Reutlingen, Germany
Reutlingen, Germany
Time filter
Source Type

Bach F.C.,University Utrecht | Miranda-Bedate A.,University Utrecht | Van Heel F.W.M.,University Utrecht | Riemers F.M.,University Utrecht | And 7 more authors.
Tissue Engineering - Part A | Year: 2017

Chronic back pain is related to intervertebral disc (IVD) degeneration and dogs are employed as animal models to develop growth factor- and cell-based regenerative treatments. In this respect, the differential effects of transforming growth factor beta-1 (TGF-β1) and bone morphogenetic protein-2 (BMP2) on canine and human chondrocyte-like cells (CLCs) derived from the nucleus pulposus of degenerated IVDs were studied. Human and canine CLCs were cultured in 3D microaggregates in basal culture medium supplemented with/without TGF-β1 (10 ng/mL) or BMP2 (100 or 250 ng/mL). Both TGF-β1 and BMP2 increased proliferation and glycosaminoglycan (GAG) deposition of human and canine CLCs. TGF-β1 induced collagen type I deposition and fibrotic (re)differentiation, whereas BMP2 induced more collagen type II deposition. In dogs, TGF-β1 induced Smad1 and Smad2 signaling, whereas in humans, it only tended to induce Smad2 signaling. BMP2 supplementation increased Smad1 signaling in both species. This altogether indicates that Smad1 signaling was associated with collagen type II production, whereas Smad2 signaling was associated with fibrotic CLC (re)differentiation. As a step toward preclinical translation, treatment with BMP2 alone and combined with mesenchymal stromal cells (MSCs) was further investigated. Canine male CLCs were seeded in albumin-based hydrogels with/without female bone marrow-derived MSCs (50:50) in basal or 250 ng/mL BMP2-supplemented culture medium. Although the results indicate that a sufficient amount of MSCs survived the culture period, total GAG production was not increased and GAG production per cell was even decreased by the addition of MSCs, implying that MSCs did not exert additive regenerative effects on the CLCs. © Copyright 2017, Mary Ann Liebert, Inc.

Muller J.,University of Tübingen | Benz K.,University of Tübingen | Ahlers M.,GELITA AG | Gaissmaier C.,TETEC AG | And 2 more authors.
Cell Transplantation | Year: 2011

Cell-based approaches using mesenchymal stromal precursor cells (MSCs) for the regeneration of intervertebral discs are attracting increased interest, even though the intervertebral disc is a very demanding environment. Implanted cells eventually face acidic pH, hypoxia, and a lack of nutrients. While the regenerative potential of MSCs for skeletal tissues has been well described, it is still questionable whether human MSCs can be prepared for prolonged survival and proper functioning and whether they can differentiate under the adverse conditions encountered in the disc. Here we examined the influence of hypoxia during expansion and differentiation on the chondrogenesis of MSCs. Chondrogenic differentiation was performed in in situ solidifying gelatin hydrogels, which represent a suitable matrix for delivering and anchoring cells within the disc tissue. To consider limitations in nutrition in the intervertebral disc, differentiation was performed at low cell concentrations in the gelatin hydrogels. Standard high-density micromass cultures served as reference controls. To determine the quality of chondrogenesis we analyzed typical marker molecules such as collagen types I, II, X, Sox-9, MIA, and aggrecan mRNA using RT-qPCR and determined protein deposition by histological stainings and biochemical methods. We could demonstrate that in gelatin-based hydrogels chondrogenic differentiation of human MSCs is possible at low cell concentrations. The quality of chondrogenic differentiation could be improved by hypoxia. Best results were obtained when the entire in vitro process, including MSC expansion and subsequent differentiation, was done under hypoxic conditions. MSCs that were expanded under reduced oxygen tension were primed for a chondrogenic differentiation. © 2011 Cognizant Comm. Corp.

Benz K.,Reutlingen University | Stippich C.,Reutlingen University | Osswald C.,TETEC AG | Gaissmaier C.,TETEC AG | And 7 more authors.
BMC Musculoskeletal Disorders | Year: 2012

