RESUMEN
Bioreactors are powerful tools with the potential to model tissue development and disease in vitro. For nearly four decades, bioreactors have been used to create tendon and ligament tissue-engineered constructs in order to define basic mechanisms of cell function, extracellular matrix deposition, tissue organization, injury, and tissue remodeling. This review provides a historical perspective of tendon and ligament bioreactors and their contributions to this advancing field. First, we demonstrate the need for bioreactors to improve understanding of tendon and ligament function and dysfunction. Next, we detail the history and evolution of bioreactor development and design from simple stretching of explants to fabrication and stimulation of two- and three-dimensional constructs. Then, we demonstrate how research using tendon and ligament bioreactors has led to pivotal basic science and tissue-engineering discoveries. Finally, we provide guidance for new basic, applied, and clinical research utilizing these valuable systems, recognizing that fundamental knowledge of cell-cell and cell-matrix interactions combined with appropriate mechanical and chemical stimulation of constructs could ultimately lead to functional tendon and ligament repairs in the coming decades.
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Reactores Biológicos/historia , Técnicas In Vitro/historia , Ligamentos/fisiología , Tendones/fisiología , Animales , Fenómenos Biomecánicos , Matriz Extracelular/fisiología , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Técnicas In Vitro/instrumentación , Ortopedia , Medicina Regenerativa , Traumatismos de los Tendones , Ingeniería de TejidosRESUMEN
Advances in mechanobiology have evolved through insights from multiple disciplines including structural engineering, biomechanics, vascular biology, and orthopaedics. In this paper, we reviewed the impact of key reports related to the study of applied loads on tissues and cells and the resulting signal transduction pathways. We addressed how technology has helped advance the burgeoning field of mechanobiology (over 33,600 publications from 1970 to 2016). We analyzed the impact of critical ideas and then determined how these concepts influenced the mechanobiology field by looking at the citation frequency of these reports as well as tracking how the overall number of citations within the field changed over time. These data allowed us to understand how a key publication, idea, or technology guided or enabled the field. Initial observations of how forces acted on bone and soft tissues stimulated the development of computational solutions defining how forces affect tissue modeling and remodeling. Enabling technologies, such as cell and tissue stretching, compression, and shear stress devices, allowed more researchers to explore how deformation and fluid flow affect cells. Observation of the cell as a tensegrity structure and advanced methods to study genetic regulation in cells further advanced knowledge of specific mechanisms of mechanotransduction. The future of the field will involve developing gene and drug therapies to simulate or augment beneficial load regimens in patients and in mechanically conditioning organs for implantation. Here, we addressed a history of the field, but we limited our discussions to advances in musculoskeletal mechanobiology, primarily in bone, tendon, and ligament tissues. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:605-619, 2018.
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Biofisica/historia , Animales , Biofisica/métodos , Historia del Siglo XIX , Historia del Siglo XX , Humanos , Mecanotransducción CelularRESUMEN
Signaling in tenocytes during development, homeostasis and injury involves multiple and redundant pathways. Given that tendons transmit mechanical forces from muscle to bone to effect movement, a key function for tenocytes is the detection of and response to mechanical stimulation. Mechanotransduction involves matrix-integrin-cytoskeleton to nucleus signaling, gap junction intercellular communication, changes in intracellular calcium (Ca(2+)), activation of receptors and their pathways, and responses to biochemical factors such as hormones, growth factors, adenosine triphosphate (ATP) and its derivatives, and neuromodulators. The primary cilium also plays a key role in the detection of mechanical signals. During development, transforming growth factor-ß (TGF-ß), bone morphogenetic protein (BMP), and hedgehog (Hh) signaling modulate tendon differentiation and formation. The response to injury is complex and varied involving not only inflammatory mediators such as interleukin-1ß but also mechanosensing. This chapter reviews the signaling pathways tenocytes use during mechanotransduction, development and in response to injury.
