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1.
Annu Rev Biomed Eng ; 26(1): 25-47, 2024 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-38166186

RESUMEN

Hyaluronan (HA) plays well-recognized mechanical and biological roles in articular cartilage and synovial fluid, where it contributes to tissue structure and lubrication. An understanding of how HA contributes to the structure of other musculoskeletal tissues, including muscle, bone, tendon, and intervertebral discs, is growing. In addition, the use of HA-based therapies to restore damaged tissue is becoming more prevalent. Nevertheless, the relationship between biomechanical stimuli and HA synthesis, degradation, and signaling in musculoskeletal tissues remains understudied, limiting the utility of HA in regenerative medicine. In this review, we discuss the various roles and significance of endogenous HA in musculoskeletal tissues. We use what is known and unknown to motivate new lines of inquiry into HA biology within musculoskeletal tissues and in the mechanobiology governing HA metabolism by suggesting questions that remain regarding the relationship and interaction between biological and mechanical roles of HA in musculoskeletal health and disease.


Asunto(s)
Ácido Hialurónico , Tendones , Ácido Hialurónico/química , Humanos , Animales , Fenómenos Biomecánicos , Tendones/fisiología , Tendones/metabolismo , Cartílago Articular/fisiología , Cartílago Articular/metabolismo , Transducción de Señal , Huesos/metabolismo , Huesos/fisiología , Líquido Sinovial/metabolismo , Líquido Sinovial/fisiología , Músculo Esquelético/fisiología , Músculo Esquelético/metabolismo , Sistema Musculoesquelético/metabolismo , Medicina Regenerativa/métodos
2.
Dev Dyn ; 252(4): 463-482, 2023 04.
Artículo en Inglés | MEDLINE | ID: mdl-36335435

RESUMEN

BACKGROUND: The interstitial extracellular matrix (ECM) is comprised of proteins and glycosaminoglycans and provides structural and biochemical information during development. Our previous work revealed the presence of transient ECM-based structures in the interstitial matrix of developing kidneys. Stromal cells are the main contributors to interstitial ECM synthesis, and the transcription factor Forkhead Box D1 (Foxd1) is critical for stromal cell function. To investigate the role of Foxd1 in interstitial ECM patterning, we combined 3D imaging and proteomics to explore how the matrix changes in the murine developing kidney when Foxd1 is knocked out. RESULTS: We found that COL26A1, FBN2, EMILIN1, and TNC, interstitial ECM proteins that are transiently upregulated during development, had a similar distribution perinatally but then diverged in patterning in the adult. Abnormally clustered cortical vertical fibers and fused glomeruli were observed when Foxd1 was knocked out. The changes in the interstitial ECM of Foxd1 knockout kidneys corresponded to disrupted Foxd1+ cell patterning but did not precede branching dysmorphogenesis. CONCLUSIONS: The transient ECM networks affected by Foxd1 knockout may provide support for later-stage nephrogenic structures.


Asunto(s)
Factores de Transcripción Forkhead , Riñón , Animales , Ratones , Matriz Extracelular/metabolismo , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Regulación de la Expresión Génica , Riñón/metabolismo
3.
Nat Methods ; 17(5): 531-540, 2020 05.
Artículo en Inglés | MEDLINE | ID: mdl-32371980

RESUMEN

Single-molecule localization microscopy is a powerful tool for visualizing subcellular structures, interactions and protein functions in biological research. However, inhomogeneous refractive indices inside cells and tissues distort the fluorescent signal emitted from single-molecule probes, which rapidly degrades resolution with increasing depth. We propose a method that enables the construction of an in situ 3D response of single emitters directly from single-molecule blinking datasets, and therefore allows their locations to be pinpointed with precision that achieves the Cramér-Rao lower bound and uncompromised fidelity. We demonstrate this method, named in situ PSF retrieval (INSPR), across a range of cellular and tissue architectures, from mitochondrial networks and nuclear pores in mammalian cells to amyloid-ß plaques and dendrites in brain tissues and elastic fibers in developing cartilage of mice. This advancement expands the routine applicability of super-resolution microscopy from selected cellular targets near coverslips to intra- and extracellular targets deep inside tissues.


