RESUMO
BACKGROUND: The transparency of the cornea is regulated by the unique organization of collagen fibrils (CFs) which is maintained by proteoglycans (PGs). The interlacing of CF lamellae in the anterior stroma provides the biomechanical properties of the cornea. OBJECTIVE: To investigate the alterations of CFs and PGs in the swollen cornea, with special reference to the anterior stroma by using electron microscopy and 3D ultrastructural tomography. METHOD: Nine healthy normal scleral corneal rings (age from 40 to 65 years) were hydrated individually in deionised water to induce swelling in the cornea. Three of them were hydrated for 2hr whereas the other three were hydrated for 48hr. The remaining three scleral normal corneal rings were used as a control.The corneas were processed for electron microscopy (EM) to study the CFs and PGs. Ultrathin sections were observed using transmission electron microscopy (JOEL 1400) and digital images of CFs, PGs and lamellae were captured using a bottom mounted Quemesa camera and iTEM Soft Imaging System. The software program 'Composer-x64, version 3.4.2.0' was used to construct individual 3D images from 120 digital images taken from -60 to + 60 degree angles. RESULTS: The 3D tomography showed the degeneration of microfibrils within the CFs of the swollen cornea. The CF diameter was significantly reduced and the interfibrillar spacing significantly increased in both the 2hr and 48hr hydrated corneas compared to the normal cornea. Within the hydrated corneas, the CF diameter was smaller and the interfibrillar spacing was increased in the middle and posterior stroma compared to the anterior stroma. The PG area in both the 2hr and the 48hr hydrated cornea was reduced in the anterior stroma, whereas it was increased in middle and posterior stroma compared to the normal cornea. The density of the PGs in both the 2hr and the 48hr samples, was reduced compared to the density of PGs in the normal cornea. CONCLUSION: The CFs, PGs and lamellae had degenerated, caused by swelling. 3D imaging demonstrated that the impairment of the microfibrils and PGs within the CF, is caused by the excessive hydration or swelling in the anterior as well as in the middle and posterior stroma. The lamellae of the anterior stroma which provides the biomechanical strength in the normal cornea, had degenerated in the swollen corneas due to the presence of the damaged CFs and PGs.
Assuntos
Colágeno/ultraestrutura , Substância Própria/ultraestrutura , Proteoglicanas/ultraestrutura , Adulto , Idoso , Edema/patologia , Tomografia com Microscopia Eletrônica , Feminino , Humanos , Imageamento Tridimensional , Masculino , Pessoa de Meia-Idade , Técnicas de Cultura de ÓrgãosRESUMO
Microfluidic devices are becoming mainstream tools to recapitulate in vitro the behavior of cells and tissues. In this study, we use microfluidic devices filled with hydrogels of mixed collagen-Matrigel composition to study the migration of lung cancer cells under different cancer invasion microenvironments. We present the design of the microfluidic device, characterize the hydrogels morphologically and mechanically and use quantitative image analysis to measure the migration of H1299 lung adenocarcinoma cancer cells in different experimental conditions. Our results show the plasticity of lung cancer cell migration, which turns from mesenchymal in collagen only matrices, to lobopodial in collagen-Matrigel matrices that approximate the interface between a disrupted basement membrane and the underlying connective tissue. Our quantification of migration speed confirms a biphasic role of Matrigel. At low concentration, Matrigel facilitates migration, most probably by providing a supportive and growth factor retaining environment. At high concentration, Matrigel slows down migration, possibly due excessive attachment. Finally, we show that antibody-based integrin blockade promotes a change in migration phenotype from mesenchymal or lobopodial to amoeboid and analyze the effect of this change in migration dynamics, in regards to the structure of the matrix. In summary, we describe and characterize a robust microfluidic platform and a set of software tools that can be used to study lung cancer cell migration under different microenvironments and experimental conditions. This platform could be used in future studies, thus benefitting from the advantages introduced by microfluidic devices: precise control of the environment, excellent optical properties, parallelization for high throughput studies and efficient use of therapeutic drugs.
