Production of mucoid microcolonies by Pseudomonas aeruginosa within infected lungs in cystic fibrosis

Direct electron microscopic examination of postmortem lung material from cystic fibrosis patients infected with Pseudomonas aeruginosa has shown that these bacterial cells form distinct fiber-enclosed microcolonies in the infected alveoli. Similar examination of bronchoscopy material from infected cystic fibrosis patients showed that the fibres of the enveloping matrix are definitely associated with the bacterial cells. The fibers of the extracellular matrix stain with ruthenium red and are therefore presumed to be polyanionic. When mucoid strains of P. aeruginosa were recovered from cystic fibrosis patients and grown in a suitable liquid medium, they were found to produce large microcolonies whose component cells were embedded in a very extensive matrix of polyanionic fibers that could be stabilized by reaction with antibodies to prevent collapse during the dehydration steps of preparation for electron microscopy. When these mucoid strains of P. aeruginosa were used to produce pulmonary infections of rats by the agar bead method, the infected alveoli contained large fiber-enclosed bacterial microcolonies. We conclude that the cells of P. aeruginosa that infect cystic fibrosis patients form microcolonies that are enveloped in a fibrous anionic matrix and that these microcolonies can be duplicated in in vitro cultures and in animal model systems.     

Mesangial cell hillocks. Nodular foci of exaggerated growth of cells and matrix in prolonged culture.

To examine the capability of glomerular mesangial cells (MCs) to produce extracellular matrix, the authors studied MCs in culture by light and electron microscopy as well as immunocytochemistry. MCs were obtained from isolated rat glomeruli and maintained up to 12 weeks in medium containing 20% fetal calf serum. MC outgrowth of primary culture and of up to three subcultures showed characteristic organization consisting of bands of elongated or stellate intertwined cells. After confluency at 10-16 days, MCs continued to grow in irregular multilayers. MCs produced extracellular matrix material within 2-4 days after plating, and large amounts of matrix accumulated with time. By 2-3 weeks, foci of exaggerated MC proliferation, matrix secretion, and necrotic cell debris formed nodular protrusions, which gradually produced large hillocks. Immunocytochemical studies of MC outgrowths were performed on culture plates or on sectioned material with the use of specific rabbit polyclonal antibodies to isolated matrix proteins and FITC-conjugated, affinity-purified second antibodies. Within 3 days of culture, MCs elaborated fibronectin and collagen Types I, III, IV, and V. With time, strands of matrix, notably in the central mass of hillocks, stained extensively for these constituents. Staining for laminin was less pronounced. Smooth muscle cell myosin was regularly found on distinct intracellular fibrils and in the extracellular material of hillocks. Electron microscopy revealed the hillocks to be composed of elongated cells on the surface and stellate cells intermingled with matrix and necrotic cell debris in the core. The results show that proliferating MCs can be maintained in homogeneous culture for a prolonged time period. MCs produce large amounts of the extracellular matrix proteins (Type IV and V collagen, fibronectin, laminin), which are found in normal glomeruli. Cultured MCs also produce interstitial collagen Types I and III. MC hillocks show the nodular accumulation of matrix similar to that seen in the mesangium of diseased glomeruli. It is concluded that the in vitro model of prolonged MC outgrowth may facilitate the investigation of factors that govern mesangial matrix production. Such a model could be used in examining the response of the mesangium to defined inflammatory or metabolic stimuli.     

Fabrication and evaluation of nanoporous alumina membranes for osteoblast culture

An understanding of osteoblast response to surface topography is essential for successful bone tissue engineering applications. Alumina has been extensively used as a substrate for bone tissue constructs. However, current techniques do not allow precise surface topography and orientation of the material. In this research, a two-step anodization process was optimized for the fabrication of nanoporous alumina membranes with uniform pore dimension and distribution. The anodization voltage can be varied to create nanoporous alumina membranes with pore sizes ranging from 30 to 80 nm in diameter. The impact of the nanoscale pores on osteoblast response was studied by evaluating cell adhesion, morphology, and matrix production. Scanning electron microscopy and atomic force microscopy were used to characterize the nanoporous alumina membranes. Osteoblast adhesion and morphology were investigated using scanning electron microscopy images and matrix production was characterized using energy dispersive spectroscopy. This research combined the advantages of using alumina, a material with proven biocompatibility and current orthopedic clinical applications, and incorporated porous features on the nanoscale which have been reported to improve osteoblast response.     

