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Biomedical subjects

M Sittinger

Publications and source records attributed to M Sittinger.

At least 19 recordsLinked to original sources
PubMed2006

Functional characterization of cell hybrids generated by induced fusion of primary porcine mesenchymal stem cells with an immortal murine cell line.

Bone marrow mesenchymal stem cells (MSC) integrate into various organs and contribute to the regeneration of diverse tissues. However, the mechanistic basis of the plasticity of MSC is not fully understood. The change of cell fate has been suggested to occur through cell fusion. We have generated hybrid cell lines by polyethylene-glycol-mediated cell fusion of primary porcine MSC with the immortal murine fibroblast cell line F7, a derivative of the GM05267 cell line. The hybrid cell lines display fibroblastic morphology and proliferate like immortal cells. They contain tetraploid to hexaploid porcine chromosomes accompanied by hypo-diploid murine chromosomes. Interestingly, many hybrid cell lines also express high levels of tissue-nonspecific alkaline phosphatase, which is considered to be a marker of undifferentiated embryonic stem cells. All tested hybrid cell lines retain osteogenic differentiation, a few of them also retain adipogenic potential, but none retain chondrogenic differentiation. Conditioned media from hybrid cells enhance the proliferation of both early-passage and late-passage porcine MSC, indicating that the hybrid cells secrete diffusible growth stimulatory factors. Murine F7 cells thus have the unique property of generating immortal cell hybrids containing unusually high numbers of chromosomes derived from normal cells. These hybrid cells can be employed in various studies to improve our understanding of regenerative biology. This is the first report, to our knowledge, describing the generation of experimentally induced cell hybrids by using normal primary MSC.

Adipogenesis↗
PubMed2004

[Molecular characterization of tissue-engineered articular chondrocyte transplants based on resorbable polymer fleece].

Three-dimensional arrangement and subsequent transplantation of chondrocytic cells in resorbable polymers has been shown to be a promising technique for the treatment of cartilaginous defects. Engineering of artificial cartilage tissue includes dedifferentiation of chondrocytes in monolayer culture, the use of biodegradable matrices and polymer scaffolds, and re-expression of chondrocytic marker genes in three-dimensional culture. The aim of this study was to characterize molecularly the phenotypic changes occurring with autologous cartilage tissue engineering. Human articular chondrocytes were isolated, cultured in medium containing human serum, and expanded up to passage 3. Chondrocytes were embedded in human fibrinogen and in polyglactin-polydioxanon fleeces and cultured three-dimensionally up to 4 weeks. Dedifferentiation of chondrocytes in monolayers and formation of cartilage tissue in vitro or after subcutaneous transplantation into nude mice was assessed by gene expression analysis of typical chondrocytic genes, histology, and immunohistochemistry. The expansion of chondrocytes with human serum resulted in the induction of type I and type III collagens, whereas cartilage-specific type II collagen, cartilage oligomeric matrix protein, cartilage link protein, and aggrecan were repressed and induced again after three-dimensional arrangement of chondrocytes in polyglactin-polydioxanon. Transplantation experiments documented the synthesis of proteoglycan and cartilage-specific type II collagen in vivo. Three-dimensional arrangement of human articular chondrocytes in resorbable polyglactin-polydioxanon fleeces supports chondrogenic differentiation and the formation of a hyaline-like cartilaginous matrix in vitro and in vivo.

Aged↗
PubMed2004

Hyaluronic acid and autologous synovial fluid induce chondrogenic differentiation of equine mesenchymal stem cells: a preliminary study.

