Histochemical analysis of extracellular matrix material during embryonic mouse lens morphogenesis in an aphakic strain of mice

Extracellular matrix material (ECM) present during early lens morphogenesis was analyzed histochemically in normal CFW mice and mutant strain aphakia by the Alcian blue 8GX, pH 2.5, Alcian blue 8GX, pH 2.5/periodic acid-Schiff combined, high-iron diamine, and van Gieson methods. At lens placode formation, the optic vesicle basal lamina in both strains was higher in sulfated glycosaminoglycan content than was the ectodermal basal lamina. In the aphakia strain, ECM components were observed intercellularly in the presumptive neural retina and lens rudiment of some specimens. This observation was peculiar to the aphakia strain. At the lens cup stage (10.5 days), the interface ECM became less uniformly dense in the CFW strain, resulting in the formation of a fibrillar structure in the widening interspace area. In contrast, the interface ECM in the mutant strain stained solidly and continuously for acidic materials, particularly sulfated glycosaminoglycans, for a full 2 days longer than in the normal strain. The optic cup and lens rudiment remained closely apposed and intercellular ECM components were observed in these tissues in most mutant specimens throughout these stages. The exact mechanism resulting in these intercellular deposits is unknown, although it is possible that they are either pulled along on the cell surface away from the interface ECM during cell shape changes related to the cell cycle or that they are secreted abnormally due to some disturbed cellular polarity. It is unclear at this time if these abnormalities of the ECM in the aphakia strain play a role in the pathogenesis of the multiple eye anomalies, or if they are a secondary effect of the gene mutation.     

The role of extracellular material in chick neurulation. II. Surface morphology of neuroepithelial cells during neural fold fusion

Changes in cell surface morphology of the neuroepithelium during fusion of neural folds in the chick were studied. As the folds were about to meet, a thick extracellular coat material (ECM) appeared between the two leading edges. Cell membranes forming the fusion area were relatively smooth and heavily coated with ECM. By contrast, the apical surface of most cells lining the wall of the neural tube was folded with much less ECM. During the contact of neural folds, ECM was displaced from the space between the two leading edges, leaving a thin, closely adherent "typical" cell surface coat. Trypsin and concanavalin A inhibited proper alignment and fusion of apposing neural folds by modifying the surface of developing neuroepithelium. Results of this study support a hypothesis that ECM may serve temporarily as an adhesive to bind together the leading edges of neural folds until establishment of more intimate contacts (junctional complexes).     

Altered expression of integrins and basement membrane proteins in malignant and pre-malignant lesions of oral mucosa

Integrins are transmembrane receptors that regulate cell-cell and cell-extracellular matrix (ECM) contact. In epithelial tissues, they interact with ECM components of the basement membrane (BM) to maintain the homeostasis and the architecture of the tissue. This interaction controls several cell functions such as adhesion, migration, proliferation, differentiation, and therefore has a key role in cancer development and metastasis. We studied the expression of integrins and ECM components of the BM by immunohistochemistry in frozen specimens of malignant squamous cell carcinoma (SCC), pre-malignant lesions of the oral mucosa (leucoplakia) and oral lichen planus. In invasive SCC, we observed altered polarity and distribution of alpha2beta1, alpha6beta4 and alpha3beta1 integrins, whereas in the in situ carcinoma alpha6beta4 and alpha3beta1 patterns only were altered. Immunostaining for ECM components such as Laminin-1 (Ln-1), Ln-5, and Collagen IV (Coll IV) was discontinuous and interrupted in invasive SCC, whereas it was normal in the in situ carcinoma. In both pre-malignant lesions and lichen planus specimens, integrins were expressed in a polarized manner in the presence of a normal BM, whereas were abnormally distributed in those tissues with altered staining patterns of the ECM components. In conclusion, we suggest that abnormal re-distribution of alpha3beta1 and alpha6beta4 integrins and expression of ECM components such as Ln-5 could play an important role in SCC invasion and metastasis.     

