MV-mimicking micelles loaded with PEG-serine-ACP nanoparticles to achieve biomimetic intra/extra fibrillar mineralization of collagen in vitro

Since their discovery, matrix vesicles (MVs) containing minerals have received considerable attention for their role in the mineralization of bone, dentin and calcified cartilage. Additionally, MVs' association with collagen fibrils, which serve as the scaffold for calcification in the organic matrix, has been repeatedly highlighted. The primary purpose of the present study was to establish a MVs–mimicking model (PEG-S-ACP/micelle) in vitro for studying the exact mechanism of MVs-mediated extra/intra fibrillar mineralization of collagen in vivo. In this study, high-concentration serine was used to stabilize the amorphous calcium phosphate (S-ACP), which was subsequently mixed with polyethylene glycol (PEG) to form PEG-S-ACP nanoparticles. The nanoparticles were loaded in the polysorbate 80 micelle through a micelle self-assembly process in an aqueous environment. This MVs–mimicking model is referred to as the PEG-S-ACP/micelle model. By adjusting the pH and surface tension of the PEG-S-ACP/micelle, two forms of minerals (crystalline mineral nodules and ACP nanoparticles) were released to achieve the extrafibrillar and intrafibrillar mineralization, respectively. This in vitro mineralization process reproduced the mineral nodules mediating in vivo extrafibrillar mineralization and provided key insights into a possible mechanism of biomineralization by which in vivo intrafibrillar mineralization could be induced by ACP nanoparticles released from MVs. Also, the PEG-S-ACP/micelle model provides a promising methodology to prepare mineralized collagen scaffolds for repairing bone defects in bone tissue engineering.     

Synthesis of fibronectin in normal and osteoarthritic articular cartilage

The content and the biosynthesis of fibronectin was examined in disease-free articular cartilage and in articular cartilage from osteoarthritic canine joints. Fibronectin content was increased in extracts of cartilage from osteoarthritic joints. Incubation of cartilage in vitro with [³H]phenylalanine and subsequent isolation of [³H]fibronectin from a gelatin affinity column and characterization by SDS-polyacrylamide gel electrophoresis and by immunoprecipitation indicated that disease-free and osteoarthritic cartilage explants synthesized fibronectin. About 50% of the [³H]fibronectin was recovered in the incubation medium. The osteoarthritic cartilage synthesized and accumulated up to 5-fold more [³H]fibronectin than disease-free cartilage.     

The Lys 280 → Gln mutation mimicking disease‐linked acetylation of Lys 280 in tau extends the structural core of fibrils and modulates their catalytic properties

Amyloid fibrillar aggregates isolated from the brains of patients with neurodegenerative diseases invariably have post‐translational modifications (PTMs). The roles that PTMs play in modulating the structures and polymorphism of amyloid aggregates, and hence their ability to catalyze the conversion of monomeric protein to their fibrillar structure is, however, poorly understood. This is particularly true in the case of tau aggregates, where specific folds of fibrillar tau have been implicated in specific tauopathies. Several PTMs, including acetylation at Lys 280, increase aggregation of tau in the brain, and increase neurodegeneration. In this study, tau‐K18 K280Q, in which the Lys 280 → Gln mutation is used to mimic acetylation at Lys 280, is shown, using HX‐MS measurements, to form fibrils with a structural core that is longer than that of tau‐K18 fibrils. Measurements of critical concentrations show that the binding affinity of monomeric tau‐K18 for its fibrillar counterpart is only marginally more than that of monomeric tau‐K18 K280Q for its fibrillar counterpart. Quantitative analysis of the kinetics of seeded aggregation, using a simple Michaelis–Menten‐like model, in which the monomer first binds and then undergoes conformational conversion to β‐strand, shows that the fibrils of tau‐K18 K280Q convert monomeric protein more slowly than do fibrils of tau‐K18. In contrast, monomeric tau‐K18 K280Q is converted faster to fibrils than is monomeric tau‐K18. Thus, the effect of Lys 280 acetylation on tau aggregate propagation in brain cells is expected to depend on the amount of acetylated tau present, and on whether the propagating seed is acetylated at Lys 280 or not.     