Asunto(s)
Encéfalo/metabolismo , Cartílago/metabolismo , Imagenología Tridimensional/métodos , Microscopía Fluorescente/métodos , Nanotecnología/métodos , Placa Amiloide/metabolismo , Imagen Individual de Molécula/métodos , Animales , Encéfalo/patología , Cartílago/patología , Núcleo Celular/metabolismo , Células Cultivadas , Interpretación de Imagen Asistida por Computador/métodos , Masculino , Ratones , Mitocondrias/metabolismo , Imagen Molecular/métodos , Poro Nuclear/metabolismo , Placa Amiloide/patología
4.
Biophys J ; 121(4): 525-539, 2022 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-35074393

RESUMEN

The mechanical behavior of tissues at the macroscale is tightly coupled to cellular activity at the microscale. Dermal wound healing is a prominent example of a complex system in which multiscale mechanics regulate restoration of tissue form and function. In cutaneous wound healing, a fibrin matrix is populated by fibroblasts migrating in from a surrounding tissue made mostly out of collagen. Fibroblasts both respond to mechanical cues, such as fiber alignment and stiffness, as well as exert active stresses needed for wound closure. Here, we develop a multiscale model with a two-way coupling between a microscale cell adhesion model and a macroscale tissue mechanics model. Starting from the well-known model of adhesion kinetics proposed by Bell, we extend the formulation to account for nonlinear mechanics of fibrin and collagen and show how this nonlinear response naturally captures stretch-driven mechanosensing. We then embed the new nonlinear adhesion model into a custom finite element implementation of tissue mechanical equilibrium. Strains and stresses at the tissue level are coupled with the solution of the microscale adhesion model at each integration point of the finite element mesh. In addition, solution of the adhesion model is coupled with the active contractile stress of the cell population. The multiscale model successfully captures the mechanical response of biopolymer fibers and gels, contractile stresses generated by fibroblasts, and stress-strain contours observed during wound healing. We anticipate that this framework will not only increase our understanding of how mechanical cues guide cellular behavior in cutaneous wound healing, but will also be helpful in the study of mechanobiology, growth, and remodeling in other tissues.


Asunto(s)
Colágeno , Fibrina , Biofisica , Análisis de Elementos Finitos , Cinética , Modelos Biológicos , Estrés Mecánico
5.
Mol Cell Proteomics ; 19(7): 1220-1235, 2020 07.
Artículo en Inglés | MEDLINE | ID: mdl-32381549

RESUMEN

Perlecan is a critical proteoglycan found in the extracellular matrix (ECM) of cartilage. In healthy cartilage, perlecan regulates cartilage biomechanics and we previously demonstrated perlecan deficiency leads to reduced cellular and ECM stiffness in vivo This change in mechanics may lead to the early onset osteoarthritis seen in disorders resulting from perlecan knockdown such as Schwartz-Jampel syndrome (SJS). To identify how perlecan knockdown affects the material properties of developing cartilage, we used imaging and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to study the ECM in a murine model of SJS, Hspg2C1532Y-Neo Perlecan knockdown led to defective pericellular matrix formation, whereas the abundance of bulk ECM proteins, including many collagens, increased. Post-translational modifications and ultrastructure of collagens were not significantly different; however, LC-MS/MS analysis showed more protein was secreted by Hspg2C1532Y-Neo cartilage in vitro, suggesting that the incorporation of newly synthesized ECM was impaired. In addition, glycosaminoglycan deposition was atypical, which may explain the previously observed decrease in mechanics. Overall, these findings provide insight into the influence of perlecan on functional cartilage assembly and the progression of osteoarthritis in SJS.


Asunto(s)
Cartílago/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Matriz Extracelular/metabolismo , Proteoglicanos de Heparán Sulfato/metabolismo , Osteocondrodisplasias/metabolismo , Animales , Proteínas de Unión al Calcio/metabolismo , Cartílago/crecimiento & desarrollo , Cartílago/ultraestructura , Moléculas de Adhesión Celular/metabolismo , Condrocitos/citología , Condrocitos/metabolismo , Cromatografía Liquida , Colágeno Tipo X/genética , Colágeno Tipo X/metabolismo , Modelos Animales de Enfermedad , Matriz Extracelular/patología , Ontología de Genes , Glicosaminoglicanos/metabolismo , Proteoglicanos de Heparán Sulfato/deficiencia , Proteoglicanos de Heparán Sulfato/genética , Ratones , Ratones Endogámicos DBA , Ratones Noqueados , Microscopía Electrónica de Transmisión , Osteoartritis/genética , Osteoartritis/metabolismo , Osteoartritis/patología , Osteocondrodisplasias/genética , Espectrometría de Masas en Tándem
6.
J Am Soc Nephrol ; 32(7): 1649-1665, 2021 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-33875569

