Migration pattern, morphology and viability of cells suspended in or sealed with fibrin glue: a histomorphologic study.

INTRODUCTION: We studied the migration pattern, morphology and viability of cells suspended in five different fibrin glues. Besides this, the behaviour of chondrocytes seeded on porous matrices comprising different collagen types sealed with fibrin glue was investigated. MATERIAL AND METHODS: In an experiment A, cell suspension (0.5x10(6) cells) was incubated with different fibrin glues. Experiment B was set up to evaluate chondrocytes migration either through a collagen I/III (Chondro-Gide, Geistlich Biomaterials, Switzerland) or collagen II matrix sealed with different fibrin glues in a perfusion chamber system. Analysis were performed by lightmicroscopy (Mayer's hematoxylin-eosin; Masson-Goldner; TUNEL test) and by transmission and scanning electron microscopy. All fibrin glues were measured for TGF-beta 1 and 2 with a specific ELISA. RESULTS: After incubation of cell suspension in autologous fibrin glue, the morphology of cells is chondrocyte-like. Spindly, process-bearing cells were seen in commercial fibrin glue. Cells suspended in commercial fibrin glue revealed a significant higher percentage of TUNEL positive cells compared to fibrin tissue adhesives mixed with autologous serum (p=0.006). The TGF-beta 1 and 2 concentration was significantly higher in partial autologous fibrin sealant (PAF) compared to their commercial counterparts (p=0.001). Cells seeded on the collagen I/III matrix retained their chondrocytic morphology, while in the type II collagen matrix the chondrocytes displayed a fibroblastic phenotype. The ratio of TUNEL positive cells for the collagen I/III matrix was significantly surpassed by the values, when a collagen II matrix was used (p=0.008). No ingrowth of cells was seen in any of the experimental conditions. CONCLUSION: Partial autologous fibrin glue and collagen I/III matrices are favourable in respect to migration pattern, morphology and viability, but definitive conclusions can only be drawn after in vivo studies. This will be addressed in future animal studies.

Matrix testing for urothelial tissue engineering.

INTRODUCTION: Surgical reconstruction of the bladder is associated with many well-known complications. Tissue engineering is under discussion as a potential therapeutic strategy and many of the proposed benefits are of special interest for children. Biomaterials play a key role in tissue engineering. Many materials have been proposed for the experimental reconstruction of the bladder and urethra. They determine the biological and mechanical characteristics of the reconstructed tissues. Most publications focus on a single material. In order to identify the most suitable biomaterial it was the aim of this study to compare biological and mechanical features of different biomaterials seeded with urothelial cells in vitro. MATERIALS AND METHODS: Commercially available biomaterials (Biogide, Ethisorb, Lyoplant, SIS, Vicryl, Xenoderm) of biologic or synthetic origin were seeded with urothelial cells. Cell-matrix constructs were cultured and investigated by scanning electron microscopy for surface structure and cell morphology. They were also subjected to extension until failure and the force required was reported as f (max). Values obtained and curve shape were compared to specimens of bladder mucosa and submucosa. RESULTS: Cell adhesion and morphology showed marked differences between materials. Cell shape varied from single spherical cells to confluent layers of flat urothelium. f (max) ranged from 0.02 N to 48.86 N for tested materials and 1.19 N for native bladder mucosa/submucosa. DISCUSSION: The materials showed marked differences in biological and mechanical features in vitro. Cells cultured on biogenic matrices were more similar to native urothelium. Most of the tested materials showed different curve shapes and higher f (max) values than native bladder mucosa.

Ifosfamide, carboplatin and etoposide combined with 41.8 degrees C whole body hyperthermia for malignant pleural mesothelioma.

We performed a phase II study combining 41.8 degrees C whole body hyperthermia with ICE chemotherapy, i.e. ifosfamide (5 g/m(2)), carboplatin (300 mg/m(2)) and etoposide (150 mg/m(2) on days 2 and 3), administered every 4 weeks, for patients with malignant pleural mesothelioma. Of 27 chemonäive, non-metastatic patients enrolled, 25 patients were evaluable for response. Overall response rate was 20% (five partial remissions; 95% CI 8.9-39.1%). Median survival time from the start of treatment for all patients was 76.6 weeks (95% CI 65.4-87.8 weeks). Progression free survival for all patients measured 29.6 weeks (95% CI 24.4-34.7 weeks). One year overall survival was 68% and 2 year overall survival was 20%. Major treatment toxicities included grade 3/4 neutropenia and thrombocytopenia in 74 and 33% of treatment cycles, respectively. One patient died due to sepsis. These promising results are consistent with continued clinical investigation; a phase III clinical trial with whole body hyperthermia as the independent variable has been initiated.

