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Tissue hydration provides articular cartilage with dynamic viscoelastic properties. Early stage osteoarthritis (OA) is marked by loss of proteoglycans and glycosaminoglycans (GAG), lowering fixed charge density, and impairing tissue osmotic function. The most common GAG replacement, chondroitin sulfate (CS), has failed to show effectiveness. Here, we investigated a synthetic polyelectrolyte, poly(styrenesulfonate) (PSS), both as a model compound to investigate polyelectrolyte transport in cartilage, and as a potential candidate to restore bulk fixed charge density in cartilage with GAG loss. Through bovine explants and histology, we determined zonal-based effective diffusion coefficients for three different molecular weights of PSS. Compared to CS, PSS was retained longer in GAG-depleted cartilage in static and compression-based desorption experiments. We explained enhanced solute performance of PSS by its more compact morphology and higher charge density by small-angle X-ray scattering. This study may improve design of GAG mimetic molecules for repairing osmotic function in OA cartilage.
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Tendon degeneration is typically described as an overuse injury with little distinction made between magnitude of load (overload) and number of cycles (overuse). Further, in vivo, animal models of tendon degeneration are mostly overuse models, where tendon damage is caused by a high number of load cycles. As a result, there is a lack of knowledge of how isolated overload leads to degeneration in tendons. A surgical model of synergist ablation (SynAb) overloads the target tendon, plantaris, by ablating its synergist tendon, Achilles. The objective of this study was to evaluate the structural and functional changes that occur following overload of plantaris tendon in a rat SynAb model. Tendon cross-sectional area (CSA) and shape changes were evaluated by longitudinal MR imaging up to 8 weeks postsurgery. Tissue-scale structural changes were evaluated by semiquantified histology and second harmonic generation microscopy. Fibril level changes were evaluated with serial block face scanning electron microscopy (SBF-SEM). Functional changes were evaluated using tension tests at the tissue and microscale using a custom testing system allowing both video and microscopy imaging. At 8 weeks, overloaded plantaris tendons exhibited degenerative changes including increases in CSA, cell density, collagen damage area fraction (DAF), and fibril diameter, and decreases in collagen alignment, modulus, and yield stress. To interpret the differences between overload and overuse in tendon, we introduce a new framework for tendon remodeling and degeneration that differentiates between the inputs of overload and overuse. In summary, isolated overload induces multiscale degenerative structural and functional changes in plantaris tendon.
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Tendão do Calcâneo , Músculo Esquelético , Ratos , Animais , Tendão do Calcâneo/patologia , Colágeno , Modelos Animais , Fibras Musculares EsqueléticasRESUMO
Tubulin and actin exist as monomeric units that polymerize to form either microtubules or filamentous actin. As the polymerization status (monomeric/polymeric ratio) of tubulin and/or actin have been shown to be important in regulating gene expression and phenotype in non-chondrocyte cells, the objective of this study was to examine the role of cytoskeletal polymerization on the chondrocyte phenotype. We hypothesized that actin and/or tubulin polymerization status modulates the chondrocyte phenotype during monolayer culture as well as in 3D culture during redifferentiation. To test this hypothesis, articular chondrocytes were grown and passaged in 2D monolayer culture. Cell phenotype was investigated by assessing cell morphology (area and circularity), actin/tubulin content, organization and polymerization status, as well as by determination of proliferation, fibroblast and cartilage matrix gene expression with passage number. Bovine chondrocytes became larger, more elongated, and had significantly (P < 0.05) increased gene expression of proliferation-associated molecules (cyclin D1 and ki67), as well as significantly (P < 0.05) decreased cartilage matrix (type II collagen and aggrecan) and increased fibroblast-like matrix, type I collagen (COL1), gene expression by passage 2 (P2). Although tubulin polymerization status was not significantly (P > 0.05) modulated, actin polymerization was increased in bovine P2 cells. Actin depolymerization, but not tubulin depolymerization, promoted the chondrocyte phenotype by inducing cell rounding, increasing aggrecan and reducing COL1 expression. Knockdown of actin depolymerization factor, cofilin, in these cells induced further P2 cell actin polymerization and increased COL1 gene expression. To confirm that actin status regulated COL1 gene expression in human P2 chondrocytes, human P2 chondrocytes were exposed to cytochalasin D. Cytochalasin D decreased COL1 gene expression in human passaged chondrocytes. Furthermore, culture of bovine P2 chondrocytes in 3D culture on porous bone substitute resulted in actin depolymerization, which correlated with decreased expression of COL1 and proliferation molecules. In 3D cultures, aggrecan gene expression was increased by cytochalasin D treatment and COL1 was further decreased. These results reveal that actin polymerization status regulates chondrocyte dedifferentiation. Reorganization of the cytoskeleton by actin depolymerization appears to be an active regulatory mechanism for redifferentiation of passaged chondrocytes.
