Sequential behaviour of extracellular matrix glycoproteins in an experimental model of hepatic fibrosis

The behaviour of extracellular matrix glycoproteins (fibronectin, laminin, basement membrane heparan-sulphate proteoglycan, type III, IV and V collagens) has been investigated in a sequential model of experimental hepatic fibrosis, using an immunofluorescence technique. The presence of some basement membrane macromolecules (such as type IV and V collagens, laminin and basement membrane heparan-sulphate proteoglycan) is detectable only in the early stages of septa formation, while type III collagen and fibronectin persist in late septa. These data suggest that hepatic fibroplasia proceeds through different steps in which stromal glycoproteins are preferentially engaged, as happens during organogenesis.     

Cryoprotectant permeability parameters for cells used in a bioengineered human corneal equivalent and applications for cryopreservation

A human corneal equivalent is being developed with applications in pharmaceutical testing and biomedical research, but the distribution of this engineered tissue, depends on successful cryopreservation. Cryopreservation of tissues depends on the presence of cryoprotectants, their addition and removal, and exposure to conditions during freezing and thawing, all of which depend on cellular membrane permeabilities to water and cryoprotectant. This study defines the permeability properties that define the rate of water and cryoprotectant movement across the plasma membrane of isolated human corneal endothelial, keratocyte, and epithelial cells. Cells were transferred from isotonic conditions (300 mosm/kg) to 0.5, 1, or 2 M dimethyl sulfoxide and propylene glycol solutions at constant temperature, and cell volumes monitored using an electronic particle counter. Histograms describing cell volume changes over time after cryoprotectant exposure allowed calculation of hydraulic conductivity (Lp), cryoprotectant permeability (Ps), and the reflection coefficient (sigma). Experimental values for Lp and Ps at 4, 13, 22, and 37 degrees C were used to determine the Arrhenius activation energy (Ea). Defining the permeability parameters and temperature dependencies allows simulation of responses of human corneal cells to addition and removal of cryoprotectants and to freezing conditions, allowing amount of supercooling, intracellular electrolyte concentration, and intracellular cryoprotectant concentration to be calculated. Simulations also show that the constituent cells in the bioengineered cornea respond differently to addition and removal of cryoprotectants and to freezing. This study has defined the requirements during cryopreservation for the corneal cells; future work will define the matrix requirements which will allow the development of a cryopreservation protocol.     

Glycerol induces reuterin production and decreases Escherichia coli population in an in vitro model of colonic fermentation with immobilized human feces

Lactobacillus reuteri ATCC 55730 is a probiotic strain that produces, in the presence of glycerol, reuterin, a broad-spectrum antimicrobial substance. This strain has been shown to prevent intestinal infections in vivo; however, its mechanisms of action, and more specifically whether reuterin production occurs within the intestinal tract, are not known. In this study, the effects of L. reuteri ATCC 55730 on intestinal microbiota and its capacity to secrete reuterin from glycerol in a novel in vitro colonic fermentation model were tested. Two reactors were inoculated with adult immobilized fecal microbiota and the effects of daily addition of L. reuteri into one of the reactors (c.10(8) CFU mL(-1)) without or with glycerol were tested on major bacterial populations and compared with addition of glycerol or reuterin alone. The addition of glycerol alone or with L. reuteri increased numbers of the Lactobacillus-Enterococcus group and decreased Escherichia coli. The addition of reuterin significantly and selectively decreased E. coli without affecting other bacterial populations. The observed decrease in E. coli concentration during the addition of glycerol (in presence or absence of L. reuteri) could be due to in situ reuterin production because 1,3-propanediol, a typical product of glycerol fermentation, was detected during the addition of glycerol.     