Invasiveness of human pleural mesothelioma cells is influenced in vitro by the three-dimensional structure of the ECM and its composition.

The grade of malignancy of a neoplasm is influenced by the invasive and metastatic potential of the tumor cells. The extracellular matrix of tissues is known to interact with many aspects of the biological behavior of tumor cells, such as differentiation and invasiveness. Therefore we studied the influence of the extracellular matrix on the morphology and invasiveness of the human biphasic pleural mesothelioma cell line MSTO-211H in vitro. The major components of the two strata encountered by a pleural mesothelioma cell leaving the epithelial community were mimicked by plating cells either on collagen I, the major component of the underlying stratum fibrosum, being encountered by cells under pathological conditions or on reconstituted basement membrane (Matrigel) in order to simulate the basement membrane of the stratum serosum of the mesothelium, which is the matrix cells have contact to under physiological conditions. Growth on collagen I leads to cell separation and invasion into the matrix, whereas growth of MSTO-211H cells on Matrigel results in the formation of a huge and dense network of cells extending throughout the whole plating area. The morphology of cell contacts between the two populations varies impressively. While cells on collagen I hardly find to each other in groups, and if so, with a broad intercellular cleft, Matrigel induces the tight approach of membranes of neighbouring cells with formation of syncytium-like structures. Administration of the main ECM components laminin and collagen IV alone and together in equimolar concentrations as present in Matrigel, does not result in any morphological changes when compared to cells growing on plastic substrates or on collagen I. Therefore, collagen I increases cell separation and invasiveness whereas an intact basement membrane seems to prevent the cells from separating and spreading, thus lowering their invasive potential.

A cell-seeded biocomposite for cartilage repair.

Chondrocytes in monolayer cultures lose their phenotype and capability to express type-II collagen, they dedifferentiate into a fibroblastic cell type. Using three-dimensional culture systems a redifferentiation of these cells may occur. In the present study we investigated the morphology and biosynthetic activity of human articular chondrocytes seeded on porous matrices of type I/III collagen (Chondrogide, Geistlich Biomaterials, Wolhusen, Switzerland). Microscopical examinations showed that chondrocytes adhere firmly to a collagen-I/III-membrane exhibiting their characteristic spherical cell shape. Cell numbers after enzymatic digestion of the membrane showed a 93% recovery of seeded cells. Immunohistological examination revealed positive staining for type-II collagen in some areas. The generated biocomposite withstands mechanical stress, keeps its size and design and does not shrink in culture. It is therefore easy to handle, can be sutured, glued or fixed with pins. This study shows, that in vitro production of autologous cartilage-like tissue could be established using a bilayer collagen type I/III fleece. This biocomposite carries active chondrocytes and is currently being evaluated in vivo in a sheep model as well as in a clinical trial for the repair of localized cartilage defects in the knee.

Hyperthermia and mafosfamide in a human-derived malignant pleural mesothelioma cell line.

PURPOSE: Diffuse malignant pleural mesothelioma is the most common primary pleural malignancy. At the beginning of the last century, this tumor was of minor incidence. Meanwhile, the use of asbestos has led to and is still leading to a rise in pleural mesothelioma incidence. There is no standard therapy for this highly aggressive disease and the development of new therapeutic strategies is imperative. METHODS: We, therefore, investigated the morphological and pharmakokinetic effects of a combined thermochemotherapy consisting of the administration of different dosages of mafosfamide with and without the application of a 1-h hyperthermia at 41.7 degrees C on the human biphasic malignant pleural mesothelioma cell line MSTO-211H. After therapy, cells were prepared for light and electron microscopy. BrdU-incorporation for the S-phase fraction, TUNEL-labeling for detection of apoptosis, and quantitative assessments using the MTT assay were performed. RESULTS: Our results demonstrate that the combination of mafosfamide with hyperthermia leads to qualitatively and quantitatively enhanced cellular damage compared to monotherapy. During combined thermochemotherapy, cell damage and death is already induced at lower mafosfamide concentrations than without hyperthermia which suggests an additive effect from hyperthermia to the action of the alkylating drug mafosfamide. Cell death thereby mostly occurs as necrotic cell death rather than as apoptosis, although in a combined thermochemotherapy apoptosis is induced temperature-dependently, when comparing temperatures from 37 degrees C to 43 degrees C. CONCLUSIONS: We suggest that the effect of substances such as ifosfamide and cyclophosfamide which are in clinical use, might be enhanced by the combination of local or regional hyperthermia in order to improve the therapeutical index of these substances in the treatment of pleural mesothelioma.

