure, the latter of which leads to premature death. Due to its role in respiration, the diaphragm is an important muscle for study. A common method for evaluating diaphragm function is ex vivo force testing, which only allows for an end point measurement. In contrast, ultrasound shear wave elastography imaging (US-SWEI) can assess diaphragm function over time; however, US-SWEI studies in dystrophic patients to date have focused on the limbs without preclinical studies. In this work, we used US-SWEI to estimate the shear wave speed (SWS) in diaphragm muscles of healthy (WT) mice, mdx mice, and mdx mice haploinsufficient for utrophin (mdx-utr) at 6 and 12 months of age. Diaphragms were then subjected to ex vivo force testing and histological analysis at 12 months of age. Between 6 and 12 months, a 23.8% increase in SWS was observed in WT mice and a 27.8% increase in mdx mice, although no significant difference was found in mdx-utr mice. Specific force generated by mdx-utr diaphragms was lower than that of WT diaphragms following twitch stimulus. A strong correlation between SWS and collagen deposition was observed, as well as between SWS and muscle fiber size. Together, these data demonstrate the ability of US-SWEI to evaluate dystrophic diaphragm functionality over time and predict the biochemical and morphological make-up of the diaphragm. Additionally, our results highlight the advantage of US-SWEI over ex vivo testing by obtaining longitudinal measurements in the same subject. STATEMENT OF SIGNIFICANCE: In DMD patients, muscles experience cycles of regeneration and degeneration that contribute to chronic inflammation and muscle weakness. This pathology only worsens with time and leads to muscle wasting, including in respiratory and cardiac muscles. Because respiratory failure is a major contributor to premature death in DMD patients, the diaphragm muscle is an important muscle to evaluate and treat over time. Currently, diaphragm function is assessed using ex vivo force testing, a technique that only allows measurement at sacrifice. In contrast, ultrasonography, particularly shear wave elasticity imaging (USSWEI), is a promising tool for longitudinal assessment; however, most US-SWEI in DMD patients aimed for limb muscles only with the absence of preclinical studies. This work broadens the applications of US-SWE imaging by demonstrating its ability to track properties and function of dystrophic diaphragm muscles longitudinally in multiple dystrophic mouse models.
Assuntos
Diafragma , Distrofia Muscular de Duchenne , Camundongos , Animais , Camundongos Endogâmicos mdx , Diafragma/diagnóstico por imagem , Diafragma/patologia , Camundongos Endogâmicos C57BL , Distrofia Muscular de Duchenne/diagnóstico por imagem , Distrofia Muscular de Duchenne/patologia , Músculo Esquelético/patologia , Elasticidade , Modelos Animais de DoençasRESUMO
Rotator cuff (RC) tendon tears are common shoulder injuries that result in irreversible and persistent degeneration of the associated muscles, which is characterized by severe inflammation, atrophy, fibrosis, and fatty infiltration. Although RC muscle degeneration strongly dictates the overall clinical outcomes, strategies to stimulate RC muscle regeneration have largely been overlooked to date. In this review, we highlight the current understanding of the cellular processes that coordinate muscle regeneration, and the roles of muscle resident cells, including immune cells, fibroadipogenic progenitors, and muscle satellite cells in the pathophysiologic regulation of RC muscles following injury. This review also provides perspectives for potential therapies to alleviate the hallmarks of RC muscle degeneration to address current limitations in postsurgical recovery.
