Three‐dimensional environments preserve extracellular matrix compartments of ovarian follicles and increase FSH‐dependent growth

In this study we performed a systematic comparative analysis of two culture environments—flat/adhesive liquid and three‐dimensional collagen gel—upon in vitro ovarian follicle development. We paid particular attention to the effects of in vitro environments upon the preservation of follicular structure and of peri‐ and intra‐follicular extracellular matrix. We show that flat/adhesive environment leads to an obvious distortion of follicle morphology, marked extracellular matrix modifications and high rates of spontaneous, i.e., FSH‐independent, follicle disruption. In contrast, three‐dimensional collagen gel environments are able to maintain follicular structure with an in vivo‐like basal lamina architecture, minimizing spontaneous disruption. Follicle distortions found in flat/adhesive culture systems include a pronounced flattening, causing the follicle horizontal diameters not to adequately reflect follicle volume. Our volume data, based on three‐axis follicle diameter measurements, indicate that three‐dimensional collagen gel environments increase follicle growth, particularly in response to FSH. This study demonstrates that preservation of both peri‐ and intra‐follicular extracellular matrix compartments during the in vitro growth and differentiation of ovarian follicles is highly desirable, and is now possible through the use of appropriate three‐dimensional collagen gel culture environments. This system allows a better understanding of the specific roles played by each of the follicle compartments during development. Mol. Reprod. Dev. 54:163–172, 1999. © 1999 Wiley‐Liss, Inc.     

The biofilm matrix

The extracellular matrix is a complex and extremely important component of all biofilms, providing architectural structure and mechanical stability to the attached population. The matrix is composed of cells, water and secreted/released extracellular macromolecules. In addition, a range of enzymic and regulatory activities can be found within the matrix. Together, these different components and activities are likely to interact and in so doing create a series of local environments within the matrix which co-exist as a functional consortium. The matrix architecture is also subject to a number of extrinsic factors, including fluctuations in nutrient and gaseous levels and fluid shear. Together, these intrinsic and extrinsic factors combine to produce a dynamic, heterogeneous microenvironment for the attached and enveloped cells.     

The Biofilm Matrix

The extracellular matrix is a complex and extremely important component of all biofilms, providing architectural structure and mechanical stability to the attached population. The matrix is composed of cells, water and secreted/released extracellular macromolecules. In addition, a range of enzymic and regulatory activities can be found within the matrix. Together, these different components and activities are likely to interact and in so doing create a series of local environments within the matrix which co-exist as a functional consortium. The matrix architecture is also subject to a number of extrinsic factors, including fluctuations in nutrient and gaseous levels and fluid shear. Together, these intrinsic and extrinsic factors combine to produce a dynamic, heterogeneous microenvironment for the attached and enveloped cells.     

The cellular matrix: a feature of tensile bearing dense soft connective tissues

The term connective tissue encompasses a diverse group of tissues that reside in different environments and must support a spectrum of mechanical functions. Although the extracellular matrix of these tissues is well described, the cellular architecture of these tissues and its relationship to tissue function has only recently become the focus of study. It now appears that tensile-bearing dense connective tissues may be a specific class of connective tissues that display a common cellular organization characterized by fusiform cells with cytoplasmic projections and gap junctions. These cells with their cellular projections are organised into a complex 3-dimensional network leading to a physically, chemically and electrically connected cellular matrix. The cellular matrix may play essential roles in extracellular matrix formation, maintenance and remodelling, mechanotransduction and during injury and healing. Thus, it is likely that it is the interaction of both the extracellular matrix and cellular matrix that provides the basis for tissue function. Restoration of both these matrices, as well as their interaction must be the goal of strategies to repair these connective tissues damaged by either injury or disease.     

Mechanical, biochemical, and extracellular matrix effects on vascular smooth muscle cell phenotype.

