Spicule and flagellated chamber formation in a growth zone of Aphrocallistes vastus (Porifera,Hexactinellida)

Three species of glass sponges (Class Hexactinellida) form massive deep‐water reefs by growing on the skeletons of past generations, with new growth largely vertical and away from sediment that buries the lower portions. Growth is therefore essential for reef health, but how glass sponges produce new skeleton or tissue is not known. We used fluorescence, light, and electron microscopy to study skeletal and tissue growth in the reef‐forming glass sponge Aphrocallistes vastus. The sponge consists of a single large tube (the osculum), usually with several side branches, each of which can function as an effective excurrent vent. New tissue forms at the tips of each of these extensions, but how this occurs in a syncytial animal, and how the tubes expand laterally as the sponge gets larger, are both unknown. The fluorescent dye PDMPO labeled more spicule types in the tips of the sponge than elsewhere, indicating growth that was concentrated at the edge of the osculum. New tissue production was tracked using the thymidine analog EdU. EdU‐labeled nuclei were found predominantly at the edge or lip of the osculum. In that region new flagellated chambers were formed from clusters of choanoblasts that spread out around the enlarging chamber. In cellular sponges clusters of choanocytes form flagellated chambers through several rounds of mitotic divisions, and also by immigration of mesohyl cells, to expand the chamber to full size. By contrast, chambers in glass sponges expand as choanoblasts produce enucleate collar bodies to fill them out. Growing chambers with enucleate structures may be an adaptation to life in the deep sea if chambers with cells, and therefore more nuclei, are costly to build.     

In vitro evaluation of natural marine sponge collagen as a scaffold for bone tissue engineering

The selection of a suitable scaffold matrix is critical for cell-based bone tissue engineering. This study aimed to identify and characterize natural marine sponges as potential bioscaffolds for osteogenesis. Callyspongiidae marine sponge samples were collected from the Fremantle coast of Western Australia. The sponge structure was assessed using scanning electron microscopy (SEM) and Hematoxylin and eosin. Mouse primary osteoblasts were seeded onto the sponge scaffold and immunostained with F-actin to assess cell attachment and aggregation. Alkaline phosphatase expression, von Kossa staining and real-time PCR were performed to examine the osteogenic potential of sponge samples. SEM revealed that the sponge skeleton possessed a collagenous fibrous network consisting of interconnecting channels and a porous structure that support cellular adhesion, aggregation and growth. The average pore size of the sponge skeleton was measured 100 to 300 μm in diameter. F-actin staining demonstrated that osteoblasts were able to anchor onto the surface of collagen fibres. Alkaline phosphatase expression, a marker of early osteoblast differentiation, was evident at 7 days although expression decreased steadily with long term culture. Using von Kossa staining, mineralisation nodules were evident after 21 days. Gene expression of osteoblast markers, osteocalcin and osteopontin, was also observed at 7, 14 and 21 days of culture. Together, these results suggest that the natural marine sponge is promising as a new scaffold for use in bone tissue engineering.     

Embryogenesis in the glass sponge Oopsacas minuta: Formation of syncytia by fusion of blastomeres

Sponges (Porifera) are unusual animals whose body plans makeinterpreting phylogenetic relationships within the group andwith other basal metazoan taxa a difficult task. Although molecularapproaches have offered new insights, some questions requirea morphological approach using detailed ultrastructural or lightmicroscopical studies of developing embryos and larvae. Glasssponges (Hexactinellida) have perhaps the most unusual bodyplan within the Metazoa because the majority of the tissue ofthe adult consists of a single giant multinucleated syncytiumthat forms the inner and outer layers of the sponge and is joinedby cytoplasmic bridges to uninucleate cellular regions. Herewe have used serial section transmission and high-resolutionscanning electron microscopy to examine when syncytia firstform in the cave-dwelling glass sponge Oopsacas minuta. We confirmthat in O. minuta blastomeres are separate until the 32-cellstage; cleavage is equal but asynchronous until a hollow blastulais formed. The sixth division yields a collection of variouslysized micromeres at the surface of the embryo and large yolk-and lipid-filled macromeres lining the blastocoel. Syncytiathen form by the fusion of micromeres to form cytoplasmic bridgeswith each other and the fusion of macromeres to form the futuremultinucleated trabecular tissue of the larva and adult sponge.The multinucleated trabecular tissue envelops and forms cytoplasmicbridges with all uninucleate cells, covering the developinglarva with a continuous syncytial epithelium. Differentiationof tissues occurs very early during embryogenesis with the separationof uninucleate and multinucleate lineages, but all cells andsyncytia are joined by cytoplasmic bridges such that there iscytoplasmic continuity throughout the entire larva. Althoughglass sponges begin life as a cellular embryo, the unusual mechanismof syncytia formation at such an early stage in developmentdistinguishes this group of animals from their closest multicellularrelatives, the Demospongiae. Most important, however, thesedata lend support to the hypothesis that the original metazoanswere cellular, not syncytial.     