Assuntos
Lesões do Manguito Rotador , Manguito Rotador , Tecido Adiposo/patologia , Fibrose , Humanos , Atrofia Muscular/patologia , Manguito Rotador/patologia , Lesões do Manguito Rotador/patologia , TendõesRESUMO
Human mesenchymal stem cells (hMSCs) are an attractive source for cell therapies because of their multiple beneficial properties, i.e. via immunomodulation and secretory factors. Microfluidics is particularly attractive for cell encapsulation since it provides a rapid and reproducible methodology for microgel generation of controlled size and simultaneous cell encapsulation. Here, we report the fabrication of hMSC-laden microcarriers based on in situ ionotropic gelation of water-soluble chitosan in a microfluidic device using a combination of an antioxidant glycerylphytate (G1Phy) compound and tripolyphosphate (TPP) as ionic crosslinkers (G1Phy:TPP-microgels). These microgels showed homogeneous size distributions providing an average diameter of 104 ± 12 µm, somewhat lower than that of control (127 ± 16 µm, TPP-microgels). The presence of G1Phy in microgels maintained cell viability over time and upregulated paracrine factor secretion under adverse conditions compared to control TPP-microgels. Encapsulated hMSCs in G1Phy:TPP-microgels were delivered to the subcutaneous space of immunocompromised mice via injection, and the delivery process was as simple as the injection of unencapsulated cells. Immediately post-injection, equivalent signal intensities were observed between luciferase-expressing microgel-encapsulated and unencapsulated hMSCs, demonstrating no adverse effects of the microcarrier on initial cell survival. Cell persistence, inferred by bioluminescence signal, decreased exponentially over time showing relatively higher half-life values for G1Phy:TPP-microgels compared to TPP-microgels and unencapsulated cells. In overall, results position the microfluidics generated G1Phy:TPP-microgels as a promising microcarrier for supporting hMSC survival and reparative activities.
Assuntos
Quitosana , Células-Tronco Mesenquimais , Microgéis , Animais , Sobrevivência Celular , Humanos , Camundongos , MicrofluídicaRESUMO
Stem cell therapies are limited by poor cell survival and engraftment. A hurdle to the use of materials for cell delivery is the lack of understanding of material properties that govern transplanted stem cell functionality. Here, we show that synthetic hydrogels presenting integrin-specific peptides enhance the survival, persistence, and osteo-reparative functions of human bone marrow-derived mesenchymal stem cells (hMSCs) transplanted in murine bone defects. Integrin-specific hydrogels regulate hMSC adhesion, paracrine signaling, and osteoblastic differentiation in vitro. Hydrogels presenting GFOGER, a peptide targeting α2ß1 integrin, prolong hMSC survival and engraftment in a segmental bone defect and result in improved bone repair compared to other peptides. Integrin-specific hydrogels have diverse pleiotropic effects on hMSC reparative activities, modulating in vitro cytokine secretion and in vivo gene expression for effectors associated with inflammation, vascularization, and bone formation. These results demonstrate that integrin-specific hydrogels improve tissue healing by directing hMSC survival, engraftment, and reparative activities.
Assuntos
Doenças Ósseas/terapia , Integrina alfa2beta1/metabolismo , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Animais , Doenças Ósseas/metabolismo , Doenças Ósseas/fisiopatologia , Medula Óssea/química , Medula Óssea/metabolismo , Regeneração Óssea , Adesão Celular , Sobrevivência Celular , Terapia Baseada em Transplante de Células e Tecidos , Humanos , Hidrogéis/química , Integrina alfa2beta1/genética , Masculino , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Peptídeos/metabolismoRESUMO
Tumor-initiating cells (TICs), a subpopulation of cancerous cells with high tumorigenic potential and stem-cell-like properties, drive tumor progression and are resistant to conventional therapies. Identification and isolation of TICs are limited by their low frequency and lack of robust markers. Here, we characterize the heterogeneous adhesive properties of a panel of human and murine cancer cells and demonstrate differences in adhesion strength among cells, which exhibit TIC properties and those that do not. These differences in adhesion strength were exploited to rapidly (~10 min) and efficiently isolate cancerous cells with increased tumorigenic potential in a label-free manner by use of a microfluidic technology. Isolated murine and human cancer cells gave rise to larger tumors with increased growth rate and higher frequency in both immunocompetent and immunocompromised mice, respectively. This rapid and label-free TIC isolation technology has the potential to be a valuable tool for facilitating research into TIC biology and the development of more efficient diagnostics and cancer therapies.