Assuntos
Movimento Celular , Colágeno , Laminina , Microfluídica , Proteoglicanas , Alicerces Teciduais , Linhagem Celular Tumoral , Colágeno/química , Colágeno/ultraestrutura , Difusão , Combinação de Medicamentos , Matriz Extracelular , Humanos , Hidrogéis , Laminina/química , Laminina/ultraestrutura , Fenômenos Mecânicos , Microfluídica/métodos , Microscopia Confocal , Metástase Neoplásica , Fenótipo , Proteoglicanas/química , Proteoglicanas/ultraestrutura , Esferoides Celulares , Alicerces Teciduais/química , Células Tumorais Cultivadas , Microambiente TumoralRESUMO
We investigate the ultrastructural features and 3D electron tomography of chameleon (Chamaeleon calyptratus) which is a native of desert environments of Saudi Arabia. The corneas of the chameleon were fixed in 2.5% glutaraldehyde containing cuprolinic blue in sodium acetate buffer for electron microscopy and tomography, and observed under a JEOL 1400 transmission electron microscope. The thin cornea (21.92 µm) contained 28-30 collagen fibril lamellae. The middle stromal lamellae (from 13 to 19) contained keratocytes with a long cell process and filled with granular material. The CF diameter increased from lamella 1 (30.44 ± 1.03) to Lamella 5 (52.83 ± 2.00) then decreased towards the posterior stoma. The percentage of large CF diameters (55-65 nm) was very high in the lamellae L14 (38.8%) and L15 (85.7%). The mean PGs area of the posterior stroma (448.21 ± 24.84 nm2 ) was significantly larger than the mean PGs area of the anterior, (309.86 ± 8.2 nm2 ) and middle stroma 245.94 ± 8.28 nm2 ). 3D electron tomography showed the distribution of PGs around and over the CF. Variable diameters of CFs in the anterior stroma may provide compact lamellae which may restrict the low wavelength of light. Variable diameters of CFs in the anterior stroma may provide compact lamellae which may restrict the low wavelength of light. This accommodation function is achieved by bending of the cornea. During bending the anterior stroma was stretched and the posterior stroma was compressed due to the presence of small CFs. The middle stroma remained stiff due to the presence of large CFs. Large proteoglycans not only maintain hydration for a longer period of time, but also act as a lubricant to neutralise the shear forces in the anterior and posterior stroma during bending.
Assuntos
Córnea/anatomia & histologia , Córnea/ultraestrutura , Lagartos/anatomia & histologia , Animais , Ceratócitos da Córnea/citologia , Tomografia com Microscopia Eletrônica/métodos , Colágenos Fibrilares/ultraestrutura , Imageamento Tridimensional/métodos , Proteoglicanas/ultraestrutura , Arábia SauditaRESUMO
Osteoarthritis and articular cartilage injuries are common conditions in human joints and a frequent cause of pain and disability. Unfortunately, cartilage is avascular and has limited capabilities for self-repair. Despite the societal impact, there is little information on the dynamic process of cartilage degeneration. We performed a series of cyclic unconfined compression tests motivated by in vivo loading conditions and designed to generate mechanical fatigue. We examined the functional (both stress-stretch and creep) responses of the tissue after recovery from a specified number of loading cycles, as well as histology and second harmonic generation microscopy images. The effect of compaction was complimented by the effect of fatigue in our unconfined compression tests. A three-way, repeated-measures mixed model ANOVA showed significant differences between loading with a physiologically relevant low magnitude, and two more severe loading magnitudes, in terms of the resulting specimen stiffness, time to equilibrium and thickness. There was a statistically significant effect of loading frequency on a specimen's time to equilibrium and significant interaction of force and frequency on specimen thickness and time to equilibrium. Increasing the number of loading cycles significantly impacted a specimen's effective stiffness and resulting thickness. We attribute permanent loss of mechanical function under cyclic loading to rearrangement and disruption of the collagen network and resulting proteoglycan (PG) aggregation, as seen in histological and second harmonic generation images, as a result of induced mechanical fatigue.