Mesenchymal stem cells (MSC) have the potential to differentiate into distinct mesenchymal tissues including cartilage, which suggest these cells as an attractive cell source for cartilage tissue engineering approaches. Our objective was to study the effects of TGF-beta1, hyaluronic acid and synovial fluid on chondrogenic differentiation of equine MSC. For that, bone marrow was aspirated from the tibia of one 18-month-old horse (Haflinger) and MSC were isolated using percoll-density centrifugation. To promote chondrogenesis, MSC were centrifuged to form a micromass and were cultured in a medium containing 10 ng/ml TGF-beta1 or 0.1mg/ml hyaluronic acid (Hylartil, Ostenil) or either 5%, 10% or 50% autologous synovial fluid as the chondrogenesis inducing factor. Differentiation along the chondrogenic lineage was documented by type II collagen and proteoglycan expression. MSC induced by TGF-beta1 alone showed the highest proteoglycan expression. Combining TGF-beta1 with hyaluronic acid could not increase the proteoglycan expression. Cultures stimulated by autologous synovial fluid (independent of concentration) and hyaluronic acid demonstrated a pronounced, but lower proteoglycan expression than cultures stimulated by TGF-beta1. The expression of cartilage-specific type II collagen was high and about the same in all stimulated cultures. In summary, hyaluronic acid and autologous synovial fluid induces chondrogenesis of equine mesenchymal stem cells, which encourage tissue engineering applications of MSC in chondral defects, as the natural environment in the joint is favorable for chondrogenic differentiation.

Animals↗
PubMed2003

[Tissue engineering: chances and challenges for application in rheumatic diseases].

Current technologies of tissue engineering offer new strategies for the treatment of cartilage and bone defects. Beyond implantation of cell suspensions, second generation products of biomaterial enforced with in vitro preformed tissues are clinically applied. Ongoing research and development focus on differentiation factors and tissue protection. In search for sources of autologous cells which are easier to collect and which may serve for more complex tissues like osteochondral implants, mesenchymal stem cells are investigated. The design of in vitro experiments, which are required for these investigations, has produced tissue engineering technologies, which may serve for pathophysiology research in inflammatory joint diseases and for exploration of treatment strategies. These together with the advances in biological therapies of rheumatic diseases are the basis of new concepts, which promise application of tissue engineering also in inflammatory joint diseases.

Animals↗
PubMed2003

Osteogenic induction of human bone marrow-derived mesenchymal progenitor cells in novel synthetic polymer-hydrogel matrices.

The aim of this project was to investigate the in vitro osteogenic potential of human mesenchymal progenitor cells in novel matrix architectures built by means of a three-dimensional bioresorbable synthetic framework in combination with a hydrogel. Human mesenchymal progenitor cells (hMPCs) were isolated from a human bone marrow aspirate by gradient centrifugation. Before in vitro engineering of scaffold-hMPC constructs, the adipogenic and osteogenic differentiation potential was demonstrated by staining of neutral lipids and induction of bone-specific proteins, respectively. After expansion in monolayer cultures, the cells were enzymatically detached and then seeded in combination with a hydrogel into polycaprolactone (PCL) and polycaprolactone-hydroxyapatite (PCL-HA) frameworks. This scaffold design concept is characterized by novel matrix architecture, good mechanical properties, and slow degradation kinetics of the framework and a biomimetic milieu for cell delivery and proliferation. To induce osteogenic differentiation, the specimens were cultured in an osteogenic cell culture medium and were maintained in vitro for 6 weeks. Cellular distribution and viability within three-dimensional hMPC bone grafts were documented by scanning electron microscopy, cell metabolism assays, and confocal laser microscopy. Secretion of the osteogenic marker molecules type I procollagen and osteocalcin was analyzed by semiquantitative immunocytochemistry assays. Alkaline phosphatase activity was visualized by p-nitrophenyl phosphate substrate reaction. During osteogenic stimulation, hMPCs proliferated toward and onto the PCL and PCL-HA scaffold surfaces and metabolic activity increased, reaching a plateau by day 15. The temporal pattern of bone-related marker molecules produced by in vitro tissue-engineered scaffold-cell constructs revealed that hMPCs differentiated better within the biomimetic matrix architecture along the osteogenic lineage.

Alkaline Phosphatase↗
PubMed2002

Human platelet supernatant promotes proliferation but not differentiation of articular chondrocytes.

