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M Sittinger

Publications and source records attributed to M Sittinger.

At least 55 records · Page 3Linked to original sources

Tissue engineering and autologous transplant formation: practical approaches with resorbable biomaterials and new cell culture techniques.

The engineering of living tissues in vivo requires new concepts in cell culture technology. In contrast to conventional cell cultures, the development of tissues depends on a three-dimensional arrangement of cells and the formation or synthesis of an appropriate extracellular matrix. Special emphasis is given to the major role of the extracellular matrix and cell differentiation in an artificial tissue. New technical approaches of in vitro tissue engineering are compared to the natural development of tissues in vivo. Current methods using resorbable biomaterials, tissue encapsulation and perfusion culture are discussed. Major consideration is given to scaffold structures of biomaterials that define a three-dimensional shape of a tissue or guide matrix formation. The different goals of tissue engineering such as in vitro models and transplant production are taken into account in the described techniques. Practical concepts comprising cell multiplication and differentiation in subsequent steps for future clinical applications are outlined.

Animals↗

[In vitro cultivation of human chondrocytes using autologous human serum supplemented culture medium: minimizing possible risk of infection with pathogens of prion diseases].

BACKGROUND: The in vitro engineering of autologous transplant might play an important role in reconstructive surgery in the near future. The amplification of isolated cells is an important part of the engineering. Because of the use of fetal calf serum (fcs) in the cellculture, there is a potential risk of transmission of prion diseases. The aim of this study was to evaluate a way of amplifying chondrocytes with autologous human serum instead of fcs and to compare the rates of proliferation. METHODS: For this purpose the isolated chondrocytes were cultured using complete medium, with 10% fcs or 10% autologous human serum being added to the medium. RESULTS: The experiments show that a culture with complete medium and autologous human serum allows a 150 to 300fold increase in the number of cells after 40 days of culture and the proliferation is up to 3fold higher than in cultures using fcs. DISCUSSION: Chondrocytes can be stimulated by autologous human serum to proliferate. It is possible to avoid fcs in the culture of chondrocytes and to minimize the risk of an infection with prions.

Adult↗

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↗

Immunohistochemical demonstration of c-myc oncogene product in middle ear cholesteatoma.

Cholesteatoma epithelium is characterized by a dysregulation with a hyperproliferative growth and altered differentiation. In a variety of cells c-myc oncogene was found to be highly linked to the control of growth and differentiation. Expression of c-myc was studied in cholesteatoma epithelium using a monoclonal antibody directed against the 67 kDa c-myc protein product and the alkaline phosphatase-antialkaline phosphatase method. For quantitative analysis a computer-linked analyzing system was used. In contrast to normal skin, keratinocytes of basal and suprabasal layers showed nuclear staining in cholesteatoma epithelium. The extent of nuclear staining of epithelial cells in the cholesteatomas studied was significantly increased. Concurrent cytoplasmic staining was observed in both skin and cholesteatoma, but with a stronger reactivity in the latter. These findings suggest participation of the c-myc oncogene in cholesteatoma epithelium.

Alkaline Phosphatase↗

[In vitro cultivation of cartilage tissue for reconstructive surgery: effect of L(+)-lactate and glycolate on cultivated human chondrocytes].

Within the scope of producing cartilage tissue in a three-dimensional culture design, the stability of the used delivery substance in-vitro tissue product has to be improved. For this, carrier materials consisting of bioresorbable polymers, e. g. poly(L[+]-lactic acid) and poly(glycolic acid) can be used. In respect of the biocompatibility of these polymers, the effect of degradation products on chondrocytes is of major interest. The available biomaterials were tested on chondrocytes in form of their monomers, glycolic acid and L(+)-lactic acid. Effects in regard of cell activity were determined with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide( MTT)test. A non-pH-effect was examined by buffering with concentrated NaOH. In a short-term testing with increasing monomer concentrations as well as in a test over a twelve-day period, L(+)-lactic acid proved to have a lower cytotoxic effect on chondrocytes than glycolic acid. Similar results were obtained with buffered culture media. Therefore, poly(L[+]-lactic acid) can be recommended for the development of chondrocytes-polymer constructs for in-vitro engineering of cartilage tissue.

Cartilage↗

[Immunohistochemical detection of c-myc proto-oncogene products in middle ear cholesteatoma].