Sponge cell aggregation: checkpoints in development indicate a high level of organismal complexity

Studies of regeneration provide insight across many scales of animal biology from the processes of cellular communication to the ecology of whole populations. Sponges are highly regenerative animals, with studies showing adults can both recover large portions of their body after predation or damage due to storms, and even reform whole individuals, via an aggregation stage, from dissociated tissues. While sponges are clearly highly regenerative, few studies actually show dissociated cells forming functional individuals. As sponges often serve as model organisms for studying the development and function of traits in metazoans, determining the universality and mechanics of their regeneration potential is important. We tested the capacity of members of seven sponge species from temperate freshwater and marine environments, from a range of taxonomic positions, and with different habits, to form functional sponges after dissociation. Development to a functional sponge progressed through a series of checkpoints: the sorting of cells and removal of debris; adhesion to a substrate and differentiation of cells; organization of cells into tissues; and regionalization of tissues. Two of the seven species tested, Spongilla lacustris and Haliclona cf. permollis, progressed through all four checkpoints, while the remaining five species progressed to various levels of development before aggregates disintegrated. Our findings highlight three important conclusions: (1) The ability of aggregates to differentiate into functional sponges is not as widespread as previously thought; (2) The species‐specific ability of aggregates to develop to functional sponges appears to be an adaptive trait; and (3) The progression of development in aggregates through checkpoints, which in later development involves formation of tissues and regionalization of tissues, highlights the complexity of the sponge body plan and suggests fundamental rules in development shared across metazoans.     

Cyclosporin A suspends transplantation reactions in the marine sponge Microciona prolifera

Sponges are the simplest extant animals but nevertheless possess self-nonself recognition that rivals the specificity of the vertebrate MHC. We have used dissociated cell assays and grafting techniques to study tissue acceptance and rejection in the marine sponge Microciona prolifera. Our data show that allogeneic, but not isogeneic, cell contacts trigger cell death and an increased expression of cell adhesion and apoptosis markers in cells that accumulate in graft interfaces. Experiments investigating the possible existence of immune memory in sponges indicate that faster second set reactions are nonspecific. Among the different cellular types, gray cells have been proposed to be the sponge immunocytes. Fluorescence confocal microscopy results from intact live grafts show the migration of autofluorescent gray cells toward graft contact zones and the inhibition of gray cell movements in the presence of nontoxic concentrations of cyclosporin A. These results suggest that cell motility is an important factor involved in sponge self/nonself recognition. Communication between gray cells in grafted tissues does not require cell contact and is carried by an extracellular diffusible marker. The finding that a commonly used immunosuppressor in human transplantation such as cyclosporin A blocks tissue rejection in marine sponges indicates that the cellular mechanisms for regulating this process in vertebrates might have appeared at the very start of metazoan evolution.     