The objective of the study was to evaluate the growth-promoting activity of human platelet supernatant on primary chondrocytes in comparison with fetal calf serum (FCS) supplemented cell culture medium. Furthermore, the differentiation potential of platelet supernatant was determined in three-dimensional artificial cartilage tissues of bovine articular chondrocytes. Proliferation of articular and nasal septal chondrocytes was assayed by incorporation of BrdU upon stimulation with ten different batches of human platelet supernatant. On bovine articular chondrocytes, all these batches were at least as growth-promoting as FCS. On nasal septal chondrocytes, nine out of ten batches revealed increased or equivalent mitogenic stimulation compared with medium supplemented with FCS. Three-dimensional culture and subsequent histological analysis of matrix formation were used to determine the differentiation properties of platelet supernatant on articular chondrocytes. Human platelet supernatant failed to induce the deposition of typical cartilage matrix components, whereas differentiation and matrix formation were apparent upon cultivation of articular chondrocytes with FCS. Proliferation assays demonstrated that human platelet supernatant stimulates growth of articular and nasal septal chondrocytes; however, platelet supernatant failed to stimulate articular chondrocytes to redifferentiate in three-dimensional chondrocyte cultures. Therefore platelet lysate may be suitable for chondrocyte expansion, but not for maturation of tissue-engineered cartilage.

Blood Platelets↗
PubMed2001

Biocompatible hydrogel supports the growth of respiratory epithelial cells: possibilities in tracheal tissue engineering.

Extensive tracheal defect reconstruction is a major challenge in plastic and reconstructive surgery. The lack of an epithelial lining on the luminal surfaces of tracheal prostheses is among the major causes of their failure. Chitosan-gelatin hydrogels were synthesized for the development of biocompatible, growth-supportive substrata for respiratory epithelial cells. We employed J774 macrophages to test the immunocompatibility of this gel. The hydrogel did not exert a cytotoxic effect on macrophages, as confirmed by tetrazolium reduction and neutral red uptake assay. Flow cytometric analysis of macrophages cultured on the hydrogel showed a comparable expression of activation markers CD11b/CD18, CD45, and CD14 to the control. Semiquantitative RT-PCR results showed an absence of upregulation of interleukin-6 (IL-6) and TNF-alpha in these macrophages with respect to the controls. Primary human respiratory epithelial cells cultured on the hydrogel showed proper attachment, normal morphology, and growth. A small proportion of cells on the hydrogel showed synchronously beating cilia. RT-PCR analysis showed that cells on the hydrogel expressed mucins 2 and 5 and cytokeratin 13, which are markers for secretory goblet and squamous cells, respectively. All these results demonstrate that the hydrogel supports the growth of a mixed population of differentiated epithelial cells. This hydrogel is suitable as a culture substratum for respiratory epithelial cells and could be used as a potential candidate for coating tracheal prostheses.

Biocompatible Materials↗
PubMed2001

Cryopreservation of artificial cartilage: viability and functional examination after thawing.

In biomedical research and in reconstructive surgery, preservation of intact tissue has been an unsolved problem. In this study, we investigated the viability of cryopreserved artificial cartilage and its synthetic activity of cartilage-specific matrix proteins after thawing for in vitro use. A polymer fleece cylinder (diameter = 3 mm; height = 3 mm) was loaded with a suspension of bovine chondrocytes (25 x 10(6)/ml) and encapsulated with fibrin glue. After a culture period of 1 week, the artificial cartilage units were frozen in a cryoprotection solution containing 10% basal medium (RPMI 1640), 10% DMSO and 80% FCS. The freezing procedure consisted of three steps: a 30-min period at +4 degrees C followed by a 24-hour storage at -80 degrees C. After that, the tissue units were transferred into liquid nitrogen (-196 degrees C) for final storage. Using histochemical staining techniques of cryogenic slices, we investigated the ability of cryopreserved artificial cartilage to produce its specific matrix after thawing. A modified MTT assay was used to determine the viability of frozen tissue units in comparison with unpreserved samples at different moments after thawing. Depending on the chondrocytes used for the formation of artificial cartilage, the viability of cryopreserved tissue varied between 65 and 85%. Both the intensity of alcian blue staining for proteoglycans and the azan staining for collagens increased proportionally with incubation time after thawing. These findings indicate that cryopreservation of small artificial cartilage units is possible with a minor loss of cell viability. Secondly, its synthetic activity of cartilage-specific matrix did not decline after the freezing process.

Animals↗
PubMed2001

Secretion of gelatinases and activation of gelatinase A (MMP-2) by human rheumatoid synovial fibroblasts.