Effects of hyaluronic acid on the morphology and proliferation of human chondrocytes in primary cell culture.

Hyaline articular cartilage is a specialised connective tissue with weight bearing and adsorbing functions. Injury or loss of which often leads to impaired joint function and severe pain. Since the self-renewing abilities of hyaline articular cartilage are limited, there is major interest in the development of bioengineered cartilaginous implants. A cell-matrix-biocomposite composed of a collagen I/III scaffold seeded with autologous chondrocytes is currently being used in clinical trials; however, in order to optimise culture conditions, we cultured human condrocytes and seeded them on type I/III collagen membranes and on Thermanox plastic coverslips with media containing 0 to 500 microg/ml Hyaluronic Acid. After 4 days, the cells were either fixed or BrdU incorporation procedures begun. HE staining clearly demonstrated that cells grown in HA form three dimensional clusters and produce secretory vesicles as opposed to the monolayer control cells with noticeably fewer secretory vesicles. BrdU incorporation revealed a noticeable increase in cell proliferation in cells grown in 100 microg/ml; however, no comparable increase in 500 micorg/ml but rather a slight depression in proliferation. Immunohistochemistry for collagen II and aggrecan revealed an obvious increase in deposition of these two substances with increased HA administration as compared to the control; however, again, the higher concentration of HA, 500 microg/ml, did not result in a further increase in production. These results suggest that HA at 100 microg/ml not only influences chondrocytes to differentiate and produce more Collagen II and aggrecan, but also increases proliferation. We, therefore, propose that the addition of HA at low to middle dosages in condrocyte culturing might help improve condrocyte redifferentation and thus, the bioengineered cartilage.

Migration of naive, effector and memory T cells: implications for the regulation of immune responses.

T cells play an important role in protective immune responses and in the pathogenesis of many diseases. Understanding the mechanisms regulating their distribution in vivo may therefore be of therapeutic value. Reviewing studies that have followed the migration of labelled naive, effector and memory T cells in healthy animals reveals that all T-cell subsets enter all organs investigated. Within the tissue, two principally different migration patterns can be identified. First, naive and memory T cells accumulate in lymphoid organs for about 48 h after injection, as the time needed for migration through lymphoid organs is longer than through non-lymphoid organs. During this time, surface molecule expression is temporarily modified. These changes are reversed before leaving the lymphoid organs and entering the blood to start a new cycle of migration. Second, effector T cells are evenly distributed throughout the body, and most die in the tissues within 24 h. However, depending on the presence of cytokines, some are able to survive and to proliferate, and thereby accumulate in defined microenvironments of the body. Analysing the principles regulating T-cell migration and survival within the tissue may lead to the development of new options for the treatment of disease.

Characteristics of human chondrocytes, osteoblasts and fibroblasts seeded onto a type I/III collagen sponge under different culture conditions. A light, scanning and transmission electron microscopy study.

Hyaline cartilage has only a limited capacity of regeneration, thus, lesions of articular cartilage can lead to early osteoarthrosis. Current concepts in conservative orthopedic therapy do not always lead to satisfying results. As one new attempt to facilitate cartilage repair, autologous transplantation of articular chondrocytes is investigated in different assays. This study was designed to create a resistible and stable cell-matrix-biocomposite with viable and biosynthetically active human chondrocytes, osteoblasts or fibroblasts. This biocomposite might serve as an implant to treat deep osteochondral defects in the knee. We collected cartilage, spongiosa and skin probes from healthy patients undergoing hip-surgery and enzymatically liberated the chondrocytes, seeded them into culture flasks and cultured them until confluent. The spongiosa and the skin samples were also placed in culture flasks and cells cultured until confluent. After 4-6 weeks, cells were trypsinized and grown on a type I/III collagen matrix (Chondrogide, Geistlich Biomaterials, Wolhusen, Switzerland) for 7 days in standard Petri dishes and in a special perfusion chamber culture system. As controls, cells were seeded onto plastic surfaces. Then scaffolds were fixed and embedded for light microscopy and electron microscopy by routine methods. Light microscopically, chondrocytes grown on the surface of the scaffold form clusters or a dense layer of sometimes rather fibroblast-like and sometimes roundish, chondrocyte-like cells. Only a few cells grow deeper into the matrix. In transmission electron microscopy, the cells have a rather chondrocyte-like morphology which emphasizes the matrix-induced redifferentiation after dedifferentiation of chondrocytes in monolayer-culture in culture flasks. Chondrocytes on plastic surfaces have a spinocellular aspect with little signs of differentiation. Grown on Chondrogide, cells are more roundish and adhere firmly to the collagen fibrils of the scaffold. Osteoblasts grown on the collagen scaffold and examined by light microscopy form a thin cell-layer on the surface of the matrix with a reticular layer of dendritic cells underneath this sheet. Transmission electron micrographs show spinocellular and flat cells on the collagen fibrils. Scanning electron micrographs show large dendritic osteoblasts on plastic and a confluent layer of flattened, dendritic cells on the collagen scaffold. Fibroblasts form a thick multi-layer of typical spinocellular cells on the collagen matrix. Fibroblasts grown on plastic surfaces and examined by scanning electron microscopy also show a dense layer of fibroblast-like cells. For all three different types of cells no morphological differences could be seen when comparing cultivation in the perfusion culture system to cultivation in standard Petri dishes, although mechanical stress is believed to induce differentiation of chondrocytes. Especially the observed partially differentiated chondrocyte-matrix biocomposite might serve as an implant to treat deep cartilage defects, whereas osteoblasts and fibroblasts seem to be less suited.