Asunto(s)
Enfermedad , Mecanotransducción Celular , Transducción de Señal , Estrés Mecánico , Tendones/metabolismo , Tenocitos/metabolismo , Animales , Comunicación Celular , Fenómenos Fisiológicos Celulares , Humanos , Tendones/citología , Tenocitos/citología , Cicatrización de HeridasRESUMEN
Tendons mainly function as load-bearing tissues in the muscloskeletal system; transmitting loads from muscle to bone. Tendons are dynamic structures that respond to the magnitude, direction, frequency, and duration of physiologic as well as pathologic mechanical loads via complex interactions between cellular pathways and the highly specialized extracellular matrix. This paper reviews the evolution and current knowledge of mechanobiology in tendon development, homeostasis, disease, and repair. In addition, we review several novel mechanotransduction pathways that have been identified recently in other tissues and cell types, providing potential research opportunities in the field of tendon mechanobiology. We also highlight current methods, models, and technologies being used in a wide variety of mechanobiology research that could be investigated in the context of their potential applicability for answering some of the fundamental unanswered questions in this field. The article concludes with a review of the major questions and future goals discussed during the recent ORS/ISMMS New Frontiers in Tendon Research Conference held on September 10 and 11, 2014 in New York City.
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Tendones/fisiología , Animales , Fenómenos Biomecánicos , Investigación Biomédica , Humanos , Soporte de PesoRESUMEN
The author started a niche biotech company in 1985 called Flexcell® to distribute an enabling technology, mechanobiology devices, to the field. He was the first University of North Carolina faculty member to start a company and stay with it as he pursued his career in academics. That was an unpopular route at that time, but a path he was driven to navigate. Those interests, merged with his training, led to the design and manufacture of mechanobiology devices such as the Flexercell® Strain Unit and the BioFlex® flexible bottom culture plates to study fundamental responses of cells to strain. Principles in these devices were also incorporated into bioreactors for tissue engineering, which are standard in the marketplace today. In this article, the major roadblocks will be chronicled that were overcome to help build the field of mechanobiology and create a small biotechnology company. Through example, the author's formula for achieving milestones will be discussed including, the DRIVE it takes to get there ["DRIVE": Determination (Confidence), Research and Development (R&D) and Risk-Taking, Innovation (Imagination) and Intellectual Property, achieving Victory, and Enterprise].
RESUMEN
Adipose-derived stem cells (ASC) are multipotent stem cells that show great potential as a cell source for osteogenic tissue replacements and it is critical to understand the underlying mechanisms of lineage specification. Here we explore the role of primary cilia in human ASC (hASC) differentiation. This study focuses on the chemosensitivity of the primary cilium and the action of its associated proteins: polycystin-1 (PC1), polycystin-2 (PC2) and intraflagellar transport protein-88 (IFT88), in hASC osteogenesis. To elucidate cilia-mediated mechanisms of hASC differentiation, siRNA knockdown of PC1, PC2 and IFT88 was performed to disrupt cilia-associated protein function. Immunostaining of the primary cilium structure indicated phenotypic-dependent changes in cilia morphology. hASC cultured in osteogenic differentiation media yielded cilia of a more elongated conformation than those cultured in expansion media, indicating cilia-sensitivity to the chemical environment and a relationship between the cilium structure and phenotypic determination. Abrogation of PC1, PC2 and IFT88 effected changes in both hASC proliferation and differentiation activity, as measured through proliferative activity, expression of osteogenic gene markers, calcium accretion and endogenous alkaline phosphatase activity. Results indicated that IFT88 may be an early mediator of the hASC differentiation process with its knockdown increasing hASC proliferation and decreasing Runx2, alkaline phosphatase and BMP-2 mRNA expression. PC1 and PC2 knockdown affected later osteogenic gene and end-product expression. PC1 knockdown resulted in downregulation of alkaline phosphatase and osteocalcin gene expression, diminished calcium accretion and reduced alkaline phosphatase enzymatic activity. Taken together our results indicate that the structure of the primary cilium is intimately associated with the process of hASC osteogenic differentiation and that its associated proteins are critical players in this process. Elucidating the dynamic role of the primary cilium and its associated proteins will help advance the application of hASC in generating autologous tissue engineered therapies in critical defect bone injuries.