The significance of syncytial tissues for the position of the hexactinellida in the metazoa

Hexactinellid sponges are metazoans in which the major tissuecomponent is a multinucleated syncytium. The preferred deepwaterhabitat of these sponges makes collection of hexactinellidsin good condition difficult, and has hindered extensive examinationof their body plan. Nonetheless, over the last three decadesa number of studies have explored their ecology, histology andphysiology. It has been shown that hexactinellids are extremelylong-lived animals. Their cytoplasm consists of a giant, multinucleatedtissue, the trabecular syncytium, which is connected via openand plugged cytoplasmic bridges to cells such as archaeocytes,choanoblasts, and cells with spherical inclusions. Because allof the sponge is cytoplasmically interconnected, electricalsignals can propagate through the animal. The effector responseis arrest of the feeding current. The perforate plugged junctionapparently allows tissues to specialize in different ways whilemaintaining limited cytoplasmic continuity. Larvae of hexactinellidsponges are already largely syncytial. Although it is not knownwhen the first syncytial tissues are formed or when perforateplugged junctions first appear during embryogenesis, evidencethat embryos are cellular until gastrulation suggests that hexactinellidsponges may have evolved from cellular sponges and that syncytialtissues are not a primitive trait of the Metazoa.     

Species specificity in cell-substrate interactions in medusae

A new system is described for the study of ECM-tissue interactions, using the ECM (called mesogloea) of various cnidarians and isolated striated muscle and endodermal tissue of jellyfish. The mesogloea consists mainly of water and collagen. It is present in all cnidarians and can be isolated without enzyme treatment. It can be used as a substrate to which cells and tissues adhere and on which they spread and migrate. Tissues of striated muscle and endoderm adhere and spread not only on mesogloea from regions they normally cover, but also from other regions of the animal. However, adhesion and spreading are highly species-specific. Species-specific adhesion is found throughout the whole mass of mesogloea even at regions where cells do not occur naturally. The cell adhesion factor can be extracted from the mesogloea so that the mesogloea no longer shows any cell adhesion properties. The extract consists mainly of a cysteine-containing collagen.     

The inhibitory properties of various sponges on Listeria spp.

Various retail and environmental sponges were tested for inhibitory properties against Listeria species and several other bacterial genera. Sterile sponges, unrinsed and rinsed in sterile distilled water or sterile neutralizing buffer, were placed on seeded plates of tryptic soy agar with 0.6% yeast extract. Plates were incubated at 30°C for 24 h and zones of inhibition measured.
The Systems Plus environmental sponge and the Technical Service Consultants Ltd sponge (sTc)© proved to be the only sponges which consistently demonstrated no inhibitory properties. Results using scanning electron microscopy showed considerable bacterial attachment to the Systems Plus sponge, further corroborating these findings.     

Selective adhesion of macrophages to denatured forms of type I collagen is mediated by scavenger receptors.

Macrophages (Mφs) are multifunctional immune cells which are involved in the regulation of immune and inflammatory responses, as well as in tissue repair and remodeling. In tissues, Mφs reside in areas which are rich in extracellular matrix (ECM), the structural component which also plays an essential role in regulating a variety of cellular functions. A major ECM protein encountered by Mφs is type I collagen, the most abundant of the fibril-forming collagens. In this study, the adhesion of RAW 264.7 murine Mphis to native fibrillar, monomeric, and denatured type I collagen was investigated. Using atomic force microscopy, structural differences between fibrillar and monomeric type I collagen were clearly resolved. When cultured on fibrillar type I collagen, Mphis adhered poorly. In contrast, they adhered significantly to monomeric, heat-denatured, or collagenase-modified type I collagen. Studies utilizing anti-beta1 and -beta2 integrin adhesion-blocking antibodies, RGD-containing peptides, or divalent cation-free conditions did not inhibit Mphi; adhesion to monomeric or denatured type I collagen. However, macrophage scavenger receptor (MSR) ligands and anti-MSR antibodies significantly blocked Mphi; adhesion to denatured and monomeric type I collagen strongly suggesting the involvement of the MSR as an adhesion molecule for denatured type I collagen. Further analysis by Western blot identified the MSR as the primary receptor for denatured type I collagen among Mphi; proteins purified from a heat-denatured type I collagen affinity column. These findings indicate that Mphis adhere selectively to denatured forms of type I collagen, but not the native fibrillar conformation, via their scavenger receptors.     