Genetic diversity of ectomycorrhizal Basidiomycetes from African and Indian tropical rain forests

Ectomycorrhizal (ECM) fungi have a worldwide distribution. However, the ecology of tropical ECM fungi is poorly documented, limiting our understanding of the symbiotic associations between tropical plants and fungi. ECM Basidiomycete diversity was investigated for the first time in two tropical rain forests in Africa (Western Upper Guinea) and in Asia (Western Ghats, India), using a fragment of the mitochondrial large subunit rRNA gene to type 140 sporocarps and 54 ectomycorrhizas. To evaluate taxonomic diversity, phylogenetic analyses were performed, and 40 sequences included from identified European specimens were used as taxonomic benchmarks. Five clades were recovered corresponding to six taxonomic groups: boletoids, sclerodermatoids, russuloids, thelephoroids, and a clade grouping the Amanitaceae and Tricholomataceae families. Our results revealed that the Russulaceae species display a great diversity with several putative new species, especially in Guinea. Other taxonomic issues at family/section levels are also briefly discussed. This study provides preliminary insights into taxonomic diversity, ECM status, and biogeographic patterns of ECM fungi in tropical two rain forest ecosystems, which appear to be as diverse as in temperate and boreal forests.     

Developmental SEM observations on an extracellular matrix in embryogenic calli ofDrosera rotundifolia andZea mays

Summary Primary embryogenic callus ofDrosera rotundifolia and long-term cultured embryogenic callus ofZea mays possess a conspicuous extracellular matrix (ECM) around and between embryogenic cells. The structural arrangement of ECM depends on the developmental stage of the embryogenic cells. Single embryoid cells were covered with, and connected by net-like material. However, surface cells of young globular embryoids were covered with a coherent layer of ECM which forms bridges with net-like material between the cells which was gradually reduced to coarse strands. When protodermis was formed on the surface of globular embryoids, the ECM disappeared completely. The ECM network was never observed on the surface of heart- and torpedo-shaped embryoids. Safranine (especially 0.1%) stabilized the structure of ECM. Digestion with pronase E and proteinase K indicated that the ECM contains proteinaceous components. Similar developmental patterns of ECM were observed in dicotyledonous and monocotyledonous examples. The ECM represents a stable morphological structure even during long-term embryogenic culture in maize.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - Dicamba 3,6-dichloro-o-anisic acid - ECM extracellular matrix - KIN kinetin - SEM scanning electron microscopy - TEM transmission electron microscopy     

Initiation of primary cell cultures from human intracranial tumors on extracellular matrix from bovine corneal endothelial cells

Tissue specimens from 105 human gliomas and 57 human meningiomas were obtained at surgery, dissociated into single cells and small cell aggregates and then plated onto plain plastic tissue culture dishes and dishes which had been precoated with an extracellular matrix (ECM) derived from bovine corneal endothelium. In 80% of the glioma cases we observed a marked improvement in initial plating efficiency, colony formation and speed of attachment when cells were plated on ECM. In 5 cases cells attached only to the ECM-coated dishes but remained afloat in the untreated dishes. In addition it could be noted that over the first 2 days, those cells which had been initiated on ECM showed more signs of morphological differentiation, i.e., extension of cytoplasmic processes or formation of fiber networks between cell groups. If adaptation occurred and proliferation began in vitro, either immediately or after a several days' lag phase, both the ECM-cultured cells as well as those which slowly had adapted to culture on plastic could be passed on to untreated culture ware and perpetuated thereon. In the case of well-differentiated low-grade gliomas where no growth in culture took place, the cultures on ECM could at least be used for initial experiments in the primary cultures (P0). Meningiomas usually attached well to both, plastic or ECM. In 50% of our cases the plating efficiency was higher on ECM but after successful initial culture, the delay until the cells on plastic reached confluence in comparison with those on ECM was 1 or 2 days. Again there were 2 cases in which the cells would not plate on plastic. Here the cells which after 1 day were still afloat plated to more than 80% within the first 2 h after transfer to ECM. In all cases the cells from plastic and ECM cultures were indistinguishable and could be passed onto untreated dishes henceforth. In later culture stages ECM offers several advantages: It is easier to shift cells to serum-free defined culture conditions, the cells will grow at a faster rate on ECM when in higher passages and the maximal number of passages possible is higher on ECM.     