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Condrócitos/fisiologia , Condrogênese/fisiologia , Citoesqueleto/fisiologia , Polimerização , Animais , Bovinos , Técnicas de Cultura de Células , Diferenciação Celular/fisiologia , Células CultivadasRESUMO
The ocular lens is a transparent flexible tissue that alters its shape to focus light from different distances onto the retina. Aside from a basement membrane surrounding the organ, called the capsule, the lens is entirely cellular consisting of a monolayer of epithelial cells on the anterior hemisphere and a bulk mass of lens fiber cells. Throughout life, epithelial cells proliferate in the germinative zone at the lens equator, and equatorial epithelial cells migrate, elongate, and differentiate into newly formed fiber cells. Equatorial epithelial cells substantially alter morphology from randomly packed cobble-stone-shaped cells into aligned hexagon-shaped cells forming meridional rows. Newly formed lens fiber cells retain the hexagonal cell shape and elongate toward the anterior and posterior poles, forming a new shell of cells that are overlaid onto previous generations of fibers. Little is known about the mechanisms that drive the remarkable morphogenesis of lens epithelial cells to fiber cells. To better understand lens structure, development, and function, new imaging protocols have been developed to image peripheral structures using whole mounts of ocular lenses. Here, methods to quantify capsule thickness, epithelial cell area, cell nuclear area and shape, meridional row cell order and packing, and fiber cell widths are shown. These measurements are essential for elucidating the cellular changes that occur during lifelong lens growth and understanding the changes that occur with age or pathology.
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Cristalino , Epitélio , Células Epiteliais , Membrana Basal , Diagnóstico por ImagemRESUMO
Actin is a central mediator of the chondrocyte phenotype. Monolayer expansion of articular chondrocytes on tissue culture polystyrene, for cell-based repair therapies, leads to chondrocyte dedifferentiation. During dedifferentiation, chondrocytes spread and filamentous (F-)actin reorganizes from a cortical to a stress fiber arrangement causing a reduction in cartilage matrix expression and an increase in fibroblastic matrix and contractile molecule expression. While the downstream mechanisms regulating chondrocyte molecular expression by alterations in F-actin organization have become elucidated, the critical upstream regulators of F-actin networks in chondrocytes are not completely known. Tropomyosin (TPM) and the RhoGTPases are known regulators of F-actin networks. The main purpose of this study is to elucidate the regulation of passaged chondrocyte F-actin stress fiber networks and cell phenotype by the specific TPM, TPM3.1, and the RhoGTPase, CDC42. Our results demonstrated that TPM3.1 associates with cortical F-actin and stress fiber F-actin in primary and passaged chondrocytes, respectively. In passaged cells, we found that pharmacological TPM3.1 inhibition or siRNA knockdown causes F-actin reorganization from stress fibers back to cortical F-actin and causes an increase in G/F-actin. CDC42 inhibition also causes formation of cortical F-actin. However, pharmacological CDC42 inhibition, but not TPM3.1 inhibition, leads to the re-association of TPM3.1 with cortical F-actin. Both TPM3.1 and CDC42 inhibition, as well as TPM3.1 knockdown, reduces nuclear localization of myocardin related transcription factor, which suppresses dedifferentiated molecule expression. We confirmed that TPM3.1 or CDC42 inhibition partially redifferentiates passaged cells by reducing fibroblast matrix and contractile expression, and increasing chondrogenic SOX9 expression. A further understanding on the regulation of F-actin in passaged cells may lead into new insights to stimulate cartilage matrix expression in cells for regenerative therapies.