Assuntos
Cartilagem Articular/fisiologia , Estresse Mecânico , Suporte de Carga , Fenômenos Biomecânicos , Colágeno/ultraestrutura , Humanos , Proteoglicanas/ultraestruturaRESUMO
Tendons are composed of collagen fibrils and proteoglycan predominantly consisting of decorin. Decorin is located on the d-band of collagen fibrils, and its glycosaminoglycan (GAG) chains have been observed between collagen fibrils with transmission electron microscopy. GAG chains have been proposed to interact with each other or with collagen fibrils, but its three-dimensional organization remains unclear. In this report, we used focused ion beam scanning electron microscopy to examine the three-dimensional organization of the GAG chain in the Achilles tendon of mature rats embedded in epoxy resin after staining with Cupromeronic blue, which specifically stains GAG chains. We used 250 serial back-scattered electron images of longitudinal sections with a 10-nm interval for reconstruction. Three-dimensional images revealed that GAG chains form a ring mesh-like structure with each ring surrounding a collagen fibril at the d-band and fusing with adjacent rings to form the planar network. This ring mesh model of GAG chains suggests that more than two GAG chains may interact with each other around collagen fibrils, which could provide new insights into the roles of GAG chains.
Assuntos
Tendão do Calcâneo/ultraestrutura , Glicosaminoglicanos/ultraestrutura , Microscopia Eletrônica de Varredura/métodos , Proteoglicanas/ultraestrutura , Tendão do Calcâneo/química , Animais , Glicosaminoglicanos/química , Imageamento Tridimensional/métodos , Masculino , Modelos Moleculares , Proteoglicanas/química , Ratos , Ratos Sprague-DawleyRESUMO
Structure of proteoglycans in the lungs and total glycosaminoglycan content in blood serum were studied on mouse model of BCG-induced granulomatous inflammation in mice (without destructive processes in the lung parenchyma and granulomas). The maximum level of sulfated glycosaminoglycans in the lungs was detected on postinfection day 30 and was related to their involvement in initiation granulomogenesis and development of granulomas. The maximum level of total glycosaminoglycans in mouse serum on postinfection day 90 coincided with minimum level of sulfated glycosaminoglycans in the lungs. This blood/lungs ratio of glycosaminoglycans can be related to the prevalence of low-molecular-weight hyaluronan fragments promoting inflammation and fibrosis in the lungs observed at the end of the experiment (postinfection day 180).
Assuntos
Glicosaminoglicanos/sangue , Granuloma/patologia , Doença Granulomatosa Crônica/patologia , Inflamação/patologia , Pulmão/patologia , Proteoglicanas/ultraestrutura , Animais , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium bovisRESUMO
Comparative analyzes of biomineralization models have being crucial for the understanding of the functional properties of biominerals and the elucidation of the processes through which biomacromolecules control the synthesis and structural organization of inorganic mineral-based biomaterials. Among calcium carbonate-containing bioceramics, egg, mollusk and echinoderm shells, and crustacean carapaces, have being fairly well characterized. However, Thoraceca barnacles, although being crustacea, showing molting cycle, build a quite stable and heavily mineralized shell that completely surround the animal, which is for life firmly cemented to the substratum. This makes barnacles an interesting model for studying processes of biomineralization. Here we studied the main microstructural and ultrastructural features of Austromegabalanus psittacus barnacle shell, characterize the occurrence of specific proteoglycans (keratan-, dermatan- and chondroitin-6-sulfate proteoglycans) in different soluble and insoluble organic fractions extracted from the shell, and tested them for their ability to crystallize calcium carbonate in vitro. Our results indicate that, in the barnacle model, proteoglycans are good candidates for the modification of the calcite crystal morphology, although the cooperative effect of some additional proteins in the shell could not be excluded.
Assuntos
Exoesqueleto/ultraestrutura , Proteoglicanas/ultraestrutura , Thoracica/ultraestrutura , Animais , Materiais Biocompatíveis/metabolismo , Carbonato de Cálcio/metabolismo , CristalizaçãoRESUMO
This issue of Matrix Biology commemorates the memory of Dick Heinegård and his exceptional contributions to identify extracellular matrix molecules and their interactions that form cartilage matrices. This tribute to him demonstrates the development of his cartilage matrix model and how this model relates to the articles in this Matrix Biology Cartilage issue.