The influence of extracellular matrix on reversible gap junction formation in vitro

In vivo gap junctions (gj) are common in the subumbrellar plate endoderm of anthomedusa. When isolated and cultivated in artificial sea water the tissue, consisting of one cell type only, forms a spheroid in which all gap junctions disappear. Gap junction (gj) formation can, however, be induced by attachment and consecutive spreading of the endodermal tissue (spheroid) on stretched extracellular matrix (ECM) material isolated from the polyp stage (with Ca2+-Mg2+-free sea water, without EDTA). Formation, and loss of gj is reversible and strictly corresponds with the alteration from the monolayer 'spread' (on stretched ECM) to 'spheroid' arrangement (no ECM) of the endodermal cells. The functional competence of induced gj is ascertained by injection of Lucifer Yellow, and the transfer of the dye is used to map the pattern of communication. The experimental conditions that result in gj formation simulate the in vivo situation of the endoderm. The influence of the ECM on gj formation, and the structural organization of the isolated endodermal tissue in this well defined in vitro system are discussed.     

Formation of organoid structures and extracellular matrix production in an intestinal epithelial cell line during long-term in vitro culture

During the long-term in vitro maintenance of an epithelial cell line established from rat duodenum (IEC-17) we have observed progressive morphological changes which, after approximately 4-5 months in culture, led to a loss of substrate adherence and to the formation of organoid structures characterized by organized layers of cells separated by continuous extracellular-like material and delimiting close lumina. The cells exhibited a defined polarity with deposition of extracellular matrix components on one side and development of microvilli on the opposite surface. The morphological changes observed did not appear to be the expression of spontaneous transformation since the cells retained a normal diploid rat karyotype and did not grow in soft agar. In this report we present the optical and electron microscopical characterization of the progressive organotypic differentiation of the cell line. Further studies are currently in progress to characterize the extracellular matrix during the process of differentiation.     

Dissecting the influence of cellular senescence on cell mechanics and extracellular matrix formation in vitro

Tissue formation and healing both require cell proliferation and migration, but also extracellular matrix production and tensioning. In addition to restricting proliferation of damaged cells, increasing evidence suggests that cellular senescence also has distinct modulatory effects during wound healing and fibrosis. Yet, a direct role of senescent cells during tissue formation beyond paracrine signaling remains unknown. We here report how individual modules of the senescence program differentially influence cell mechanics and ECM expression with relevance for tissue formation. We compared DNA damage-mediated and DNA damage-independent senescence which was achieved through over-expression of either p16^Ink4a or p21^Cip1 cyclin-dependent kinase inhibitors in primary human skin fibroblasts. Cellular senescence modulated focal adhesion size and composition. All senescent cells exhibited increased single cell forces which led to an increase in tissue stiffness and contraction in an in vitro 3D tissue formation model selectively for p16 and p21-overexpressing cells. The mechanical component was complemented by an altered expression profile of ECM-related genes including collagens, lysyl oxidases, and MMPs. We found that particularly the lack of collagen and lysyl oxidase expression in the case of DNA damage-mediated senescence foiled their intrinsic mechanical potential. These observations highlight the active mechanical role of cellular senescence during tissue formation as well as the need to synthesize a functional ECM network capable of transferring and storing cellular forces.     

Effect of extracellular matrix proteins on in vitro testosterone production by rat Leydig cells

The aim of this study was to detect the effect of extracellular matrix (ECM) proteins on rat Leydig cell shape, adhesion, expression of integrin subunits and testosterone production, in vitro. Leydig cells isolated from adult rats were cultured on plates uncoated or coated with different concentrations of laminin-1, fibronectin, or type IV collagen in the presence or absence of hCG for 3 or 24 hr. A significant increase of cell adhesion and of alpha3, alpha5, and beta1 integrin subunit expression was observed when cells were cultured on ECM proteins, compared to those grown on uncoated plates. Leydig cells cultured on glass coverslips coated with ECM proteins for 24 hr exhibited elongated shapes with long cell processes (spreading), while cells cultured on uncoated plates showed few cell processes. A significant decrease in testosterone production was observed when basal and hCG-stimulated Leydig cells were cultured for 3 or 24 hr on plates coated with type IV collagen (12 and 24 microg/cm(2)) compared to uncoated plates. A significant though a slighter decrease in testosterone production was also observed in cells cultured on plates coated with fibronectin (12 and 24 microg/cm(2)), compared to uncoated plates. Laminin-1 did not modify testosterone production under basal or hCG stimulated conditions. These results suggest that ECM proteins are able to modulate Leydig cell steroidogenesis, in vitro.     