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Carcinogênese/patologia , Adesão Celular , Separação Celular/métodos , Hidrodinâmica , Neoplasias/fisiopatologia , Células-Tronco Neoplásicas/patologia , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Progressão da Doença , Feminino , Proteínas de Fluorescência Verde/metabolismo , Humanos , Células MCF-7 , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Microfluídica , Transdução de Sinais , Estresse MecânicoRESUMO
Skeletal muscle possesses efficient ability to regenerate upon minor injuries, but its capacity to regenerate is severely compromised with traumatic injuries and muscle-associated diseases. Recent evidence suggests that skeletal muscle regeneration can be enhanced by transplantation of muscle satellite cells (MuSCs) or treatment with pro-myogenic factors, such as Wingless-type MMTV Integrated 7a (Wnt7a) protein. Although direct intramuscular injection is the simplest method to deliver MuSCs and Wnt7a for regenerative therapy, direct injections are not viable in many clinical cases where structural integrity is severely compromised. To address this challenge, we evaluated the feasibility of co-delivering pro-myogenic factors, such as Wnt7a, and MuSCs using a synthetic poly(ethylene glycol) (PEG)-based hydrogel to the affected skeletal muscles. The Wnt7a release rate can be controlled by modulating the polymer density of the hydrogel, and this release rate can be further accelerated through the proteolytic degradation of the hydrogel. Treating cryo-injured tibialis anterior (TA) muscles with Wnt7a-loaded hydrogels resulted in an improved regenerative response by day 14, measured by increased muscle fiber cross-sectional area, bulk TA mass, and the number of Pax7+ MuSCs at the injury site, compared to the TA muscles treated with Wnt7a-free hydrogels. Co-delivery of Wnt7a and primary MuSCs using the synthetic hydrogel to the cryo-injured TA muscles significantly increased cellular migration during the engraftment process. This work provides a synthetic biomaterial platform for advancing treatment strategies of skeletal muscle conditions where direct intramuscular injection may be challenging. Finally, the current outcomes establish an important foundation for future applications in treating severe muscle trauma and diseases, where the endogenous repair capacity is critically impaired. STATEMENT OF SIGNIFICANCE: Skeletal muscle injuries and diseases cause debilitating health consequences, including disability and diminished quality of life. Treatment using protein and stem cell-based therapeutics may help regenerate the affected muscles, but direct intramuscular injection may not be feasible in severe muscle injuries due to the gravely damaged tissue structure. In chronic muscle diseases, such as Duchenne muscular dystrophy, local treatment of the diaphragm, a muscle critical for respiration, may be necessary but direct injection is difficult due to its thin dimensions. To address this challenge, this work presents a synthetic and bioactive muscle "patch" that enables concurrent administration of proteins and muscle stem cells for accelerated muscle healing.
Assuntos
Hidrogéis/química , Músculo Esquelético/fisiologia , Regeneração/fisiologia , Proteínas Wnt/genética , Animais , Linhagem Celular , Movimento Celular , Proliferação de Células , Cruzamentos Genéticos , Feminino , Proteínas de Fluorescência Verde/genética , Humanos , Masculino , Maleimidas/química , Camundongos , Camundongos Endogâmicos C57BL , Fibras Musculares Esqueléticas/fisiologia , Polietilenoglicóis/química , Proteínas Recombinantes/genética , Células Satélites de Músculo Esquelético/fisiologiaRESUMO
Because of their immunomodulatory activities, human mesenchymal stem cells (hMSCs) are being explored to treat a variety of chronic conditions such as inflammatory bowel disorders and graft-vs-host disease. Treating hMSCs with IFN-γ prior to administration augments these immunomodulatory properties; however, this ex vivo treatment limits the broad applicability of this therapy due to technical and regulatory issues. In this study, we engineered an injectable synthetic hydrogel with tethered recombinant IFN-γ that activates encapsulated hMSCs to increase their immunomodulatory functions and avoids the need for ex vivo manipulation. Tethering IFN-γ to the hydrogel increases retention of IFN-γ within the biomaterial while preserving its biological activity. hMSCs encapsulated within hydrogels with tethered IFN-γ exhibited significant differences in cytokine secretion and showed a potent ability to halt activated T-cell proliferation and monocyte-derived dendritic cell differentiation compared to hMSCs that were pre-treated with IFN-γ and untreated hMSCs. Importantly, hMSCs encapsulated within hydrogels with tethered IFN-γ accelerated healing of colonic mucosal wounds in both immunocompromised and immunocompetent mice. This novel approach for licensing hMSCs with IFN-γ may enhance the clinical translation and efficacy of hMSC-based therapies.