Cholesteatoma epithelium is characterised by a keratinocyte dysregulation with a hyperproliferative growth and altered differentiation. In a variety of cells c-myc oncogene was found to be highly linked to the control of growth and differentiation. The expression of c-myc was studied in cholesteatoma epithelium using a monoclonal antibody directed against the 67 kD c-myc protein product and the alkaline phosphatase-anti-alkaline phosphatase-method. Furthermore for quantitative analysis we used a computer aided analysing system. In contrast to normal skin the keratinocytes of suprabasal layers showed a nuclear staining in cholesteatoma epithelium. The incidence of nuclear stainings of epithelial cells in cholesteatoma was significantly increased. Simultaneously, cytoplasmatic staining was observed in both skin and cholesteatoma with a stronger reactivity in the latter. Our findings suggest a participation of the c-myc oncogene in the reported dysregulation of cholesteatoma epithelium.

Biopsy↗

[Tissue engineering: artificial tissue replacement containing vital components].

Tissue engineering as a new field of research has gained increasing importance in recent years. The interdisciplinary field combines, biomaterials cell biology, and cell culture bio-engineering technology. The main focus of tissue engineering is the synthesis of artificial constructs or tissues based on vital cells or cell matrix. Biomaterials provide a three-dimensional structure to shape or guide tissue development. Isolated cartilage cells from a patient can form new tissues when suspended in non-woven resorbable polymers for reconstructive surgery. To achieve sufficient amounts of autologous cells for transplant formation, cells from biopsies have to be multiplied in monolayer culture. Dedifferentiated and undifferentiated mesenchymal cells may be used for bone and cartilage engineering. High cell densities in three-dimensional cultures require perfusion techniques to stabilize culture conditions. Morphogenetic factors such as BMP (bone morphogenetic protein) are thought to play a key role in inducing and controlling phenotypic tissue formation. In conclusion, modern in vitro approaches open new avenues for the development of vital tissue replacements for the clinic. Tissues can be repaired with the patient's own cells eventually leaving no residual artificial materials. Tissue engineering further provides new approaches for in vitro models of the extracellular matrix or diseases which mainly affect this matrix such as rheumatoid arthritis or osteoarthritis. This article describes recent developments in connective tissue engineering and discusses the potential for human tissue repair and reconstructive surgery.

Animals↗

Engineering of cartilage tissue using bioresorbable polymer fleeces and perfusion culture.

Replacement of injured or diseased skeletal tissues by either autograft or allograft cartilage has increased steadily during recent decades. The ideal method is to use autologous cartilage; however, this is extremely limited due to the scarcity of donor sites. We present a new approach to the in vitro formation of cartilage grafts for autologous grafting in reconstructive surgery. Bioresorbable polymer fleeces of polylactic acid were used as temporary cell carrier matrices to establish three-dimensional cultures of human chondrocytes. The polymer surface was coated with poly-L-lysine before cell integration. These cell-polymer tissue constructs were encapsulated with low melting point agarose and then placed in perfusion culture chambers to provide a constant supply of nutrients into the cultures. The culture medium consisted of Ham's F12 supplemented with 2% fetal calf serum and 50 micrograms/ml ascorbic acid. The cell-polymer tissues were harvested and frozen for toloudine and alcian blue staining as well as electron microscopic examination after different periods of time in culture. A monoclonal antibody specific for collagen type II was used to characterize the cell phenotype. With this culture procedure chondrocytes maintained a differentiated phenotype with synthesis of collagen and proteoglycan. Collagen fibrils with clear cross-striation were evident in electron microscopic images. The results show that our organotypic cell culture method allows the in vitro production of bioartificial cartilage for transplantation.

Biomedical Engineering↗

[Cultivation of human cartilage tissue in a 3-dimensional perfusion culture chamber: characterization of collagen synthesis].