Immunoelectron microscopic evidence for release of eosinophil granule matrix protein onto microfilariae of Onchocerca volvulus in the skin after exposure to amocarzine

The involvement of eosinophils in the host reaction to microfilariae (mf) of Onchocerca volvulus was studied by immunohistochemistry and immunoelectron microscopy. Skin biopsies were obtained from patients after transepidermal administration of the microfilaricide amocarzine. At 20–28 h after the application of amocarzine, mf were degenerated or dead and a marked eosinophil-parasite adherence (EPA) reaction was seen, with intense staining for intra- and extracellular eosinophil granule proteins such as eosinophil cationic protein (ECP) surrounding the mf. Immunoelectron microscopically the eosinophil granule matrix in intact and necrotic eosinophils was specifically labeled, whereas granules whose matrix had dissolved showed no specific gold particle binding. As specific labeling was seen on lowly electron-dense material adjacent to matrix-depleted granules, the material was regarded as released eosinophil granule matrix material. Intact and necrotic eosinophils, matrix-containing as well as matrix-depleted eosinophil granules, and released eosinophil granule matrix material were observed on the surface of damaged mf and between collagen fibers. The coincidence of mf degeneration, EPA reaction, and release of eosinophil granule matrix material on damaged mf and collagen fibers indicated a role of eosinophils and eosinophil granule matrix protein in the host reaction to mf after amocarzine application. Received: 3 March 1998 / Accepted: 13 March 1998     

Endothelial-smooth muscle interactions in vitro: effects of high pH,flowing medium and extracellular matrix.

The interactions of cultured bovine aortic and human umbilical or saphenous vein endothelium with cultured fibroblasts or smooth muscle cells were studied using light microscopy, scanning electron microscopy, and a radioisotope adhesion assay. (1) Resuspended fibroblasts or smooth muscle cells readily ''overgrew'' confluent endothelial monolayers under static culture conditions, but not when cultures were exposed to a continuously stirred medium. (2) Exposure of cultured endothelial or smooth muscle cells to a moderately alkaline environment alters the disposition of pericellular concanavalin. A positive extracellular material. This does not affect the initial adhesion of endothelial or smooth muscle cells, but does affect cell spreading. (3) Endothelial adhesion to cultured smooth muscle cells involves both adhesion and spreading. Recently subcultured or rapidly proliferating smooth muscle cells support initial adhesion, but not spreading. Spreading appears to require the establishment of a suitable extracellular matrix, and this is inhibited both by a flowing medium and by an alkaline extracellular environment.     

猪脂肪干细胞与脱细胞真皮基质的生物相容性

背景：脱细胞真皮基质已广泛应用于器官或组织缺损的修补。 目的：观察猪脂肪源性干细胞与脱细胞真皮基质的相容性。 方法：酶消化法原代培养猪脂肪源性干细胞,利用流式细胞仪和多向诱导分化方法鉴定脂肪干细胞,将其与脱细胞真皮基质共培养。 结果与结论：实验成功培养出猪脂肪源性干细胞,流式细胞仪检测结果显示阳性表达CD44和CD105,不表达CD34和CD45;在诱导培养基条件下,可向脂肪细胞、成骨细胞分化。苏木精-伊红染色及扫描电镜发现干细胞能在脱细胞真皮基质表面黏附生长。脂肪源性干细胞与脱细胞真皮基质具有良好的相容性,将两者体外复合培养后,有望将复合物植入体内进行缺损组织、器官的修复。     

Ultrastructure of psammoma bodies of meningioma in tissue culture.

An 8-day-old tissue culture of a human meningioma was studied by electron microscopy. Psammoma bodies were detected in all stages of evolution, affording a unique opportunity for observing the genesis of these structures. Matrix vesicles appeared instrumental in the calcification of a granular extracellular material. Although matrix vesicles are described in both physiologic and pathologic calcification, they have not been previously reported in the very few ultrastructural studies of psammoma bodies in meningiomas.     

Endothelial-smooth muscle interactions in vitro: effects of high pH, flowing medium and extracellular matrix

The interactions of cultured bovine aortic and human umbilical or saphenous vein endothelium with cultured fibroblasts or smooth muscle cells were studied using light microscopy, scanning electron microscopy, and a radioisotope adhesion assay. (1) Resuspended fibroblasts or smooth muscle cells readily 'overgrew' confluent endothelial monolayers under static culture conditions, but not when cultures were exposed to a continuously stirred medium. (2) Exposure of cultured endothelial or smooth muscle cells to a moderately alkaline environment alters the disposition of pericellular concanavalin. A positive extracellular material. This does not affect the initial adhesion of endothelial or smooth muscle cells, but does affect cell spreading. (3) Endothelial adhesion to cultured smooth muscle cells involves both adhesion and spreading. Recently subcultured or rapidly proliferating smooth muscle cells support initial adhesion, but not spreading. Spreading appears to require the establishment of a suitable extracellular matrix, and this is inhibited both by a flowing medium and by an alkaline extracellular environment.     