Assuntos
Hidrogéis/farmacologia , Imunomodulação/efeitos dos fármacos , Interferon gama/farmacologia , Células-Tronco Mesenquimais/imunologia , Cicatrização/efeitos dos fármacos , Animais , Humanos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/patologia , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos Endogâmicos C57BLRESUMO
Laminin incorporation into biological or synthetic hydrogels has been explored to recapitulate the dynamic nature and biological complexity of neural stem cell (NSC) niches. However, the strategies currently explored for laminin immobilization within three-dimensional (3D) matrices do not address a critical aspect influencing cell-matrix interactions, which is the control over laminin conformation and orientation upon immobilization. This is a key feature for the preservation of the protein bioactivity. In this work, we explored an affinity-based approach to mediate the site-selective immobilization of laminin into a degradable synthetic hydrogel. Specifically, a four-arm maleimide terminated poly(ethylene glycol) (PEG-4MAL) macromer was functionalized with a mono-PEGylated recombinant human N-terminal agrin (NtA) domain, to promote high affinity binding of laminin. Different NtA concentrations (10, 50 and 100 µM) were used to investigate the impact of NtA density on laminin incorporation, hydrogel biophysical properties, and biological outcome. Laminin was efficiently incorporated for all the conditions tested (laminin incorporation >95%), and the developed hydrogels revealed mechanical properties (average storage modulus (G') ranging from 187 to 256 Pa) within the values preferred for NSC proliferation and neurite branching and extension. Affinity-bound laminin PEG-4MAL hydrogels better preserve laminin bioactivity, compared to unmodified hydrogels and hydrogels containing physically entrapped laminin, this effect being dependent on NtA concentration. This was evidenced by the 10 µM NtA-functionalized PEG-4MAL gels incorporating laminin that support enhanced human NSC proliferation and neurite extension, compared to the latter. Overall, this work highlights the potential of the proposed engineered matrices to be used as defined 3D platforms for the establishment of artificial NSC niches and as extracellular matrix-mimetic microenvironments to support human NSC transplantation.
Assuntos
Engenharia , Hidrogéis/química , Hidrogéis/farmacologia , Laminina/química , Maleimidas/química , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Crescimento Neuronal/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Fenótipo , Polietilenoglicóis/químicaRESUMO
Human intestinal organoids (HIOs) represent a powerful system to study human development and are promising candidates for clinical translation as drug-screening tools or engineered tissue. Experimental control and clinical use of HIOs is limited by growth in expensive and poorly defined tumor-cell-derived extracellular matrices, prompting investigation of synthetic ECM-mimetics for HIO culture. Since HIOs possess an inner epithelium and outer mesenchyme, we hypothesized that adhesive cues provided by the matrix may be dispensable for HIO culture. Here, we demonstrate that alginate, a minimally supportive hydrogel with no inherent cell instructive properties, supports HIO growth in vitro and leads to HIO epithelial differentiation that is virtually indistinguishable from Matrigel-grown HIOs. In addition, alginate-grown HIOs mature to a similar degree as Matrigel-grown HIOs when transplanted in vivo, both resembling human fetal intestine. This work demonstrates that purely mechanical support from a simple-to-use and inexpensive hydrogel is sufficient to promote HIO survival and development.