In reconstructive head and neck surgery, there is a great need for cartilage transplants. Sufficient autologous graft is often not available. Heterologous cartilage is used frequently, although there is danger of transmitting viral infections and resorption rates are high. We have developed a three-dimensional model for the formation of cartilage in vitro. The aim of this study was to characterize the collagen synthesis under these culture conditions. Human chondrocytes were isolated by digesting septal cartilage matrix in the presence of type II collagenase, hyaluronidase, and Dnase II in Ham's F12 medium. The resulting cells were kept in monolayer culture for one week and then suspended in 2% ultra-low-melting agarose (1:1). The cell-agarose conglomerate was encapsulated with a 3% ultra-low-melting agarose solution and placed in a perfusion culture chamber. A permanent flow of fresh medium (Ham's F-12 supplemented with 50 micrograms/ml ascorbic acid and 2% fetal calf serum) was provided by a peristaltic pump which delivered 1 ml/h with on/off intervals of 30 min. Samples were recovered after two weeks. Using electron microscopy abundant collagen fibril formation was shown. The collagen fibrils were identified histologically as cartilage specific type II collagen. No mRNA expression of collagen type X was observed using in situ hybridization. The cells appeared in a round cell shape with round nucleus and only slight variations in form and size. The present results indicate that the chondrocytes maintain their differentiated phenotype and continue to synthesize typical matrix products in this three-dimensional perfusion culture chamber.(ABSTRACT TRUNCATED AT 250 WORDS)

Cartilage↗

Engineering of cartilage tissue using bioresorbable polymer carriers in perfusion culture.

Bioresorbable polymer fleeces with a high internal surface area were used as temporary matrices to establish three-dimensional cultures of isolated human articular chondrocytes. The polymer surface was coated with poly-L-lysine to support cell attachment. The resulting cell-polymer tissues were cultured in perfusion culture chambers to achieve a constant supply of nutrients by diffusion. Retention and accumulation of extracellular matrix components synthesized by the chondrocytes were improved by encapsulation of the cell-polymer integrate in agarose gel. The cell-polymer tissues formed abundant collagen fibrils in vitro with a typical cross-triation clearly visible in electron microscopy analysis. Chondrocytes and intercellular matrix stained positively with monoclonal antibodies specific for differentiated chondrocytes and type II collagen. Synthesis of proteoglycans and collagen was also evident by further analysis with alcian blue and azan staining of cell-polymer tissue sections. The presented experimental tissue culture technique offers a novel concept for the in vitro formation of vital cartilage implants for reconstructive surgery or treatment of destructive joint diseases and possibly for the in vitro engineering of human tissues in general, with applications in drug testing and replacement of animal experiments.

Adult↗

[Culture of human cartilage tissue using a perfusion chamber].

In the field of otolaryngology cartilage grafting is commonly performed to reconstruct skeletal defects. Knowledge of chondrocyte growth and differentiation can now be used to engineer cartilage tissue for grafting. The first condition is that chondrocytes maintain their differentiated phenotype besides being able to produce a new cartilage matrix. The target of this study was to develop a three-dimensional culture system for in-vitro formation of vital cartilage transplants. Chondrocytes were isolated by digesting the cartilage matrix with collagenase and hyaluronidase. After embedding in "low-melting" agarose, the chondrocytes were placed into a perfusion culture chamber to provide a constant supply of nutrients to the cultures. The peristaltic pump was operated with on/off intervals of 30 min. Ham's F12 supplemented with 2% FCS and 50 micrograms/ml ascorbic acid was employed as culture medium. Monoclonal antibodies specific to collagens type I and type II were used to characterise cells and matrix synthesis. Synthesis of proteoglycans and collagens was achieved using toluidine blue and azan staining. Under the described culture conditions, the chondrocytes maintained a differentiated phenotype (expression of collagen type II) with synthesis of collagens and proteoglycans. An accumulation of matrix products was achieved pericellularly. After 2-8 weeks the obtained tissue exhibited an excellent histological appearance showing the typical features of cartilage tissue. The results show that the perfusion chamber allows a quick in-vitro fabrication of a piece of pure cartilage tissue for transplantation.

Cartilage↗

Humoral immune response against minor collagens type IX and XI in patients with cartilage graft resorption after reconstructive surgery.

OBJECTIVES: The humoral immune response against a broad spectrum of cartilage antigens (cellular and matrix antigens) was studied in a group of patients who showed resorption and/or rejection of transplanted cartilage in nasal surgery. METHODS: Sera were obtained from patients with successful and unsuccessful cartilage grafting in the nose, from age and sex-matched healthy donors and from patients with rheumatoid arthritis. Antibodies to cartilage components were analysed by the following methods: (1) indirect immunofluorescence on cartilage sections, (2) ELISA using cultured human chondrocytes, isolated chondrocyte membranes and purified collagens type I, II, III, VI, IX and XI, and (3) immunoblotting with purified collagens and chondrocyte cell membranes. RESULTS: In the cartilage grafting group showing resorption problems, levels of anti-collagen antibodies were significantly higher against native collagen types IX (p < 0.002) and XI (p < 0.002) compared with the non-resorption group and the normal donors. Both transplantation groups revealed elevated reactivities against isolated chondrocytes in the ELISA. In contrast, no reactivity was detectable against collagens type II, III, and VI and chondrocyte cell membranes by both ELISA and immunoblotting. CONCLUSIONS: These data demonstrate for the first time the existence of a humoral immune response, primarily directed against the so called 'minor cartilage collagens', in patients showing cartilage resorption. Autoreactivities to collagen which are typical of inflammatory rheumatic diseases may also play an important role in the repeated failure of cartilage grafting.