The caddisfly Ceraclea fulva and the freshwater sponge Ephydatia fluviatilis: a successful relationship

The association between the aquatic phases of the caddisfly Ceraclea fulva (Trichoptera, Leptoceridae) and the freshwater sponge Ephydatia fluviatilis (Porifera, Spongillidae) has been investigated by means of scanning and transmission electron microscopy (SEM, TEM). Ceraclea fulva habitually feeds on sponges and builds its case by using the siliceous spicules of the sponge, which are arranged side by side, inter-crossed, cemented with silk, and organised in layers. In the newly hatched larva, the case is strengthened exclusively by cemented siliceous spicules, while during growth, the insect adds sponge fragments to it. The fine organisation of the sponge tissues growing on the case proves that the sponge is functional. Inter-spaced, small protrusions, derived from the outermost compact silk layer, form a series of "bridges" enhancing case/sponge adhesion. Tube-case shape varies according to the aquatic developmental phase of the insect: in the mature larva and pupa, this shelter carries larger sponge fragments dorsally. The caddisfly acts as carrier of the sponge, thus facilitating its dispersal and the colonisation of new habitats. This justifies regarding this association as a successful mutualistic relationship, and not as a unilateral parasitic behaviour on the part of the insect.     

Sponge white patch disease affecting the Caribbean sponge Amphimedon compressa

We report on a novel sponge disease, hereafter termed 'sponge white patch' (SWP), affecting the Caribbean sponge species Amphimedon compressa. SWP is characterized by distinctive white patches of variable size that are found irregularly on the branches of diseased sponges. Nearly 20% of the population of A. compressa at Dry Rocks Reef, Florida, USA, showed symptoms of SWP at the time of investigation (November 2007-July 2010). Approximately 21% of the biomass of SWP individuals was bleached, as determined by volume displacement. Scanning electron microscopy analysis showed severe degradation of bleached tissues. Transmission electron microscopy of the same tissues revealed the presence of a spongin-boring bacterial morphotype that had previously been implicated in sponge disease (Webster et al. 2002; Mar Ecol Prog Ser 232:305-309). This particular morphotype was identified in 8 of 9 diseased A. compressa individuals investigated in this study. A close relative of the aforementioned disease-causing alphaproteobacterium was also isolated from bleached tissues of A. compressa. However, whether the spongin-boring bacteria are true pathogens or merely opportunistic colonizers remains to be investigated. Molecular fingerprinting by denaturing gradient gel electrophoresis (DGGE) demonstrated a distinct shift from the microbiota of healthy A. compressa to a heterogeneous mixture of environmental bacteria, including several phylotypes previously implicated in sponge stress or coral disease. Nevertheless, tissue transplantation experiments conducted in the field failed to demonstrate infectivity from diseased to healthy sponges, leaving the cause of SWP in A. compressa to be identified.     

Progress towards a controlled culture of the marine sponge Pseudosuberites andrewsi in a bioreactor

Explants of the tropical sponge Pseudosuberites andrewsi were fed with the marine diatom Phaeodactylum tricornotum. The food was supplied either as intact algae or as a filtered crude extract. Growth (measured as an increase in underwater weight) was found in both experiments. The explants fed with intact algae increased to an average underwater weight of 255% of the initial weight in 45-60 days. The explants fed with crude extract increased to an average of 200% of the initial weight in 30 days. These results show that it is possible to grow a sponge using a single microorganism species as a food source. In addition, it was demonstrated that sponges are also capable of growing on non-particulate food. Therefore, this study is an important step forward towards the development of controlled, in vivo sponge cultures.     

