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Biomedical subjects

M Sittinger

Publications and source records attributed to M Sittinger.

At least 19 recordsLinked to original sources
PubMed1998

Tissue engineering: generation of differentiated artificial tissues for biomedical applications.

A new field in biomedical science has been established. Cell biologists, engineers, and surgeons now work within a team. Artificial connective, epithelial, or neuronal tissues are being constructed using living cells and different kinds of biomaterials. Numerous companies and laboratories are presenting dynamic developments in this field. Prognoses predict that, at the beginning of the coming century, the industry of tissue engineering will reach the importance of the present genetic technology. An enormous demand for organ and tissue transplants motivates research activities and drives the acquisition of innovative techniques and creative solutions. At the front of this development is the creation of artificial skin for severely burned patients and the generation of artificial cartilage for implantation in articular joint diseases. Future challenges are the construction of liver organoids and the development of an artificial kidney on the basis of cultured cells. In this paper we show strategies, needs, tools, and equipment for tissue engineering. The presupposition for all projects is the induction, development, and maintenance of differentiation within the tissue under in vitro conditions. As experiments in conventional culture dishes continued to fail, new cell and tissue culture methods had to be developed. Tissues are cultured under conditions as close as possible to their natural environment. To optimize adherence or embedding, cells are grown on novel tissue carriers and on individually selected biomatrices or scaffolds. The tissues are subsequently transferred into different types of containers for permanent perfusion with fresh culture medium. This guarantees constant nutrition of the developing tissue and prevents the accumulation of harmful metabolites. An organo-typical environment for epithelial cells, for example, is obtained in gradient containers, which are permanently superfused at the apical and basal sides with different media. Long term experiments result in cultured tissues in a quality thus far unreached.

Biomedical Engineering↗
PubMed1997

Development of in vitro model systems for destructive joint diseases: novel strategies for establishing inflammatory pannus.

OBJECTIVE: To establish a novel 3-dimensional (3-D) in vitro model for the investigation of destructive processes in rheumatoid arthritis (RA). METHODS: Two distinct culture systems were developed, consisting of RA synovial membrane and articular cartilage explants or interactive RA synovial cell/chondrocyte cultures embedded in 3-D fibrin matrices. The expression of proteolytic enzymes, chondrocyte matrix architecture, and matrix degradation parameters was analyzed by immunohistochemistry. RESULTS: Of 28 RA explant cultures, 16 displayed an invasion of synovial tissue into the cartilage explants, compared with 1 of 8 osteoarthritis explants. The expression of collagenase and vascular cell adhesion molecule 1 could be demonstrated at the cartilage-pannus junction. Of 20 interactive cell cultures, 18 revealed invasive behavior and remained vital for extended periods of time. CONCLUSION: The models presented allow us to study distinct aspects of destructive joint diseases under in vitro conditions that resemble human pathology. Moreover, our model is able to supplement animal experiments in basic research and drug testing.

Animals↗
PubMed1997

[Proliferative potential of nasal septum chondrocytes for in vitro culture of cartilage transplants].

BACKGROUND: Recent developments in the field of tissue engineering provide novel approaches in tissue repair and reconstructive surgery using the patients own cells. Isolated chondrocytes form new cartilage when seeded in appropriate scaffolds. Usually the number of cells from a cartilage biopsy is not sufficient. The present study investigates the potential of cell amplification of human nasal chondrocytes in monolayer culture. METHODS: Nasal cartilage cells from seven healthy patients with age between 16 and 60 years were enzymatically isolated with collagenase and hyaluronidase. Subsequently, cells were seeded in 75 cm2 culture flasks. After confluency, cultures were trypsinized, counted, and again seeded at a concentration of 5 x 10(4) cells/ml. Dulbecco's MEM supplemented with 10% FCS was used as culture medium. RESULTS: After enzymatic digest, an average of 5 x 10(5) cells per patient were isolated. At least 85% of the cells were vital. Within four to eight weeks, the cells number was increased 10(3) to 10(5) fold. No correlation between the proliferative activity and the age of the patient was observed in this study. DISCUSSION: The observed increase in cell number resembles about 10 to 20 cell doublings. Although the doubling time appears to be longer during the second month, no definite limit of proliferative activity was seen during the time of study. Proliferating chondrocytes in monolayer lose their tissue-specific phenotype. For the de novo formation of cartilage transplants, redifferentiation of the expanded cells has to be stimulated. CONCLUSION: This study shows that human nasal chondrocytes can be expanded sufficiently in monolayer for the engineering of autologous cartilage transplants.