RESUMEN

BACKGROUND: The extracellular matrix (ECM) is a network of proteins and glycosaminoglycans that provides structural and biochemical cues to cells. In the kidney, the ECM is critical for nephrogenesis; however, the dynamics of ECM composition and how it relates to 3D structure during development is unknown. METHODS: Using embryonic day 14.5 (E14.5), E18.5, postnatal day 3 (P3), and adult kidneys, we fractionated proteins based on differential solubilities, performed liquid chromatography-tandem mass spectrometry, and identified changes in ECM protein content (matrisome). Decellularized kidneys were stained for ECM proteins and imaged in 3D using confocal microscopy. RESULTS: We observed an increase in interstitial ECM that connects the stromal mesenchyme to the basement membrane (TNXB, COL6A1, COL6A2, COL6A3) between the embryo and adult, and a transient elevation of interstitial matrix proteins (COL5A2, COL12A1, COL26A1, ELN, EMID1, FBN1, LTBP4, THSD4) at perinatal time points. Basement membrane proteins critical for metanephric induction (FRAS1, FREM2) were highest in abundance in the embryo, whereas proteins necessary for integrity of the glomerular basement membrane (COL4A3, COL4A4, COL4A5, LAMB2) were more abundant in the adult. 3D visualization revealed a complex interstitial matrix that dramatically changed over development, including the perinatal formation of fibrillar structures that appear to support the medullary rays. CONCLUSION: By correlating 3D ECM spatiotemporal organization with global protein abundance, we revealed novel changes in the interstitial matrix during kidney development. This new information regarding the ECM in developing kidneys offers the potential to inform the design of regenerative scaffolds that can guide nephrogenesis in vitro.

7.
Adv Funct Mater ; 31(35)2021 Aug 26.
Artículo en Inglés | MEDLINE | ID: mdl-34840547

RESUMEN

Cells embedded in the extracellular matrix of tissues play a critical role in maintaining homeostasis while promoting integration and regeneration following damage or disease. Emerging engineered biomaterials utilize decellularized extracellular matrix as a tissue-specific support structure; however, many dense, structured biomaterials unfortunately demonstrate limited formability, fail to promote cell migration, and result in limited tissue repair. Here, we developed a reinforced composite material of densely packed acellular extracellular matrix microparticles in a hydrogel, termed tissue clay, that can be molded and crosslinked to mimic native tissue architecture. We utilized hyaluronic acid-based hydrogels, amorphously packed with acellular articular cartilage tissue particulated to ~125-250 microns in diameter and defined a percolation threshold of 0.57 (v/v) beyond which the compressive modulus exceeded 300kPa. Remarkably, primary chondrocytes recellularized particles within 48 hours, a process driven by chemotaxis, exhibited distributed cellularity in large engineered composites, and expressed genes consistent with native cartilage repair. We additionally demonstrated broad utility of tissue clays through recellularization and persistence of muscle, skin, and cartilage composites in a subcutaneous in vivo mouse model. Our findings suggest optimal strategies and material architectures to balance concurrent demands for large-scale mechanical properties while also supporting recellularization and integration of dense musculoskeletal and connective tissues. TABLE OF CONTENTS ENTRY: We present a new design framework for regenerative articular cartilage scaffolds using acellular extracellular matrix particles, packed beyond a percolation threshold, and crosslinked within chondroinductive hydrogels. Our results suggest that the architecture and the packing, rather than altering the individual components, creates a composite material that can balance mechanics, porosity to enable migration, and tissue specific biochemical interactions with cells. Moreover, we provide a technique that we show is applicable to other tissue types.