Cross-linked collagen surface for cell culture that is stable,uniform, and optically superior to conventional surfaces

Summary A new type of collagen surface for use with cultures of peripheral nervous system cells is described. Collagen is derivatized to plastic culture dishes by a cross-linking reagent, 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide-metho-p-toluenesulfonate (carbodiimide), to form a uniform and durable surface for cell attachment and growth that allows dry storage, long-term culture, and improved microscopy. Surfaces of collagen derivatized to plastic were compared to surfaces of adsorbed or ammonia-polymerized collagen in terms of collagen binding and detachment, growth by dorsal root ganglion cells, and electron microscopy appearances. Derivatized collagen surfaces retained more collagen and showed much less evidence of degradation and cellular damage over periods of many weeks than did conventional adsorbed surfaces. Long-term survival of cells on derivatized collagen was far superior to that on the other surfaces, with almost 90% of cultures still viable after 10 wk. Transmission electron microscopy showed an organized layer of single fibrils that supported cell growth well, and scanning electron microscopy demonstrated an increased uniformity of derivatized collagen surfaces compared to ammoniated collagen surfaces. Applications for this improved substrate surface are discussed. This work was supported by the Leopold Schepp Foundation, the Dysautonomia Foundation, National Institutes of Health Grants NS14768 and NS11237, and Institutional Core Grant HD06276.     

Accelerated wound repair, cell proliferation, and collagen accumulation are produced by a cartilage-derived growth factor

Cartilage-derived growth factor (CDGF), a cationic polypeptide of approximately 18,000 mol wt, was prepared from bovine articular cartilage; other sources were bovine and human scapular and costal cartilage. Previous studies have shown that CDGF stimulates the proliferation of cultured mouse fibroblasts as well as chondrocytes and endothelial cells from various sources. In this study, CDGF was shown to stimulate dose-dependently the accumulation of DNA and collagen by rat embryo fibroblasts and a population of fibroblasts derived from granulation tissue. CDGF also stimulated the proliferation of cultured bovine capillary endothelial cells dose-dependently. To evaluate the effects of CDGF in vivo, we implanted polyvinyl alcohol sponges subcutaneously in rats. 6 d postimplantation, sponges were injected with 300 micrograms of partially purified CDGF, a dose which takes into account the cell numbers in the sponges as compared with cell cultures. CDGF rapidly disappeared from the sponges and only approximately 10% of the initial dose was present at 4 h. Despite its transient presence, CDGF caused a relative increase in sponge DNA content of 2.6-fold at 48 h and 2.4-fold at 72 h. We repeated the sponge experiment by using 500-ng injections of CDGF purified to near homogeneity by heparin-Sepharose chromatography. Purified CDGF caused significant increases in sponge collagen, protein, and DNA content at 48 and 72 h after a single injection. The effects of CDGF were abolished by heat and unaffected by reduction of disulfide linkages. Morphologically, CDGF did not evoke an inflammatory response, and its effect on proliferating endothelial cells and fibroblasts was, therefore, probably direct. However, increases in DNA content of sponges could not be fully accounted for by increased DNA synthesis, which suggests that recruitment may be an important component of the in vivo response. Taken together, the effects of CDGF on cultured cells and granulation tissue suggest that the sustained presence of CDGF in vivo may greatly enhance its effects upon wound repair.     

Characterization of a synthetic peptide from type IV collagen that promotes melanoma cell adhesion, spreading, and motility

The adhesion and motility of tumor cells on basement membranes is a central consideration in tumor cell invasion and metastasis. Basement membrane type IV collagen directly promotes the adhesion and migration of various tumor cell types in vitro. Our previous studies demonstrated that tumor cells adhered and spread on surfaces coated with intact type IV collagen or either of the two major enzymatically purified domains of this protein. Only one of these major domains, the pepsin-generated major triple helical fragment, also supported tumor cell motility in vitro, implicating the involvement of the major triple helical region in type IV collagen-mediated tumor cell invasion in vivo. The present studies extend our previous observations using a synthetic peptide approach. A peptide, designated IV-H1, was derived from a continuous collagenous region of the major triple helical domain of the human alpha 1(IV) chain. This peptide, which has the sequence GVKGDKGNPGWPGAP, directly supported the adhesion, spreading, and motility of the highly metastatic K1735 M4 murine melanoma cell line, as well as the adhesion and spreading of other cell types, in a concentration-dependent manner in vitro. Furthermore, excess soluble peptide IV-H1, or polyclonal antibodies directed against peptide IV-H1, inhibited type IV collagen-mediated melanoma cell adhesion, spreading, and motility, but had no effect on these cellular responses to type I collagen. The full complement of cell adhesion, spreading, and motility promoting activities was dependent upon the preservation of the three prolyl residues in the peptide IV-H1 sequence. These studies indicate that peptide IV-H1 represents a cell-specific adhesion, spreading, and motility promoting domain that is active within the type IV collagen molecule.     