Lethal granuloma disintegration in mycobacteria-infected TNFRp55-/- mice is dependent on T cells and IL-12.

Genetically susceptible, TNFRp55 gene-deficient (TNFRp55-/-) mice succumb to infection with Mycobacterium avium. Before their death, M. avium-infected TNFRp55-/- mice develop granulomatous lesions that, in contrast to granulomas in wild-type syngeneic mice, undergo acute disintegration. To determine the factors involved in these events, we depleted T cell subsets or neutralized the inflammatory cytokines IFN-gamma, IL-12, or TNF in TNFRp55-/- mice infected i.v. with M. avium. Infected TNFRp55-/- mice treated with a control mAb became moribund between days 26 and 34 postinfection, showing widespread inflammatory cell apoptosis within disintegrating granulomas. In contrast, TNFRp55-/- mice depleted of either CD4+ or CD8+ cells after granuloma initiation stayed healthy until at least day 38 postinfection and showed no signs of granuloma destruction. Neutralization of IL-12, but not of IFN-gamma or TNF, also protected M. avium-infected TNFRp55-/- mice from granuloma decomposition and from premature death. Treatment with dexamethasone or with a specific inhibitor of inducible NO synthase did not prevent granuloma dissolution or death of TNFRp55-/- mice. In conclusion, granuloma disintegration in TNFRp55-/- mice is a lethal event that is dependent on IL-12 and that is mediated by an excess of T cells.

Development of a biocomposite to fill out articular cartilage lesions. Light, scanning and transmission electron microscopy of sheep chondrocytes cultured on a collagen I/III sponge.

The regenerative capacity of hyaline articular cartilage is limited. Thus, lesions of this tissue are a proarthrotic factor, and up to now the conservative treatment of cartilage lesions and arthrosis does not yield satisfying results. Therefore, autologous transplantation of articular chondrocytes is being investigated in a variety of different assays. The aim of our study was to create a mechanically stable cell-matrix implant with viable and active chondrocytes which could serve to fill out articular lesions created in the knees of sheep. For this purpose, articular cartilage was collected from knee lesions, chondrocytes were liberated enzymatically and seeded in culture flasks and cultured till confluency. Cells were then trypsinized and grown on a type I/III collagen matrix (Chondro-Gide, Geistlich Biomaterials, Wolhusen, Switzerland) for 3, 6 and 10 days before being fixed and embedded for electron microscopy by routine methods. Scanning electron microscopy was performed after dehydration in acetone, critical point drying and sputter-coating with gold-paladium. Light microscopically, clusters of chondrocytes can be seen on the surface of the matrix with a few cells growing into the matrix. Transmission electron microscopic photographs yield a rather differentiated chondrocyte-like appearance, which is evidence of a matrix-induced redifferentiation after dedifferentiation during the growth period in the culture flasks. Scanning electron microscopic results show large, flattened chondrocytes without signs of differentiation on plastic, whereas chondrocytes grown on the Chondro-Gide sponge show a more roundish aspect wrapping firmly around the collagen fibrils, exhibiting numerous contacts with the matrix. This cell-matrix biocomposite can now serve to fill out articular cartilage lesions created in the knees of sheep.

Different types of pulmonary granuloma necrosis in immunocompetent vs. TNFRp55-gene-deficient mice aerogenically infected with highly virulent Mycobacterium avium.