Assuntos
Matriz Extracelular/ultraestrutura , Pesquisa Biomédica/história , Cartilagem/citologia , Cartilagem/ultraestrutura , História do Século XX , História do Século XXI , Humanos , Masculino , Proteoglicanas/ultraestruturaRESUMO
The primary structural components of articular cartilage are the zonally differentiated interconnected network of collagen fibrils and proteoglycans, the latter having the potential to bind large amounts of water. Both components exist in a coupled relationship that gives rise to its remarkable mechanical properties. The response of cartilage to compression is governed both by the degree to which the hydrated proteoglycans are constrained within this fibrillar network and the ease with which the matrix fluid can be displaced. The functional properties of cartilage are therefore closely linked to the integrity of the fibrillar network. Our current understanding of this network has been derived via studies conducted at the macro, micro, and ultrastructural levels. Of particular interest to joint researchers and clinicians are issues relating to how the network structure varies both directionally and with zonal depth, how its integrity is maintained via mechanisms of fibril interconnectivity, and how it is modified by ageing, degeneration, and trauma. Physical models have been developed to explore modes of interconnectivity. Combined micromechanical and structural studies confirm the critical role that this interconnectivity must play but detailed descriptions at the molecular level remain elusive. Current computationally based models of cartilage have in some cases implemented the fibrillar component, albeit simplistically, as a separate structure. Considering how important a role fibril network interconnectivity plays in actual tissue structure and mechanical behavior, and especially how it changes with degeneration, a major challenge facing joint tissue modellers is how to incorporate such a feature in their models.
Assuntos
Cartilagem Articular/fisiologia , Cartilagem Articular/ultraestrutura , Colágeno/fisiologia , Colágeno/ultraestrutura , Humanos , Modelos Biológicos , Proteoglicanas/fisiologia , Proteoglicanas/ultraestruturaRESUMO
OBJECTIVE: This study assessed the micro-morphological changes in demineralized dentine scaffold following incubation with recombinant dentine matrix protein 1 (rDMP1). DESIGN: Extracted human molar crowns were sectioned into 6 beams (dimensions: 0.50mm×1.70mm×6.00mm), demineralized and incubated overnight in 3 different media (n=4): rDMP1 in bovine serum albumin (BSA), BSA and distilled water. Samples were placed in a chamber with simulated physiological concentrations of calcium and phosphate ions at constant pH 7.4. Samples were immediately processed for transmission electron microscopy (TEM) and field emission-scanning electron microscopy (FE-SEM) after 1 and 2 weeks. RESULTS: Analysis of the scaffold showed that decalcification process retained the majority of endogenous proteoglycans and phosphoproteins. rDMP1 treated samples promoted deposition of amorphous calcium phosphate (ACP) precursors and needle shaped hydroxyapatite crystals surrounding collagen fibrils. The BSA group presented ACP bound to collagen with no needle-like apatite crystals. Samples kept in distilled water showed no evidence of ACP and crystal apatite. Results from rDMP1 immobilized on dentine matrix suggests that the acidic protein was able to bind to collagen fibrils and control formation of amorphous calcium phosphate and its subsequent transformation into hydroxyapatite crystals after 2 weeks. CONCLUSION: These findings suggest a possible bio-inspired strategy to promote remineralization of dentine for reparative and regenerative purposes.
Assuntos
Dentina/ultraestrutura , Proteínas da Matriz Extracelular/química , Fosfoproteínas/química , Desmineralização do Dente/patologia , Remineralização Dentária/métodos , Adolescente , Adulto , Cálcio/química , Fosfatos de Cálcio/química , Colágeno/metabolismo , Cristalografia , Durapatita/química , Humanos , Concentração de Íons de Hidrogênio , Processamento de Imagem Assistida por Computador/métodos , Microscopia Eletrônica , Microscopia Eletrônica de Varredura/métodos , Microscopia Eletrônica de Transmissão/métodos , Fosfatos/química , Fosfoproteínas/ultraestrutura , Ácidos Fosfóricos/química , Ligação Proteica , Proteoglicanas/ultraestrutura , Proteínas Recombinantes , Albumina Sérica/química , Água/química , Adulto JovemRESUMO
The extracellular matrix of tendon is mainly composed of discontinuous Type-I collagen fibrils and small leucine rich proteoglycans (PG). Macroscopic tendon behaviors like stiffness and strength are determined by the ultrastructural arrangement of these components. When a tendon is submitted to load, the collagen fibrils both elongate and slide relative to their neighboring fibrils. The role of PG glycosaminoglycan (GAG) sidechains in mediating inter-fibril load sharing remains controversial, with competing structure-function theories suggesting that PGs may mechanically couple neighboring collagen fibrils (cross-linking them to facilitate fibril stretch) or alternatively isolating them (promoting fibril gliding). In this study, we sought to clarify the functional role of GAGs in tensile tendon mechanics by directly investigating the mechanical response of individual collagen fibrils within their collagen network in both native and GAG depleted tendons. A control group of Achilles tendons from adult mice was compared with tendons in which GAGs were enzymatically depleted using chondroitinase ABC. Tendons were loaded to specific target strains, chemically fixed under constant load, and later sectioned for morphological analysis by an atomic force microscope (AFM). Increases in periodic banding of the collagen fibrils (D-period) or decreases in fibril diameter was considered to be representative of collagen fibril elongation and the mechanical contribution of GAGs at the ultrascale was quantified on this basis. At high levels of applied tendon strain (10%), GAG depleted tendons showed increased collagen stretch (less fibril sliding). We conclude that the hydrophilic GAGs seem thus not to act as mechanical crosslinks but rather act to promote collagen fibril sliding under tension.