Absence of Filamin A Prevents Cells from Responding to Stiffness Gradients on Gels Coated with Collagen but not Fibronectin

Cell types from many tissues respond to changes in substrate stiffness by actively remodeling their cytoskeletons to alter spread area or adhesion strength, and in some cases changing their own stiffness to match that of their substrate. These cell responses to substrate stiffness are linked to substrate-induced changes in the state, localization, and amount of numerous proteins, but detailed evidence for the requirement of specific proteins in these distinct forms of mechanical response are scarce. Here we use microfluidics techniques to produce gels with a gradient of stiffness to show the essential function of filamin A in cell responses to mechanical stimuli and dissociate cell spreading and stiffening by contrasting responses of a pair of human melanoma-derived cell lines that differ in expression of this actin cross-linking protein. M2 melanoma cells null for filamin A do not alter their adherent area in response to increased substrate stiffness when they link to the substrate only through collagen receptors, but change adherent area normally when bound through fibronectin receptors. In contrast, filamin A-replete A7 cells change adherent area on both substrates and respond more strongly to collagen I-coated gels than to fibronectin-coated gels. Strikingly, A7 cells alter their stiffness, as measured by atomic force microscopy, to match the elastic modulus of the substrate immediately adjacent to them on the gradient. M2 cells, in contrast, maintain a constant stiffness on all substrates that is as low as that of A7 cells on the softest gels examined (1000 Pa). Comparison of cell spreading and cell stiffening on the same gradient substrates shows that cell spreading is uncoupled from stiffening. At saturating collagen and fibronectin concentrations, adhesion of M2 cells is reduced compared to that of A7 cells to an extent approximately equal to the difference in adherent area. Filamin A appears to be essential for cell stiffening on collagen, but not for cell spreading on fibronectin. These results have implications for different models of cell protrusion and adhesion and identify a key role for filamin A in altering cellular stiffness that cannot be compensated for by other actin cross-linkers in vivo.     

Scanning electron microscope evidence for diatom uptake by two Antarctic sponges

Scanning electron microscopy (SEM) investigation of two Antarctic sponges, Phorbas glaberrima and Tedania charcoti, showed that the exopinacoderm effects a direct uptake of benthic diatoms which settle on the sponge surface. In P. glaberrima, planktonic diatoms were also observed penetrating through the inhalant system, the primary way of feeding in sponges. Benthic diatoms which accumulate in the mesohyl underneath the exopinacoderm help to strengthen the sponge cortex and may be an alimentary source during oligotrophic periods in the Antarctic environment.     

The removal of extracellular fibronectin from areas of cell-substrate contact

Immunofluorescent labeling for fibronectin was largely excluded from sites of closest contact between spreading chicken gizzard fibroblasts and the substratum. This was observed by double immunofluorescent labeling of fixed cells for fibronectin and vinculin, a smooth muscle intracellular protein that is specifically associated with focal adhesion plaques, in conjunction with interference-reflection microscopy. When the cells were plated on a fibronectin-coated substratum they adhered to its surface and rapidly spread on it. The immunofluorescent labeling for fibronectin in those cultures (after fixation and triton permeabilization) was usually absent from the newly formed, vinculin-containing focal adhesion plaques. We have found, however, that the accessibility to the cell-substrate gap at the focal adhesion plaques is limited and therefore a more direct approach was adopted. We have found that cells spreading on a substrate coated with rhodamine-labeled fibronectin progressively removed the underlying protein from the substrate. The removal of fibronectin involved at least two distinct mechanisms. Part of the substrate-associated fibronectin was removed from small areas and displaced toward the cell center. The arrowhead-shaped areas from which fibronectin was removed often coincided with vinculin-rich focal contacts. We observed, however, many areas where focal contacts were found over unperturbed fibronectin carpet, as well as fibronectin-free areas with no overlapping focal contacts. The possibilities that fibronectin is actively displaced from areas of cell-substrate contact, that the focal adhesion plaques are transiently associated with these areas and their implications on the dynamics of cell spreading and locomotion are discussed. The second route of fibronectin removal from the substrate was endocytosis. The rhodamine-labeled fibronectin was found in the cells in a partial or transient association with clathrin-containing structures.     