The vascular smooth muscle cell (VSMC) is surrounded by a complex extracellular matrix that provides and modulates a variety of biochemical and mechanical cues that guide cell function. Conventional two-dimensional monolayer culture systems recreate only a portion of the cellular environment, and therefore there is increasing interest in developing more physiologically relevant three-dimensional culture systems. This review brings together recent studies on how mechanical, biochemical, and extracellular matrix stimulation can be applied to study VSMC function and how the combination of these factors leads to changes in phenotype. Particular emphasis is placed on in vitro experimental studies in which multiple stimuli are combined, especially in three-dimensional culture systems and in vascular tissue engineering applications. These studies have provided new insight into how VSMC phenotype is controlled, and they have underscored the interdependence of biochemical and mechanical signaling. Future improvements in creating more complex in vitro culture environments will lead to a better understanding of VSMC biology, new treatments for vascular disease, as well as improved blood vessel substitutes.     

Matrix proteins associated with bone calcification are present in human vascular smooth muscle cells grownin vitro

Summary Atherosclerotic lesions are composed of cellular elements that have migrated from the vessel lumen and wall to form the cellular component of the developing plaque. The cellular elements are influenced by various growth-regulatory molecules, cytokines, chemoattractants, and vasoregulatory molecules that regulate the synthesis of the extracellular matrix composing the plaque. Because vascular smooth muscle cells (VSMC) constitute the major cellular elements of the atherosclerotic plaque and are thought to be responsible for the extracellular matrix that becomes calcified in mature plaques, immunostaining for collagenous and noncollagenous proteins typically associated with bone matrix was conducted on VSMC grownin vitro. VSMC obtained from human aorta were grown in chambers on glass slides and immunostained for procollagen type I, bone sialoprotein, osteonectin, osteocalcin, osteopontin, decorin, and biglycan. VSMC demonstrated an intense staining for procollagen type I, and a moderately intense staining for the noncollagenous proteins, bone sialoprotein and osteonectin, two proteins closely associated with bone mineralization. Minimal immunostaining was noted for osteocalcin, osteopontin, decorin, and biglycan. The presence in VSMC of collagenous and noncollagenous proteins associated with bone mineralization suggest that the smooth muscle cells in the developing atherosclerotic plaque play an important role in the deposition of the extracellular matrix involved in calcification of developing lesions.     

Lipids in the developing enamel of the rat incisor. Parallel histochemical and biochemical investigations

Morphologic examination of the developing enamel of rat incisors showed the presence of cell processes and remnants. Histochemical investigation of rapid-frozen freeze-substituted samples, using p-phenylenediamine or a phosphotungstic acid chromic acid mixture, revealed osmiophilic components which were extractable in chloroform-methanol solution and were located inside the tubule-like structures of the extracellular matrix. The presecretory cell-rich and developing enamel zones underwent quantitative and qualitative lipid analysis. Comparison of the biochemical data as well as of the morphological observations, suggests a cellular origin for enamel lipids randomly adsorbed by extracellular matrix components during enamel processing. The nature of the material which appeared as an osmiophilic intra-tubular filling is still unresolved.     

Topography of Extracellular Matrix Mediates Vascular Morphogenesis and Migration Speeds in Angiogenesis

The extracellular matrix plays a critical role in orchestrating the events necessary for wound healing, muscle repair, morphogenesis, new blood vessel growth, and cancer invasion. In this study, we investigate the influence of extracellular matrix topography on the coordination of multi-cellular interactions in the context of angiogenesis. To do this, we validate our spatio-temporal mathematical model of angiogenesis against empirical data, and within this framework, we vary the density of the matrix fibers to simulate different tissue environments and to explore the possibility of manipulating the extracellular matrix to achieve pro- and anti-angiogenic effects. The model predicts specific ranges of matrix fiber densities that maximize sprout extension speed, induce branching, or interrupt normal angiogenesis, which are independently confirmed by experiment. We then explore matrix fiber alignment as a key factor contributing to peak sprout velocities and in mediating cell shape and orientation. We also quantify the effects of proteolytic matrix degradation by the tip cell on sprout velocity and demonstrate that degradation promotes sprout growth at high matrix densities, but has an inhibitory effect at lower densities. Our results are discussed in the context of ECM targeted pro- and anti-angiogenic therapies that can be tested empirically.     