Adult↗

Antigen presenting cell function of class II positive human nasal chondrocytes.

It is postulated that class II positive chondrocytes may be actively involved in the destruction or rejection of vital transplanted cartilage grafts. To investigate whether human nasal chondrocytes may also function as accessory cells in ongoing immune reactions with cartilage destruction, mixed leukocyte-chondrocyte cultures and antigen presentation assays were performed. Freshly isolated HLA class II antigen negative chondrocytes obtained from nasal septa were not stimulatory to autologous resting T lymphocytes. HLA class II positive chondrocytes treated with gamma-interferon were able to present antigens to autologous activated T cells derived from an antigen (tetanus) specific T cell line. Upon incubation with activated T cells, initially class II negative changed their phenotype resulting in the expression of class II antigens and enabling them to effectively present antigen. These results suggest an active role of chondrocytes in the rejection of cartilage grafts.

Antigen-Presenting Cells↗

Effect of growth factors on cell proliferation by human nasal septal chondrocytes cultured in monolayer.

In the field of reconstructive surgery, autologous cartilage grafting is commonly performed to reconstruct skeletal defects. Because of the limited supply of fresh autologous cartilage many investigators concentrate on in vitro production of cartilage tissue. Several growth factors regulate the metabolism and activation of cartilage cells. In order to enhance the culture conditions for cartilage cells, the aim of our investigations was to characterize the influence of transforming growth factor (TGF)-beta, epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) on the proliferation of differentiated human nasal septal chondrocytes. The isolated cells were cultured in monolayer using DMEM with and without 10% FCS. The cell proliferation was assessed using tritiated thymidine. We measured an increase of the proliferation rates when the different growth factors were added. The most important stimulatory effect was due to bFGF and the less to EGF. If all growth factors were added together a fivefold increase in the proliferative activity of the cells was achieved. The effects were further enhanced by factors present in fetal calf serum. We conclude that the culture conditions for cell expansion for cartilage engineering can be optimized employing growth factors.

Adult↗

Antibodies to the minor cartilage collagen type IX in otosclerosis.

The presence of antibodies to collagens type I, II, III, VI, IX, and XI was studied in patients with otosclerosis, using enzyme-linked immunosorbent assays. Levels of antibodies to collagens type II and IX were significantly higher in these patients as compared to sex- and age-matched control subjects, whereas no differences were found between the levels of antibodies to collagens type I, III, VI, and XI. These observations for the first time document the presence of autoantibodies against a minor collagen type IX in patients with otosclerosis and support a possible role for collagen autoimmunity in the etiology of otosclerosis.

Adult↗

[Cartilage-specific autoimmunity in otosclerosis].

The otic capsule of patients with otosclerosis contains premature bone with numerous cartilaginous remnants. Some investigators have proposed that the pathogenesis of otosclerosis is related to these cartilaginous rests in the otic capsule. In this study we investigated the presence of a humoral immune reaction against cartilage-specific antigens using ELISA-methods in patients with otosclerosis. Concomitantly, 8 age- and sex-matched healthy blood donors, free of any symptoms of autoimmune disease, served as controls. The following antigen substrates were used: collagen (I, II, III, VI, IX and XI), chondrocytes and chondrocyte membranes. Findings then showed that the levels of antibodies to collagens type II and IX as well as to chondrocytes were higher in the otosclerosis patients than in the control subjects. The high titer of antibodies against chondrocytes was not accompanied by an increase in antibodies against the chondrocyte membranes. To our knowledge these observations represent the first evidence for the existence of autoantibodies against minor collagens and chondrocyte-specific antigens and support a possible role for a cartilage-specific autoimmunity in the etiopathogenesis of otosclerosis.

Adult↗