PubMed Health⌕ Search

Biomedical subjects

J Aigner

Publications and source records attributed to J Aigner.

35 records · Page 2Linked to original sources
PubMed1997

[Vital long-term preservation of human gingiva in perfusion culture].

Perfusion culture offers the advantage of keeping gingiva alive for a long time as an stable explant according to cell biological parameters. To investigate the suitability of cultured human gingival explants for transplantations the biopsies were put into a newly developed perfusion chamber and cultured for at least 21 days. Gingiva explants were derived from healthy donors undergoing surgical removal of molar teeth. The tissue pieces were cultured without prior proteolytic desintegration or subculture. Immediately after excision a morphological and immunohistochemical analysis of the tissue was carried out and the distribution pattern of cytokeratin and vimentin was examined. Gingival explants cultured for 7, 14 and 21 days in serum-free keratinocyte growth medium in perfusion culture were analyzed in the same way. The morphology of the cultured explant (21 days) was well preserved from stratum basale up to stratum corneum. As proved by immunohistochemical incubation with antibodies to CK 5/6, CK 14 and CK 19, a tissue-specific cytokeratin (CK) expression pattern was maintained during the whole perfusion period. After 7 days of culture vimentin was synthesized in the fibroblast layer and was found in small quantities in each layer of the epithelium. In contrast to conventional cultures, where dissociation of the tissue and a subculture interruption is usually needed for long-term culture, this is not necessary for perfusion cultured tissue. The use of perfusion-cultured gingival explants as autogenous transplants is investigated herein.

Gingiva↗
PubMed1997

Culture of embryonic renal collecting duct epithelia in a gradient container.

During organogenesis the ampullar epithelium of the renal collecting duct acts as an inducer which generates all of the nephron anlagen. As development proceeds, one part of the collecting duct cells in the ampullar tip retain their inducer capability, while others develop into the functional epithelium consisting of principal and intercalated (IC) cells. The events leading from the embryonic inducer to the mature tissue are unknown. We investigated the maturation of embryonic collecting duct epithelium derived from neonatal rabbit kidney under in vitro conditions. To prevent dedifferentiation the epithelia were cultured on kidney-specific support material within a tissue carrier. Apical and basal compartments of the epithelia were simulated in a gradient culture container. The two sides of the epithelium were each constantly perfused with a different medium. During the 14-day incubation the tissue was not subcultured. The development of collecting duct cell features was investigated with morphological and immunohistochemical methods. Both light and electron microscopy revealed morphologically intact epithelia following gradient culture. The polarized cells rested on a uniformly developed basement membrane. The continuous application of aldosterone during the culture modulated the development of collecting duct cell characteristics. Both basal and luminal administration of aldosterone initiated differentiation in the embryonic epithelia. Using the sodium (Na) channel blocker amiloride, it was demonstrated that Na channels are involved in the differentiation of the IC cell phenotype.

Aldosterone↗
PubMed1997

PCDAmp1, a new antigen at the interface of the embryonic collecting duct epithelium and the nephrogenic mesenchyme.

In the neonatal rabbit kidney nephrogenesis is not yet terminated. The ampullar collecting duct epithelium acts as an inducer that generates the nephron anlagen, however, to date the morphogenic mechanisms involved are unknown. A presupposition for successful nephron induction is the close tissue interaction between the basal aspect of the ampullar collecting duct epithelium and the surrounding mesenchyme. To gain new insights in this area we raised monoclonal antibodies (mabs), to identify specific structures localized at the tissue interface. With the generated mab CDAmp1 we found an intensive immunohistochemical reaction between the basal aspect of the ampullar collecting duct epithelium and the mesenchyme. The label was most concentrated at the ampullar tip and continuously decreased in the shaft region. In the maturing collecting duct of the neonatal kidney and in the adult renal collecting duct no immunohistochemical reaction was found. The binding pattern of mab CDAmp1 is different from that of all known collecting duct cell markers and from antibodies against known basement membrane compounds such as laminin or collagen type IV. Under in vitro conditions immunoreactivity with mab CDAmp1 was obtained using embryonic collecting duct epithelia and perfusion culture. The antigen was present in specimens treated with Iscove's modified Dulbecco's Medium (IMDM) containing 10% fetal bovine serum. Omittance of serum or hormonal treatment with aldosterone, insulin or vitamin D3 led to the disappearance of the newly detected antigen, while characteristics of the differentiated collecting duct cells were up-regulated. We conclude that the expression of PCDAmp1 is a characteristic feature of the embryonic parts of the collecting duct epithelium. It may play a pivotal role during nephron induction.