The immunopathogenesis of mycobacterial infections frequently involves the formation of caseating granulomas which cause tissue destruction and, in the case of tuberculosis (TB), may lead to cavity formation. Both intravenous and aerosol models of Mycobacterium tuberculosis infection in mice do not reflect the pulmonary lesions characteristic of TB patients. Using both low-dose (10(2) colony-forming units, cfu) and high-dose (10(5) cfu) aerosol infection with a highly virulent strain of Mycobacterium avium (TMC724) in C57BL/6 mice, it is now shown that these mice are capable of developing centrally caseating necrosis in lung granulomas after approximately 4 months of infection. In contrast, mice infected intravenously with the high dose never developed this type of lesion, although bacterial counts in their lungs reached levels comparable to those attained by aerosol-infected mice (10(10) cfu). To study the relevance of events signalled by tumour necrosis factor (TNF) in this model, TNFRp55 gene-deficient and syngeneic C57BL/6 immunocompetent mice were infected with 10(5) cfu M. avium via aerosol. In gene-deficient mice, newly formed pulmonary granulomas acutely disintegrated, showing signs of apoptotic cell death and neutrophil influx, and TNFRp55 knock-out mice all succumbed to infection just beyond the stage of granuloma initiation. Aerogenic infection with M. avium in mice is a suitable model to study the immunopathogenesis of granuloma necrosis because it closely mimicks the histopathology of mycobacterial infections in humans, including TB. Furthermore, the use of TNFRp55 gene-deficient mice in this model establishes a role for TNF in maintaining the integrity of a developing pulmonary granuloma.

Differences in morphology and cytoskeleton of MCF-7 and MX-1 cells after therapy with OH-tamoxifen and the pure estrogen antagonist ZM 182780. An immunofluorescence and scanning electron microscopic study.

The adjuvant endocrine therapy of breast cancer with non-steroidal antiestrogens of the triphenylethylene-type such as tamoxifen is clinically well established, and pure steroidal antiestrogens are being introduced in clinical trials to circumvent the probable occurrence of tamoxifen resistance. Nevertheless, there do still remain some unsolved questions about the exact mechanisms of these substances. We therefore investigated the different effects of 4-OH-Tamoxifen (OHT), a non-steroidal antiestrogen, versus ZM 182780, a pure steroidal antiestrogen, on the morphology and on the cytoskeleton of MCF-7 (estrogen receptor-positive) and MX-1 (estrogen receptor-negative) cells. For this purpose cells were treated for 2, 5 and 7 days with OHT, ZM182780 and different concentrations of beta-estradiol. Interestingly, in scanning electron microscopy, MCF-7 cells showed more differentiation by forming three-dimensional structures such as acini or tubule-like structures under ZM 182780 therapy than with OHT. As expected, MX-1 cells showed no effects after ZM 182780-therapy, but OHT led to a decrease in the number of these cells and produced a fibroblast-like appearance of the estrogen receptor-negative MX-1 cells. The following immunocytochemical experiments on the tubulin, vimentin, cytokeratin and actin cytoskeleton surprisingly did not show marked differences within the morphologically differentiated ZM 182780-treated population compared to the control group of MCF-7 cells. Only the OHT-treated cells of both, the ER(+) and the ER(-) cells, showed a rearrangement of actin filaments and cytokeratin which appeared even more pronounced within the ER(-) MX-1 cells. No experimental group showed morphologically detectable changes in tubulin or vimentin distribution. These data suggest a non ER-mediated OHT-effect on the cytoskeleton that also affects the ER(-) cell line MX-1.

[New therapy procedure for localized cartilage defects. Encouraging results with autologous chondrocyte implantation]. / Neues Therapieverfahren für lokalisierte Knorpeldefekte. Ermutigende Resultate mit der autologen Chondrozytenimplantation.

Owing to the poor regenerative capacity of cartilage, cartilaginous defects are considered to represent pre-arthrotic factors. In addition to autologous and allogenic osteochondral fragments, proliferative tissue, such as periosteum and perichondrium are increasingly being used as graft material. The aim of treatment is to eliminate the defect and to restore the load-bearing capacity and function of the affected joint. A new, recently introduced, approach aims to stimulate the formation of new cartilage via autologous cultured chondrocyte implantation (ACI). The rationale for this treatment is the restoration of loadable hyaline or hyaline-like articular cartilage. Although long-term results are not yet available, clinical follow-up data obtained so far are encouraging. In addition to existing methods of treating cartilaginous defects, this article describes a modified method of transplantation of autologous chondrocytes. With this method the periosteal flap used to cover a defect is replaced by an absorbable collagenl/III membrane (Chondrogide, Geistlich Wolhusen, Switzerland) that is used as a carrier for the patient's own chondrocytes. After placement in the defect, the membrane is fixed in place with fibrin glue (MACI).