Perlecan-enriched intercellular space of junctional epithelium provides primary infrastructure for leukocyte migration through squamous epithelial cells

The aim of this study was to demonstrate the presence of intraepithelial stroma represented by extracellular matrix (ECM) deposits in the junctional epithelium to clarify its function as a scaffold for leukocyte migration through epithelial cells. Twenty-three biopsy specimens from the gingiva including the junctional epithelium were examined to determine comparative protein and gene level expression profiles for keratin and ECM molecules between the junctional epithelium and the gingival epithelium using immunohistochemistry and in situ hybridization. Intraepithelial leukocyte types and frequencies were also determined and compared between the junctional and gingival epithelia. In the junctional epithelium, which was positive for keratin 19, perlecan was strongly deposited in intercellular space of the whole epithelial layer, while it was faintly positive around the parabasal layer of the gingival epithelium. Perlecan mRNA signals were enhanced to a greater degree in both epithelial and inflammatory cells within the junctional epithelium. In the junctional epithelium, greater numbers of neutrophils and macrophages were found as compared with the gingival epithelium. Our results showed that perlecan is the primary ECM molecule comprising intraepithelial stroma of the junctional epithelium, in which leukocytes may migrate on ECM scaffolds in intercellular space toward the surface of the gingival sulci or pockets.     

Remineralisation study of artificial root caries lesions after fluoride treatment. An in vitro study using electric caries monitor and transversal micro-radiography

Aims: To evaluate and compare remineralisation of root caries lesions after in vitro treatment with various fluoride (F) agents using an Electric Caries Monitor (ECM) and Transversal Micro‐Radiography (TMR). Materials: Permanent human teeth were extracted and root surface specimens were sectioned, prepared (n = 35), and randomly allocated into seven different experimental groups (groups 1–7). Methods: Root surfaces were demineralised in an acidified gel (pH = 5.0) for 3 weeks followed by various F treatments and stored in a standardised remineralising solution at 37°C for 6 weeks. The root surfaces were treated twice daily with different dentifrice slurries for 2 min, either with a neutral placebo dentifrice without F (group 5); or a neutral sodium fluoride (NaF) 1400 p.p.m. F dentifrice (group 1); or a neutral 1250 p.p.m. F dentifrice (group 6); or an acid dentifrice (pH 4.7) with 1400 p.p.m. F containing amine fluoride (AmF) (groups 3 and 4) or a 1250 p.p.m. (pH 4.7) AmF dentifrice (group 6). In groups 1, 2, 5, 6, and 7, the root surfaces were additionally rinsed for 2 min with a neutral non‐F placebo solution. In groups 3 and 4, rinsing were performed for 2 min with an acid (pH 4.7) 250 p.p.m. F solution, containing 125 p.p.m. F as AmF and 125 p.p.m. F as potassium fluoride (KF), once or twice per day respectively. ECM was used to measure electrical resistance on root surfaces at baseline and after 3 and 6 weeks respectively. TMR technique was used to measure and compare root surface lesion depths and mineral loss. Results: Six weeks daily treatment with a dentifrice slurry containing AmF followed by rinsing with a combination of equal amounts of AmF and KF solution twice a day showed a statistical significant higher ECM values compared with the other groups. TMR data measuring lesion depths and mineral loss reduction supported the results of the ECM findings. Conclusions: Daily application of a dentifrice slurry containing 1400 p.p.m. F as AmF combined with twice daily rinsing with a 250 p.p.m. F solution containing equal amount of AmF and KF significantly remineralise primary root caries lesions in vitro. ECM and TMR are valuable complementary methods in order to analyse the remineralisation processes.     