Assuntos
Colágeno/metabolismo , Glicosaminoglicanos/metabolismo , Proteoglicanas/metabolismo , Tendões/fisiologia , Animais , Fenômenos Biomecânicos , Colágeno/química , Colágeno/ultraestrutura , Feminino , Glicosaminoglicanos/química , Glicosaminoglicanos/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão , Nanotecnologia , Proteoglicanas/química , Proteoglicanas/ultraestrutura , Tendões/metabolismo , Tendões/ultraestrutura , Suporte de Carga/fisiologiaRESUMO
INTRODUCTION: The uniform distribution of collagen fibrils and proteoglycans maintain the transparency of normal cornea. We describe the ultrastructural features of camel cornea including collagen fibrils and proteoglycans (PGs). METHODS: Camel corneas (of 6-, 8-, and 10-month-old animals) were fixed in 2.5% glutaraldehyde containing cuprolinic blue in sodium acetate buffer and processed for electron microscopy. The 'AnalySIS LS Professional' program was used to analyze the collagen fibril diameter. RESULTS: The camel cornea consists of four layers: the epithelium (227 µm), stroma (388 µm), Descemet's membrane (DM), and endothelium. The epithelium constituted 36% of the camel cornea, whereas corneal stroma constituted 62% of the corneal thickness (629 µm). The PGs in the posterior stroma were significantly larger in number and size compared with the anterior and middle stroma. The collagen fibril diameter was 25 nm and interfibrillar spacing 40 nm. Fibrillar structures are present throughout the DM. CONCLUSION: The structure of the camel cornea is very different from human and other animals. The unique structure of the cornea might be an adaptation to help the camel to survive in a hot and dry climate. The camel cornea may also be a good model to study the effect of hot and dry climates on the cornea.
Assuntos
Camelus/anatomia & histologia , Colágeno/ultraestrutura , Córnea/ultraestrutura , Proteoglicanas/ultraestrutura , AnimaisRESUMO
PURPOSE: The stroma is the major part of the cornea, in which collagen fibrils and proteoglycans are distributed uniformly. We describe the ultrastructure of corneal layers, collagen fibrils (CF), and proteoglycans (PGs) in the tree shrew cornea. METHODS: Tree shrew corneas (5, 6, and 10 week old animals) and normal human corneas (24, 25, and 54 years old) were fixed in 2.5% glutaraldehyde containing cuprolinic blue in a sodium acetate buffer. The tissue was processed for electron microscopy. The 'iTEM Olympus Soft Imaging Solutions GmbH' program was used to measure the corneal layers, collagen fibril diameters and proteoglycan areas. RESULTS: The tree shrew cornea consists of 5 layers: the epithelium, Bowman's layer, stroma, Descemet's membrane, and endothelium. The epithelium was composed of squamous cells, wing cells and basal cells. The Bowman's layer was 5.5±1.0 µm thick and very similar to a normal human Bowman's layer. The stroma was 258±7.00 µm thick and consisted of collagen fibril lamellae. The lamellae were interlaced with one another in the anterior stroma, but ran parallel to one another in the middle and posterior stroma. Collagen fibrils were decorated with proteoglycan filaments with an area size of 390 ±438 nm(2). The collagen fibril had a minimum diameter of 39±4.25 nm. The interfibrillar spacing was 52.91±6.07 nm. Within the collagen fibrils, very small electron-dense particles were present. CONCLUSIONS: The structure of the tree shrew cornea is very similar to that of the normal human cornea. As is the case with the human cornea, the tree shrew cornea had a Bowman's layer, lamellar interlacing in the anterior stroma and electron-dense particles within the collagen fibrils. The similarities of the tree shrew cornea with the human cornea suggest that it could be a good structural model to use when studying changes in collagen fibrils and proteoglycans in non-genetic corneal diseases, such as ectasia caused after LASIK (laser-assisted in situ keratomileusis).