Animals↗
PubMed1997

[Realistic imaging of cell systems using confocal laser scanning microscopy exemplified by 3-dimensional chondrocyte culture].

BACKGROUND: We have developed a three-dimensional model for tissue engineering of cartilage. Chondrocytes were isolated and first multiplied in conventional monolayer cultures. Then the cells are seeded with or without agarose on special absorbable scaffolds that provided stability and enabled three-dimensional cell distribution of the tissue-engineered cartilage. The aim of the study was to investigate the possibility of avoiding agarose in tissue engineering because of the potential risk of causing an inflammatory process in later human implantation. METHOD: For the first time we investigated cell distribution combined with vitality directly in the cell carrier under the conditions described by using confocal laser-scanning microscopy. Working with unfixed cells, this method enables the reconstruction of three-dimensional cell cultures with suitable cell markers that closely simulates the physiologic situation, thereby exceeding each other method. RESULTS: It was evident that agarose had no positive effect on cell distribution and vitality. CONCLUSION: Further experiments concerning the effect of agarose on synthesis of cartilage-specific matrix are in progress.

Cartilage↗
PubMed1996

Endotoxin-inducible granulocyte-mediated hepatocytotoxicity requires adhesion and serine protease release.

In primary cultures of Kupffer cells and hepatocytes, human granulocytes potentiated toxicity of endotoxin about 1000-fold. Granulocyte elastase activity was found to correlate with toxicity. The serine protease inhibitors alpha1-antitrypsin, eglin C, and aprotinin protected against toxicity. Tumor necrosis factor-alpha (TNF-alpha) induced cytotoxicity and elastase release, whereas neutralization of TNF-alpha blocked both events. We conclude that TNF-alpha formed by Kupffer cells activates granulocytes. Experiments in cultures where cells were separated by membranes permeable to mediators indicated that cell contact is needed for toxicity. Scanning electron microscopy showed granulocytes adhering to and interdigitating with hepatocytes. Using liver cells from ICAM-1-deficient mice had no effect on toxicity. However, neutralizing CD31 inhibited toxicity and elastase release but not granulocyte adhesion. Our findings demonstrate that adhesion of granulocytes is a necessary but not sufficient condition for the synergistic interaction of endotoxin-stimulated liver macrophages and granulocytes in the proteolytic killing of hepatocytes.

Androstadienes↗
PubMed1996

Approach to an organo-typical environment for cultured cells and tissues.

If cells or tissues are taken out of an organ and put in culture, normally they lose morphological, physiological and biochemical features. This dedifferentiation process starts during the isolation procedure and continues during the whole culture period. It is caused by the stagnant liquid condition and the inadequate anchorage of cells at the bottom of tissue culture plasticware. The use of filters as basement membrane substitutes and the coating of cultureware with extracellular matrix proteins improve the environmental factors for cultured cells but do not consider the paracrine influence of cytokines or the nutritional needs of individual cell types. To limit cellular dedifferentiation in culture, we constructed a new system, which adapts, as far as possible, cell and tissue cultures to an organo-typical environment. The system is based on a compatible cell carrier arrangement, which allows individual selection of supports for optimal cell anchorage and differentiation. The cell carriers are placed in a newly constructed container, which is permanently perfused with fresh culture medium. The system runs outside an incubator with simple laboratory tools; only a peristaltic pump, a warming table and pH-stabilized media are necessary. Without any subculturing, acute and chronic influences of drugs or the quality of medical implantation grafts can be studied over months.

Cell Adhesion↗
PubMed1995

Development of renal podocytes cultured under medium perifusion.