Pancreatic tumor cells influence the composition of the extracellular matrix

The malignant behavior of cancers depends on the microenvironmental context. We investigated compositional alterations of the extracellular matrix (ECM) in pancreatic cancer, with special emphasis on the proteoglycans decorin, lumican, and versican. Compared with normal controls (n=18), marked overexpression of these proteoglycans was observed in pancreatic cancer tissues (n=30) by quantitative RT-PCR (p<0.0001). Immunohistochemistry revealed abundance of proteoglycans in the ECM of pancreatic cancer specimens, whereas tumor cells themselves were devoid of either decorin, lumican or versican. RT-PCR confirmed pancreatic stellate cells (PSCs) as the major source of these proteins. Interestingly, TGFbeta1 and conditioned medium derived from pancreatic cancer cell lines synergistically suppressed the expression of known anti-tumor factors decorin and lumican, but stimulated the expression of pro-metastatic factor versican in cultured PSCs. These findings indicate that malignant cells can actively influence the composition of the ECM through TGFbeta1 and other soluble factors, altering their microenvironment in a tumor-favorable way.     

Temperature-dependent cell detachment on Pluronic gels

Cell culture on gels made of poly(ethylene oxide) and poly(propylene oxide) (Pluronic), which has a lower critical solution temperature around 30 degrees C, could be performed for 48 h. However, the Pluronic gels were highly hydrophilic and tended to dissolve in the culture medium. We achieved temperature-dependent detachment of cells from Pluronic gels containing or lacking extracellular matrix (ECM) by cooling the gels to 4 degrees C. Using normal human umbilical vein endothelial cells (HUVECs) grown on and released from Pluronic gels lacking ECM, we further found that the expression ratio of the surface markers CD34 and CD105 was twofold higher than for cells grown on polystyrene and removed with trypsin. In addition, the expression ratios for CD34 and CD105 on HUVECs cultivated on the Pluronic gels containing higher concentrations of ECM were lower, which may be due to ECM coating of the cell surface and, thus, interference with antibody binding. In summary, temperature-dependent detachment of cells from Pluronic gels allows the isolation of cells under mild conditions. This can be a powerful tool for surface marker analysis by flow cytometry.     

Human bronchial epithelial cells secrete laminin 5, express hemidesmosomal proteins, and assemble hemidesmosomes.

Epithelial cells attach to the basement membrane through adhesive contacts between the basal cells of the epithelium and the proteins of the extracellular matrix (ECM). The hemidesmosome (HD) is a specialized cell-ECM contact, that mediates the attachment of the epithelial cell basal surface to the ECM. In bronchial epithelial cells, the protein components that constitute the HD have not been demonstrated. Using immunohistochemical techniques, we determined that normal human bronchial epithelial (NHBE) cells express the HD cell surface integrin alpha6beta4 and produce laminin 5, the ECM protein associated with HDs. Furthermore, expression of the HD-associated structural proteins, bullous pemphigoid antigens 1 (BPAG 1) and 2 (BPAG 2), was demonstrated in NHBE cells by immunofluorescence microscopy and immunoblot analyses. In addition, we confirmed the presence of laminin 5 in the basement membrane (BM) of bronchial epithelial biopsy specimens and of BP230, BP180, and the alpha6beta4 integrin heterodimer at the site of bronchial epithelial cell-ECM interaction in vivo. Finally, using electron microscopy, we were able to demonstrate intact HDs in a glutaraldehyde-fixed NHBE cell monolayer. These findings suggest that bronchial epithelium forms HDs and that the laminin 5-alpha6beta4 integrin interaction may be important in stabilizing epithelial cell adhesion to the BM in the lung.     

Vocal fold tissue failure: preliminary data and constitutive modeling

In human voice production (phonation), linear small-amplitude vocal fold oscillation occurs only under restricted conditions. Physiologically, phonation more often involves large-amplitude oscillation associated with tissue stresses and strains beyond their linear viscoelastic limits, particularly in the lamina propria extracellular matrix (ECM). This study reports some preliminary measurements of tissue deformation and failure response of the vocal fold ECM under large-strain shear The primary goal was to formulate and test a novel constitutive model for vocal fold tissue failure, based on a standard-linear cohesive-zone (SL-CZ) approach. Tissue specimens of the sheep vocal fold mucosa were subjected to torsional deformation in vitro, at constant strain rates corresponding to twist rates of 0.01, 0.1, and 1.0 rad/s. The vocal fold ECM demonstrated nonlinear stress-strain and rate-dependent failure response with a failure strain as low as 0.40 rad. A finite-element implementation of the SL-CZ model was capable of capturing the rate dependence in these preliminary data, demonstrating the model's potential for describing tissue failure. Further studies with additional tissue specimens and model improvements are needed to better understand vocal fold tissue failure.     