Assuntos
Lâmina Limitante Anterior/ultraestrutura , Substância Própria/ultraestrutura , Lâmina Limitante Posterior/ultraestrutura , Endotélio Corneano/ultraestrutura , Adulto , Fatores Etários , Animais , Colágeno/ultraestrutura , Doenças da Córnea/patologia , Dilatação Patológica/etiologia , Dilatação Patológica/patologia , Matriz Extracelular/ultraestrutura , Humanos , Ceratomileuse Assistida por Excimer Laser In Situ/efeitos adversos , Microscopia Eletrônica , Pessoa de Meia-Idade , Proteoglicanas/ultraestrutura , MusaranhosRESUMO
The skeletal muscle extracellular matrix (ECM) plays an important role in muscle fiber force transmission, maintenance, and repair. In both injured and diseased states, ECM adapts dramatically, a property that has clinical manifestations and alters muscle function. Here we review the structure, composition, and mechanical properties of skeletal muscle ECM; describe the cells that contribute to the maintenance of the ECM; and, finally, overview changes that occur with pathology. New scanning electron micrographs of ECM structure are also presented with hypotheses about ECM structurefunction relationships. Detailed structurefunction relationships of the ECM have yet to be defined and, as a result, we propose areas for future study.
Assuntos
Matriz Extracelular/fisiologia , Matriz Extracelular/ultraestrutura , Músculo Esquelético/fisiologia , Músculo Esquelético/ultraestrutura , Animais , Fenômenos Biomecânicos , Colágeno/fisiologia , Colágeno/ultraestrutura , Glicosaminoglicanos/fisiologia , Glicosaminoglicanos/ultraestrutura , Humanos , Camundongos , Microscopia Eletrônica de Varredura , Modelos Animais , Proteoglicanas/fisiologia , Proteoglicanas/ultraestrutura , CoelhosRESUMO
The need to identify inhibitors of cancer invasion has driven the development of quantitative in vitro invasion assays. The most common assays used are based on the original Boyden assay system. Today commercially available plastic inserts for multi-well plates, which possess a cell-permeable membrane, as typified by Transwell(®) Permeable Supports, permit accurate repeatable invasion assays. When placed in the well of a multi-well tissue culture plate these inserts create a two-chamber system separated by the cell-permeable membrane. To create an invasion assay the pores in the membrane are blocked with a gel composed of extracellular matrix that is meant to mimic the typical matrices that tumour cells encounter during the invasion process in vivo. By placing the cells on one side of the gel and a chemoattractant on the other side of the gel, invasion is determined by counting those cells that have traversed the cell-permeable membrane having invaded towards the higher concentration of chemoattractant. In this chapter, in addition to protocols for performing Transwell invasion assays, there is consideration of the limitations of current assay designs with regard to available matrices and the absence of tumour microenvironment cells.