BACKGROUND: In the past, podocytes have been described as highly susceptible to dedifferentiation under cell culture. Whether this process resulted from insufficient culture conditions or whether it was a consequence of missing cellular interactions remained unclear. A further reason could be that podocytes within the maturing kidney are irreversibly growth-arrested at a very early point of development because proliferating cells have been detected at the S-shaped body stage but not at the capillary loop stage or in the maturing glomeruli. These were important reasons that hindered the establishment of podocyte cell culture systems. EXPERIMENTAL DESIGN: The aim of our present study was to culture podocytes under the most organotypic conditions possible to maintain typical cellular characteristics. Cortex explants of neonatal rabbit kidneys consisting of nephrogenic tissue were used as a source for podocytes. No serum additives were given for the whole culture period of 13 days. An organ-specific environment was obtained by keeping the podocytes within the surrounding renal tissue and by ensuring a permanent exchange of medium. RESULTS: mAb were used to characterize podocytes and the other glomerular cell types. Cultured podocytes and parietal cells of Bowman's capsule were identified by EnPo 1. Ks 19.2.105, a marker for cytokeratin 19, was used to discriminate among these epithelial cells because cytokeratin 19 is expressed by the parietal cells of Bowman's capsule but not by podocytes. The Ab EC1 specifically detected endothelial cells. Glomerular endothelium cultured under medium perifusion expressed these typical Ag and thus could be unequivocally discriminated. Furthermore, by means of the proliferation marker Ki-67, it could be demonstrated that glomerulus-like structures developed under culture by proliferation of visceral and parietal cells of Bowman's capsule. CONCLUSIONS: A culture model is presented that allows the maintenance of developing podocytes within the organ-specific tissue environment and under permanent medium perifusion.

Animals↗
PubMed1995

Transitional differentiation patterns of principal and intercalated cells during renal collecting duct development.

The developing renal collecting duct epithelium of neonatal rabbits exhibits 3 different zones. The ampullary tip epithelium acts as an embryonic inducer and is responsible for the generation of all of the nephron anlagen. It pilots the whole microarchitecture of the kidney. In the ampullary neck epithelium multiple cell divisions cause the elongation of the embryonic collecting duct so that the organ can grow. Finally, the cells in the ampullar shaft transdifferentiate into the functional collecting duct epithelium (CD) consisting of Principal (P) and various kinds of Intercalated (IC) cells. It is unknown by which morphogenic mechanisms the ampullar cells develop into the heterogeneously composed collecting duct epithelium. Using both morphological and immunohistochemical methods, we investigated the transdifferentiation patterns leading from the ampullar epithelium to the P and IC cells in the neonatal kidney. An electron microscope analysis of the cortico-medullary course of the developing collecting duct revealed that conspicuous morphological alterations start in the neck of the ampulla. The lumen of the neck region is narrowed to a slit. While most of the cells in the ampullar tip exhibit few, short microvilli, the neck cells bear numerous, extremely long microvilli at their apical cell poles. All of the neck cells exhibit the same cytoplasmic staining pattern and the same number of mitochondria. Farther down in the shaft, clearly recognizable P and IC cells are found. Thus, differentiation into P and IC cells starts with a transitional precursor cell type in the ampullar neck. Perfusion culture experiments with the embryonic collecting duct epithelium made it possible to generate transitional and differentiated cell types for the first time under in vitro conditions. The cultured epithelial cells showed characteristics common to both P and IC cells. Immunohistochemical findings revealed that morphological differentiation starts before the functional properties of P and IC cells can be detected.

Animals↗
PubMed1994

Interrelationship of renal vascular development and nephrogenesis.

Within the cortex region of the neonatal rabbit kidney the developing microvasculature was investigated by means of two endothelium-detecting antibodies (EnPo 1 and EC1). Rows of antibody-labelled cells were found within tissue regions that had previously been described as avascular. We conclude that these vessel-like structures detected by EnPo 1 and EC1 are capillary precursors without lumina. Furthermore, beneath the fibrous capsule within the morphologically homogeneous mesenchyme two cell populations can be discriminated by use of differential antigen expression. The EnPo 1 antigen, which is abundant on endothelial cells and podocytes at different developmental stages, was detected on a subpopulation of mesenchymal cells. These cells were exclusively detected surrounding the tip of the collecting duct ampulla. Due to the unique specificity of EC1 and EnPo 1 the process of microvascular development can be readily followed on serial optical sections gained by laser scan microscopy. (1) Adjacent to EnPo 1-positive mesenchymal cell islets vessel-like structures are found that are in contact with the differentiated vasculature. (2) The renal vesicle is enclosed by a network of vessel-like structures establishing contact with differentiated vessels. (3) No guidance of invading capillary sprouts toward the developing glomerulus and nephron is required, since vascular elements already accompany the earliest detectable nephron stage.