Genes involved in matrix formation in Pseudomonas aeruginosa PA14 biofilms

Pseudomonas aeruginosa forms diverse matrix-enclosed surface-associated multicellular assemblages (biofilms) that aid in its survival in a variety of environments. One such biofilm is the pellicle that forms at the air-liquid interface in standing cultures. We screened for transposon insertion mutants of P. aeruginosa PA14 that were unable to form pellicles. Analysis of these mutants led to the identification of seven adjacent genes, named pel genes, the products of which appear to be involved in the formation of the pellicle's extracellular matrix. In addition to being required for pellicle formation, the pel genes are also required for the formation of solid surface-associated biofilms. Sequence analyses predicted that three pel genes encode transmembrane proteins and that five pel genes have functional homologues involved in carbohydrate processing. Microscopic and macroscopic observations revealed that wild-type P. aeruginosa PA14 produces a cellulase-sensitive extracellular matrix able to bind Congo red; no extracellular matrix was produced by the pel mutants. A comparison of the carbohydrates produced by the wild-type strain and pel mutants suggested that glucose was a principal component of the matrix material. Together, these results suggest that the pel genes are responsible for the production of a glucose-rich matrix material required for the formation of biofilms by P. aeruginosa PA14.     

Detection of chondroitin sulfates and decorin in developing fetal and neonatal rat lung

Chondroitin sulfates and their related proteoglycans are components of extracellular matrix that act as key determinants of growth and differentiation characteristics of developing lungs. Changes in their immunohistochemical distribution during progressive organ maturation were examined with monospecific antibodies to chondroitin sulfate, a nonbasement membrane chondroitin sulfate proteoglycan, and the specific chondroitin sulfate-containing proteoglycan decorin in whole fetuses and lungs from newborn and adult rats. Alveolar and airway extracellular matrix immunostained heavily in the prenatal rat for both chondroitin sulfate and chondroitin sulfate proteoglycan, whereas decorin was confined to developing airways and vessels. These sites retained their respective levels of reactivity with all antibodies through 1-10 days postnatal but thereafter became progressively more diminished and focal in alveolar regions. The heavy staining seen early in development was interpreted to reflect a significant and wide distribution of chondroitin sulfates, chondroitin sulfate proteoglycans, and decorin in rapidly growing tissues, whereas the reduced and more focal reactivity observed at later time points coincided with known focal patterns of localization of fibrillar elements of the extracellular matrix and a more differentiated state.     

Localization of soluble endogenous lectins and their ligands at specific extracellular sites

Soluble lectins of chicken, rat, frog, and the cellular slime mold, Dictyostelium discoideum, were purified and specific antibodies raised against these proteins were used to immunohistochemically localize the lectins in and around the tissues in which they were synthesized. Within cells, some of these soluble lectins (chicken-lactose-lectin-II in intestinal goblet cells, discoidin II in prespore cells) appear to be concentrated within vesicles whereas others (e.g., rat beta-galactoside lectin in pulmonary alveolar and smooth muscle cells) appear to be free in the cytoplasm. All of these lectins are eventually secreted to extracellular sites in developing or adult tissues. The sites include mucin (chicken-lactose-lectin-II in intestine); developing extracellular matrix (chicken-lactose-lectin-I in muscle; Xenopus laevis lectin in blastula stage embryos); slime (discoidin I); developing spore coat (discoidin II); and a specialized extracellular matrix, elastic fibers (rat beta-galactoside lectin in lung). In cases where this has been studied in detail (discoidin I, discoidin II, and chicken-lactose-lectin-II), the lectin is associated with a complementary extracellular ligand, at least transiently. Lectin-ligand interactions presumably confer specialized properties in these particular extracellular domains.     