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Actinas , Desdiferenciação Celular , Condrócitos , Fibras de Estresse , Tropomiosina , Condrócitos/metabolismo , Condrócitos/citologia , Fibras de Estresse/metabolismo , Animais , Actinas/metabolismo , Tropomiosina/metabolismo , Tropomiosina/genética , Fenótipo , Células Cultivadas , Proteína cdc42 de Ligação ao GTP/metabolismo , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição SOX9/genética , Transativadores/metabolismo , Transativadores/genéticaRESUMO
BACKGROUND: Bioengineered cartilage is a developing therapeutic to repair cartilage defects. The matrix must be rich in collagen type II and aggrecan and mechanically competent, withstanding compressive and shearing loads. Biomechanical properties in native articular cartilage depend on the zonal architecture consisting of 3 zones: superficial, middle, and deep. The superficial zone chondrocytes produce lubricating proteoglycan-4, whereas the deep zone chondrocytes produce collagen type X, which allows for integration into the subchondral bone. Zonal and chondrogenic expression is lost after cell number expansion. Current cell-based therapies have limited capacity to regenerate the zonal structure of native cartilage. HYPOTHESIS: Both passaged superficial and deep zone chondrocytes at high density can form bioengineered cartilage that is rich in collagen type II and aggrecan; however, only passaged superficial zone-derived chondrocytes will express superficial zone-specific proteoglycan-4, and only passaged deep zone-derived chondrocytes will express deep zone-specific collagen type X. STUDY DESIGN: Controlled laboratory study. METHODS: Superficial and deep zone chondrocytes were isolated from bovine joints, and zonal subpopulations were separately expanded in 2-dimensional culture. At passage 2, superficial and deep zone chondrocytes were seeded, separately, in scaffold-free 3-dimensional culture within agarose wells and cultured in redifferentiation media. RESULTS: Monolayer expansion resulted in loss of expression for proteoglycan-4 and collagen type X in passaged superficial and deep zone chondrocytes, respectively. By passage 2, superficial and deep zone chondrocytes had similar expression for dedifferentiated molecules collagen type I and tenascin C. Redifferentiation of both superficial and deep zone chondrocytes led to the expression of collagen type II and aggrecan in both passaged chondrocyte populations. However, only redifferentiated deep zone chondrocytes expressed collagen type X, and only redifferentiated superficial zone chondrocytes expressed and secreted proteoglycan-4. Additionally, redifferentiated deep zone chondrocytes produced a thicker and more robust tissue compared with superficial zone chondrocytes. CONCLUSION: The recapitulation of the primary phenotype from passaged zonal chondrocytes introduces a novel method of functional bioengineering of cartilage that resembles the zone-specific biological properties of native cartilage. CLINICAL RELEVANCE: The recapitulation of the primary phenotype in zonal chondrocytes could be a possible method to tailor bioengineered cartilage to have zone-specific expression.
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Cartilagem Articular , Condrócitos , Humanos , Animais , Bovinos , Condrócitos/metabolismo , Agrecanas/metabolismo , Colágeno Tipo II/metabolismo , Colágeno Tipo X/metabolismo , Diferenciação Celular , Células Cultivadas , Engenharia Tecidual/métodosRESUMO
OBJECTIVE: The superficial zone (SZ) of articular cartilage is responsible for distributing shear forces for optimal cartilage loading and contributes to joint lubrication through the production of proteoglycan 4 (PRG4). PRG4 plays a critical role in joint homeostasis and is chondroprotective. Normal PRG4 production is affected by inflammation and irregular mechanical loading in post-traumatic osteoarthritis (PTOA). THe SZ chondrocyte (SZC) phenotype, including PRG4 expression, is regulated by the actin cytoskeleton in vitro. There remains a limited understanding of the regulation of PRG4 by the actin cytoskeleton in native articular chondrocytes. The filamentous (F)-actin cytoskeleton is a potential node in crosstalk between mechanical stimulation and cytokine activation and the regulation of PRG4 in SZCs, therefore developing insights in the regulation of PRG4 by actin may identify molecular targets for novel PTOA therapies. MATERIALS AND METHODS: A comprehensive literature search on PRG4 and the regulation of the SZC phenotype by actin organization was performed. RESULTS: PRG4 is strongly regulated by the actin cytoskeleton in isolated SZCs in vitro. Biochemical and mechanical stimuli have been characterized to regulate PRG4 and may converge upon actin cytoskeleton signaling. CONCLUSION: Actin-based regulation of PRG4 in native SZCs is not fully understood and requires further elucidation. Understanding the regulation of PRG4 by actin in SZCs requires an in vivo context to further potential of leveraging actin arrangement to arthritic therapeutics.