8.
Adv Funct Mater ; 31(1)2021 Jan 04.
Artículo en Inglés | MEDLINE | ID: mdl-34764824

RESUMEN

Accurately replicating and analyzing cellular responses to mechanical cues is vital for exploring metastatic disease progression. However, many of the existing in vitro platforms for applying mechanical stimulation seed cells on synthetic substrates. To better recapitulate physiological conditions, a novel actuating platform is developed with the ability to apply tensile strain on cells at various amplitudes and frequencies in a high-throughput multi-well culture plate using a physiologically-relevant substrate. Suspending fibrillar fibronectin across the body of the magnetic actuator provides a matrix representative of early metastasis for 3D cell culture that is not reliant on a synthetic substrate. This platform enables the culturing and analysis of various cell types in an environment that mimics the dynamic stretching of lung tissue during normal respiration. Metabolic activity, YAP activation, and morphology of breast cancer cells are analyzed within one week of cyclic stretching or static culture. Further, matrix degradation is significantly reduced in breast cancer cell lines with metastatic potential after actuation. These new findings demonstrate a clear suppressive cellular response due to cyclic stretching that has implications for a mechanical role in the dormancy and reactivation of disseminated breast cancer cells to macrometastases.

9.
Small ; 17(6): e2006699, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33470544

RESUMEN

Reciprocal interactions between the cell nucleus and the extracellular matrix lead to macroscale tissue phenotype changes. However, little is known about how the extracellular matrix environment affects gene expression and cellular phenotype in the native tissue environment. Here, it is hypothesized that enzymatic disruption of the tissue matrix results in a softer tissue, affecting the stiffness of embedded cell and nuclear structures. The aim is to directly measure nuclear mechanics without perturbing the native tissue structure to better understand nuclear interplay with the cell and tissue microenvironments. To accomplish this, an atomic force microscopy needle-tip probe technique that probes nuclear stiffness in cultured cells to measure the nuclear envelope and cell membrane stiffness within native tissue is expanded. This technique is validated by imaging needle penetration and subsequent repair of the plasma and nuclear membranes of HeLa cells stably expressing the membrane repair protein CHMP4B-GFP. In the native tissue environment ex vivo, it is found that while enzymatic degradation of viable cartilage tissues with collagenase 3 (MMP-13) and aggrecanase-1 (ADAMTS-4) decreased tissue matrix stiffness, cell and nuclear membrane stiffness is also decreased. Finally, the capability for cell and nucleus elastography using the AFM needle-tip technique is demonstrated. These results demonstrate disruption of the native tissue environment that propagates to the plasma membrane and interior nuclear envelope structures of viable cells.


Asunto(s)
Núcleo Celular , Matriz Extracelular , Proteína ADAMTS4 , Membrana Celular , Complejos de Clasificación Endosomal Requeridos para el Transporte , Células HeLa , Humanos , Metaloproteinasa 13 de la Matriz , Microscopía de Fuerza Atómica
10.
Connect Tissue Res ; 62(1): 53-71, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-32856502

RESUMEN

The muscle-tendon interface is an anatomically specialized region that is involved in the efficient transmission of force from muscle to tendon. Due to constant exposure to loading, the interface is susceptible to injury. Current treatment methods do not meet the socioeconomic demands of reduced recovery time without compromising the risk of reinjury, requiring the need for developing alternative strategies. The extracellular matrix (ECM) present in muscle, tendon, and at the interface of these tissues consists of unique molecules that play significant roles in homeostasis and repair. Better, understanding the function of the ECM during development, injury, and aging has the potential to unearth critical missing information that is essential for accelerating the repair at the muscle-tendon interface. Recently, advanced techniques have emerged to explore the ECM for identifying specific roles in musculoskeletal biology. Simultaneously, there is a tremendous increase in the scope for regenerative medicine strategies to address the current clinical deficiencies. Advancements in ECM research can be coupled with the latest regenerative medicine techniques to develop next generation therapies that harness ECM for treating defects at the muscle-tendon interface. The current work provides a comprehensive review on the role of muscle and tendon ECM to provide insights about the role of ECM in the muscle-tendon interface and discusses the latest research techniques to explore the ECM to gathered information for developing regenerative medicine strategies.