In monolayer cultures human rheumatoid synovial fibroblasts (HRSF) secrete gelatinase A (MMP-2) and, unlike other human fibroblasts, to a minor extent also gelatinase B (MMP-9) as inactive proenzymes. In this regard HRSF resemble the fibrosarcoma cell line HT-1080. Unlike HT-1080, however, HRSF do not increase the secretion of MMP-9 in response to phorbol-12-myristate-13-acetate. This indicates that in HRSF the protein kinase C pathway for an enhanced MMP-9 secretion is inactive. None of the substances used in our study increased MMP-9 secretion, but some of them inhibited MMP-9 secretion. The secretion of MMP-2 could not be enhanced either, not even by dbcAMP, which has been reported to be effective in Sertoli and peritubular cells. Activation of MMP-2 in HRSF could be induced by treatment with concanavalin A (ConA) or cytochalasin D, as was shown for other cell types. This activation was not accompanied by a significant change in the amount of secreted TIMP-1 and TIMP-2. In contrast to reports on human skin fibroblasts, however, the activation of MMP-2 could not be induced in HRSF by treatment of the cells with monensin or sodium orthovanadate. Moreover, monensin was shown to act as an inhibitor of ConA- or cytochalasin D-mediated activation. Additionally, and in contrast to a report on a rat fibroblast cell line, MMP-2 activation is not mediated via the MAP kinase pathway in HRSF: PD 98059, a specific inhibitor of MAP kinase kinase, did not inhibit the activation of MMP-2. Similarly ineffective were PD 169316, an inhibitor for p38 MAP kinase, other inhibitors for protein kinases as lavendustin A, Gö 6983, wortmannin, rapamycin, as well as the protein tyrosine kinase inhibitors herbimycin A and genistein. Only staurosporin, a broad spectrum inhibitor of protein kinases, and the ionophores monensin and A 23187 effectively inhibited MMP-2 activation in HRSF. Our results demonstrate that MMP-2 can be activated by quite different pathways, and that different cells, even when belonging to the fibroblast family, do not necessarily use the same activating pathways.

Arthritis, Rheumatoid↗
PubMed2001

[Establishment of an in vitro model for rheumatoid arthritis as test system for therapeutical substances].

In our Tissue Engineering group a 3D in vitro model for rheumatoid Arthritis (in vitro pannus) was established with the aim to develop a standardized drug-screening test to analyze the effects of drugs and different biological substances. The advanced model consists of chondrocyte pellet cultures interacting with rheumatoid arthritis (RA) synovial cell cultures. To establish interactive 3D co-cultures defined rheumatoid arthritis synovial cell populations were centrifuged directly on chondrocyte pellet cultures. Histochemical stainings during time of co-culture revealed obvious invasion by RA synovial cell populations into the chondrocyte matrix. Gene expression analysis showed a downregulation of collagen type II expression in chondrocytes within 2 weeks after co-culture with RA synovial cells. Those interactive co-cultures allow the study of single cell populations as well as the cellular interactions in this system under in vitro conditions. Thus, the established co-culture model may be suitable for routine screening tests, which can be useful in supplementing animal experiments in basic research and drug testing.

Animal Testing Alternatives↗
PubMed2001

In vitro expression of cartilage-specific markers by chondrocytes on a biocompatible hydrogel: implications for engineering cartilage tissue.

Natural cartilage tissue has a limited self-regenerative capacity; thus, strategies to replenish the lost cartilage are desired in reconstructive and plastic surgery. Tissue-engineered cartilage using biodegradable polymeric scaffolds is one such approach gaining wide attention. We have earlier demonstrated the biocompatible nature and ability of chitosan-gelatin hydrogel to maintain differentiated populations of respiratory epithelial cells. The aim of the present study was to evaluate its suitability as a substratum for inducing chondrocyte growth and differentiation. Electron microscopic (SEM) analysis of freeze-dried hydrogels showed a highly porous morphology with interconnections as seen in cross section. Chondrocytes were observed to attach and exhibited a differentiated phenotype with proper cell-cell contact on three-dimensional freeze-dried hydrogels. When cultured on two-dimensional hydrogel films they showed higher growth rates (4-6%) compared with a polystyrene (TCPS) control until 6 days (p > 0.05), which slowed down after 10 days. Immunofluorescent microscopic studies revealed that chondrocytes on hydrogel films exhibited comparable expression of beta1 integrin (CD29) to TCPS controls, indicating the ability of the hydrogel substrate to maintain normal expression of beta1 integrin. RT-PCR analysis of chondrocytes grown on hydrogel films showed that chondrocytes express the mRNA for extracellular matrix proteins such as collagen type IIalpha1 (COL IIalpha1), COL III, COL IXalpha3. Expression of COL I was less prominent than COL II as indication of differentiation. Expression of COL X could not be detected, suggesting an absence of chondrocyte hypertrophy. Chondrocytes also showed weak mRNA expression of aggrecan, a cartilage-specific proteoglycan. All of these results point out the ability of the chitosan-gelatin hydrogel to induce the expression of mRNAs for cartilage-specific extracellular matrix proteins by nasal septal chondrocytes. This hydrogel needs to be further evaluated for its ability to support chondrocyte-specific marker expression to explore the possibility of forming a tissue resembling natural cartilage in vitro.