Asunto(s)
Tejido Adiposo/citología , Diferenciación Celular/fisiología , Cilios/fisiología , Células Madre Multipotentes/fisiología , Osteogénesis/fisiología , Análisis de Varianza , Cilios/ultraestructura , Técnicas de Silenciamiento del Gen , Ingeniería Genética/métodos , Humanos , Microscopía Fluorescente , Interferencia de ARN , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Canales Catiónicos TRPP/genética , Canales Catiónicos TRPP/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Mechanical stimuli play important roles in proliferation and differentiation of connective tissue cells, and development and homeostatic maintenance of tissues. However, excessive mechanical loading to a tissue can injure cells and disrupt the matrix, as occurs in tendinopathy. Tendinopathy is a common clinical problem in athletes and in many occupational settings due to overuse of the tendon. Moreover, interleukin (IL)-1ß is generally considered to be a "bad" cytokine, activating NF-κb and cell death and inducing matrix metalloproteinase (MMPs 1, 2, 3) expression and matrix destruction. However, activated NF-κB can also drive a cell survival pathway. We have reported that cyclic strain induced tenocyte death in three-dimensional (3D) cultures, and IL-1ß could promote cell survival under strain. Therefore, it was hypothesized that 1) cyclic strain could induce cell death in tenocytes as observed in pathologic tendons in vivo; 2) a gene expression profile indicative of tendinopathy could be identified; and 3) low-dose IL-1ß could protect cells from strain-induced, tendinopathy-like changes. Human tenocytes were cultured in 3D type I collagen hydrogels and subjected to 3.5% elongation at 1 Hz for 1 h/day for up to 5 days with or without IL-1ß. Real-time RT-PCR data showed that cyclic strain regulated the expression of tendinopathy marker genes in a manner similar to that found in pathological tendons from patients and that addition of IL-1ß reversed the gene expression changes to control levels. Results of further studies showed that IL-1ß may modulate cell survival through upregulating the expression of connexin 43, which is involved in the modulation of cell death/survival in a variety of cells and tissues. The elucidation of the mechanisms underlying strain-induced cell death and recovery from strain injury will facilitate our understanding of the pathogenesis of tendinopathy and may lead to the discovery of new molecular targets for early diagnosis and treatment of tendinopathy.
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Fibroblastos/fisiología , Interleucina-1beta/metabolismo , Mecanotransducción Celular/fisiología , Tendones/fisiología , Supervivencia Celular/fisiología , Células Cultivadas , Módulo de Elasticidad/fisiología , Humanos , Estrés MecánicoRESUMEN
Understanding the mechanisms that regulate mechanosensitivity in osteoblasts is important for controlling bone homeostasis and the development of new drugs to combat bone loss. It is believed that prestress or force generation (the tensile stress within the cell body) plays an important role in regulating cellular mechanosensitivity. In the present study, a three-dimensional (3D) collagen culture was used to monitor the change in prestress of the osteoblast-like cells. Collagen hydrogel compaction has been used as an indicator of the change in the degree of cell prestress. Previous results in this model demonstrated that extracellular ATP reduced the mechanosensitivity of osteoblasts by reducing cellular prestress. To elucidate the potential mechanisms involved in this process, the signaling pathways downstream of P2 purinoceptors involved in regulating the compaction of type I collagen gels were investigated. By using specific inhibitors to these signaling pathways, we found that ATP-induced reduction in collagen gel compaction rate is dependent on mitogen-activated protein kinase (MAKP) and NF-kappaB pathways. However, blocking protein kinase C with GF109203X did not change the compaction kinetics in the presence of ATPgammaS. Moreover, blocking cyclic AMP (cAMP), phosphatidylinositol-3 kinase (PI3K), calmodulin (CaM) or L-type voltage sensitive calcium channels did not affect ATP's ability to reduce collagen gel compaction. The results from the present and previous studies indicate that extracellular ATP may act as a negative feedback modulator in the mechanotransduction system since mechanical stimuli increase ATP release from stimulated cells.