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Purpose: Epithelial cells in the equatorial region of the ocular lens undergo a remarkable transition from randomly packed cells into precisely aligned and hexagon-shaped cells organized into meridional rows. We investigated the function of nonmuscle myosin IIA (encoded by Myh9) in regulating equatorial epithelial cell alignment to form meridional rows during secondary fiber cell morphogenesis. Methods: We used genetic knock-in mice to study a common human Myh9 mutation, E1841K, in the rod domain. The E1841K mutation disrupts bipolar filament assembly. Lens shape, clarity, and stiffness were evaluated, and Western blots were used to determine the level of normal and mutant myosins. Cryosections and lens whole mounts were stained and imaged by confocal microscopy to investigate cell shape and organization. Results: We observed no obvious changes in lens size, shape, and biomechanical properties (stiffness and resilience) between the control and nonmuscle myosin IIA-E1841K mutant mice at 2 months of age. Surprisingly, we found misalignment and disorder of fiber cells in heterozygous and homozygous mutant lenses. Further analysis revealed misshapen equatorial epithelial cells that cause disorientation of the meridional rows before fiber cell differentiation in homozygous mutant lenses. Conclusions: Our data indicate that nonmuscle myosin IIA bipolar filament assembly is required for the precise alignment of the meridional rows at the lens equator and that the organization of lens fiber cells depends on the proper patterning of meridional row epithelial cells. These data also suggest that lens fiber cell organization and a hexagonal shape are not required for normal lens size, shape transparency, or biomechanical properties.
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Cristalino , Miosina não Muscular Tipo IIA , Camundongos , Animais , Humanos , Miosina não Muscular Tipo IIA/genética , Diferenciação Celular/fisiologia , Células Epiteliais , MutaçãoRESUMO
Articular cartilage derives its load-bearing strength from the mechanical and physiochemical coupling between the collagen network and negatively charged proteoglycans, respectively. Current disease modeling approaches and treatment strategies primarily focus on cartilage stiffness, partly because indentation tests are readily accessible. However, stiffness measurements via indentation alone cannot discriminate between proteoglycan degradation versus collagen degradation, and there is a lack of methods to monitor physiochemical contributors in full-stack tissue. To decouple these contributions, here, we developed a platform that measures tissue swelling in full-depth equine cartilage explants using piezoresistive graphene strain sensors. These piezoresistive strain sensors are embedded within an elastomer bulk and have sufficient sensitivity to resolve minute, real-time changes in swelling. By relying on simple DC resistance measurements over optical techniques, our platform can analyze multiple samples in parallel. Using these devices, we found that cartilage explants under enzymatic digestion showed distinctive swelling responses to a hypotonic challenge and established average equilibrium swelling strains in healthy cartilage (4.6%), cartilage with proteoglycan loss (0.5%), and in cartilage with both collagen and proteoglycan loss (-2.6%). Combined with histology, we decoupled the pathologic swelling responses as originating either from reduced fixed charge density or from loss of intrinsic stiffness of the collagen matrix in the superficial zone. By providing scalable and in situ monitoring of cartilage swelling, our platform could facilitate regenerative medicine approaches aimed at restoring osmotic function in osteoarthritic cartilage or could be used to validate physiologically relevant swelling behavior in synthetic hydrogels.
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Cartilagem Articular , Grafite , Animais , Cavalos , Cartilagem Articular/metabolismo , Modelos Biológicos , Colágeno/metabolismo , Proteoglicanas/metabolismoRESUMO
OBJECTIVES: Mechanical loading is crucial for tendon matrix homeostasis. Under-stimulation of tendon tissue promotes matrix degradation and ultimately tendon failure. In this study, we examined the expression of tendon matrix molecules and matrix-degrading enzymes (matrix metalloproteinases) in stress-deprived tail tendons and compared to tendons that were mechanically loaded by a simple restraining method. DATA DESCRIPTION: Isolated mouse tail fascicles were either floated or restrained by magnets in cell culture media for 24 h. The gene expression of tendon matrix molecules and matrix metalloproteinases in the tendon fascicles of mouse tails were examined by real-time RT-PCR. Stress deprivation of tail tendons increase Mmp3 mRNA levels. Restraining tendons represses these increases in Mmp3. The gene expression response to restraining was specific to Mmp3 at 24 h as we did not observe mRNA level changes in other matrix related genes that we examined (Col1, Col3, Tnc, Acan, and Mmp13). To elucidate, the mechanisms that may regulate load transmission in tendon tissue, we examined filamentous (F-)actin staining and nuclear morphology. As compared to stress deprived tendons, restrained tendons had greater staining for F-actin. The nuclei of restrained tendons are smaller and more elongated. These results indicate that mechanical loading regulates specific gene expression potentially through F-actin regulation of nuclear morphology. A further understanding on the mechanisms involved in regulating Mmp3 gene expression may lead to new strategies to prevent tendon degeneration.