ECM Protein Nanofibers and Nanostructures Engineered Using Surface-initiated Assembly

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber diameter, composition and organization. In addition to providing structural support, the physical and chemical properties of the ECM play an important role in multiple cellular processes including adhesion, differentiation, and apoptosis. In vivo, the ECM is assembled by exposing cryptic self-assembly (fibrillogenesis) sites within proteins. This process varies for different proteins, but fibronectin (FN) fibrillogenesis is well-characterized and serves as a model system for cell-mediated ECM assembly. Specifically, cells use integrin receptors on the cell membrane to bind FN dimers and actomyosin-generated contractile forces to unfold and expose binding sites for assembly into insoluble fibers. This receptor-mediated process enables cells to assemble and organize the ECM from the cellular to tissue scales. Here, we present a method termed surface-initiated assembly (SIA), which recapitulates cell-mediated matrix assembly using protein-surface interactions to unfold ECM proteins and assemble them into insoluble fibers. First, ECM proteins are adsorbed onto a hydrophobic polydimethylsiloxane (PDMS) surface where they partially denature (unfold) and expose cryptic binding domains. The unfolded proteins are then transferred in well-defined micro- and nanopatterns through microcontact printing onto a thermally responsive poly(N-isopropylacrylamide) (PIPAAm) surface. Thermally-triggered dissolution of the PIPAAm leads to final assembly and release of insoluble ECM protein nanofibers and nanostructures with well-defined geometries. Complex architectures are possible by engineering defined patterns on the PDMS stamps used for microcontact printing. In addition to FN, the SIA process can be used with laminin, fibrinogen and collagens type I and IV to create multi-component ECM nanostructures. Thus, SIA can be used to engineer ECM protein-based materials with precise control over the protein composition, fiber geometry and scaffold architecture in order to recapitulate the structure and composition of the ECM in vivo.     

Cytoskeletal elements in cotton seed hair developmentin vitro: Their possible regulatory role in cell wall organization

Summary The localization and orientation of cytoskeletal elements in developing cotton fibres were studied by the indirect immunofluorescence and the dry cleaving technique. Microtubules are transversely arranged to the cell axis, most probably in a flat helix, in the cortex of expanding fibres. Since the innermost deposited cellulose microfibrils always show primarily the same orientation it is postulated that the microtubules control the transverse deposition of the cellulose fibrils. Little further cell expansion takes place during secondary wall formation and the microfibril pattern corresponds to that of the cortical microtubules,e.g., in the steepness of their helicoidal turns. Microtubules with a length of 7–20 m were observed, probably they are longer. The importance of microtubule length on microfibril deposition is discussed. The density of microtubule packing is in the range of 8–14 m^-1 as in other comparable cell types. In contrast to the microtubules, actin filaments are most likely longitudinally oriented during different phases of fibre development. The dry cleaving technique reveals numerous coated pits in the plasma membrane which are not crossed by microtubules. They seem to be linked to the latter by filamentous structures.     

Adhesion,proliferation, and adipogenesis in primary rat cell cultures: effects of collagenous substrata,fibronectin, and serum

Summary The effects of collagenous substrata, fibronectin, and fetal bovine serum on the adhesion, proliferation, and adipogenesis of rat stromal-vascular cells are reported. There was no effect on initial stromal-vascular cell-attachment by fetal bovine serum or fibronectin. The number of cells attached to a hydrated collagen-gel was almost twice (P<0.04) the number attached to dried collagen-gel or dried denatured collagen-gel. Total number of cells after 5 days in culture was similar among the collagenous substrata and among the treatments with or without fibronectin in the growth media. Total number of cells increased significantly (P<0.02) with 10% FBS. Adipocytic formation was inhibited by hydrated collagen-gel (P<0.02) compared to dried collagen-gel or dried, denatured collagenous substrata. An interaction occurred between dried, denatured gel and fetal bovine serum so that total formation of adipocytes increased by increasing the level of fetal bovine serum (P<0.07). Adipocytic formation was inhibited by hydrated collagen-gel at all levels of fetal bovine serum. The percentage of cells that converted to adipocytes was significantly lower (P<0.01) on hydrated collagen-gel compared to dried, denatured or dried collagen-gel. Percentage of conversion was not significantly different among levels of fetal bovine serum, although this percentage increased as fetal bovine serum level increased. Adipocytic conversion was not different between fibronectin-treated or untreated cells. Morphology of stromal vascular cells was similar on dried collagen and dried, denatured collagen-gel, but tended to remain bipolar on hydrated collagen-gel. These studies indicate that fetal bovine serum in combination with the extracellular matrix (dried, denatured collagen) increased the differentiation of rat stromal-vascular cells into adipocytes, and that hydrated collagen inhibited differentiation.     