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Adenosina Trifosfato/metabolismo , Colágeno/metabolismo , Sistema de Señalización de MAP Quinasas , FN-kappa B/metabolismo , Células 3T3 , Adenosina Trifosfato/farmacología , Adenilil Ciclasas/metabolismo , Animales , Secuencia de Bases , Calmodulina/metabolismo , Colágeno/genética , Cartilla de ADN/genética , Líquido Extracelular/metabolismo , Geles , Expresión Génica/efectos de los fármacos , Cinética , Mecanotransducción Celular , Ratones , Modelos Biológicos , Quinasa de Cadena Ligera de Miosina/metabolismo , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Receptores Purinérgicos P2/metabolismo , Estrés MecánicoRESUMEN
We investigated the effects of two types of cyclic tensile strain, continuous and rest inserted, on osteogenic differentiation of human adipose-derived adult stem cells (hASCs). The influence of these mechanical strains was tested on two hASC lines having different mineral deposition potential, with one cell line depositing approximately nine times as much calcium as the other hASC line after 14 days of culture in osteogenic medium on tissue culture plastic. Results showed that both continuous (10% strain, 1 Hz) and rest inserted cyclic tensile strain (10% strain, 1 Hz, 10 s rest after each cycle) regimens increased the amount and rate of calcium deposition for both high and low calcium depositing hASC lines as compared to unstrained controls. The response was similar for both types of tensile strain for a given cell line, however, cyclic tensile strain had a much stronger osteogenic effect on the high calcium depositing hASC line, suggesting that mechanical loading has a greater effect on cell lines that already have an innate ability to produce bone as compared to cell lines that do not. This is the first study to investigate the osteodifferentiation effects of cyclic tensile strain on hASCs and the first to show that both continuous (10%, 1 Hz) and rest inserted (10%, 1 Hz, 10 s rest) cyclic tensile strain accelerate hASC osteodifferentiation and increase calcium accretion.
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Modelos Biológicos , Osteogénesis/fisiología , Tejido Adiposo/citología , Células Madre Adultas , Calcio/metabolismo , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Humanos , Células Madre Mesenquimatosas , Estrés Mecánico , Resistencia a la Tracción , Ingeniería de Tejidos/métodosRESUMEN
Titin (also called connectin) is a major protein in sarcomere assembly as well as providing elastic return of the sarcomere postcontraction in cardiac and striated skeletal muscle tissues. In addition, it has been speculated that titin is associated with nuclear functions, including chromosome and spindle formation, and regulation of muscle gene expression. In the present study, a short isoform of titin was detected in a human osteoblastic cell line, MG-63 cells, by both immunostaining and Western blot analysis. Confocal images of titin staining showed both cytoplasmic and nuclear localization in a punctate pattern. Therefore, we hypothesized that human titin may contain a nuclear localization signal (NLS). A functional NLS, 200-PAKKTKT-206, located in a low-complexity, titin-specific region between Z2 and Z repeats, was found by sequentially deleting segments of the NH(2)-terminal sequence in conjunction with an enhanced green fluorescent protein reporter system and confirmed by site-directed mutagenesis. Overexpression of titin's amino terminal fragment (Z1Z2Zr) in human osteoblasts (MG-63) increased cell proliferation by activating the Wnt/beta-catenin pathway. RT-PCR screens of tissue panels demonstrated that residues 1-206 were ubiquitously expressed at low levels in all tissues and cell types analyzed. Our data implicate a dual role for titin's amino terminal region, i.e., a novel nuclear function promoting cell division in addition to its known structural role in Z-line assembly.