Importance of size and sulfation of heparin in release of basic fibroblast growth factor from the vascular endothelium and extracellular matrix.

We have characterized the importance of size, sulfation, and anticoagulant activity of heparin in release of basic fibroblast growth factor (bFGF) from the subendothelial extracellular matrix (ECM) and the luminal surface of the vascular endothelium. For this purpose, 125I-bFGF was first incubated with ECM and confluent endothelial cell cultures, or administered as a bolus into the blood of rats, the immobilized 125I-bFGF was then subjected to release by various chemically modified species of heparin and size-homogeneous oligosaccharides derived from depolymerized heparin. Both totally desulfated and N-desulfated heparin failed to release the ECM-bound bFGF. Likewise, substitution of N-sulfate groups of heparin and low molecular weight heparin (fragmin) by acetyl or hexanoyl residues resulted in an almost complete inhibition of bFGF release by these polysaccharides. The presence of O-sulfate groups in heparin increased but was not critical for release of ECM-bound bFGF. Similar structural requirements were identified for release of 125I-bFGF bound to low-affinity sites on the surface of vascular endothelial cells. Oligosaccharides derived from depolymerized heparin and containing as little as 8-10 sugar units were, on a weight basis, equivalent to whole heparin in their ability to release bFGF from ECM. Low-sulfate oligosaccharides were less effective releasers of bFGF as compared to medium- and high-sulfate fractions of the same size oligosaccharides. Heparin fractions with high and low affinity to antithrombin III exhibited a similar high bFGF-releasing activity despite a 200-fold difference in their anticoagulant activities.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural and Ultrastructural Analysis of the Cervical Discs of Young and Elderly Humans

Several studies describing the ultrastructure and extracellular matrix (ECM) of intervertebral discs (IVDs) involve animal models and specimens obtained from symptomatic individuals during surgery for degenerative disease or scoliosis, which may not necessarily correlate to changes secondary to normal aging in humans. These changes may also be segment-specific based on different load patterns throughout life. Our objective was to describe the ECM and collagen profile of cervical IVDs in young (G1 - <35 years) and elderly (G2 - >65 years) presumably-asymptomatic individuals. Thirty cervical discs per group were obtained during autopsies of presumably-asymptomatic individuals. IVDs were analyzed with MRI, a morphological grading scale, light microscopy, scanning electron microscopy (SEM) and immunohistochemistry (IHC) for collagen types I, II, III, IV, V, VI, IX and X. Macroscopic degenerative features such as loss of annulus-nucleus distinction and fissures were found in both groups and significantly more severe in G2 as expected. MRI could not detect all morphological changes when compared even with simple morphological inspection. The loose fibrocartilaginous G1 matrix was replaced by a denser ECM in G2 with predominantly cartilaginous characteristics, chondrocyte clusters and absent elastic fibers. SEM demonstrated persistence of an identifiable nucleus and Sharpey-type insertion of cervical annulus fibers even in highly-degenerated G2 specimens. All collagen types were detected in every disc sector except for collagen X, with the largest area stained by collagens II and IV. Collagen detection was significantly decreased in G2: although significant intradiscal differences were rare, changes may occur faster or earlier in the posterior annulus. These results demonstrate an extensive modification of the ECM with maintenance of basic ultrastructural features despite severe macroscopic degeneration. Collagen analysis supports there is not a “pathologic” collagen type and changes are generally similar throughout the disc. Understanding the collagen and ultrastructural substrate of degenerative changes in the human disc is an essential step in planning restorative therapies.     