Immunocytochemical localization of collagen types I,III, IV,and fibronectin in the human dermis

Summary The distribution of collagen types I, III, IV, and of fibronectin has been studied in the human dermis by light and electron-microscopic immunocytochemistry, using affinity purified primary antibodies and tetramethylrhodamine isothiocyanate-conjugated secondary antibodies. Type I collagen was present in all collagen fibers of both papillary and reticular dermis, but collagen fibrils, which could be resolved as discrete entities, were labeled with different intensity. Type III collagen codistributed with type I in the collagen fibers, besides being concentrated around blood vessels and skin appendages. Coexistence of type I and type III collagens in the collagen fibrils of the whole dermis was confirmed by ultrastructural double-labelling experiments using colloidal immunogold as a probe. Type IV collagen was detected in all basement membranes. Fibronectin was distributed in patches among collagen fibers and was associated with all basement membranes, while a weaker positive reaction was observed in collagen fibers. Ageing caused the thinning of collagen fibers, chiefly in the recticular dermis. The labeling pattern of both type I and III collagens did not change in skin samples from patients of up to 79 years of age, but immunoreactivity for type III collagen increased in comparison to younger skins. A loss of fibronectin, likely related to the decreased morphogenetic activity of tissues, was observed with age.     

Local production of fibronectin by ectopic human retinal cells

Summary The distribution of fibronectin mRNA and fibronectin in adult human retina and epiretinal membranes was investigated by in situ hybridisation and immunohistochemical techniques. The cells in normal adult retina contained little or no fibronectin mRNA and the retina only showed fibronectin immunoreactivity in retinal vessels. The cells in detached neuroretina did not contain fibronectin message but the vitreoretinal interface of the detached retina exhibited variable fibronectin immunoreactivity. Retinal glia, retinal pigment epithelium and fibroblast-like cells in membranes at the vitreoretinal juncture (epiretinal membranes) showed variable labelling with the fibronectin mRNA probe and all the membranes immunostained for fibronectin. No difference could be detected between membrane cell types in the intensity of labelling with the mRNA probe or for fibronectin immunoreactivity. The results indicate that cells in situ in attached and detached adult human retina do not produce fibronectin. Although fibronectin at the vitreoretinal juncture in retinal detachment is probably partly derived from plasma fibronectin resulting from breakdown of the blood-retinal barrier, ectopic retinal cells produce fibronectin and contribute to the glycoprotein in epiretinal membranes.     

金黄滴虫细胞核微丝系统的初步观察

金黄滴虫细胞核内经常存在着许多直径约为7nm的微丝。这些徽丝大多组合成走向不定的徽丝束,微丝束交织而成遍布核内的网架。核被下面微丝束较多,它们的存在常使核被外凸而成隆脊。核内微丝与核内结构如核仁、染色质等似乎都是相连的。有些微丝横跨核被,一端位于核内,另一端位于核周腔中,并靠近叶绿体。核周腔和内质网腔中也存在着微丝和另一种纤维,印管状纤维。用细胞松弛素B处理后,细胞核、核周腔和内质网腔中的微丝均消失,细胞核的形态也发生变化,似乎微丝网架有支持细胞核的作用。核内微丝可能是在内质网中组装,然后经核周腔进入核内的。     

Bufonid nucleocytoplasmic hybrids arrested at the early gastrula stage lack a fibronectin-containing fibrillar extracellular matrix

Summary Most hybrids betweenBufo bufo andB. calamita obtained by nuclear transplantation become arrested at the early gastrula stage. In both parental controls and the hybrid embryos, the presence and distribution of extracellular matrix was analysed with fluorescent wheat germ agglutinin and by immunolabelling with antibodies directed against fibronectin. InB. bufo andB. calamita gastrulae and in the few hybrids that complete gastrulation, the inner surface of the blastocoel roof is covered by a fibronectin-rich fibrillar matrix. In nucleocytoplasmic hybrids whose development was arrested at the gastrula stage, the fibronectin-containing extracellular matrix was either totally absent or poorly developed and disorganized.