Christiane Goepfert

RGD-peptides for tissue engineering of articular cartilage

One keypoint in the development of a biohybrid implant for articular cartilage defects is the specific binding of cartilage cells to a supporting structure. Mimicking the physiological adhesion process of chondrocytes to the extracellular matrix is expected to improve cell adhesion of in vitro cultured chondrocytes. Our approach involves coating of synthetic scaffolds with tailor-made, cyclic RGD-peptides, which bind to specific integrin receptors on the cell surface. In this study we investigated the expression pattern of integrins on the cell surface of chondrocytes and their capability to specifically bind to RGD-peptide coated materials in the course of monolayer cultivation. Human chondrocytes expressed integrins during a cultivation period of 20 weeks. Receptors proved to be functionally active as human and pig chondrocytes attached to RGD-coated surfaces. A competition assay with soluble RGD-peptide revealed binding specificity to the RGD-entity. Chondrocyte morphology changed with increasing amounts of cyclic RGD-peptides on the surface.

Redifferentiation of chondrocytes and cartilage formation under intermittent hydrostatic pressure

Since articular cartilage is subjected to varying loads in vivo and undergoes cyclic hydrostatic pressure during periods of loading, it is hypothesized that mimicking these in vivo conditions can enhance synthesis of important matrix components during cultivation in vitro. Thus, the influence of intermittent loading during redifferentiation of chondrocytes in alginate beads, and during cartilage formation was investigated. A statistically significant increased synthesis of glycosaminoglycan and collagen type II during redifferentiation of chondrocytes embedded in alginate beads, as well as an increase in glycosaminoglycan content of tissue-engineered cartilage, was found compared to control without load. Immunohistological staining indicated qualitatively a high expression of collagen type II for both cases.

Cartilage engineering from mesenchymal stem cells.

Mesenchymal progenitor cells known as multipotent mesenchymal stromal cells or mesenchymal stem cells (MSC) have been isolated from various tissues. Since they are able to differentiate along the mesenchymal lineages of cartilage and bone, they are regarded as promising sources for the treatment of skeletal defects. Tissue regeneration in the adult organism and in vitro engineering of tissues is hypothesized to follow the principles of embryogenesis. The embryonic development of the skeleton has been studied extensively with respect to the regulatory mechanisms governing morphogenesis, differentiation, and tissue formation. Various concepts have been designed for engineering tissues in vitro based on these developmental principles, most of them involving regulatory molecules such as growth factors or cytokines known to be the key regulators in developmental processes. Growth factors most commonly used for in vitro cultivation of cartilage tissue belong to the fibroblast growth factor (FGF) family, the transforming growth factor-beta (TGF-β) super-family, and the insulin-like growth factor (IGF) family. In this chapter, in vivo actions of members of these growth factors described in the literature are compared with in vitro concepts of cartilage engineering making use of these growth factors.

Relationship between physical, biochemical and biomechanical properties of tissue-engineered cartilage-carrier-constructs.

Three-dimensional cartilage-carrier-constructs were produced according to a standard protocol from chondrocytes of an adult mini-pig. Physical parameters (height and weight) correlated very well with total DNA content (r2=0.86, re. 0.94). The relation between DNA content and glycosaminoglycan content was less but still significant. No significant relationship was found between the elasticity module and the DNA content, even if the elasticity module increased slightly at higher DNA content. With respect to later implantation, selection of a construct for implantation based on the weight, which can be determined non-invasive and under sterile conditions, seems to be justifiable.

Evaluation of cartilage specific matrix synthesis of human articular chondrocytes after extended propagation on microcarriers by image analysis.

BACKGROUND Cell-based technologies for the repair of cartilage defects usually rely on the expansion of low numbers of chondrocytes isolated from biopsies of healthy cartilage. Proliferating chondrocytes are known to undergo dedifferentiation characterized by downregulation of collagen type II and proteoglycan production, and by upregulation of collagen type I synthesis. Re-expression of cartilage specific matrix components by expanded chondrocytes is therefore critical for successful cartilage repair. METHODS Human articular chondrocytes were expanded on microcarriers Cytodex 3. The growth area was increased by adding empty microcarriers. Added microcarriers were colonized by bead-to-bead transfer of the cells. The chondrocytes were harvested from the microcarriers and characterized by their ability to synthesize collagen type II when cultivated in alginate beads using chondrogenic growth factors. A semi-automatic image analysis technique was developed to determine the fractions of collagen type II and type I positive cells. RESULTS The expansion of human articular chondrocytes on microcarriers yielded high cell numbers and propagation rates compared to chondrocytes expanded in flask culture for one passage. The proportion of collagen type II positive cells compared to collagen type I synthesizing cells was increased compared to chondrocytes expanded using conventional methods. The matrix synthesis upon treatment with chondrogenic factors IGF-I and BMP-7 was enhanced whereas TGF-ss had an inhibitory effect on microcarrier expanded chondrocytes. CONCLUSIONS Expanding human articular chondrocytes on microcarriers omitting subcultivation steps leads to superior ratios of collagen type II to type I forming cells compared to the expansion in conventional monolayer culture.