Asunto(s)
Medicina Regenerativa , Tendones , Matriz Extracelular , Músculos
11.
J Proteome Res ; 19(10): 3955-3967, 2020 10 02.
Artículo en Inglés | MEDLINE | ID: mdl-32830507

RESUMEN

The myotendinous junction is a highly interdigitated interface designed to transfer muscle-generated force to tendon. Understanding how this interface is formed and organized, as well as identifying tendon- and muscle-specific extracellular matrix (ECM), is critical for designing effective regenerative therapies to restore functionality to damaged muscle-tendon units. However, a comparative analysis of the ECM proteome across this interface has not been conducted. The goal of this study was to resolve the distribution of ECM proteins that are uniformly expressed as well as those specific to each of the muscle, tendon, and junction tissues. The soleus muscles from 5-month-old wild-type C57BL/6 mice were harvested and dissected into the central muscle (M) away from tendon, the junction between muscle and tendon (J) and the tendon (T). Tissues were processed by either homogenizing in guanidine hydrochloride or fractionating to isolate the ECM from more soluble intracellular components and then analyzed using liquid chromatography-tandem mass spectrometry. Overall, we found that both tissue processing methods generated similar ECM profiles. Many ECM were found across the muscle-tendon unit, including type I collagen and associated fibril-regulating proteins. The ECM identified exclusively in M were primarily related to the basal lamina, whereas those specific to T and J tissue included thrombospondins and other matricellular ECM. Type XXII collagen (COL22A1) was restricted to J, and we identified COL5A3 as a potential marker of the muscle-tendon interface. Immunohistochemical analysis of key proteins confirmed the restriction of some basal lamina proteins to M, tenascin-C to T, and COL22A1 to J. COL5A3, PRELP, and POSTN were visualized in the tissue surrounding the junction, suggesting that these proteins play a role in stabilizing the interface. This comparative map provides a guide for tissue-specific ECM that can facilitate the spatial visualization of M, J, and T tissues and inform musculoskeletal regenerative therapies.


Asunto(s)
Proteoma , Tendones , Animales , Colágeno , Matriz Extracelular , Proteínas de la Matriz Extracelular , Ratones , Ratones Endogámicos C57BL
12.
Connect Tissue Res ; 61(3-4): 278-291, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32186210

RESUMEN

Osteoarthritis (OA) is typically managed in late stages by replacement of the articular cartilage surface with a prosthesis as an effective, though undesirable outcome. As an alternative, hydrogel implants or growth factor treatments are currently of great interest in the tissue engineering community, and scaffold materials are often designed to emulate the mechanical and chemical composition of mature extracellular matrix (ECM) tissue. However, scaffolds frequently fail to capture the structure and organization of cartilage. Additionally, many current scaffold designs do not mimic processes by which structurally sound cartilage is formed during musculoskeletal development. The objective of this review is to highlight methods that investigate cartilage ontogenesis with native and model systems in the context of regenerative medicine. Specific emphasis is placed on the use of cartilage explant cultures that provide a physiologically relevant microenvironment to study tissue assembly and development. Ex vivo cartilage has proven to be a cost-effective and accessible model system that allows researchers to control the culture conditions and stimuli and perform proteomics and imaging studies that are not easily possible using in vivo experiments, while preserving native cell-matrix interactions. We anticipate our review will promote a developmental biology approach using explanted tissues to guide cartilage tissue engineering and inform new treatment methods for OA and joint damage.


Asunto(s)
Cartílago Articular/metabolismo , Matriz Extracelular/metabolismo , Modelos Biológicos , Osteoartritis/metabolismo , Regeneración , Animales , Cartílago Articular/patología , Matriz Extracelular/patología , Humanos , Osteoartritis/patología , Osteoartritis/terapia , Medicina Regenerativa , Técnicas de Cultivo de Tejidos
13.
Dev Biol ; 435(2): 122-129, 2018 03 15.
Artículo en Inglés | MEDLINE | ID: mdl-29352963

RESUMEN

The extracellular matrix (ECM) plays a crucial role in embryogenesis, serving both as a substrate to which cells attach and as an active regulator of cell behavior. However, little is known about the spatiotemporal expression patterns and 3D structure of ECM proteins during embryonic development. The lack of suitable methods to visualize the embryonic ECM is largely responsible for this gap, posing a major technical challenge for biologists and tissue engineers. Here, we describe a method of viewing the 3D organization of the ECM using a polyacrylamide-based hydrogel to provide a 3D framework within developing murine embryos. After removal of soluble proteins using sodium dodecyl sulfate, confocal microscopy was used to visualize the 3D distribution of independent ECM networks in multiple developing tissues, including the forelimb, eye, and spinal cord. Comparative analysis of E12.5 and E14.5 autopods revealed proteoglycan-rich fibrils maintain connections between the epidermis and the underlying tendon and cartilage, indicating a role for the ECM during musculoskeletal assembly and demonstrating that our method can be a powerful tool for defining the spatiotemporal distribution of the ECM during embryogenesis.