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Actinas , Metaloproteinase 3 da Matriz , Estresse Mecânico , Tendões , Animais , Camundongos , Fenômenos Magnéticos , Metaloproteinase 3 da Matriz/genética , RNA Mensageiro/genética , Tendões/fisiologiaRESUMO
Actin is a central mediator of the chondrocyte phenotype. Monolayer expansion of articular chondrocytes on tissue culture polystyrene, for cell-based repair therapies, leads to chondrocyte dedifferentiation. During dedifferentiation, chondrocytes spread and filamentous (F-)actin reorganizes from a cortical to a stress fiber arrangement causing a reduction in cartilage matrix expression and an increase in fibroblastic matrix and contractile molecule expression. While the downstream mechanisms regulating chondrocyte molecular expression by alterations in F-actin organization have become elucidated, the critical upstream regulators of F-actin networks in chondrocytes are not completely known. Tropomyosin (TPM) and the RhoGTPases are known regulators of F-actin networks. The purpose of this study is to elucidate the regulation of passaged chondrocyte F-actin stress fiber networks and cell phenotype by the specific TPM, TPM3.1, and the RhoGTPase, CDC42. Our results demonstrated that TPM3.1 associates with cortical F-actin and stress fiber F-actin in primary and passaged chondrocytes, respectively. In passaged cells, we found that TPM3.1 inhibition causes F-actin reorganization from stress fibers back to cortical F-actin and also causes an increase in G/F-actin. CDC42 inhibition also causes formation of cortical F-actin. However, CDC42 inhibition, but not TPM3.1 inhibition, leads to the re-association of TPM3.1 with cortical F-actin. Both TPM3.1 and CDC42 inhibition reduces nuclear localization of myocardin related transcription factor, which is known to suppress dedifferentiated molecule expression. We confirmed that TPM3.1 or CDC42 inhibition partially redifferentiates passaged cells by reducing fibroblast matrix and contractile expression, and increasing chondrogenic SOX9 expression. A further understanding on the regulation of F-actin in passaged cells may lead into new insights to stimulate cartilage matrix expression in cells for regenerative therapies.
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In osteoarthritis (OA), a disease characterized by progressive articular cartilage degradation and calcification, the articular chondrocyte phenotype changes and this correlates with actin cytoskeleton alterations suggesting that it regulates gene expression essential for proper phenotype. This study reports that OA is associated with the loss of adseverin, an actin capping and severing protein. Adseverin deletion (Adseverin-/-) in mice compromised articular chondrocyte function, by reducing F-actin and aggrecan expression and increasing apoptosis, Indian hedgehog, Runx2, MMP13, and collagen type X expression, and cell proliferation. This led to stiffer cartilage and decreased hyaline and increased calcified cartilage thickness. Together, these changes predisposed the articular cartilage to enhanced OA severity in Adseverin-/- mice who underwent surgical induction of OA. Adseverin-/- chondrocyte RNA sequencing and in vitro studies together suggests that adseverin modulates cell viability and prevents mineralization. Thus, adseverin maintains articular chondrocyte phenotype and cartilage tissue homeostasis by preventing progression to hypertrophic differentiation in vivo. Adseverin may be chondroprotective and a potential therapeutic target.