Extracellular matrix: functions in the nervous system

An astonishing number of extracellular matrix glycoproteins are expressed in dynamic patterns in the developing and adult nervous system. Neural stem cells, neurons, and glia express receptors that mediate interactions with specific extracellular matrix molecules. Functional studies in vitro and genetic studies in mice have provided evidence that the extracellular matrix affects virtually all aspects of nervous system development and function. Here we will summarize recent findings that have shed light on the specific functions of defined extracellular matrix molecules on such diverse processes as neural stem cell differentiation, neuronal migration, the formation of axonal tracts, and the maturation and function of synapses in the peripheral and central nervous system.     

Collagen synthesis by normal and bromodeoxyuridine-modulated cells in myogenic culture

Connective tissue appears to play an important role in the development, function, and pathology of vertebrate skeletal muscle. In order to analyze the source of the extracellular matrix in developing muscle, hydroxyproline assays and fine-structure analyses were made on clonal cultures derived from embryonic musculature. The cells in fibroblast cultures synthesized and accumulated an extensive extracellular matrix of nonstriated reticular-like microfibrils. In myoblast cultures, no extracellular matrix was evident by electron microscopy, although hydroxyproline assays demonstrated that the cells synthesized and released a soluble collagenous protein into the medium. When grown with fibroblasts, multinucleated myofibers exhibited a complete basement membrane consisting of both basal and reticular laminae. Bromodeoxyuridine-treated myoblasts, in addition to exhibiting morphological and cytological alterations, synthesized and elaborated an extracellular matrix identical to that in fibroblast cultures. The potential for a similar alteration or “modulation” of myoblast expression in situ is discussed in regard to its possible relation to fibrosis in muscular dystrophy.     

Ontogeny of the basal lamina in the sea urchin embryo

The patterns of expression for several extracellular matrix components during development of the sea urchin embryo are described. An immunofluorescence assay was employed on paraffin-sectioned material using (i) polyclonal antibodies against known vertebrate extracellular matrix components: laminin, fibronectin, heparan sulfate proteoglycan, collagen types I, III, and IV; and (ii) monoclonal antibodies generated against sea urchin embryonic components. Most extracellular matrix components studied were found localized within the unfertilized egg in granules (0.5-2.0 micron) distinct from the cortical granules. Fertilization initiated trafficking of the extracellular matrix (ECM) components from within the egg granules to the basal lamina of the developing embryo. The various ECM components arrived within the developing basal lamina at different times, and not all components were unique to the basal lamina. Two ECM components were not found within the egg. These molecules appeared de novo at the mesenchyme blastula stage, and remained specific to the mesoderm through development. The reactivity of antibodies to vertebrate ECM antigens with components of the sea urchin embryo suggests the presence of immunologically similar ECM molecules between the phyla.     

Contractile forces regulate cell division in three-dimensional environments

Physical forces direct the orientation of the cell division axis for cells cultured on rigid, two-dimensional (2D) substrates. The extent to which physical forces regulate cell division in three-dimensional (3D) environments is not known. Here, we combine live-cell imaging with digital volume correlation to map 3D matrix displacements and identify sites at which cells apply contractile force to the matrix as they divide. Dividing cells embedded in fibrous matrices remained anchored to the matrix by long, thin protrusions. During cell rounding, the cells released adhesive contacts near the cell body while applying tensile forces at the tips of the protrusions to direct the orientation of the cell division axis. After cytokinesis, the daughter cells respread into matrix voids and invaded the matrix while maintaining traction forces at the tips of persistent and newly formed protrusions. Mechanical interactions between cells and the extracellular matrix constitute an important mechanism for regulation of cell division in 3D environments.     