Extracellular matrix of sea urchin and other marine invertebrate embryos

The extracellular matrix surrounding the sea urchin embryo (outer ECM) contains fibers and granules of various sizes which are organized in recognizable patterns as shown by ultrastructural studies, particularly stereoimaging techniques. The use of the ruthenium red method for retaining and staining the ECM, with modifications of the Luft (Anatomical Record 171:347-368, 1971) method for invertebrate embryos, allows for the clarification of certain structures, particularly fiber compaction in the interzonal region, and microvillus-associated bodies. The inner ECM in the sea urchin embryo includes the basal lamina and blastocoel matrix. Stereoimages show that the fibers which are loosely distributed in the blastocoel matrix become compacted around the periphery of the blastocoel to form the basal lamina. The ruthenium red method was also used on a number of marine invertebrate embryos and larvae, representing different phyla, to facilitate comparisons between their surface coats. The similarities observed in the specimens shown suggest that ECMs are widely found on marine invertebrate eggs, embryos, and larvae, and that they resemble vertebrate ECMs and may, therefore, have similar functions.     

Comparative lipid analysis of purified plasma membranes and shed extracellular membrane vesicles from normal murine thymocytes and leukemic GRSL cells

The lipid fluidity in purified plasma membranes (PM) of murine leukemic GRSL cells, as measured by fluorescence polarization, is much higher than in PM of normal thymocytes. This was found to be due to relatively low contents of cholesterol and sphingomyelin and a high amount of unsaturated fatty acyl chains, especially linoleic acid, in the phospholipids. PM from GRSL cells contain markedly more phosphatidylethanolamine than those from thymocytes. For both GRSL cells and thymocytes the detailed lipid composition of isolated PM was compared with that of the corresponding shed extracellular membranes (ECM), which were isolated from the ascites fluid and from thymus cell suspensions, respectively. The somewhat decreased lipid fluidity of thymocyte ECM as compared to their PM, can be ascribed to the increased cholesterol/phospholipid molar ratio (0.88 vs. 0.74). No other major differences were found between the lipid composition of these membranes. In contrast, significant differences were found between PM and ECM from GRSL cells. In this system a much lower lipid fluidity of the shed ECM was found, due to the much increased cholesterol/phospholipid molar ratio (3.5-fold) and sphingomyelin (9-fold) content, as compared to the PM. Further, the ECM contain relatively more lysophosphatidylethanolamine and less phosphatidylcholine and -inositol. ECM contain a higher amount of polyunsaturated fatty acids, especially in the phosphatidylethanolamine and lysophosphatidylethanolamine classes. On the other hand, the fatty acids of phosphatidylcholine and lysophosphatidylcholine are more saturated than in PM. In particular, ECM of GRSL cells contain less oleic and linoleic acid residues and more arachidonic acid and 22:polyunsaturated fatty acid residues than PM. The possible relevance of these differences with respect to the mechanism of shedding of vesicles from the cell surface, is discussed.     

Ultrastructural Study of Changes in Cell Morphology and Extracellular Matrix in Prospective Endodermal Cells of Newt Embryos (Cynops pyrrhogaster) before and during Gastrulation

The cell morphology, cell-to-cell contact behavior and extracellular matrix (ECM) of inner cells (prospective endodermal cells) of newt ( Cynops pyrrhogaster ) embryos were examined from the morula to gastrula stage by light and electron microscopy. The inner cells showed increased cell-to-cell contact from the early blastula to early gastrula stage. The cells formed blebs (5–15 μm in diameter) during the blastula stage, and started to form filopodia and lamellipodia before gastrulation. Alcian blue and lanthanum nitrate treatment revealed ECM components on the cell surface in the early blastula stage and these components increased in amount from the late blastula to early gastrula stage. It is suggested that the increase in ECM components on the cell surface may have some relation with changes in cell-to-cell contact and formation of processes on the cell surface. Besides the cell surface ECM components, glycogen-like granules were observed in intercellular spaces. From the distribution of granules in gastrulae, it is suggested that these may be important in maintaining intercellular spaces for migration of invaginating cells.     