Assuntos
Ensaios de Migração Celular/instrumentação , Invasividade Neoplásica , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Ensaios de Migração Celular/métodos , Colágeno/química , Colágeno/ultraestrutura , Colágeno Tipo I/química , Colágeno Tipo I/ultraestrutura , Combinação de Medicamentos , Géis , Humanos , Laminina/química , Laminina/ultraestrutura , Proteoglicanas/química , Proteoglicanas/ultraestrutura , Coloração e Rotulagem/métodosRESUMO
OBJECT: To investigate the relationship of the different diffusion tensor imaging (DTI) parameters (ADC, FA, and first eigenvector (EV)) to the constituents (proteoglycans and collagen), the zonal arrangement of the collagen network, and mechanical loading of articular cartilage. MATERIAL AND METHODS: DTI of eight cartilage-on-bone samples of healthy human patellar cartilage was performed at 17.6 T. Three samples were additionally imaged under indentation loading. After DTI, samples underwent biomechanical testing, safranin-O staining for semiquantitative proteoglycan estimation, and scanning electron microscopy (SEM) for depicting collagen architecture. RESULTS: From the articular surface to the bone-cartilage interface, ADC continuously decreased and FA increased. Cartilage zonal heights calculated from EVs strongly correlated with SEM-derived zonal heights (P < 0.01, r (2)=0.87). Compression reduced ADC in the superficial 30% of cartilage and increased FA in the superficial 5% of cartilage. Reorientation of the EVs indicative of collagen fiber reorientation under the indenter was observed. No significant correlation was found between ADC, FA, and compressive stiffness. CONCLUSIONS: Correlating ADC and FA with proteoglycan and collagen content suggests that diffusion is dominated by different depth-dependent mechanisms within cartilage. Knowledge of the spatial distribution of the DTI parameters and their variation contributes to form a database for future analysis of defective cartilage.
Assuntos
Fenômenos Biomecânicos/fisiologia , Cartilagem Articular/anatomia & histologia , Cartilagem Articular/citologia , Imagem de Tensor de Difusão/métodos , Microscopia Eletrônica de Varredura/métodos , Adulto , Anisotropia , Cartilagem Articular/ultraestrutura , Colágeno/análise , Difusão , Imagem de Difusão por Ressonância Magnética/métodos , Humanos , Processamento de Imagem Assistida por Computador/métodos , Patela/anatomia & histologia , Patela/citologia , Proteoglicanas/análise , Proteoglicanas/ultraestruturaRESUMO
Corneal transparency is fundamental to the visual system, and is directly related to the ordered collagen fibril architecture that the cornea maintains. Proteoglycans, through their protein core and highly anionic glycosaminoglycan side chains, are thought to regulate the collagen organisation in the corneal stroma. To understand the inter-relationships between proteoglycans and collagen fibrils in the cornea, adult mouse corneas were treated with cuprolinic blue and three-dimensional reconstructions of the anterior, mid and posterior corneal stroma were obtained. The reconstructions show regular diameters of collagen fibrils throughout the cornea and uniform interfibrillar spacing within each region. Both longitudinal and transverse reconstructions were obtained to establish a clear picture of proteoglycan organisation, yet no distinct regular pattern or symmetry of proteoglycan orientation was observed. Large, electron-dense proteoglycans (possibly chondroitin sulphate/dermatan sulphate proteoglycans) interconnecting two or often three adjacent collagen fibrils are seen, whilst another sub-population of smaller proteoglycans (of the keratan sulphate variety) interconnect only neighbouring fibrils. The reconstructions suggest a complex interaction between proteoglycans and collagen, which allows for the dynamic control of collagen fibril architecture in the cornea.
Assuntos
Colágeno , Substância Própria , Tomografia com Microscopia Eletrônica/métodos , Conformação Proteica , Proteoglicanas , Animais , Colágeno/metabolismo , Colágeno/ultraestrutura , Substância Própria/química , Substância Própria/metabolismo , Camundongos , Modelos Moleculares , Proteoglicanas/metabolismo , Proteoglicanas/ultraestruturaRESUMO
Interactions between collagens and proteoglycans help define the structure and function of extracellular matrices. The cornea, which contains proteoglycans with keratan sulphate or chondroitin/dermatan sulphate glycosaminoglycan chains, is an excellent model system in which to study collagen-proteoglycan structures and interactions. Here, we present the first three-dimensional electron microscopic reconstructions of the cornea, and these include corneas from which glycosaminoglycans have been selectively removed by enzymatic digestion. Our reconstructions show that narrow collagen fibrils associate with sulphated proteoglycans that appear as extended, variable-length linear structures. The proteoglycan network appears to tether two or more collagen fibrils, and thus organize the matrix with enough spatial specificity to fulfill the requirements for corneal transparency. Based on the data, we propose that the characteristic pseudohexagonal fibril arrangement in cornea is controlled by the balance of a repulsive force arising from osmotic pressure and an attractive force due to the thermal motion of the proteoglycans.