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Proteínas Musculares/química , Proteínas Quinasas/química , Transporte Activo de Núcleo Celular , Animales , Línea Celular , Proliferación Celular , Chlorocebus aethiops , Conectina , Cricetinae , Regulación de la Expresión Génica/fisiología , Silenciador del Gen , Humanos , Ratones , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Estructura Terciaria de Proteína , Proteínas Wnt/metabolismo , alfa Catenina/metabolismo , beta Catenina/metabolismoRESUMEN
Tendon overuse injuries are a major source of clinical concern. Cyclic loading causes material damage and induces biochemical responses in tendon. The purpose of this study was to examine the biochemical and biomechanical tendon response after applying cyclical loading over varying durations. Avian flexor digitorum profundus tendons were loaded (3 or 12 MPa) to a fixed number of cycles across either 1 or 12 days in vitro. The tendon response evaluations included biomechanical data gathered during loading and subsequent failure testing. Evaluations also included cellular viability, cell death, and proteoglycan, collagen, collagenase, and prostaglandin E(2) (PGE(2)) content measurements obtained from tissue specimens and media samples. Significant strains (up to 2%) accumulated during loading. Loading to 12 MPa significantly reduced maximum stress (33% and 27%) and energy density (42% and 50%) when applied across 1 or 12 days, respectively. Loading to 3 MPa also caused a 40% reduction in energy density, but only when applied across 12 days. Cell death and collagenase activity increased significantly with increasing magnitude and duration. However, no differences occurred in cell viability or collagen content. Glycosaminoglycan content increased 50% with load magnitude, while PGE(2) production increased 2.5-fold with loading magnitude and 11-fold with increased duration. Mechanical fatigue-induced mechanical property changes were exhibited by the tendons in response to increased loading magnitude across just 1 day. However, when the same loading was applied over a longer period, most outcomes were magnified substantially, relative to the short duration regimens. This is presumably due to the increased response time for the complex cellular response to loading. A key contributor may be the inflammatory mediator, PGE(2), which exhibited large magnitude and duration dependent increases to cyclic loading.
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Trastornos de Traumas Acumulados/fisiopatología , Traumatismos de los Tendones/fisiopatología , Tendones/fisiopatología , Animales , Fenómenos Biomecánicos , Muerte Celular , Pollos , Colagenasas/metabolismo , Dinoprostona/metabolismo , Femenino , Glicosaminoglicanos/metabolismo , Técnicas de Cultivo de Órganos , Estrés Mecánico , Tendones/citología , Factores de Tiempo , Soporte de PesoRESUMEN
Varieties of cell-matrix or cell-cell adhesions are associated with the actin cytoskeleton. However, for gap junctions, which are both channels and adhesions, there has been little evidence for such an association. The purpose of this study was to determine if connexin 43 (Cx43) associates with actin and to determine if this association is altered under mechanical load in tenocytes, a mechanically sensitive cell. Avian tenocytes were subjected to multiple cyclic strain regimens and then fixed and examined immunohistochemically at various times poststrain to determine where Cx43 protein was localized within the cells in relation to actin filaments. Four percent of tenocytes had colocalization of actin filaments and Cx43, which was significantly increased with 5% cyclic strain. To confirm this phenomenon, human tenocytes and COS-7 cells were also subjected to cyclic strain and then fixed at the same times after strain. As with avian tenocytes, Cx43 was colocalized with actin in human tenocytes and COS-7 cells. The colocalization increased significantly after cyclic strain in human tenocytes but not in COS-7 cells. The lack of detectable changes in COS-7 cells may indicate that they are less mechanosensitive than tenocytes perhaps due to the less robust actin cytoskeleton seen in the COS-7 cells when compared to the tenocytes. Furthermore, inhibiting myosin II activity greatly reduced the immunohistochemically-detectable Cx43 on actin filaments. Connexins may associate with actin to stabilize gap junctions at the plasma membrane, ensuring that tenocytes remain coupled during periods of prolonged or intense mechanical loading.
