Toward biomimetic materials in bone regeneration: functional behavior of mesenchymal stem cells on a broad spectrum of extracellular matrix components.

Bone defect treatments can be augmented by mesenchymal stem cell (MSC) based therapies. MSC interaction with the extracellular matrix (ECM) of the surrounding tissue regulates their functional behavior. Understanding of these specific regulatory mechanisms is essential for the therapeutic stimulation of MSC in vivo. However, these interactions are presently only partially understood. This study examined in parallel, for the first time, the effects on the functional behavior of MSCs of 13 ECM components from bone, cartilage and hematoma compared to a control protein, and hence draws conclusions for rational biomaterial design. ECM components specifically modulated MSC adhesion, migration, proliferation, and osteogenic differentiation, for example, fibronectin facilitated migration, adhesion, and proliferation, but not osteogenic differentiation, whereas fibrinogen enhanced adhesion and proliferation, but not migration. Subsequently, the integrin expression pattern of MSCs was determined and related to the cell behavior on specific ECM components. Finally, on this basis, peptide sequences are reported for the potential stimulation of MSC functions. Based on the results of this study, ECM component coatings could be designed to specifically guide cell functions.

Development of a high-throughput screening assay based on the 3-dimensional pannus model for rheumatoid arthritis.

The 3-dimensional (3-D) pannus model for rheumatoid arthritis (RA) is based on the interactive co-culture of cartilage and synovial fibroblasts (SFs). Besides the investigation of the pathogenesis of RA, it can be used to analyze the active profiles of antirheumatic pharmaceuticals and other bioactive substances under in vitro conditions. For a potential application in the industrial drug-screening process as a transitional step between 2-dimensional (2-D) cell-based assays and in vivo animal studies, the pannus model was developed into an in vitro high-throughput screening (HTS) assay. Using the CyBitrade mark-Disk workstation for parallel liquid handling, the main cell culture steps of cell seeding and cultivation were automated. Chondrocytes were isolated from articular cartilage and seeded directly into 96-well microplates in high-density pellets to ensure formation of cartilage-specific extracellular matrix (ECM). Cell seeding was performed automatically and manually to compare both processes regarding accuracy, reproducibility, consistency, and handling time. For automated cultivation of the chondrocyte pellet cultures, a sequential program was developed using the CyBio Control software to minimize shear forces and handling time. After 14 days of cultivation, the pannus model was completed by coating the cartilage pellets with a layer of human SFs. The effects due to automation in comparison to manual handling were analyzed by optical analysis of the pellets, histological and immunohistochemical staining, and real-time PCR. Automation of this in vitro model was successfully achieved and resulted in an improved quality of the generated pannus cultures by enhancing the formation of cartilage-specific ECM. In addition, automated cell seeding and media exchange increased the efficiency due to a reduction of labor intensity and handling time.

Emulsion-based synthesis of PLGA-microspheres for the in vitro expansion of porcine chondrocytes.

The in vitro cell expansion of autologous chondrocytes is of high interest in regenerative medicine since these cells can be used to treat joint cartilage defects. In order to preserve chondrocyte phenotype, while optimizing adhesion on microspheres, several processing parameters for the microsphere synthesis were varied. In this study three different polylactide-co-glycolides were used with differing lactide-glycolide ratios (85:15 and 50:50) and differing inherent viscosities. An emulsion route was established, where the polymer was dissolved in chloroform and then injected into a stirred polyvinyl alcohol-water solution at different polymer concentrations and different stirring velocities to produce microspheres with varying diameters. The sphere size distribution and morphology was analyzed using image processing software on SEM pictures. Based on previous experiments with commercial microspheres, three optimum samples were selected for further investigations. The degradation of the microspheres was determined in a long-term experiment in culture medium for 3 months. Adherent cells were characterized after 3 and 5 days by FDA+EB vital staining and in SEM.

A microcarrier-based cultivation system for expansion of primary mesenchymal stem cells.

Microcarrier cultures have been shown to allow extensive cell expansion of tissue engineering relevant cells, such as chondrocytes, while maintaining their phenotype. Our aim was to investigate the in vitro three-dimensional expansion of porcine bone-marrow-derived primary mesenchymal stem cells (MSC) using commercially available Cytodex type 1, type 2, and type 3 microcarriers. In comparison, the Cytodex type 1 microcarriers showed the best results for adherence with over 80% adherent cells after 3 h of incubation, analyzed by the Poisson distribution. Different start cell densities ranging from 1 to 3 x 106 cells per 100 cm2 had only a minor influence on adhesion. The proliferation was examined on Cytodex type 1 microcarriers over a cultivation time of 28 days, which could reveal cell growth and proof of cells recolonizing freshly added microcarriers. Scanning electron microscopy displayed appropriate cell morphology and confirmed cell proliferation. After enzymatic harvest from microcarriers, the osteogenic and chondrogenic differentiation of these cells was induced and shown by relevant histochemistry, such as von Kossa and Alcian blue staining. Totaling the results, we have shown that the three-dimensional expansion of MSC on microcarriers represents a beneficial alternative to the conventional two-dimensional monolayer cultivation method.

Gene expression profiling of human articular cartilage grafts generated by tissue engineering.

Cartilage tissue engineering is applied clinically to cover and regenerate articular cartilage defects. In this study autologous human cartilage tissue engineering grafts based on bioresorbable polyglactin/polydioxanone scaffolds were analyzed on the broad molecular level. RNA from freshly isolated, primary and expanded adult articular chondrocytes and from three-dimensional cartilage grafts were used for gene expression profiling using oligonucleotide microarrays. The capacity of cartilage grafts to form cartilage matrix was evaluated after subcutaneous transplantation into nude mice. Gene expression profiling showed reproducibly the regulation of 905 genes and documented that chondrocytes undergo fundamental changes during cartilage tissue engineering regarding chondrocyte metabolism, growth, and differentiation. Three-dimensional assembly of expanded, dedifferentiated chondrocytes initiated the re-differentiation of cells that was accompanied by the reversal of the expression profile of multiple players of the transforming growth factor (TGF) signaling pathway including growth and differentiation factor-5 and inhibitor of differentiation-1 as well as by the induction of typical cartilage-related matrix genes such as type II collagen and cartilage oligomeric matrix protein. Cartilage grafts formed a cartilaginous matrix after transplantation into nude mice. Three-dimensional tissue culture of expanded articular chondrocytes initiates chondrocyte re-differentiation in vitro and leads to the maturation of cartilage grafts towards hyaline cartilage in vivo.