Streptococcal protein FOG, a novel matrix adhesin interacting with collagen I in vivo

Group G streptococcus (GGS) is a human pathogen of emerging clinical significance. It causes skin and soft tissue infections, occasionally resulting in life-threatening conditions such as sepsis and necrotizing fasciitis. We recently identified FOG, a novel surface protein of GGS with fibrinogen binding and immune evasion properties. Here we investigated the role of FOG in streptococcal primary adhesion to host tissue. A FOG-expressing clinical isolate adhered more efficiently to human skin biopsies ex vivo and to the murine dermis in vivo than a FOG-deficient strain. Scanning and transmission electron microscopy of skin specimens exhibited that this property was assigned to the ability of FOG to interact with collagen I, a major interstitial component of the dermis. Overlay experiments with human skin extracts and radiolabeled FOG followed by matrix-assisted laser desorption/ionization time of flight mass spectrometry analysis identified both the alpha1- and alpha2-chains of collagen I as targets for FOG. Transmission electron microscopy of the molecular complexes revealed thread-like FOG molecules binding via their NH2 termini to distinct sites on collagen I monomers and fibrils. The results demonstrate that FOG is important for GGS adhesion in vivo, implying a pathogenic role for this surface protein.     

支架材料对棕色脂肪源性干细胞向起搏细胞诱导分化的影响

目的观察不同三维支架材料对棕色脂肪来源干细胞（BADSCs）诱导分化成起搏细胞的效果,为构建生物起搏器提供实验依据。 方法将培养7 d的原代BADSCs分别种植到胶原海绵、明胶海绵和透明质酸水凝胶3种不同的材料中,在不同时间用光镜和扫描电镜观察细胞-支架复合体中细胞形态学的变化,免疫荧光染色检测心肌细胞、起搏细胞相关蛋白的表达。采用单因素方差分析。 结果细胞在3种支架上均能存活、增殖,LIVE/DEAD检测显示,培养3 d的胶原海绵、明胶海绵和透明质酸水凝胶3种细胞-支架复合物死细胞率分别为（46.35±1.50）%、（47.00±1.60）%和（1.76±1.08）%,其中细胞在透明质酸水凝胶中死亡率最低,并且细胞-透明质酸水凝胶复合物可自发性地搏动,三组比较差异具有统计学意义（F = 37.56,P < 0.05）。培养至2周时,胶原海绵、明胶海绵和透明质酸水凝胶中Connexin45细胞阳性率分别为（10.67±1.25）%、（13.67±1.25）%和（21.00±1.60）%,差异有统计学意义（F = 9.435,P < 0.01）,HCN2细胞阳性率分别为（11.00±1.60）%、（14.00±2.16）%和（34.33±3.68）%,差异有统计学意义（F = 17.52,P < 0.01）,HCN4细胞阳性率分别为（18.67±2.05）%、（13.00±1.60）%和（66.00±2.94）%,差异有统计学意义（F = 27.96,P < 0.01）,Sr细胞阳性率分别为（13.00±1.63）%、（14.33±1.24）%和（75.33±3.30）%,差异有统计学意义（F = 36.40,P < 0.01）,水凝胶中Connexin45、HCN2、HCN4和Sr的细胞阳性率均高于胶原海绵和明胶海绵,差异均具有统计学意义（P < 0.05）。 结论BADSCs在胶原海绵、明胶海绵和透明质酸水凝胶中均能很好地生长和分化,但透明质酸水凝胶更适用于组织工程化起搏器的构建。