Asunto(s)
Desarrollo Embrionario , Matriz Extracelular/ultraestructura , Microscopía Confocal/métodos , Adhesión del Tejido/métodos , Resinas Acrílicas , Animales , Detergentes/farmacología , Epidermis/ultraestructura , Proteínas de la Matriz Extracelular/efectos de los fármacos , Proteínas de la Matriz Extracelular/ultraestructura , Colorantes Fluorescentes , Miembro Anterior/embriología , Miembro Anterior/ultraestructura , Formaldehído , Hidrogeles , Ratones , Ratones Endogámicos C57BL , Morfogénesis , Polímeros , Proteoglicanos/análisis , Dodecil Sulfato de Sodio/farmacología , Manejo de Especímenes , Coloración y Etiquetado/métodos , Tendones/embriología , Tendones/ultraestructura , Fijación del Tejido
14.
J Biomech Eng ; 141(8)2019 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-30874718

RESUMEN

During chondrogenesis, tissue organization changes dramatically. We previously showed that the compressive moduli of chondrocytes increase concomitantly with extracellular matrix (ECM) stiffness, suggesting cells were remodeling to adapt to the surrounding environment. Due to the difficulty in analyzing the mechanical response of cells in situ, we sought to create an in silico model that would enable us to investigate why cell and ECM stiffness increased in tandem. The goal of this study was to establish a methodology to segment, quantify, and generate mechanical models of developing cartilage to explore how variations in geometry and material properties affect strain distributions. Multicellular geometries from embryonic day E16.5 and postnatal day P3 murine cartilage were imaged in three-dimensional (3D) using confocal microscopy. Image stacks were processed using matlab to create geometries for finite element analysis using ANSYS. The geometries based on confocal images and isolated, single cell models were compressed 5% and the equivalent von Mises strain of cells and ECM were compared. Our simulations indicated that cells had similar strains at both time points, suggesting that the stiffness and organization of cartilage changes during development to maintain a constant strain profile within cells. In contrast, the ECM at P3 took on more strain than at E16.5. The isolated, single-cell geometries underestimated both cell and ECM strain and were not able to capture the similarity in cell strain at both time points. We expect this experimental and computational pipeline will facilitate studies investigating other model systems to implement physiologically derived geometries.

15.
J Lipid Res ; 58(10): 2061-2070, 2017 10.
Artículo en Inglés | MEDLINE | ID: mdl-28754825

RESUMEN

Protein post-translational modifications (PTMs) serve to give proteins new cellular functions and can influence spatial distribution and enzymatic activity, greatly enriching the complexity of the proteome. Lipidation is a PTM that regulates protein stability, function, and subcellular localization. To complement advances in proteomic identification of lipidated proteins, we have developed a method to image the spatial distribution of proteins that have been co- and post-translationally modified via the addition of myristic acid (Myr) to the N terminus. In this work, we use a Myr analog, 12-azidododecanoic acid (12-ADA), to facilitate fluorescent detection of myristoylated proteins in vitro and in vivo. The azide moiety of 12-ADA does not react to natural biological chemistries, but is selectively reactive with alkyne functionalized fluorescent dyes. We find that the spatial distribution of myristoylated proteins varies dramatically between undifferentiated and differentiated muscle cells in vitro. Further, we demonstrate that our methodology can visualize the distribution of myristoylated proteins in zebrafish muscle in vivo. Selective protein labeling with noncanonical fatty acids, such as 12-ADA, can be used to determine the biological function of myristoylation and other lipid-based PTMs and can be extended to study deregulated protein lipidation in disease states.


Asunto(s)
Diferenciación Celular , Ácido Mirístico/metabolismo , Imagen Óptica , Procesamiento Proteico-Postraduccional , Animales , Línea Celular , Ácidos Láuricos/metabolismo , Ratones , Proteómica
16.
Dev Biol ; 418(2): 242-7, 2016 10 15.
Artículo en Inglés | MEDLINE | ID: mdl-27578148