A computational model of in vitro angiogenesis based on extracellular matrix fibre orientation

Recent interest in the process of vascularisation within the biomedical community has motivated numerous new research efforts focusing on the process of angiogenesis. Although the role of chemical factors during angiogenesis has been well documented, the role of mechanical factors, such as the interaction between angiogenic vessels and the extracellular matrix, remains poorly understood. In vitro methods for studying angiogenesis exist; however, measurements available using such techniques often suffer from limited spatial and temporal resolutions. For this reason, computational models have been extensively employed to investigate various aspects of angiogenesis. This paper outlines the formulation and validation of a simple and robust computational model developed to accurately simulate angiogenesis based on length, branching and orientation morphometrics collected from vascularised tissue constructs. Microvessels were represented as a series of connected line segments. The morphology of the vessels was determined by a linear combination of the collagen fibre orientation, the vessel density gradient and a random walk component. Excellent agreement was observed between computational and experimental morphometric data over time. Computational predictions of microvessel orientation within an anisotropic matrix correlated well with experimental data. The accuracy of this modelling approach makes it a valuable platform for investigating the role of mechanical interactions during angiogenesis.     

Absence of SPARC in lens epithelial cells results in altered adhesion and extracellular matrix production in vitro

The matricellular protein SPARC (also known as osteonectin and BM-40) is expressed abundantly in lens epithelium. That SPARC-null mice exhibit early cataractogenesis, indicates a role for SPARC in the maintenance of lens transparency. Comparison of cultured wild-type and SPARC-null lens epithelial cells revealed significant changes in adhesion to different substrates. SPARC-null lens cells displayed enhanced attachment and spreading, focal adhesion formation, and resistance to trypsin detachment in comparison to wild-type cells. In the absence of SPARC, there was increased deposition of the ECM protein laminin-1 (LN-1). Proteins associated with focal adhesions were increased in SPARC-null versus wild-type lens cells: levels of alpha6-integrin heterodimers, talin, and paxillin phosphorylated on tyrosine were enhanced significantly, as was the association of beta1-integrin with talin and paxillin. Restoration of the wild-type phenotype in SPARC-null cultures was accomplished through genetic rescue by stable transfection of SPARC cDNA. Our findings indicate that SPARC is counter-adhesive for murine lens epithelial cells and demonstrate that multiple factors contribute to this activity. We also identify SPARC as a modulator of LN-1 secretion and deposition by these cells, an activity important in epithelial cell-ECM interactions in the ocular lens.     

P-glycoprotein expression in extracellular matrix formation of chondrogenic differentiation of human adult stem cells

Mesenchymal stem cell (MSC) has been known as a good source of progenitor for multiple connective tissue including cartilage, muscle, adipocyte, and bone. P-glycoproteins (P-gps) also known as ABCB1 that exports diverse substrates are the product of the multidrug resistance-1 (MDR-1) gene. P-gp expression has been reported in chondrosarcoma and hypertrophic chondrocyte in the human growth plate. This study was designed to investigate the expression of P-gp during chondrogenic differentiation of adult human stem cells. Bone marrow samples were obtained from nine human donors after informed consent. The isolated mononuclear cells (MNCs) were incubated as one pellet/tube and 0.5ml chondrogenic medium in the presence of 10ng/ml of TGF-beta 1 and TGF-beta 3 for 28 days. The expression of surface P-gps was analyzed by flow cytometry and quantitative RT-PCR was performed for the detection of mRNA expression of MDR-1 and type II collagen gene. Total collagen and glycosaminoglycan (GAG) contents of the pellets were measured. Surface P-gp expression of the MSCs was decreased during chondrogenic differentiation. MDR-1 gene was decreased 10-fold after the 2-week incubation whereas type II collagen gene was increased 491-fold after the 4-week incubation in chondrogenic medium. The total amount of collagen and GAG were increased during pellet culture. This study has demonstrated a decrease in expression of P-gp and down regulation of MDR-1 gene consistently by flow cytometry and quantitative RT-PCR, but an increased expression of type II collagen on MSC during chondrogenesis.     