The ultrastructure of the bone-hydroxyapatite interface in vitro.

Rat bone marrow cells were cultured on plasma-sprayed hydroxyapatite (HA). The cells formed a mineralized extracellular matrix (ECM) that exhibited several characteristics of bone tissue. The interface between this mineralized ECM and the HA was studied at the ultrastructural level with scanning and transmission electron microscopy and x-ray microanalysis. Initially, the deposition of a globular, afibrillar matrix was observed on HA. This was followed by the integration of collagen fibers in this matrix and their subsequent mineralization. At the bone-HA interface two distinctly different interfacial structures were observed. An electron-dense layer with a thickness of 20-60 nm was regularly present, which contained both organic and inorganic material and was rich in glycosaminoglycans. The interfaces differed however, in the presence or absence of an amorphous zone which was free of collagen fibers and had an average thickness of 0.7-0.8 microns. It was frequently seen interposed between the electron-dense layer and the hydroxyapatite. Similar interfacial structures have also been described in the in vivo environment, where they were referred to as lamina limitans-like or cement linelike. From the results of this study, it can be concluded that the described in vitro system is a suitable model to study bone-biomaterial interactions.     

Basement membrane heparan sulfate proteoglycan is the main proteoglycan synthesized by glomerular epithelial cells in culture.

The production and distribution of basement membrane-type heparan sulfate proteoglycans (BM HSPG) were investigated in a mouse glomerular epithelial cell line. Confluent cell monolayers were radiolabeled with [35S]sulfate or [35S]cysteine. Proteoglycans were isolated from the medium and cell layers by ion exchange chromatography and their nature determined by enzyme digestion (chondroitinase ABC) or degradative treatment (nitrous acid). It was found that more than 80% of the proteoglycans in both the cell layer and medium were heparan sulfate proteoglycans (HSPG) based on their susceptibility to nitrous acid degradation. More than half of the HSPG in the cell layer could be precipitated with an antiserum that specifically recognizes BM HSPG; only 10% of those released into the medium were precipitated with this antiserum. When immunoprecipitates of [35S] sulfate-labeled proteoglycans were analyzed by SDS-PAGE, the mature proteoglycans ran as a broad band at the top of the gel. When immunoprecipitates of [35S]cysteine-labeled proteoglycans were similarly analyzed, a 250 kd precursor core protein band was seen in addition to the mature proteoglycan. When BM HSPG were localized by immunofluorescence and immunoelectron microscopy (immunoperoxidase), they were found intracellularly in biosynthetic compartments (ER and Golgi cisternae) and extracellularly in deposits of basement membrane-like matrix located beneath and between the cells. These results indicate that l) BM HSPG are the predominant type of proteoglycans made by glomerular epithelial cells in culture; 2) these HSPG are assembled into a loosely organized matrix that is deposited beneath and between the cells; and 3) this cell type produces a higher proportion of BM HSPG than other cultured epithelial cells studied previously.     

Giant-cell fibroblastoma: a case report emphasising the presence of hyperplastic subplasmalemmal linear densities in continuity with granular matrices in the extracellular space

The histological, immunohistochemical and ultrastructural features of a case of giant-cell fibroblastoma from the soft tissues of the chest wall in a 48-year-old female are described with special reference to the cell surface and matrix. Subplasmalemmal linear densities (SLDs) characterised cell surfaces, and exhibited excessive development of the dense external component: foci of identical dense material were present in the matrix. The nature of these dense foci, both the external component of the SLD and those free in the extracellular space, was investigated by light microscope immunostaining for fibronectin, laminin and collagen IV. All three proteins stained vessels. There was weaker but positive staining for tumour cell surfaces and matrix, consistent with the widely dispersed nature of the dense foci. Given their fine structural appearance, these dense foci can be referred to as granular matrices. Given also that the matrix protein immunostaining pattern is consistent with the distribution of these granular matrices as observed by electron microscopy, they may be provisionally interpreted as a kind of basement-membrane-related granular matrix. The presence of these proteins emphasises the point that, while giant-cell fibroblastoma fibroblasts lack a lamina, they nevertheless bear basement-membrane-related proteins organised, however, in a non-laminate fashion. The observations reinforce the need to qualify immunostaining results by ultrastructural investigation in order to understand the organisation of immuno-detected proteins and are discussed in terms of their diagnostic and possible biological significance.     