Animals↗
PubMed1994

Maturation of renal collecting duct cells in vivo and under perifusion culture.

The embryonic collecting duct epithelium of neonatal kidney undergoes profound functional changes during maturation. In its initial state as inductor epithelium it appears homogeneous, but differentiates into a heterogeneously composed collecting duct epithelium consisting of principal and intercalated cells. The mechanism of this terminal differentiation process is unknown. We used morphological and immunohistochemical methods to investigate the maturation of the collecting duct system in neonatal rabbit kidney and under organotypic culture conditions. The new perifusion culturing method allowed us to follow the differentiation of the ampullary collecting duct epithelium under conditions as close as possible to the situation within the organ. With this technique we were able to induce a differentiation process similar to that in the in situ situation. This process led to the appearance of a mixed cell population consisting of principal and intercalated-like cells, respectively. A continuous perifusion of the medium made it possible to stabilize the microenvironment under culture conditions and thus to maintain the heterogeneous composed collecting duct epithelium in a differentiated status over long periods of time.

Animals↗
PubMed1993

Histochemical markers reveal an unexpected heterogeneous composition of the renal embryonic collecting duct epithelium.

The ampullary collecting duct epithelium acts as an inductor in the embryonic and neonatal kidney. It induces the formation of all nephron generations and thus determines the whole architecture of the kidney. As the organ matures, the collecting duct epithelium itself transdifferentiates. The ampullary inductor epithelium, which appears homogeneous as revealed by light microscopy, develops into the well-known heterogeneous epithelium of the mature collecting duct consisting of light principal and dark intercalated cells. Up to now the mechanisms initiating and regulating this transdifferentiation step are unknown. Only very few data are available concerning functional characteristics of the ampullary epithelial cells of neonatal rabbit kidney. Therefore, a characterization of the collecting duct ampullary cells was carried out by means of immunohistological techniques using a set of different monoclonal antibodies and the lectin peanut agglutinin. All epithelial cells within the ampullary tip and neck were positive for cytokeratin 19, an intermediate filament protein. On the other hand, the monoclonal antibody CD 7 revealed a clear cut boundary between the ampullary neck and the ampullary tip region. Furthermore, after incubation with the monoclonal antibody BO-7 specifically reacting with intercalated cells of the mature collecting duct, both labeled and unlabeled cells were observed within the whole ampullary epithelium. These results were confirmed by scanning electron microscopical investigations which revealed two distinct epithelial cell populations. Thus, an unexpected heterogeneity of the ampullary epithelium could be demonstrated.

Amino Acid Sequence↗
PubMed1993

Aldosterone modulates PNA binding cell isoforms within renal collecting duct epithelium.

To investigate the differentiation of the ampullary collecting duct cells into adult principal and intercalated cells, the embryonic cortex of newborn New Zealand rabbit kidney was isolated and brought in culture. With this culture technique the ampullary cells formed a polarized collecting duct epithelium which was kept under permanent exchange of medium and in the presence of aldosterone, arginine vasopressin and/or insulin. After 14 days of perfusion culture the epithelia showed light and dark cells resembling the principal and intercalated cells of the adult collecting duct. The differentiation from embryonic into adult collecting duct cells was controlled by applying the monoclonal antibody CD 7. Independent of the hormonal treatment all of the epithelial cells matured in culture and expressed the CD 7 antigen. This corresponded with the situation found within the adult kidney, where the CD 7 antigen was localized in all principal and intercalated (IC) cells, whereas the embryonic ampullary epithelium in the neonatal kidney remained negative. A differentiation feature of the beta-type intercalated cell was investigated by labeling the cultured epithelia with peanut agglutinin (PNA). In contrast to the CD 7 antigen the development of PNA binding was highly dependent of time and individual hormone administration. While in control epithelia only 8% of PNA positive cells were found, aldosterone induced epithelia revealed 72% PNA labeled cells. The combination of aldosterone and insulin increased the number of PNA-positive cells to 90%. By scanning electron microscopy it could further be shown that several isoforms of cells were reactive with PNA. Thus, in culture the PNA label is not restricted to the typical beta-type IC cells.

Aldosterone↗
PubMed1992

Characterization of an endothelial protein in the developing rabbit kidney.