Adolescent↗
PubMed1997

[Transplantation of in vitro cultured cartilage materials: characterization of matrix synthesis].

BACKGROUND: Recently a three-dimensional model for the formation of cartilage in vitro was developed. The aim of this study was to investigate the amount and quality of newly synthesized matrix after graftig in vitro engineered cartilage into athymic nude mice. MATERIAL AND METHODS: Group I received transplants consisting of human chondrocytes, agarose, and E 200 (a bioabsorbable polymer fleece that offers mechanical stability. Ethicon Inc). Group II received chondrocytes and agarose only. At intervals of six, 12, and 24 weeks after subcutaneous transplantation we used azan blue staining and antibodies against collagen type I, collagen type II, and chondroitin-4sulfate to characterize the matrix synthesis. A quantitative analysis was performed using the computer image analyzing software photoshop (Adobe Inc). RESULTS: In group I, the amounts of newly synthesized cartilage specific collagen type II and chondroitin-4 sulfate increased progressively. Twenty-four weeks after transplantation, these amounts were comparable to the original human cartilage from which the chondrocytes were derived. Collagen type I was detected only in small quantities in the periphery of the transplants. Gross examination revealed sufficient mechanical stability and unremarkable changes in size and form. In contrast to this, group II transplants showed markedly smaller amounts of cartilage specific matrix components as collagen type II and chondroitin-4 sulfate and at the same time greater amounts of collagen type I. It was found both in the periphery and in central parts of the transplants. There was a remarkable loss of volume in all transplants and mechanical stability was poor. CONCLUSIONS: The absorbable cell carrier E 200 not only offers mechanical stability to in vitro engineered cartilage but also had a positive effect on the development of cartilage in our experiments. In conclusion, in vitro engineered cartilage is a promising pathway for the replacement of cartilage defects.

Adolescent↗
PubMed1997

Artificial tissues in perfusion culture.

In the stagnant environment of traditional culture dishes it is difficult to generate long term experiments or artificial tissues from human cells. For this reason a perfusion culture system with a stable supply of nutrients was developed. Human chondrocytes were seeded three-dimensionally in resorbable polymer fleeces. The cell-polymer tissues were then mounted in newly developed containers (W.W. Minuth et al, Biotechniques, 1996) and continuously perfused by fresh medium for 40 days. Samples from the effluate were analyzed daily, and the pH of the medium and glucose concentration remained stable during this period. The lactid acid concentration increased from 0.17 mg/ml to 0.35 mg/ml, which was influenced by the degradation of the resorbable polymer fibers used as three dimensional support material for the cells. This perfusion system proved to be reliable especially in long term cultures. Any components in the culture medium of the cells could be monitored without disturbances as caused by manual medium replacement. These results suggest the described perfusion culture system to be a valuable and convenient tool for many applications in tissue engineering, especially in the generation of artificial connective tissue.

Adult↗
PubMed1996

Resorbable polyesters in cartilage engineering: affinity and biocompatibility of polymer fiber structures to chondrocytes.