Extracellular matrix organization in developing muscle: correlation with acetylcholine receptor aggregates

Monoclonal antibodies recognizing laminin, heparan sulfate proteoglycan, fibronectin, and two apparently novel connective tissue components have been used to examine the organization of extracellular matrix of skeletal muscle in vivo and in vitro. Four of the five monoclonal antibodies are described for the first time here. Immunocytochemical experiments with frozen-sectioned muscle demonstrated that both the heparan sulfate proteoglycan and laminin exhibited staining patterns identical to that expected for components of the basal lamina. In contrast, the remaining matrix constituents were detected in all regions of muscle connective tissue: the endomysium, perimysium, and epimysium. Embryonic muscle cells developing in culture elaborated an extracellular matrix, each antigen exhibiting a unique distribution. Of particular interest was the organization of extracellular matrix on myotubes: the build-up of matrix components was most apparent in plaques overlying clusters of an integral membrane protein, the acetylcholine receptor (AChR). The heparan sulfate proteoglycan was concentrated at virtually all AChR clusters and showed a remarkable level of congruence with receptor organization; laminin was detected at 70-95% of AChR clusters but often was not completely co-distributed with AChR within the cluster; fibronectin and the two other extracellular matrix antigens occurred at approximately 20, 8, and 2% of the AChR clusters, respectively, and showed little or no congruence with AChR. From observations on the distribution of extracellular matrix components in tissue cultured fibroblasts and myogenic cells, several ideas about the organization of extracellular matrix are suggested. (a) Congruence between AChR clusters and heparan sulfate proteoglycan suggests the existence of some linkage between the two molecules, possibly important for regulation of AChR distribution within the muscle membrane. (b) The qualitatively different patterns of extracellular matrix organization over myotubes and fibroblasts suggest that each of these cell types uses somewhat different means to regulate the assembly of extracellular matrix components within its domain. (c) The limited co-distribution of different components within the extracellular matrix in vitro and the selective immune precipitation of each antigen from conditioned medium suggest that each extracellular matrix component is secreted in a form that is not complexed with other matrix constituents.     

Properties of Jack bean α-mannosidase in the presence of hyaluronan

An enzymatic reaction within a mesh-like structure constructed using hyaluronan was investigated in order to understand the influence of specific reaction environments in a living body on the reaction. This mesh-like structure, which mimicked extracellular matrix conditions, was found to accelerate glycohydrolysis by Jack bean α-mannosidase.     

Properties of Jack Bean α-Mannosidase in the Presence of Hyaluronan

An enzymatic reaction within a mesh-like structure constructed using hyaluronan was investigated in order to understand the influence of specific reaction environments in a living body on the reaction. This mesh-like structure, which mimicked extracellular matrix conditions, was found to accelerate glycohydrolysis by Jack bean α-mannosidase.     

Extracellular matrix ligand and stiffness modulate immature nucleus pulposus cell-cell interactions

The nucleus pulposus (NP) of the intervertebral disc functions to provide compressive load support in the spine, and contains cells that play a critical role in the generation and maintenance of this tissue. The NP cell population undergoes significant morphological and phenotypic changes during maturation and aging, transitioning from large, vacuolated immature cells arranged in cell clusters to a sparse population of smaller, isolated chondrocyte-like cells. These morphological and organizational changes appear to correlate with the first signs of degenerative changes within the intervertebral disc. The extracellular matrix of the immature NP is a soft, gelatinous material containing multiple laminin isoforms, features that are unique to the NP relative to other regions of the disc and that change with aging and degeneration. Based on this knowledge, we hypothesized that a soft, laminin-rich extracellular matrix environment would promote NP cell-cell interactions and phenotypes similar to those found in immature NP tissues. NP cells were isolated from porcine intervertebral discs and cultured in matrix environments of varying mechanical stiffness that were functionalized with various matrix ligands; cellular responses to periods of culture were assessed using quantitative measures of cell organization and phenotype. Results show that soft (<720 Pa), laminin-containing extracellular matrix substrates promote NP cell morphologies, cell-cell interactions, and proteoglycan production in vitro, and that this behavior is dependent upon both extracellular matrix ligand and substrate mechanical properties. These findings indicate that NP cell organization and phenotype may be highly sensitive to their surrounding extracellular matrix environment.