Improved long-term storage of hybridomas at -80 degrees C using a bovine milk derivative

A medium comprising 40% bovine milk fraction and 10% DMSO (medium A) was used for the long-term storage of hybridomas at -80 degrees C. The viability of the cells, their growth recovery and ability to secrete antibody were studied and the results were compared to those obtained after storage in a medium containing 40% fetal calf serum and 10% of DMSO (medium B). Hybridomas have been kept for 2 years in medium A; the viability of such cells was 75%, the cells were healthy (electron microscopy), they rapidly proliferated when they were cultured in RPMI supplemented with 10% FCS or with 9% milk fraction + 1% FCS and they released measurable levels of antibody. In contrast, hybridomas stored under the same conditions but in medium B died after 6 months.     

Elastofibroma: An in Vivo Model of Abnormal Neoelastogenesis

Thirteen cases of elastofibroma have been studied by conventional light and electron microscopy, as well as by histochemistry and immunohisto-chemistry. By light microscopy elastinophilic material appeared as huge fibers crossing collagen bundles. Immunohistochemistry demonstrated a strong positivity for elastin in numerous and circumscribed areas of the extracellular matrix. By electron microscopy, collagen consisted of 40-50-nm wide fibrils, and elastin was made of large aggregates of moderately electron-dense material surrounding a very thin, apparently normal, elastin core. At high magnification these aggregates consisted of short tubules, often in regular arrays, surrounded by microfibrils and microfilaments. These data, associated with selective digestions on thin sections with elastase, purified collagenase, hyaluronidase, and chondroitinase ABC, revealed that elastic fibers in elastofibroma seem to be made of true elastin surrounded by an enormous amount of hydrophilic material, in which some elastin, chondroitin sulfates, and collagenase type-VII sensitive material are aggregated forming a rather ordered array of short tubules.     

用聚乳酸海绵材料构建组织工程真皮

目的 利用多孔聚乳酸海绵材料作为真皮支架材料，研究其在组织工程真皮构建中的作用及意义。方法 采用盐溶法制作出多孔聚乳酸海绵材料，再接种皮肤成纤维细胞，形成细胞—支架结构物；利用细胞计数、组织染色、电镜和免疫组化等检测手段，观察细胞在材料上的生长、增殖及分泌情况。结果 成纤维细胞在支架材料上状态良好，且能缓慢增殖。胞外基质丰富，胶原分泌旺盛。结论 聚乳酸能支持皮肤成纤维细胞正常的生理代谢和分泌，是理想的组织工程真皮支架材料。     

A New Tool Improves Diagnostic Test Performance for Transmission EM Evaluation of Axonemal Dynein Arms

Abstract

Diagnosis of primary ciliary dyskinesia (PCD) by identification of dynein arm loss in transmission electron microscopy (TEM) images can be confounded by high background noise due to random electron-dense material within the ciliary matrix, leading to diagnostic uncertainty even for experienced morphologists. The authors developed a novel image analysis tool to average the axonemal peripheral microtubular doublets, thereby increasing microtubular signal and reducing random background noise. In a randomized, double-blinded study that compared two experienced morphologists and three different diagnostic approaches, they found that use of this tool led to improvement in diagnostic TEM test performance.     

Ultrastructure of gallstones produced in mice fed a high cholesterol diet

Mice fed a high cholesterol-cholic acid diet for two to six months develop gallstones; these were studied by transmission electron microscopy after glutaraldehyde-digitonin fixation. Examination of the contents of mouse gallbladders presents views of layered structures and surrounding amorphous material. We interpret these images of gallstones to suggest that they may arise by cohesion of material rich in cholesterol to form more ordered structures. Gallbladder contents of mice fed the diet for five to six months were found to contain occasional crystals and rectangular areas similar to those observed in thin sections of human gallstones (unpublished observations). Recent findings that human gallstones can be dissolved with chenodeoxycholic acid are discussed, with reference to their applicability to studies of gallstones in mice.     