The resorbable polymers polyglycolic acid (PGA) and polylactic acid (PLA) are gaining increasing importance in tissue engineering and cell transplantation. The present investigation was focused on the biocompatibility and cell retaining behavior of PGA/poly-L-lactide (PLLA) (90/10) and PLLA nonwoven structures for the in vitro development of chondrocyte-polymer constructs. The effect of the relevant monomers to chondrocytes was analyzed. Type II collagen and poly-L-lysine were compared to improve loading of PGA/PLLA and PLLA polymer nonwovens with chondrocytes. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zoliumbrom ide (MTT) test was applied for quantification. At concentrations above 2 mg/mL, glycolic acid was more cytotoxic than lactic acid. As shown by pH equilibration, the cytotoxic effect is not due merely to the acidity of the alpha-hydroxy acids. Regarding the degradation products, glycolic acid, and L(+) lactic acid, nonwovens of PLLA are more biocompatible with chondrocytes than nonwovens of polyglycolide. Collagen type II and poly-L-lysine generally improved cell seeding on resorbable polymers in tissue engineering; however, their efficiency varies depending on the type of fiber structure.

Adult↗
PubMed1996

Monoclonal antibodies against human chondrocytes.

Cell-specific antigens are mainly found in cells or membrane surfaces rather than in the surrounding matrix. However, until now it was not possible to produce antibodies specific for cellular structures of chondrocytes. In 1989, Lance (Immunol. Lett. 21:63-73; 1989) first established specific monoclonal antibodies for human articular chondrocytes tested only by immunofluorescence. Studies describing the specificity of these five antibodies (HUMC 1-5) and their relevance for immunohistological analysis of cartilage tissue were not available until now. Therefore, the aim of the following study was to investigate the distribution of HUMC 1, 2, 3, 4, and 5 in mesenchymal cells in vivo and in vitro immunohistochemically. Further investigations concentrate on the localization of chondrocyte specific antigens using immunoelectron microscopy. Immunohistological studies showed positive immunostainings with all five antibodies in human chondrocytes in vivo and in vitro. A cross-reaction with human fibroblasts and osteoblasts for the antibodies HUMC 2 and HUMC 5 was observed. Furthermore, a parallel loss of immunoreactivity for HUMC 1, HUMC 3, and HUMC 4 was observed in cultured chondrocytes indicating that the specific antigens vanish during differentiation observed in vitro. Subsequent immunoblot analysis employing collagens as antigens did not show any reactivity. Using immunoelectron microscopy, gold particle labeling was observed in intracytoplasmatic vesicles of isolated chondrocytes. Our results indicate that HUMC 1, HUMC 3, and HUMC 4 are specific for cartilage cells and might be suitable for immunohistological analysis of different cartilage tissues and pathologically altered chondrocytes.

Antibodies, Monoclonal↗
PubMed1996

[Tissue engineering of human cartilage tissue for reconstructive surgery using biocompatible resorbable fibrin gel and polymer carriers].

Current practical approaches in cartilage engineering still face problems with three dimensional cell distribution or require components for cell immobilization, raising biocompatibility problems. In this study, we present a new model using cells cross-linked by fibrin within biocompatible resorbable polymers. Both components have been in clinical use for a long time. Immunohistochemical procedures showed that this model provides optimal requirements for in vitro cartilage production. Immunochemically, cartilage-specific extracellular components such as proteoglycan, chondroitin sulfate and collagen II were characterized. Histomorphological methods showed a mechanically stable tissue compound that lasted for at least 5 weeks. This model may be the first to provide all biocompatible requirements for in vitro production of autologous cartilage transplants for reconstructive surgery.

Biocompatible Materials↗
PubMed1996

Encapsulation of artificial tissues in polyelectrolyte complexes: preliminary studies.

The in vitro engineering of vital tissues from isolated cells requires primarily the synthesis of a new intercellular matrix. Structural components of the extracellular matrix are large molecules such as collagens and proteoglycans. To retain and accumulate new matrix molecules within three-dimensional cell cultures, chondrocyte-polymer constructs were encapsulated in polyelectrolyte complex membranes. Further, these membranes might also be relevant for other applications where cells or tissues have to be isolated from their environment by semipermeable structures.

Cells, Cultured↗
PubMed1996

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↗
PubMed1996

[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↗
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

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↗
PubMed1995

[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↗
PubMed1995

[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↗
PubMed1995

[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↗
PubMed1995

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↗