Aggrecans↗
PubMed2000

Emerging strategies of bone and joint repair.

The advances in biomedicine over the past decade have provided revolutionary insights into molecules that mediate cell proliferation and differentiation. Findings on the complex interplay of cells, growth factors, matrix molecules and cell adhesion molecules in the process of tissue patterning have vitalized the revolutionary approach of bioregenerative medicine and tissue engineering. Here we review the impact of recent work in this interdisciplinary field on the treatment of musculoskeletal disorders. This novel concept combines the transplantation of pluripotent stem cells, and the use of specifically tailored biomaterials, arrays of bioactive molecules and gene transfer technologies to direct the regeneration of pathologically altered musculoskeletal tissues.

Bone Diseases↗
PubMed2000

Matrix-mixed culture: new methodology for chondrocyte culture and preparation of cartilage transplants.

For cartilage engineering a variety of biomaterials were applied for 3-dimensional chondrocyte embedding and transplantation. In order to find a suitable carrier for the in vitro culture of chondrocytes and the subsequent preparation of cartilage transplants we investigated the feasibility of a combination of the well-established matrices fibrin and alginate. In this work human articular chondrocytes were embedded and cultured either in alginate, a mixture of alginate and fibrin, or in a fibrin gel after the extraction of the alginate component (porous fibrin gel) over a period of 30 days. Histomorphological analysis, electron microscopy, and immunohistochemistry were performed to evaluate the phenotypic changes of the chondrocytes, as well as the quality of the newly formed cartilaginous matrix. Our experiments showed that a mixture of 0.6% alginate with 4.5% fibrin promoted sufficient chondrocyte proliferation and differentiation, resulting in the formation of a specific cartilage matrix. Alginate served as a temporary supportive matrix component during in vitro culture and can be easily removed prior to transplantation. The presented tissue engineering method on the basis of a mixed alginate-fibrin carrier offers the opportunity to create stable cartilage transplants for reconstructive surgery.

Alginates↗
PubMed2000

Mechanical quality of tissue engineered cartilage: results after 6 and 12 weeks in vivo.

Traumatic events are a primary cause for local lesions of articular cartilage. If treated early, restoration of the initial joint geometry and integrity may be achieved. In large defects, sufficient material is not available to bridge the affected area. Heterologeous transplantation is not well accepted due to the risk of infection and immune response. Alternatives are cartilage-like structures, which may be cultured in vitro and transplanted into the defect site. Critical to the success of these new tissues are their mechanical properties. Goals of this study were to generate a hyaline-like cartilage structure, to evaluate its performance in vivo and to verify that its cellular and material properties meet those of native cartilage. Hyaline-like cartilage specimens were generated in vitro and implanted in the backs of nude mice. Specimens were explanted after 6 and 12 weeks, mechanically tested using an indentation test and histologically examined. In mechanical testing, stiffness and failure load significantly increased between weeks 6 and 12. At 12 weeks, mechanical properties of the hyaline-like cartilage were comparable to those of native nasal septal cartilage. Compared to native articular cartilage, the engineered tissue achieved up to 30-50% in strength and mechanical stiffness. In histological examination, specimens showed neocartilage formation. The mechanical testing procedure proved to be sufficiently sensitive to identify differences in properties between cartilage specimens of different origin and at different stages of healing. As an adjunct to histological analysis, mechanical testing may be a valuable tool for judging the utility of engineered cartilage prior to a broad clinical usage.