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Cartilagem Articular , Osteoartrite , Camundongos , Animais , Proteínas dos Microfilamentos/metabolismo , Condrócitos , Proteínas Hedgehog/metabolismo , Osteoartrite/genética , Diferenciação Celular , Cartilagem Articular/metabolismo , Actinas/metabolismoRESUMO
The transparent ocular lens in the anterior chamber of the eye is responsible for fine focusing of light onto the retina. The lens is entirely cellular with bulk of the tissue composed of fiber cells, and the anterior hemisphere of the lens is covered by a monolayer of epithelial cells. Lens epithelial cells are important for maintaining fiber cell homeostasis and for continual growth of the lens tissue throughout life. Cataracts, defined as any opacity in the lens, remain the leading cause of blindness in the world. Following cataract surgery, lens epithelial cells can undergo a process of epithelial-to-mesenchymal transition (EMT), leading to secondary cataracts due to posterior capsular opacification (PCO). Since the epithelial cells make up only a small fraction of the lens, specialized techniques are required to study lens epithelial cell biology and pathology. Studies using native lens epithelial cells often require pooling of samples to obtain enough cells to make sufficient samples for traditional molecular biology techniques. Here, we provide detailed protocols that enable the study of native mouse lens epithelial cells, including immunostaining of the native lens epithelium in flat mounts, extraction of RNA and proteins from pairs of lens epithelial monolayers, and isolation of lens epithelial cells for primary culture. These protocols will enable researchers to gain better insight on representative molecular expression and cellular structure of lens epithelial cells. We also provide comparative data between native, primary culture, and immortalized lens epithelial cells and discuss the advantages and disadvantages of each technique presented.
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Actin is a central mediator between mechanical force and cellular phenotype. In tendons, it is speculated that mechanical stress deprivation regulates gene expression by reducing filamentous (F)-actin. However, the mechanisms regulating tenocyte F-actin remain unclear. Tropomyosins (Tpms) are master regulators of F-actin. There are more than 40 Tpm isoforms, each having the unique capability to stabilize F-actin subpopulations. We investigated F-actin polymerization in stress-deprived tendons and tested the hypothesis that stress fiber-associated Tpm(s) stabilize F-actin to regulate cellular phenotype. Stress deprivation of mouse tail tendon down-regulated tenogenic and up-regulated protease (matrix metalloproteinase-3) mRNA levels. Concomitant with mRNA modulation were increases in G/F-actin, confirming reduced F-actin by tendon stress deprivation. To investigate the molecular regulation of F-actin, we identified that tail, Achilles, and plantaris tendons express three isoforms in common: Tpm1.6, 3.1, and 4.2. Tpm3.1 associates with F-actin in native and primary tenocytes. Tpm3.1 inhibition reduces F-actin, leading to decreases in tenogenic expression, increases in chondrogenic expression, and enhancement of protease expression in mouse and human tenocytes. These expression changes by Tpm3.1 inhibition are consistent with tendinosis progression. A further understanding of F-actin regulation in musculoskeletal cells could lead to new therapeutic interventions to prevent alterations in cellular phenotype during disease progression.
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Actinas , Tendinopatia , Humanos , Camundongos , Animais , Actinas/metabolismo , Tendinopatia/metabolismo , Tendões/metabolismo , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fenótipo , Peptídeo Hidrolases/metabolismo , Tropomiosina/metabolismoRESUMO
Whole mount imaging of the lens allows for high spatial resolution visualization of lens epithelial structures by using small molecule fluorescent probes. However, the visualization of specific proteins in lens epithelial cells within whole lenses remains a challenge as the capsule that surrounds the lens does not allow penetration of antibodies. Here we describe a whole mount imaging method that allows us to overcome this challenge by digesting the lens capsules of paraformaldehyde fixed lenses using collagenase. This method enables the penetration of antibodies for effective visualization of proteins in the epithelium of whole lenses.â¢A limitation to lens whole mount imaging is the ability to visualize specific proteins as the collagen capsule surrounding the lens impedes the penetration of antibodiesâ¢This protocol helps overcome this limitation by a light collagenase digestion of the capsule of fixed lenses prior to immunostainingâ¢This method allows for the imaging of specific proteins in the epithelium of the whole lens tissue.