Assuntos
Colágeno/ultraestrutura , Córnea , Tomografia com Microscopia Eletrônica/métodos , Proteoglicanas/ultraestrutura , Animais , Bovinos , Condroitinases e Condroitina Liases/metabolismo , Córnea/química , Córnea/ultraestrutura , Glicosaminoglicanos/metabolismo , Glicosaminoglicanos/ultraestrutura , Glicosídeo Hidrolases/metabolismo , Modelos Moleculares , Proteoglicanas/metabolismoRESUMO
OBJECTIVE: Osteoarthritis (OA) is characterized by the changes in structure and composition of articular cartilage. However, it is not fully known, what is the depth-wise change in two major components of the cartilage solid matrix, i.e., collagen and proteoglycans (PGs), during OA progression. Further, it is unknown how the depth-wise changes affect local tissue strains during compression. Our aim was to address these issues. METHODS: Data from the previous microscopic and biochemical measurements of the collagen content, distribution and orientation, PG content and distribution, water content and histological grade of normal and degenerated human patellar articular cartilage (n=73) were reanalyzed in a depth-wise manner. Using this information, a composition-based finite element (FE) model was used to estimate tissue function solely based on its composition and structure. RESULTS: The orientation angle of collagen fibrils in the superficial zone of cartilage was significantly less parallel to the surface (P<0.05) in samples with early degeneration than in healthy samples. Similarly, PG content was reduced in the superficial zone in early OA (P<0.05). However, collagen content decreased significantly only at the advanced stage of OA (P<0.05). The composition-based FE model showed that under a constant stress, local tissue strains increased as OA progressed. CONCLUSION: For the first time, depth-wise point-by-point statistical comparisons of structure and composition of human articular cartilage were conducted. The present results indicated that early OA is primarily characterized by the changes in collagen orientation and PG content in the superficial zone, while collagen content does not change until OA has progressed to its late stage. Our simulation results suggest that impact loads in OA joint could create a risk for tissue failure and cell death.
Assuntos
Cartilagem Articular/metabolismo , Cartilagem Articular/fisiopatologia , Colágeno/análise , Osteoartrite do Joelho/metabolismo , Osteoartrite do Joelho/fisiopatologia , Proteoglicanas/análise , Adulto , Cadáver , Colágeno/ultraestrutura , Análise de Elementos Finitos , Análise de Fourier , Humanos , Microscopia de Polarização , Pessoa de Meia-Idade , Modelos Biológicos , Osteoartrite do Joelho/classificação , Proteoglicanas/ultraestrutura , Estresse MecânicoRESUMO
PURPOSE: This study examined the role of epidermal growth factor receptor (EGFR) signalling on the organization and remodelling of collagen fibrils (CFs) and proteoglycans (PGs) in the stroma of diabetic rat cornea. METHODS: Diabetes was induced in female Wistar rats (n = 5) by streptozotocin (STZ) injection (55 mg/kg). Treatment with a selective inhibitor of EGFR tyrosine kinase, AG1478, was started on the same day as the induction of diabetes and administered every other day for 4 weeks. Corneas were fixed in 4% paraformaldehyde at 4 degrees to allow for analysis of CF diameters and in 2.5% glutaraldehyde in sodium acetate buffer containing cuprolinic blue to enable the study of PG distribution. AnalySIS soft imaging software was used to analyse CFs and PGs. RESULTS: Epithelial thickness, and median diameter and area fraction of CF in corneal stroma were decreased in diabetic rat cornea compared with normal cornea (p < 0.001), whereas the median PG area and area fractions were significantly increased (p < 0.001). Treatment with AG1478, although it had no action on normal cornea, prevented these diameter and area fraction changes in CFs and PGs. The cornea of AG1478-treated diabetic rats showed a slight increase in CF diameter and area fraction and a decreased number density. CONCLUSIONS: These data show that the distribution of corneal stroma CFs and PGs was altered after 4 weeks of diabetes and that, furthermore, treatment with an EGFR signalling inhibitor normalized these abnormalities. The data suggest that EGFR plays an important role in the development of diabetes-induced corneal remodelling.