A new protein (EnPo 1 antigen) abundant on endothelial cells and glomerular podocytes has been characterized by means of the mouse monoclonal antibody EnPo 1. Following electrophoretical separation of rabbit kidney homogenates EnPo 1 recognized a protein with a molecular weight of 110 kDa and an isoelectric point of 5.9 in Western blots. Using immunohistological techniques, the EnPo 1 antigen has been localized in high concentrations on glomerular podocytes of different developmental stages. Furthermore, the EnPo 1 antigen was expressed on endothelial cells of all adult rabbit organs tested so far. Detailed analysis of neonatal rabbit kidney revealed the abundance of EnPo 1 antigen on both differentiated vessels as well as on immature endothelial cells and endothelium of the microvasculature. Thus, for the first time a marker for in situ investigations of angiogenic processes within the mammalian kidney is available. Analysis of kidney cryosections by confocal laser scan microscopy revealed a direct connection between mature and differentiating vessels in the outer kidney cortex. Furthermore, two differentially organized cell populations discriminated by their EnPo 1 binding pattern were localized in the embryonic renal cortex. Morphologically, these cells were not distinguishable from other mesenchymal cells.

Animals↗
PubMedDate not supplied

Behavior of tissue-engineered human cartilage after transplantation into nude mice.

Cartilage lacks the ability to regenerate structural defects. Therefore, autologous grafting has been used routinely to replace cartilaginous lesions. Because tissue engineering of human cartilage with the help of bioresorbable polymer scaffolds is possible in experimental models, the demand for the clinical application grows. In this study we present an analysis of the behavior of transplants made of human chondrocyte pools, agarose and the resorbable polymer scaffold Ethisorb and a preliminary comparison with transplants made of single patients' cells and Ethisorb but without the additional ingredient agarose. Chondrocytes were isolated from the matrix of human septal cartilage by enzymatic digestion. The pool cells were kept in monolayer culture for 2 weeks, the single patients' cells for 3-4 weeks. Chondrocyte pools were suspended in agarose and seeded into the resorbable polymer scaffold Ethisorb. Single patients' cells were seeded without agarose. All cell-polymer constructs were kept in perfusion culture for 10-14 days and transplanted subcutaneously into thymusaplastic nude mice. Additionally we implanted Ethisorb fleeces embedded in agarose without chondrocytes. After 6, 12 and 24 weeks the animals were sacrificed and the specimens were explanted and analyzed histochemically and immunohistochemically. Polymer scaffolds not seeded with chondrocytes did not show cartilage formation. Resorption was complete after 12 weeks in vivo. Transplants from cell pools remained mechanically stable over 24 weeks apart from four transplants that were resorbed completely. Cartilage formation was observed in all pool-specimens with the presence of chondronic structures and a homogeneous matrix containing hyaline cartilage-specific matrix molecules such as collagen type II. Single patients' transplants showed hyaline cartilage matrix synthesis and mechanical stability as well. Chondrocyte pools are a suitable method to study cartilage engineering of human cells in vitro and in vivo in experimental models. Under clinical conditions it is, however, necessary to study the generation of cartilage from single patients' cells. We showed that it is possible without additional ingredients such as agarose. However, variations in the preliminary results show that the clinical application with human cells is more difficult than one would expect when using human chondrocyte pools. Further studies need to be performed to find out which individual factors influence the in vitro engineered cartilage's fate in vivo.

Journal Article↗
PubMedDate not supplied

Tissue engineering of autologous cartilage transplants for rhinology.

In reconstructive surgery there is increasing demand for cartilage transplants to fill defects, especially nose and/or outer ear defects. Tissue engineering is one of the most modern pathways to generate autologous cartilage transplants. Isolated chondrocytes obtained from a tiny patient's biopsy were seeded on bioresorbable preshaped cell carriers to provide a 3-dimensional cell arrangement as in vivo. The combined use of these cell carriers in form of a non-woven mesh and a constant medium perfusion was performed to generate a cartilage-like cell-polymer-construct, which was finally subcutanously implanted in nude mice for full maturation. After explantation of 6 months, expression of cartilage specific extracellular matrix molecules was obvious by using histochemical and immunohistochemical methods. These data show that tissue engineering with isolated multiplied human chondrocytes from a tiny biopsy seeded on bioresorbable polymer is a promising system to generate autologous cartilage transplants for replacements in reconstructive surgery.

Animals↗