Detecting microdamage in bone

Fatigue-induced microdamage in bone contributes to stress and fragility fractures and acts as a stimulus for bone remodelling. Detecting such microdamage is difficult as pre-existing microdamage sustained in vivo must be differentiated from artefactual damage incurred during specimen preparation. This was addressed by bulk staining specimens in alcohol-soluble basic fuchsin dye, but cutting and grinding them in an aqueous medium. Nonetheless, some artefactual cracks are partially stained and careful observation under transmitted light, or epifluorescence microscopy, is required. Fuchsin lodges in cracks, but is not site-specific. Cracks are discontinuities in the calcium-rich bone matrix and chelating agents, which bind calcium, can selectively label them. Oxytetracycline, alizarin complexone, calcein, calcein blue and xylenol orange all selectively bind microcracks and, as they fluoresce at different wavelengths and colours, can be used in sequence to label microcrack growth. New agents that only fluoresce when involved in a chelate are currently being developed--fluorescent photoinduced electron transfer (PET) sensors. Such agents enable microdamage to be quantified and crack growth to be measured and are useful histological tools in providing data for modelling the material behaviour of bone. However, a non-invasive method is needed to measure microdamage in patients. Micro-CT is being studied and initial work with iodine dyes linked to a chelating group has shown some promise. In the long term, it is hoped that repeated measurements can be made at critical sites and microdamage accumulation monitored. Quantification of microdamage, together with bone mass measurements, will help in predicting and preventing bone fracture failure in patients with osteoporosis.     

A novel osteochondral implant.

A novel implant for the use as an osteochondral graft was designed. This implant was prepared by stepwise formation of calcium phosphate crystals within the matrix of a lyophilised collagen sponge. Chondrocytes were then grown on this material to create the osteochondral implant. The implant was characterized with light microscopy, scanning electron microscopy (SEM), electron diffraction crystallography (EDX), and IR. It was observed with IR that the implant had a peak, that was not found so distinctly in its components, at 1400 cm(-1), implying a strong interaction of the two main ingredients of the implant, calcium phosphate and collagen. This strong interaction was also shown in the graft degradation test while the untreated collagen sponge degraded rapidly (in one day) the mineral loaded implant was able to maintain its integrity for two weeks. In the chondrocyte culture medium degradation of the implant was shown by a decrease of the calcium content and calcium to phosphorous ratio. Also, EDX revealed the presence of sulfur one and two weeks after incubation, an element not found among the components of the implant, possibly due to the development of an extracellular matrix. SEM showed that the form of the crystals of calcium phosphate differed depending on whether they were prepared on the template, collagen, or in the absence of a template. The chondrocytes appeared to be growing in number on the implant and their shapes were morphologically normal. The chondrocyte loaded collagen-calcium phosphate composite could thus be considered a potential tissue engineered osteochondral implant.     

异种脱细胞真皮替代睑板材料重建眼睑的可行性

背景：眼睑后层重建是眼睑重建的重点和难点,其中睑板替代物更是研究的焦点。异种脱细胞真皮作为一种新型的组织工程材料,在国内外烧伤整形领域,正得到广泛的研究和应用。 目的：观察异种(猪)脱细胞真皮植入兔眼睑后的组织相容性极其组织病理学变化。 方法：剥取健康小白猪全层皮肤20 cm×20 cm,制备异种(猪)脱细胞真皮基质。同时制备兔睑板全层缺损模型并植入脱细胞真皮基质,观察大体情况,并分别于第1,2,3周取移植交界处眼睑组织光镜下观察组织学的改变。 结果与结论：大体观察未见明显排斥反应及眼睑的变形;光镜下1周时可见局部炎症细胞浸润,2周时炎症细胞减少,3周时正常纤维组织长入,逐渐分割代替植入的胶原纤维,炎症反应消失。提示异种脱细胞真皮免疫原性低,并可引导新生胶原的生长,是一种良好的睑板替代物。     

The effect of sodium ascorbate on the mechanical properties of hyaluronan-based vascular constructs

Esterified hyaluronic acid (HYAFF) is routinely used for clinical tissue engineering applications such as skin and cartilage. In a previous study we developed a technique for in vitro generation of cylindrical constructs from cellularized HYAFF flat sheets. In the present investigation we studied the possibility to improve mechanical properties of this vascular construct by the addition of sodium ascorbate (SA). Non-woven HYAFF flat sheets were seeded with porcine aortic smooth muscle cells (SMCs) and cultured for 14 or 28 days with standard medium or medium added with SA. In selected experiments HYAFF sheets seeded with SMCs were wrapped to obtain cylindrical shape and then cultured in control medium or SA added medium for up to 28 days. We estimated cell viability for flat sheets, and performed histological examination, analysis of extracellular matrix (ECM) deposition and mechanical tests on tubular constructs. The number of viable cells and ECM deposition increased with time in constructs cultured in the presence of SA, as compared to control group. Moreover, SA improved mechanical properties of the vascular construct lowering material stiffness and increasing tensile strength as compared to untreated controls. The addition of SA to the medium improved cell proliferation and ECM synthesis on this biodegradable material, which leads to the formation of well organized, mechanical resistant tissue-engineered structure.