Adult↗
PubMed2000

Preparation of a pure autologous biodegradable fibrin matrix for tissue engineering.

Parallel to the growing role of tissue engineering, the need for cell embedding materials, which allow cells to stabilise in a three-dimensional distribution, has increased. Although several substances have been tested, fibrin is thus far the only one that permits the clinical application of cultured tissue. To date, autologous fibrinogen has usually been polymerised with bovine thrombin, which can cause severe immunological side effects. The objective of this study was to explore the practicability of obtaining autologous thrombin from a single patient in an adequate concentration and amount. Fibrinogen was cryoprecipitated from 200 ml of freshly-frozen plasma. Thrombin was isolated from the supernatant through ion-exchange chromatography. The thrombin was first bound to Sephadex A-50 and then eluated using 2 ml of a salt buffer (2.0 M NaCl in 0.015 M trisodiumcitrate, pH 7.0). The activity of the thrombin (51 NIH x ml(-1) to 414 NIH x ml(-1) reached levels comparable to those in commercially available fibrin glues (4-500 NIH x ml(-1)). The study has shown that it is possible to obtain a sufficient amount of autologous thrombin from a single donor to create a fibrin matrix of high efficiency without the risk of immunological and infectious side effects.

Absorbable Implants↗
PubMed2000

[Protection of autogenous cartilage transplants from resorption using membrane encapsulation].

In reconstruction of cartilage defects, autogenous transplantation is known as a reliable and experienced method. Although a clinical application has not been reported until now, tissue engineering permits in vitro production of autogenous cartilage transplants. Nevertheless, in both methods the cartilage is exposed to individually varying resorptive mechanisms. Among other methods for in vivo tissue protection, the encapsulation with a semipermeable polyelectrolytecomplex membrane could guarantee sufficient protection against resorptive influences. Human septal cartilage was encapsulated (group 1) with polyelectrolytecomplex membranes and subcutaneously implanted on the back of thymusaplastic nude mice. Cartilage implants without encapsulation (group 2) were used as control. Scanning electron microscopy and histochemical investigations were performed 1, 4, 8, 12 and 16 weeks after implantation. Group 1 showed no signs of resorption and chronic inflammation at all. In contrast, group 2 presented, correlating to the time of implanta-tion, increasing signs of cell death and fibrotic transformation, representing an increased activity of resorption. In conclusion, tissue encapsulation with a polyelectrolytecomplex membrane could ensure a sufficient protection of human cartilage transplants from resorptive influences. For the plastic-reconstructive surgeon the desired result becomes more calculable.

Animals↗
PubMed2000

[Chondrocyte transplantation in PGLA/polydioxanone fleece].

The transplantation of chondrogenic cells in a supportive carrier structure proved to be a promising alternative for the treatment of cartilage defects. In the study presented we focused on the transplantation of allogeneic chondrocytes in a biodegradable polymer scaffold (PGLA/Polydioxanon) in articular cartilage defects in a rabbit defect model. Isolated allogeneic chondrocytes embedded in a PGLA polymer scaffold were transplanted into osteochondrogenic defects of the patellar groove and compared with empty defects and transplants of polymer scaffolds without cells. The histological and histochemical analysis was performed after 4 and 12 weeks. The transplant integration and the architecture of the newly formed cartilage were evaluated with a semiquantitative score. After 4 weeks the development of a hyaline-like cartilage tissue of the cell-polymer-transplants was observed, after 12 weeks the defects were nearly completely filled with hyaline-like cartilage. The biodegradation of the polymer construct did not affect the histological structure of the transplant area. Defects of the groups with empty defect and polymer transplants without cells revealed no or insufficient healing indices. The study demonstrated that biodegradable polymers served as suitable carriers for the chondrocyte transplantation, which is due to the in-vitro establishment of a semi-solid cartilage transplant and the resulting effective transplant fixation into the defect. In-vivo the polymer cell transplants seem to provide a supportive microenvironment for the development of hyaline cartilage. The controlled release of morphogenic factors or bioactive molecules and the use of pluripotent mesenchymal progenitor cells opens new perspectives for the optimization of cartilage repair procedures.

Animals↗