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Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Conexina 43/metabolismo , Tendones/citología , Actinas/análisis , Adulto , Animales , Aves , Células COS , Células Cultivadas , Preescolar , Chlorocebus aethiops , Conexina 43/análisis , Femenino , Fibroblastos/química , Uniones Comunicantes/química , Uniones Comunicantes/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Miosina Tipo II/metabolismo , Estrés Mecánico , Tendones/químicaRESUMEN
Strain magnitudes within tenocytes undergoing substrate tensile strain are not well defined. It was hypothesized that strain magnitudes at the cellular level would reflect those of the applied substrate (equibiaxial or uniaxial) strain. A vacuum-operated device was used to apply equibiaxial or uniaxial tension to a flexible substrate upon which tenocytes were cultured in monolayer. Images of tenocytes labeled with Fura-2, to detect free intracellular calcium ions, and MitoFluor Green, to detect mitochondria, were taken prior to strain and for 20 min during application of static strain. A custom-written, texture correlation program computed strain magnitudes in the cell based on the change in pixel pattern displacements between images of non-strained and strained cells. On average, cellular strain was approximately 37+/-8% and 63+/-11% of the applied equibiaxial and uniaxial substrate strain, respectively. The largest cell strains were detected in cells oriented parallel to the direction of applied uniaxial tensile strain. However, strain magnitudes within a cell were heterogeneous. The variance in strain magnitude within and among tenocytes is dependent on cell orientation, cell stiffness, cytoskeleton organization, subcellular organelles, or placement and type of cell-substrate contacts. Results of the present study indicate that cultured tenocytes experience a moderate fraction of the applied substrate strain.
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Tendones/citología , Tendones/fisiología , Animales , Fenómenos Biomecánicos , Calcio/metabolismo , Células Cultivadas , Pollos , Técnicas In Vitro , Mitocondrias/metabolismo , Modelos Biológicos , Modelos Estadísticos , Estrés Mecánico , Resistencia a la TracciónRESUMEN
Bone remodeling is a localized process, but regulated by systemic signals such as hormones, cytokines, and mechanical loading. The mechanism by which bone cells convert these systemic signals into local signals is not completely understood. It is broadly accepted that the "prestress" in cytoskeleton of cells affects the magnitude of cellular responses to mechanical stimuli. Prestress derives from stiff cytoskeletal proteins and their connections within the cell and from cell contractility upon attaching to matrix. In an in vitro model of three-dimensional gel compaction, the relative cellular prestress levels in the same matrix environment were determined by matrix compaction rate: a greater compaction rate resulted in a higher level of prestress. In the present study, the effects of ATP on the prestress of osteoblasts were studied using mouse MC3T3-E1 cells grown in three-dimensional bioartificial tissues (BATs). ATP (> or =100 microM) reduced the compaction rate of BATs in a dose-dependent manner. ADP, 2'-(or 3')-O-(4-benzoylbenzoyl) ATP, and UTP, but not alpha,beta-methylene ATP, also reduced the compaction rate but to a lesser extent. Pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid tetrasodium did not block the effect of ATP on BAT compaction rate. These results indicate that both P2X and P2Y receptors are involved in ATP-induced reduction of BAT compaction rate. Steady fluid flow and RT-PCR results showed that ATP reduced cell attachment on type I collagen by downregulating the expression of integrin alpha(1). These results suggest a potential role for P2 receptors in matrix remodeling and repair and as a potential drug target in treatment of bone diseases.
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Adenosina Trifosfato/fisiología , Remodelación Ósea/fisiología , Osteoblastos/fisiología , Adenosina Trifosfato/metabolismo , Animales , Fenómenos Biomecánicos , Adhesión Celular , Técnicas de Cultivo de Célula , Línea Celular , Supervivencia Celular , Colágeno Tipo I , Líquido Extracelular , Perfilación de la Expresión Génica , Hidrólisis , Cinética , Ratones , Nucleótidos/fisiología , Receptores Purinérgicos P2/fisiología , Estrés MecánicoRESUMEN
Cellular responses to mechanical stimuli are regulated by interactions with the extracellular matrix, which, in turn, are strongly influenced by the degree of cell stiffness (Young's modulus). It was hypothesized that a more elastic cell could better withstand the rigors of remodeling and mechanical loading. It was further hypothesized that interleukin-1beta (IL-1beta) would modulate intracellular cytoskeleton polymerization and regulate cell stiffness. The purpose of this study was to investigate the utility of IL-1beta to alter the Young's modulus of human tenocytes. Young's modulus is the ratio of the stress to the strain, E = stress/strain = (F/A)/(deltaL/L0), where L0 is the equilibrium length, deltaL is the length change under the applied stress, F is the force applied, and A is the area over which the force is applied. Human tenocytes were incubated with 100 pM recombinant human IL-1beta for 5 days. The Young's modulus was reduced by 27-63%. Actin filaments were disrupted in >75% of IL-1beta-treated cells, resulting in a stellate shape. In contrast, immunostaining of alpha-tubulin showed increased intensity in IL-1beta-treated tenocytes. Human tenocytes in IL-1beta-treated bioartificial tendons were more tolerant to mechanical loading than were untreated counterparts. These results indicate that IL-1beta reduced the Young's modulus of human tenocytes by disrupting the cytoskeleton and/or downregulating the expression of actin and upregulating the expression of tubulins. The reduction in cell modulus may help cells to survive excessive mechanical loading that may occur in damaged or healing tendons.