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Purpose: Epithelial to mesenchymal transition (EMT) is a cause of anterior and posterior subcapsular cataracts. Central to EMT is the formation of actin stress fibers. Selective targeting of actin stress fiber-associated tropomyosin (Tpm) in epithelial cells may be a means to prevent stress fiber formation and repress lens EMT. Methods: We identified Tpm isoforms in mouse immortalized lens epithelial cells and epithelial and fiber cells from whole lenses by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) followed Sanger sequencing. We focused on the role of one particular tropomyosin isoform, Tpm3.1, in EMT. To induce EMT, we treated cells or native lenses with TGFß2. To test the function of Tpm3.1, we exposed cells or whole lenses to a Tpm3.1-specific chemical inhibitor, TR100, as well as investigated lenses from Tpm3.1 knockout mice. We examined stress fiber formation by confocal microscopy and assessed EMT progression by analysis of alpha-smooth muscle actin (αSMA) mRNA (real-time RT-PCR), and protein (Western immunoassay [WES]). Results: Lens epithelial cells express eight Tpm isoforms. Cell culture studies showed that TGFß2 treatment results in the upregulation of Tpm3.1, which associates with actin in stress fibers. TR100 prevents stress fiber formation and reduces αSMA in TGFß2-treated cells. Using an ex vivo lens culture model, TGFß2 treatment results in stress fiber formation at the basal regions of the epithelial cells. Genetic knockout of Tpm3.1 or treatment of lenses with TR100 prevents basal stress fiber formation and reduces epithelial αSMA levels. Conclusions: Targeting specific stress fiber associated tropomyosin isoform, Tpm3.1, is a means to repress lens EMT.
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Actinas/genética , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal/fisiologia , Cristalino/citologia , Fibras de Estresse/metabolismo , Tropomiosina/metabolismo , Animais , Western Blotting , Sobrevivência Celular , Células Epiteliais/efeitos dos fármacos , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Fator de Crescimento Transformador beta2/farmacologiaRESUMO
Culture of human osteoblast-like MG-63 cells within collagen gels results in the generation of intrinsic stress. Release of such collagen gels from attachment results in gel contraction and enhanced MMP-1, MMP-3, and alpha2 integrin mRNA levels. To understand the potential role of microtubules and signaling pathways involved in MG-63 cell-mediated contraction and gene expression, cells were cultured in collagen gels. After 24 h collagen gels were released, then immediately treated with nocodazole or specific protein kinase inhibitors. Contraction was assessed, RNA isolated, and real-time PCR analysis performed. Treatment with high concentrations of a microtubule depolymerization agent, nocodazole, enhanced early contraction and led to elevated mRNA levels for MMP-3, whereas low concentrations inhibited contraction at later time points and did not affect mRNA levels. ROCK inhibitor treatment (Y27632) inhibited collagen gel contraction and led to depressed mRNA levels. The ERK1/2 inhibitor U0126 did not affect contraction, but treatment led to depressed MMP-1, MMP-3, and alpha2 mRNA levels. The p38MAPK inhibitor SB203580 modestly affected contraction, but did not affect mRNA levels. These results suggest the potential role of cytoskeletal integrity and multiple kinase signaling pathways in specific bone-remodeling events.
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Colágeno/metabolismo , Citoesqueleto/metabolismo , Géis/metabolismo , Transdução de Sinais/fisiologia , Linhagem Celular Tumoral , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Integrina alfa2/genética , Integrina alfa2/metabolismo , Metaloproteinase 1 da Matriz/genética , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/genética , Metaloproteinase 3 da Matriz/metabolismo , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Nocodazol/farmacologia , Osteossarcoma , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Moduladores de Tubulina/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismoRESUMO
Chondrocytes dedifferentiate as a result of monolayer culture for cell number expansion. This is associated with the development of an elongated shape, increased actin polymerization, development of stress fibres, and expression of contractile molecules. Given the changes in actin status with dedifferentiation, the hypothesis of this study was that adseverin, an actin severing and capping protein, plays a role in regulating chondrocyte phenotype and function. This study reports that serial passaging of articular chondrocytes in monolayer culture resulted in loss of adseverin protein expression as early as Day 14 of culture and remained repressed in Passage 2 (P2) cells. Knockdown of adseverin by siRNA in primary chondrocytes promoted an increase in cell size and an elongated shape, actin stress fibres, decreased G-/F-actin ratio, and increased number of actin-free barbed ends. The cells also showed increased expression of the contractile genes and proteins, vinculin and α-smooth muscle actin, and increased ability to contract collagen gels. These are all features of dedifferentiation. These effects were due to adseverin as adseverin overexpression following transfection of the green fluorescent protein-adseverin plasmid partially reversed all of these changes in P2 chondrocytes. Furthermore, sox9 and aggrecan chondrogenic gene expression was upregulated, and collagen type I genes expression was downregulated with adseverin overexpression. The change in aggrecan mRNA expression had functional consequence as these cells exhibited increased total proteoglycan synthesis. These findings demonstrate that adseverin regulates features indicative of redifferentiation in passaged articular chondrocytes through modulation of the actin cytoskeleton status and potentially may regulate the maintenance of phenotype in primary chondrocytes.