Atropa belladonna L. water extract: modulator of extracellular matrix formation in vitro and in vivo

Previously, we found that treatment of cutaneous wounds with Atropa belladonna L. (AB) revealed shortened process of acute inflammation as well as increased tensile strength and collagen deposition in healing skin wounds (Gál et al. 2009). To better understand AB effect on skin wound healing male Sprague-Dawley rats were submitted to one round full thickness skin wound on the back. In two experimental groups two different concentrations of AB extract were daily applied whereas the control group remained untreated. For histological evaluation samples were removed on day 21 after surgery and stained for wide spectrum cytokeratin, collagen III, fibronectin, galectin-1, and vimentin. In addition, in the in vitro study different concentration of AB extract were used to evaluate differences in HaCaT keratinocytes proliferation and differentiation by detection of Ki67 and keratin-19 expressions. Furthermore, to assess ECM formation of human dermal fibroblasts on the in vitro level fibronectin and galectin-1 were visualized. Our study showed that AB induces fibronectin and galectin-1 rich ECM formation in vitro and in vivo. In addition, the proliferation of keratinocytes was also increased. In conclusion, AB is an effective modulator of skin wound healing. Nevertheless, further research is needed to find optimal therapeutic concentration and exact underlying mechanism of action.     

Mesenchymal stem cells differentiate into keratinocytes and express epidermal kallikreins: Towards an in vitro model of human epidermis

Epidermal differentiation is a complex process in which keratinocytes go through morphological and biochemical changes in approximately 15 to 30 days. Abnormal keratinocyte differentiation is involved in the pathophysiology of several skin diseases. In this scenario, mesenchymal stem cells (MSCs) emerge as a promising approach to study skin biology in both normal and pathological conditions. Herein, we have studied the differentiation of MSC from umbilical cord into keratinocytes. MSC were cultured in Dulbecco's modified Eagle's medium (DMEM) (proliferation medium) and, after characterization, differentiation was induced by culturing cells in a defined keratinocyte serum-free medium (KSFM) supplemented with epidermal growth factor (EGF) and calcium chloride ions. Cells cultivated in DMEM were used as control. Cultures were evaluated from day 1 to 23, based on the cell morphology, the expression of p63, involucrin and cytokeratins (KRTs) KRT5, KRT10 and KRT14, by quantitative polymerase chain reaction, Western blot analysis or immunofluorescence, and by the detection of epidermal kallikreins activity. In cells grown in keratinocyte serum-free medium with EGF and 1.8 mM calcium, KRT5 and KRT14 expression was shown at the first day, followed by the expression of p63 at the seventh day. KRT10 expression was detected from day seventh while involucrin was observed after this period. Data showed higher kallikrein (KLK) activity in KSFM-cultured cells from day 11th in comparison to control. These data indicate that MSC differentiated into keratinocytes similarly to that occurs in the human epidermis. KLK activity detection appears to be a good methodology for the monitoring the differentiation of MSC into the keratinocyte lineage, providing useful tools for the better understanding of the skin biology.     

Inactivation of the Pseudomonas putida KT2440 dsbA gene promotes extracellular matrix production and biofilm formation

To identify genes essential to biofilm formation in Pseudomonas putida KT2440, 12 mutants defective in oxidative stress-related or metabolic pathway-related genes were evaluated. Of them, only the dsbA mutant lacking the disulfide bond isomerase exhibited significantly increased attachment to the polystyrene surface. Visual evaluation by extracellular matrix staining and scanning electron microscopy indicated that the KT2440-Δ dsbA strain displays enhanced extracellular matrix production, rugose colony morphology on agar plates and floating pellicles in static culture. Accordingly, we propose that deletion of the dsbA gene may stimulate production of the extracellular matrix, resulting in those phenotypes. In addition, the lack of detectable fluorescence in the KT2440-Δ dsbA under UV light as well as in both the wild type and the KT2440-Δ dsbA when grown on Luria–Bertani plates containing ferrous iron suggests that the fluorescent molecule may be a fluorescent siderophore with its synthesis/secretion controlled by DsbA in KT2440. These phenotypic defects observed in the dsbA mutant were complemented by the full-length KT2440 and Escherichia coli dsbA genes. In contrast to the role of DsbA in other bacteria, our results provide the first evidence that disruption of P. putida KT2440 dsbA gene overproduces the extracellular matrix and thus promotes biofilm formation.     