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Forma de la Célula/efectos de los fármacos , Forma de la Célula/fisiología , Interleucina-1/fisiología , Tendones/citología , Citoesqueleto de Actina/química , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/fisiología , Actinas/metabolismo , Anciano , Anciano de 80 o más Años , Fenómenos Biomecánicos , Supervivencia Celular/fisiología , Células Cultivadas , Preescolar , Elasticidad/efectos de los fármacos , Femenino , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/fisiología , Regulación de la Expresión Génica/fisiología , Humanos , Masculino , Microtúbulos/química , Microtúbulos/efectos de los fármacos , Microtúbulos/fisiología , Tendones/efectos de los fármacos , Tendones/fisiología , Tubulina (Proteína)/metabolismo , Soporte de PesoRESUMEN
The mouse has become the most important model organism for the study of human physiology and disease. However, until the recent generation of mice lacking the enzyme gulanolactone oxidase (Gulo), the final enzyme in the ascorbic acid biosynthesis pathway, examination of the role of ascorbic acid in various biochemical processes using this model organism has not been possible. In the mouse, similar to most mammals but unlike humans who carry a mutant copy of this gene, Gulo produces ascorbic acid from glucose. We report here that, although ascorbic acid is essential for survival, its absence does not lead to measurable changes in proline hydroxylation. Vitamin C deficiency had no significant effect on the hydroxylation of proline and collagen production during tumor growth or in angiogenesis associated with tumor or mammary gland growth. This suggests that factors other than ascorbic acid can support proline hydroxylation and collagen synthesis in vivo. Furthermore, the failure of Gulo-/- mice to thrive on a vitamin C-deficient diet therefore suggests that ascorbic acid plays a critical role in survival other than the maintenance of the vasculature.
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Ácido Ascórbico/fisiología , Colágeno/biosíntesis , Glándulas Mamarias Animales/irrigación sanguínea , Neoplasias Mamarias Experimentales/irrigación sanguínea , Animales , Ácido Ascórbico/genética , Ácido Ascórbico/metabolismo , Femenino , Hidroxilación , Hidroxiprolina/metabolismo , Glándulas Mamarias Animales/crecimiento & desarrollo , Glándulas Mamarias Animales/metabolismo , Neoplasias Mamarias Experimentales/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Neovascularización Patológica/metabolismo , Neovascularización Fisiológica , Prolina/metabolismo , Prolina/farmacocinética , Piel/metabolismo , Pérdida de PesoRESUMEN
Stiffness is an important mechanical property of connective tissues, especially for tissues subjected to cyclic strain in vivo, such as tendons. Therefore, modulation of material properties of native or engineered tissues is an important consideration for tissue repair. Interleukin 1-beta (IL-1beta) is a cytokine most often associated in connective tissues with induction of matrix metalloproteinases and matrix destruction. However, IL-1beta may also be involved in constructive remodeling and confer a cell survival value to tenocytes. In this study, we investigated the effects of IL-1beta on the properties of human tenocyte-populated bioartificial tendons (BATs) fabricated in a novel three-dimensional (3D) culture system. IL-1beta treatment reduced the ultimate tensile strength and elastic modulus of BATs and increased the maximum strain. IL-1beta at low doses (1, 10 pM) upregulated elastin expression and at a high dose (100 pM) downregulated type I collagen expression. Matrix metalloproteinases, which are involved in matrix remodeling, were also upregulated by IL-1beta. The increased elasticity prevented BATs from rupture caused by applied strain. The results in this study suggest that IL-1beta may act as a defense/survival factor in response to applied mechanical loading. The balance between cell intrinsic strain and external matrix strain is important for maintaining the integrity of tendons.