RESUMEN

The pericellular matrix (PCM) is a component of the extracellular matrix that is found immediately surrounding individual chondrocytes in developing and adult cartilage, and is rich in the proteoglycan perlecan. Mutations in perlecan are the basis of several developmental disorders, which are thought to arise from disruptions in the mechanical stability of the PCM. We tested the hypothesis that defects in PCM organization will reduce the stiffness of chondrocytes in developing cartilage by combining a murine model of Schwartz-Jampel syndrome, in which perlecan is knocked down, with our novel atomic force microscopy technique that can measure the stiffness of living cells and surrounding matrix in embryonic and postnatal tissues in situ. Perlecan knockdown altered matrix organization and significantly decreased the stiffness of both chondrocytes and interstitial matrix as a function of age and genotype. Our results demonstrate that the knockdown of a spatially restricted matrix molecule can have a profound influence on cell and tissue stiffness, implicating a role for outside-in mechanical signals from the PCM in regulating the intracellular mechanisms required for the overall development of cartilage.


Asunto(s)
Cartílago/fisiopatología , Proteínas de la Matriz Extracelular/deficiencia , Proteoglicanos de Heparán Sulfato/deficiencia , Animales , Fenómenos Biomecánicos , Cartílago/crecimiento & desarrollo , Cartílago/patología , Condrocitos/patología , Condrocitos/fisiología , Modelos Animales de Enfermedad , Matriz Extracelular/patología , Matriz Extracelular/fisiología , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/fisiología , Femenino , Técnicas de Silenciamiento del Gen , Proteoglicanos de Heparán Sulfato/genética , Proteoglicanos de Heparán Sulfato/fisiología , Masculino , Ratones , Ratones Endogámicos DBA , Ratones Noqueados , Microscopía de Fuerza Atómica , Osteocondrodisplasias/genética , Osteocondrodisplasias/patología , Osteocondrodisplasias/fisiopatología , Embarazo
17.
J Shoulder Elbow Surg ; 24(1): 111-9, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-25193488

RESUMEN

BACKGROUND: A persistent atrophy of muscle fibers and an accumulation of fat, collectively referred to as fatty degeneration, commonly occur in patients with chronic rotator cuff tears. The etiology of fatty degeneration and function of the residual rotator cuff musculature have not been well characterized in humans. We hypothesized that muscles from patients with chronic rotator cuff tears have reduced muscle fiber force production, disordered myofibrils, and an accumulation of fat vacuoles. METHODS: The contractility of muscle fibers from biopsy specimens of supraspinatus muscles of 13 patients with chronic full-thickness posterosuperior rotator cuff tears was measured and compared with data from healthy vastus lateralis muscle fibers. Correlations between muscle fiber contractility, American Shoulder and Elbow Surgeons (ASES) scores, and tear size were analyzed. Histology and electron microscopy were also performed. RESULTS: Torn supraspinatus muscles had a 30% reduction in maximum isometric force production and a 29% reduction in normalized force compared with controls. Normalized supraspinatus fiber force positively correlated with ASES score and negatively correlated with tear size. Disordered sarcomeres were noted, along with an accumulation of lipid-laden macrophages in the extracellular matrix surrounding supraspinatus muscle fibers. CONCLUSIONS: Patients with chronic supraspinatus tears have significant reductions in muscle fiber force production. Force production also correlates with ASES scores and tear size. The structural and functional muscle dysfunction of the residual muscle fibers is independent of the additional area taken up by fibrotic tissue. This work may help establish future therapies to restore muscle function after the repair of chronically torn rotator cuff muscles.


Asunto(s)
Miofibrillas/ultraestructura , Manguito de los Rotadores/patología , Traumatismos de los Tendones/patología , Tejido Adiposo/patología , Anciano , Matriz Extracelular/patología , Matriz Extracelular/ultraestructura , Femenino , Humanos , Macrófagos/patología , Masculino , Microscopía Electrónica de Transmisión , Persona de Mediana Edad , Contracción Muscular/fisiología , Fibras Musculares Esqueléticas/patología , Fibras Musculares Esqueléticas/fisiología , Fibras Musculares Esqueléticas/ultraestructura , Atrofia Muscular/patología , Atrofia Muscular/fisiopatología , Miofibrillas/patología , Lesiones del Manguito de los Rotadores , Sarcómeros/patología , Sarcómeros/ultraestructura
18.
ACS Biomater Sci Eng ; 10(3): 1418-1434, 2024 03 11.
Artículo en Inglés | MEDLINE | ID: mdl-38319825