Background: Cell-based approaches towards restoration of prolapsed or degenerated intervertebral discs are hampered by a lack of measures for safe administration and placement of cell suspensions within a treated disc. In order to overcome these risks, a serum albumin-based hydrogel has been developed that polymerizes after injection and anchors the administered cell suspension within the tissue. Methods. A hydrogel composed of chemically activated albumin crosslinked by polyethylene glycol spacers was produced. The visco-elastic gel properties were determined by rheological measurement. Human intervertebral disc cells were cultured in vitro and in vivo in the hydrogel and their phenotype was tested by reverse-transcriptase polymerase chain reaction. Matrix production and deposition was monitored by immuno-histology and by biochemical analysis of collagen and glycosaminoglycan deposition. Species specific in situ hybridization was performed to discriminate between cells of human and murine origin in xenotransplants. Results: The reproducibility of the gel formation process could be demonstrated. The visco-elastic properties were not influenced by storage of gel components. In vitro and in vivo (subcutaneous implants in mice) evidence is presented for cellular differentiation and matrix deposition within the hydrogel for human intervertebral disc cells even for donor cells that have been expanded in primary monolayer culture, stored in liquid nitrogen and re-activated in secondary monolayer culture. Upon injection into the animals, gels formed spheres that lasted for the duration of the experiments (14 days). The expression of cartilage- and disc-specific mRNAs was maintained in hydrogels in vitro and in vivo, demonstrating the maintenance of a stable specific cellular phenotype, compared to monolayer cells. Significantly higher levels of hyaluronan synthase isozymes-2 and -3 mRNA suggest cell functionalities towards those needed for the support of the regeneration of the intervertebral disc. Moreover, mouse implanted hydrogels accumulated 5 times more glycosaminoglycans and 50 times more collagen than the in vitro cultured gels, the latter instead releasing equivalent quantities of glycosaminoglycans and collagen into the culture medium. Matrix deposition could be specified by immunohistology for collagen types I and II, and aggrecan and was found only in areas where predominantly cells of human origin were detected by species specific in situ hybridization. Conclusions: The data demonstrate that the hydrogels form stable implants capable to contain a specifically functional cell population within a physiological environment. © 2012 Benz et al; licensee BioMed Central Ltd.

Benz K.,Reutlingen University | Stippich C.,Reutlingen University | Fischer L.,Harlan Laboratories Ltd | Mohl K.,Harlan Laboratories Ltd | And 9 more authors.
European Spine Journal | Year: 2012

Purpose Regenerative repair is a promising new approach in treating damaged intervertebral discs. An experimental scheme was established for autologous and/or allogenic repair after massive disc injury. Methods Disc healing was promoted in 11 animals by injecting in vitro expanded autologous/homologous disc cells 2 weeks after stab injury of lumbar discs L1-2. The following control discs were used in our sheep injury model: L2-3, vehicle only; L3-4, injury only; L4-5, undamaged; and lumbar discs from four non-experimental animals. Disc cells were suspended in a biologically supportive albumin/hyaluronan two-component hydrogel solution that polymerizes when inserted in order to anchor cells at the injection site. The parameters studied were MRI, DNA, glycosaminoglycan, collagen content, histology, immunohistology for collagens type I, II and aggrecan, and mRNA expression of GAPDH, β-actin, collagen type I, II, X, aggrecan, lubricin, and IL-1β. Results All parameters demonstrated almost complete healing of the injured discs after 6 months, when compared with data from both the endogenous non-injured controls as well as from the healthy animals. Conclusion Sheep experience spontaneous recovery from disc injury. The process of endogenous repair can be enhanced by means of hydrogel-supported cells. © Springer-Verlag 2012.

Bach F.C.,University Utrecht | de Vries S.A.H.,TU Eindhoven | Riemers F.M.,University Utrecht | Boere J.,University Utrecht | And 11 more authors.
European Cells and Materials | Year: 2016

During intervertebral disc (IVD) maturation, notochordal cells (NCs) are replaced by chondrocyte-like cells (CLCs) in the nucleus pulposus, suggesting that NCs play a role in maintaining tissue health. Affirmatively, NC-conditioned medium (NCCM) exerts regenerative effects on CLC proliferation and extracellular matrix (ECM) production. The aim of this study was to identify NC-secreted substances that stimulate IVD regeneration. By mass spectrometry of porcine, canine and human NCCM, 149, 170 and 217 proteins were identified, respectively, with 66 proteins in common. Mainly ECM-related proteins were identified, but also organelle-derived and membrane-bound vesicle proteins. To determine whether the effect of NCCM was mediated by soluble and/or pelletable factors, porcine and canine NCCM were separated into a soluble (NCCM-S; peptides and proteins) and pelletable (NCCM-P; protein aggregates and extracellular vesicles) fraction by ultracentrifugation, and tested on bovine and canine CLCs in vitro, respectively. In each model, NCCM-S exerted a more pronounced anabolic effect than NCCM-P. However, glycosaminoglycan (GAG) uptake from the medium into the carrier gel prevented more definite conclusions. While the effect of porcine NCCM-P on bovine CLCs was negligible, canine NCCM-P appeared to enhance GAG and collagen type II deposition by canine CLCs. In conclusion, porcine and canine NCCM exerted their anabolic effects mainly through soluble factors, but also the pelletable NCCM factors showed moderate regenerative potential.Although the regenerative potential of NCCM-P should not be overlooked, future studies should focus on unraveling the protein-based regenerative mechanism from NCCM produced from isolated NCs, e.g. by NCCM fractionation and pathway blocking studies. © 2016, AO Research Institute. All rights reserved.