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Órganos Bioartificiales , Interleucina-1beta/administración & dosificación , Técnicas de Cultivo de Órganos/métodos , Tendones/citología , Tendones/fisiología , Ingeniería de Tejidos/métodos , Células Cultivadas , Fuerza Compresiva/efectos de los fármacos , Fuerza Compresiva/fisiología , Relación Dosis-Respuesta a Droga , Elasticidad , Análisis de Falla de Equipo , Humanos , Estrés Mecánico , Tendones/efectos de los fármacos , Resistencia a la Tracción/efectos de los fármacos , Resistencia a la Tracción/fisiologíaRESUMEN
An in vitro model was used to investigate the effect of mechanical stimuli on adaptation to load and calcium signaling in aligned medial collateral ligament cells (MCL). This model used a patterned silicone membrane to align the cells parallel with the direction of the microgrooves. Alignment created an architecture that simulated a degree of cell orientation in native ligament tissue. It was hypothesized that aligned ligament cells would be more efficient at calcium wave propagation than cells that were randomly oriented. It was further hypothesized that calcium wave propagation would be greater among cells that were both aligned and subjected to mechanical stretch compared to cells that were aligned but not stretched. Rat MCL cells were loaded with Fura-2AM, a calcium-binding dye, and mechanically indented using a micropipette tip. A ratio-imaging fluorescence technique was used to quantitate the calcium (Ca2+) response. It was concluded that stretching ligament cells prior to stimulation increased their sensitivity to load and their ability to propagate a calcium wave. However, the ability of aligned cells to propagate this wave was not significantly different when compared to nonaligned cells. Treatment of cultures with inhibitors such as apyrase and suramin significantly reduced the number of cells recruited in the calcium response. Hence, it was concluded that ATP released from mechanically stimulated cells was a principal mediator responsible for the rise in intracellular calcium in ligament cells. Further, purinoceptor activation may amplify the signal to alert and recruit more cells in a response to mechanical stimulation.
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Reactores Biológicos , Técnicas de Cultivo de Célula/instrumentación , Ligamentos/citología , Ligamentos/fisiología , Mecanotransducción Celular/fisiología , Estimulación Física/instrumentación , Adaptación Fisiológica/fisiología , Animales , Materiales Biocompatibles/química , Señalización del Calcio/fisiología , Comunicación Celular/fisiología , Técnicas de Cultivo de Célula/métodos , Células Cultivadas , Elasticidad , Estimulación Física/métodos , Ratas , Ratas Sprague-Dawley , Estrés Mecánico , Propiedades de SuperficieRESUMEN
Sympathetic efferent nerves are present in tendons, but their function within tendon is unknown. alpha(1)-Adrenoceptors are expressed by a variety of cell types. In the presence of norepinephrine (NE), adrenoceptors activate G(q/11) signaling pathways that subsequently increase intracellular Ca(2+) concentration ([Ca(2+)](ic)). It was hypothesized that avian tendon cells express functional adrenoceptors that respond to NE by increasing [Ca(2+)](ic). Avian tendon cells were analyzed for mRNA expression of alpha(1)-adrenoceptors by RT-PCR. Avian tendons expressed the alpha(1A)- and alpha(1B)-adrenoceptor subtypes. Furthermore, both tendon surface epitenon cells and internal fibroblasts infused with a Ca(2+)-sensitive dye, fura 2, and stimulated with NE responded by increasing [Ca(2+)](ic). KMD-3213, an alpha(1A)-adrenoceptor antagonist, significantly reduced the Ca(2+) response. Other adrenoceptor antagonists had no effect on the Ca(2+) response. The absence of extracellular Ca(2+) also significantly reduced the response to NE, indicating that Ca(2+) influx contributed to the rise in [Ca(2+)](ic). This study provides the first evidence that tendon cells express adrenoceptors and that the NE-induced Ca(2+) response is coupled to the alpha(1A)-adrenoceptor subtype.