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Cartilagem Articular/citologia , Condrócitos/metabolismo , Gelsolina/metabolismo , Proteínas dos Microfilamentos/metabolismo , Animais , Bovinos , Diferenciação Celular/genética , Forma Celular , Tamanho Celular , Condrócitos/citologia , Condrogênese/genética , Regulação da Expressão Gênica , Fenótipo , Polimerização , RatosRESUMO
BACKGROUND: Autologous chondrocyte implantation, which uses passaged chondrocytes, commonly leads to the formation of fibrocartilage. When chondrocytes are passaged to increase cell numbers, they lose their phenotype and ability to form hyaline cartilage. The use of transforming growth factor ß (TGFß) to redifferentiate passaged chondrocytes has been validated in vitro; however, it is unknown if redifferentiated chondrocytes will enhance defect repair when implanted in vivo. Furthermore, fibrin gel is used in orthopaedic surgery as a fixative and scaffold and could be an appropriate carrier to enhance retention of cells in the repair site. PURPOSE: To investigate if passaged redifferentiated chondrocytes in fibrin gel have the ability to form cartilage tissue and if these redifferentiated cells will enhance the formation of hyaline cartilage in vivo when implanted into critical-size osteochondral defects. STUDY DESIGN: Controlled laboratory study. METHODS: Rabbit and human chondrocytes were serially passaged twice in monolayer culture. Twice-passaged cells were used directly (dedifferentiated) or redifferentiated in high-density culture with TGFß3. Dedifferentiated or redifferentiated cells were mixed with fibrin gel to form fibrin clots, which were cultured in vitro to assess the use of fibrin gel as a scaffold or implanted in vivo in a critical-size osteochondral defect in New Zealand White rabbit knee joints. Rabbits were sacrificed 6 weeks after implantation, and tissues were assessed histologically and by immunohistochemistry. RESULTS: Redifferentiation of passaged chondrocytes by means of 3-dimensional culture in the presence of TGFß3 improved the formation of cartilaginous tissues in vitro, and culture in fibrin gel did not affect the cell phenotype. Implantation of dedifferentiated cells in vivo resulted in fibrocartilaginous repair tissues. Redifferentiated chondrocyte implants resulted in granulation tissues containing the hyaline cartilage marker collagen type 2. CONCLUSION: Redifferentiated chondrocytes will maintain their chondrogenic differentiation in fibrin clots. Implanted redifferentiated chondrocytes show a different reparative response than dedifferentiated chondrocytes and do not appear to enhance repair at an early time point. Another study of longer duration is required to assess tissue maturation over time. CLINICAL RELEVANCE: Redifferentiation of passaged chondrocytes with TGFß3 before implantation does not improve defect repair in the first 6 weeks.
Assuntos
Artroplastia Subcondral/métodos , Condrócitos/fisiologia , Condrogênese , Fibrina/uso terapêutico , Cartilagem Hialina/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Cartilagem Articular/citologia , Cartilagem Articular/lesões , Diferenciação Celular , Células Cultivadas , Condrócitos/transplante , Colágeno/metabolismo , Feminino , Humanos , Articulação do Joelho , Masculino , Projetos Piloto , Coelhos , Engenharia Tecidual , Fator de Crescimento Transformador beta , Fator de Crescimento Transformador beta3RESUMO
Life-long eye lens function requires an appropriate gradient refractive index, biomechanical integrity and transparency. We conducted an extensive study of wild-type mouse lenses 1-30 months of age to define common age-related changes. Biomechanical testing and morphometrics revealed an increase in lens volume and stiffness with age. Lens capsule thickness and peripheral fiber cell widths increased between 2 to 4 months of age but not further, and thus, cannot account for significant age-dependent increases in lens stiffness after 4 months. In lenses from mice older than 12 months, we routinely observed cataracts due to changes in cell structure, with anterior cataracts due to incomplete suture closure and a cortical ring cataract corresponding to a zone of compaction in cortical lens fiber cells. Refractive index measurements showed a rapid growth in peak refractive index between 1 to 6 months of age, and the area of highest refractive index is correlated with increases in lens nucleus size with age. These data provide a comprehensive overview of age-related changes in murine lenses, including lens size, stiffness, nuclear fraction, refractive index, transparency, capsule thickness and cell structure. Our results suggest similarities between murine and primate lenses and provide a baseline for future lens aging studies.