Assuntos
Alginatos/farmacologia , Hidrogéis/farmacologia , Intestinos/efeitos dos fármacos , Organoides/efeitos dos fármacos , Células-Tronco Pluripotentes/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Colágeno/farmacologia , Combinação de Medicamentos , Epitélio/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Humanos , Laminina/farmacologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteoglicanas/farmacologia , Engenharia Tecidual/métodosRESUMO
IMPACT STATEMENT: The goal of this study was to determine the threshold for a critically sized, nonhealing muscle defect by characterizing key components in the balance between fibrosis and regeneration as a function of injury size in the mouse quadriceps. There is currently limited understanding of what leads to a critically sized muscle defect and which muscle regenerative components are functionally impaired. With the substantial increase in preclinical VML models as testbeds for tissue engineering therapeutics, defining the critical threshold for VML injuries will be instrumental in characterizing therapeutic efficacy and potential for subsequent translation.
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Doenças Musculares/patologia , Doenças Musculares/terapia , Miofibrilas/fisiologia , Junção Neuromuscular/citologia , Músculo Quadríceps/citologia , Músculo Quadríceps/lesões , Engenharia Tecidual , Animais , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Quadríceps/fisiologia , Alicerces Teciduais , CicatrizaçãoRESUMO
Critical limb ischemia, the most severe form of peripheral artery disease, leads to extensive damage and alterations to skeletal muscle homeostasis. Although recent research has investigated the tissue-specific responses to ischemia, the role of the muscle stem cell in the regeneration of its niche components within skeletal muscle has been limited. To elucidate the regenerative mechanism of the muscle stem cell in response to ischemic insults, we explored cellular interactions between the vasculature, neural network, and muscle fiber within the muscle stem cell niche. Using a surgical murine hindlimb ischemia model, we first discovered a significant increase in subsynaptic nuclei and remodeling of the neuromuscular junction following ischemia-induced denervation. In addition, ischemic injury causes significant alterations to the myofiber through a muscle stem cell-mediated accumulation of total myonuclei and a concomitant decrease in myonuclear domain size, possibly to enhance the transcriptional and translation output and restore muscle mass. Results also revealed an accumulation of total mitochondrial content per myonucleus in ischemic myofibers to compensate for impaired mitochondrial function and high turnover rate. Taken together, the findings from this study suggest that the muscle stem cell plays a role in motor neuron reinnervation, myonuclear accretion, and mitochondrial biogenesis for skeletal muscle regeneration following ischemic injury.
Assuntos
Extremidades/irrigação sanguínea , Isquemia/metabolismo , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/patologia , Junção Neuromuscular , Animais , Modelos Animais de Doenças , Isquemia/etiologia , Camundongos , Mitocôndrias Musculares/metabolismo , Mioblastos/metabolismo , RegeneraçãoRESUMO
Muscle satellite cells (MuSCs) play a central role in muscle regeneration, but their quantity and function decline with comorbidity of trauma, aging, and muscle diseases. Although transplantation of MuSCs in traumatically injured muscle in the comorbid context of aging or pathology is a strategy to boost muscle regeneration, an effective cell delivery strategy in these contexts has not been developed. We engineered a synthetic hydrogel-based matrix with optimal mechanical, cell-adhesive, and protease-degradable properties that promotes MuSC survival, proliferation, and differentiation. Furthermore, we establish a biomaterial-mediated cell delivery strategy for treating muscle trauma, where intramuscular injections may not be applicable. Delivery of MuSCs in the engineered matrix significantly improved in vivo cell survival, proliferation, and engraftment in nonirradiated and immunocompetent muscles of aged and dystrophic mice compared to collagen gels and cell-only controls. This platform may be suitable for treating craniofacial and limb muscle trauma, as well as postoperative wounds of elderly and dystrophic patients.