Capacitively coupled electric fields accelerate proliferation of osteoblast-like primary cells and increase bone extracellular matrix formation in vitro

Over the last few years, electric and electromagnetic fields have gained more and more significance in the therapy of bone fracture healing and bone disease. Yet, the underlying mechanisms on a cellular and molecular level are not completely understood. In the present study we have investigated the effects of capacitively coupled, pulsed electric fields on cellular proliferation, alkaline phosphatase activity, and matrix protein synthesis of osteoblast-like primary cells in vitro. Cells were derived from bovine periosteum and electrically stimulated by saw-tooth pulses of 100 V external voltage and 16 Hz frequency. This corresponds to an electric field of 6 kV/m across the cell membranes as could be shown by computer simulation. Field application caused acceleration of cell culture development. A significant increase of proliferation concurrent with an enhancement of alkaline phosphatase activity was observed in sub-confluent cultures. Exposure of confluent osteoblast-like primary cells to electric fields resulted in enhanced synthesis and secretion of extracellular matrix-related proteins. These findings suggest that capacitively coupled electric fields accelerate bone cell proliferation and differentiation in vitro and enhance the synthesis of cells leading to promoted matrix formation and maturation.     

Influence of substrate composition on human embryonic stem cell differentiation and extracellular matrix production in embryoid bodies

Stem cells reside in specialized niches in vivo. Specific factors, including the extracellular matrix (ECM), in these niches are directly responsible for maintaining the stem cell population. During development, components of the stem cell microenvironment also control differentiation with precise spatial and temporal organization. The stem cell microenvironment is dynamically regulated by the cellular component, including stem cells themselves. Thus, a mechanism exists whereby stem cells modify the ECM, which in turn affects the fate of the stem cell. In this study, we investigated whether the type of ECM initially adsorbed to the culture substrate can influence the composition of the ECM deposited by human embryonic stem cells (hESCs) differentiating in embryoid bodies, and whether different ECM composition and deposition profiles elicit distinct differentiation fates. We have shown that the initial ECM environment hESCs are exposed to affects the fate decisions of those cells and that this initial ECM environment is constantly modified during the differentiation process. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:212–219, 2015     

Anti-inflammatory potential of human corneal stroma-derived stem cells determined by a novel in vitro corneal epithelial injury model

BACKGROUND An in vitro injury model mimicking a corneal surface injury was optimised using human corneal epithelial cells(hCEC).AIM To investigate whether corneal-stroma derived stem cells(CSSC) seeded on an amniotic membrane(AM) construct manifests an anti-inflammatory, healing response.METHODS Treatment of hCEC with ethanol and pro-inflammatory cytokines were compared in terms of viability loss, cytotoxicity, and pro-inflammatory cytokine release, in order to generate the in vitro injury. This resulted in an optimal injury of 20%(v/v) ethanol for 30 s with 1 ng/mL interleukin-1(IL-1) beta. Co-culture experiments were performed with CSSC alone and with CSSC-AM constructs.The effect of injury and co-culture on viability, cytotoxicity, IL-6 and IL-8 production, and IL1 B, TNF, IL6, and CXCL8 mRNA expression were assessed.RESULTS Co-culture with CSSC inhibited loss of hCEC viability caused by injury. Enzyme linked immunosorbent assay and polymerase chain reaction showed a significant reduction in the production of IL-6 and IL-8 pro-inflammatory cytokines, and reduction in pro-inflammatory cytokine mRNA expression during co-culture with CSSC alone and with the AM construct. These results confirmed the therapeutic potential of the CSSC and the possible use of AM as a cell carrier for application to the ocular surface.CONCLUSION CSSC were shown to have a potentially therapeutic anti-inflammatory effectwhen treating injured hCEC, demonstrating an important role in corneal regeneration and wound healing, leading to an improved knowledge of their potential use for research and therapeutic purposes.