Identification of molecular markers for articular cartilage.

OBJECTIVE The aim of the current study was to identify molecular markers for articular cartilage (AC) that can be used as tools for the quality control of tissue engineered (TE) cartilage. DESIGN A genome-wide expression analysis was performed using RNA isolated from articular and growth plate (GP) cartilage, both extracted from the knee joints of 6 weeks old minipigs. After confirming the specific expression for selected genes by RT-PCR, these were used as molecular markers for the quality control of TE cartilage. RESULTS Albeit several known chondrocyte markers were expressed to a similar extent in articular and GP cartilage, our genome-wide expression analysis led us to identify genes being selectively expressed in either GP or articular chondrocytes. These findings led us to perform a RT-PCR expression analysis for the corresponding genes to demonstrate the absence of GP-specific markers in TE cartilage, while common or AC markers were expressed. CONCLUSIONS Taken together, these results provide important novel insights into chondrocyte biology in general and AC in particular. In addition, it is reasonable to speculate, that some of the identified genes play distinct roles in the regulation of articular chondrocyte differentiation and/or function, thereby raising the possibility that they may serve as targets for non-operative therapies of osteoarthritis (OA).

Improving In Vitro Generated Cartilage-Carrier-Constructs by Optimizing Growth Factor Combination

The presented study is focused on the generation of osteochondral implants for cartilage repair, which consist of bone substitutes covered with in vitro engineered cartilage. Re-differentiation of expanded porcine cells was performed in alginate gel followed by cartilage formation in high-density cell cultures. In this work, different combinations of growth factors for the stimulation of re-differentiation and cartilage formation have been tested to improve the quality of osteochondral implants. It has been demonstrated that supplementation of the medium with growth factors has significant effects on the properties of the matrix. The addition of the growth factors IGF-I (100 ng/mL) and TGF-β1 (10 ng/mL) during the alginate culture and the absence of any growth factors during the high-density cell culture led to significantly higher GAG to DNA ratios and Young’s Moduli of the constructs compared to other combinations. The histological sections showed homogenous tissue and intensive staining for collagen type II.

Technical Strategies to Improve Tissue Engineering of Cartilage-Carrier-Constructs

Technical aspects play an important role in tissue engineering. Especially an improved design of bioreactors is crucial for cultivation of artificial three-dimensional tissues in vitro. Here formation of cartilage-carrier-constructs is used to demonstrate that the quality of the tissue can be significantly improved by using optimized culture conditions (oxygen concentration, growth factor combination) as well as special bioreactor techniques to induce fluid-dynamic, hydrostatic or mechanical load during generation of cartilage.

In Vitro Generation of Cartilage-Carrier-Constructs on Hydroxylapatite Ceramics with Different Surface Structures

Tissue engineering approaches for healing cartilage defects are partly limited by the inability to fix cartilage to bone during implantation. To overcome this problem, cartilage can be - already in vitro - generated on a ceramic carrier which serves as bone substitute. In this study, the influence of a hydroxylapatite carrier and its surface structure on the quality of tissue engineered cartilage was investigated. Application of the carrier reduced significantly biomechanical and biochemical properties of the generated tissue. In addition, slight changes in the quality of the formed matrix, in the adhesive strength between cartilage and biomaterial and in attachment and proliferation of a chondrocyte monolayer could be observed for commercial grade carriers, with respect to modified topographies obtained by smooth grinding/polishing. These first results demonstrated an influence of the carrier and its surface structure, but further research is needed for explaining the described effects and for optimization of cartilage-carrier-constructs.

3D-Bioreactor culture of human hepatoma cell line HepG2 as a promising tool for in vitro substance testing.

Future developments in pharmaceutical research and regulatory requirements such as the European REACH program require high numbers of animal experiments. As a result of ethical concerns, cell culture tests with human cell lines or primary cells are considered as an alternative. However, current testing protocols using 2D cell cultures in Petri dishes are not equivalent to animal trials. 3D tissue cultures may overcome fundamental obstacles in the development of new therapeutic agents. Many new candidates of therapeutic agents are intended as agonists or antagonists of specific receptors on human cells. For these substances, organ-like test systems based on human cells are mandatory. In some cases, new pharmaceuticals lead to unexpected adverse reactions even after successful animal trials. It is assumed that 3D test systems based on human cells might help to overcome these problems.