Stumm M.,BG Berlin Genetics GmbH | Boger E.,TETEC AG | Gaissmaier C.G.,TETEC AG | Osswald C.,TETEC AG | And 5 more authors.
Osteoarthritis and Cartilage | Year: 2012

Objective: In vitro expansion is an important step to acquire sufficient cells in human tissue engineering technologies. The high number of chondrocytes needed for human articular cartilage implants requires in vitro expansion of the primary cells, bearing a theoretical risk of in vitro induced changes in the genomes. To gain more insights into this situation, model cultures were prepared and analyzed. Design: 25 chondrocyte cell DNA samples from nine donors were analyzed by array comparative genomic hybridization (aCGH) on whole genome level and 28 chondrocyte cell samples from 16 individuals were analyzed by fluorescence in situ hybridization (FISH) on single cell level. The expanded cells were further characterized upon the chondrocytic mRNA phenotype by reverse-transciptase polymerase chain reaction (RT-PCR). Results: The molecular karyotyping results revealed autosomal stability, but all male samples analyzed by aCGH displayed a variable loss of the Y-chromosome. These data were confirmed by FISH-experiments and suggest an age dependant effect toward the loss of the Y-chromosome in cultured chondrocytes. RT-PCR data for the mRNAs from collagen types I, II, and aggrecan and the pro-inflammatory cytokine interleukin-1ß (IL-1ß) did not reveal any correlation of transcriptional activity in cultures with Y-chromosome losses, nor were there statistically significant differences between cells from female and male donors. Conclusions: While cells of male origin may suffer from an age-related loss of the Y-chromosome, there was no indication of a functional impairment. The data suggest some caution toward applying proliferative steps when considering chondrocytes from elderly male patients for tissue engineering approaches. © 2012 Osteoarthritis Research Society International.

Benz K.,Reutlingen University | Stippich C.,Reutlingen University | Freudigmann C.,Reutlingen University | Mollenhauer J.A.,Reutlingen University | And 3 more authors.
Journal of Translational Medicine | Year: 2013

Background: The discovery of mesenchymal stem cells (MSCs) or MSC-like cells in cartilage tissue does not tie in well with the established view that MSCs derive from a perivascular niche. The presence of MSCs may raise concerns about specificity and application safety, particularly in terms of the regulatory site. The aim of the present study was to investigate the benefits or possible risks of the MSC-like properties of cells isolated from cartilage in the context of autologous chondrocyte implantation.Methods: Chondrocytic cells were isolated from cartilage or intervertebral disc tissue. Flow cytometry was used to analyze the expression of cell surface antigens. MSC-like cells were either enriched or depleted by means of magnetic cell sorting (MACS) involving the monoclonal antibodies W5C5/SUSD2 and W8B2/MSCA-1. We addressed the issues of prolonged expansion of such cells as well as the influence of culture medium as a trigger for selecting a single cell type. Established protocols were used to study in vitro differentiation. In addition to histological and biochemical assessment, the acquired phenotypes were also evaluated on the mRNA transcript level.Results: In the studied cells, we found strongly analogous expression of antigens typically expressed on MSCs, including CD49e, CD73, CD90, CD105, CD140b and CD166. The expression of W5C5 and W8B2 antigens in cartilage cell sub-populations did not correlate with multi-potency. We demonstrated that a chondroid precursor, but not a bona fide multipotent mesenchymal, cell type can be obtained under established in vitro culture conditions. The culture media used for expansion influenced the cell phenotype.Conclusions: The risk of adverse adipose or osseous differentiation is not posed by expanded chondrocyte cultures, even after enrichment of putative MSC-like cell populations by MACS. It is possible that this limited " stemness" in chondrocytes, expanded for use in ACI, may instead be beneficial as it allows re-differentiation under appropriate conditions despite prolonged times in culture. © 2013 Benz et al.; licensee BioMed Central Ltd.

Loading TETEC AG collaborators
Loading TETEC AG collaborators