Quartz crystal microbalance biosensor study of endothelial cells and their extracellular matrix following cell removal: Evidence for transient cellular stress and viscoelastic changes during detachment and the elastic behavior of the pure matrix

A quartz crystal microbalance (QCM) cell biosensor utilizing living endothelial cells (ECs) or human breast cancer cells (MCF-7) adhering to the gold QCM surface was used to study the relative contributions of the cells and their underlying extracellular matrix (ECM) to the measured QCM Deltaf and DeltaR shifts. The ECM represents a natural biomaterial that is synthesized by the cells to enable their attachment to surfaces. We followed the detachment of the ECs or MCF-7 cells from their ECM using a nonproteolytic method and were able to apportion the total frequency, Deltaf, decrease of the biosensor into contributions from cell attachment and from the intact underlying ECM. We also demonstrated that the Deltaf shift remaining after EC removal corresponds to ECM as determined by light microscopic visualization of the stained protein. During the process of cell detachment, we observed a novel transient increase in viscoelastic behavior expressed as a transient increase in the motional resistance, DeltaR, parameter. Then we showed via a simulation experiment using ECs stained with fluorescent rhodamine-labeled phalloidin, an actin stain, that the transient viscoelastic increase correlated with cellular stress exhibited by the cells during removal with ethylene glycol bis(2-aminoethyl ether)-N,N,N',N'- tetraacetic acid. Prior to cells lifting from their ECM, the attached ECs rearrange their actin microfilaments first into peripheral stress fibers and second into internal aggregates, to maintain cell-cell connectivity, retain their spread morphology, and attempt to adhere more tightly to their underlying ECM. The decrease in DeltaR following its transient rise corresponds to cells finally losing their attachment focal points and lifting from the ECM. We also characterized the normalized f shifts, -Delta(Deltaf)(ECM)/attached cell and -Delta(Deltaf)(cells)/attached cell, as a function of varying the number of adherent cells. Finally, we demonstrate that the underlying native ECM biomaterial, from which all cells have been removed, does not exhibit any significant level of energy dissipation, in contrast to the cells when they are attached to the ECM.     

Morphologic complexity of the pericellular matrix in the annulus of the human intervertebral disc

The pericellular region of the extracellular matrix (ECM) contains collagens, proteoglycans and other noncollagenous matrix proteins. Although such specialized pericellular ECM has been well studied in articular cartilage, little is known about the pericellular matrix in the disc. In the study reported here, pericellular matrix was studied in annulus tissue from 52 subjects ranging in age from 17-74 years. In aging/degenerating intervertebral discs, cells were identified that formed a distinctive cocoon of encircling pericellular ECM. Immunohistochemical studies identified types I, II, III and VI collagen in these pericellular sites with diverse morphological features. Similar types of changes in the pericellular matrix were observed in both surgical specimens and control donor discs. Results indicate the need for future studies to address why such specialized matrix regions form around certain disc cells and to determine the consequences of these unusual matrix regions on annular lamellar organization and function.     

Three-dimensional modeling of mechanical forces in the extracellular matrix during epithelial lumen formation

Mechanical interactions between cells and extracellular matrix (ECM) mediate epithelial cyst formation. This work relies on the combination of numerical modeling with live cell imaging, to piece together a novel nonintrusive method for determining three-dimensional (3D) mechanical forces caused by shape changes of a multicellular aggregate at the early stages of epithelial cyst formation. We analyzed the evolution of Madin-Darby canine kidney cells in 3D cultures using time-lapse microscopy, with type I collagen gel forming the ECM. The evolving 3D interface between the ECM and the cell aggregate was obtained from microscopy images, and the stress on the surface of a proliferating aggregate and in the surrounding ECM was calculated using the finite element method. The viscoelastic properties of the ECM (a needed input for the finite element method solver) were obtained through oscillatory shear flow experiments on a rheometer. For validation purpose, the forces exerted by an aggregate on a force-sensor array were measured and compared against the computational results.