RESUMEN

Protein adsorption after biomaterial implantation is the first stage of the foreign body response (FBR). However, the source(s) of the adsorbed proteins that lead to damaged associated molecular patterns (DAMPs) and induce inflammation have not been fully elucidated. This study examined the effects of different protein sources, cell-derived (from a NIH/3T3 fibroblast cell lysate) and serum-derived (from fetal bovine serum), which were compared to implant-derived proteins (after a 30 min subcutaneous implantation in mice) on activation of RAW 264.7 cells cultured in minimal (serum-free) medium. Both cell-derived and serum-derived protein sources when preadsorbed to either tissue culture polystyrene or medical-grade silicone induced RAW 264.7 cell activation. The combination led to an even higher expression of pro-inflammatory cytokine genes and proteins. Implant-derived proteins on silicone explants induced a rapid inflammatory response that then subsided more quickly and to a greater extent than the studies with in vitro cell-derived or serum-derived protein sources. Proteomic analysis of the implant-derived proteins identified proteins that included cell-derived and serum-derived, but also other proteinaceous sources (e.g., extracellular matrix), suggesting that the latter or nonproteinaceous sources may help to temper the inflammatory response in vivo. These findings indicate that both serum-derived and cell-derived proteins adsorbed to implants can act as DAMPs to drive inflammation in the FBR, but other protein sources may play an important role in controlling inflammation.


Asunto(s)
Reacción a Cuerpo Extraño , Proteómica , Ratones , Animales , Células RAW 264.7 , Macrófagos , Inflamación , Proteínas , Siliconas
19.
iScience ; 27(2): 108838, 2024 Feb 16.
Artículo en Inglés | MEDLINE | ID: mdl-38303699

RESUMEN

The extracellular matrix (ECM) is an integral part of multicellular organisms, connecting different cell layers and tissue types. During morphogenesis and growth, tissues undergo substantial reorganization. While it is intuitive that the ECM remodels in concert, little is known regarding how matrix composition and organization change during development. Here, we quantified ECM protein dynamics in the murine forelimb during appendicular musculoskeletal morphogenesis (embryonic days 11.5-14.5) using tissue fractionation, bioorthogonal non-canonical amino acid tagging, and mass spectrometry. Our analyses indicated that ECM protein (matrisome) composition in the embryonic forelimb changed as a function of development and growth, was distinct from other developing organs (brain), and was altered in a model of disease (osteogenesis imperfecta murine). Additionally, the tissue distribution for select matrisome was assessed via immunohistochemistry in the wild-type embryonic and postnatal musculoskeletal system. This resource will guide future research investigating the role of the matrisome during complex tissue development.

20.
FASEB J ; 26(6): 2538-45, 2012 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-22415307

RESUMEN

During forelimb regeneration in the newt Notophthalmus viridescens, the dynamic expression of a transitional matrix rich in hyaluronic acid, tenascin-C, and fibronectin controls muscle cell behavior in vivo and in vitro. However, the influence of extracellular matrix (ECM) remodeling on tissue stiffness and the cellular response to mechanical variations during regeneration was unknown. By measuring the transverse stiffness of tissues in situ, we found undifferentiated regenerative blastemas were less stiff than differentiated stump muscle (13.3±1.6 vs. 16.6±1.2 kPa). To directly determine how ECM and stiffness combine to affect skeletal muscle fragmentation, migration, and fusion, we coated silicone-based substrates ranging from 2 to 100 kPa with matrices representative of transitional (tenascin-C and fibronectin) and differentiated environments (laminin and Matrigel). Using live-cell imaging, we found softer tenascin-C-coated substrates significantly enhanced migration and fragmentation of primary newt muscle cells. In contrast, stiffer substrates coated with laminin, Matrigel, or fibronectin increased differentiation while suppressing migration and fragmentation. These data support our in vivo observations that a transitional matrix of reduced stiffness regulates muscle plasticity and progenitor cell recruitment into the regenerative blastema. These new findings will enable the determination of how biochemical and mechanical cues from the ECM control genetic pathways that drive regeneration.


Asunto(s)
Músculo Esquelético/fisiología , Regeneración/fisiología , Animales , Fenómenos Biomecánicos , Diferenciación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Colágeno/metabolismo , Combinación de Medicamentos , Matriz Extracelular/fisiología , Fibronectinas/metabolismo , Laminina/metabolismo , Mioblastos/fisiología , Notophthalmus viridescens , Proteoglicanos/metabolismo , Células Madre/citología , Tenascina/metabolismo
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