Peter Adamietz

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.

Isolation and partial characterization of the ADP-ribosylated nuclear proteins from Ehrlich ascites tumor cells

Abstract The (ADP-ribose)n protein conjugates formed by incubation of Ehrlich ascites tumor cell nuclei with 1 mM (3H)NAD were isolated by chromatography on boronate cellulose columns with a yield of >85%. Possible contamination by glycoproteins was excluded by rechromatography after specific release of the (ADP-ribose)n residues from their acceptors. Dodecyl sulfate gel electrophoresis revealed numerous protein bands which coincided with the (3H)ADP-ribose bands obtained by fluorography of the gels. 40% of the acceptor proteins were identified as the nucleosomal core histones. Most of these histones, however, appeared in the non-histone fraction because of extensive modification by poly(ADP-ribose). Drastic changes in properties were also seen in the true non-histone proteins which comprised 60% of the total conjugated protein. Besides several prominent acceptor proteins (Mr = 12,000; 31,000; 125,000) numerous proteins were detected indicating a considerable heterogeneity of non-histone acceptors.

Perfusion cultures and modelling of oxygen uptake with three-dimensional chondrocyte pellets

Chondrocyte pellets were cultivated in a perfused flow chamber and supplied with medium by a constant flow rate from a conditioning vessel. In this conditioning vessel the medium was aerated and used medium was exchanged semi-continuously. The higher amount of DNA and glycosaminoglycane (GAG) in these pellets compared to control cultures under stationary conditions showed a positive effect of the reactor system, compared to standard culture conditions. A diffusion reaction model was applied to calculate the oxygen uptake of the cell pellet and to describe the oxygen profile within the pellet. The model included diffusion within the cell pellet and oxygen uptake of the cells. Calculated data were compared to experimental data obtained by tissue engineered chondrocyte cell pellets. Model calculations agreed rather well with experimental data.

Present and Future Therapies of Articular Cartilage Defects

AbstractBackground: Until today, no universally successful therapy to treat substantial articular cartilage defects has been available. Numerous therapeutic approaches can only improve clinical symptoms of joint lesions, but cannot stimulate the regenerative and reactive capacity of the biological tissue in the defect, and, thus, cannot restore an articular surface capable of functional load bearing. Some other therapeutic options promised impressing results at the beginning, but did not withstand the process of a closer investigation. Even after laborious, invasive and expensive therapies, patients still complain about pain, joint effusions, restricted movement, or articular blockage. Established and Novel Therapies: The aim of all therapeutic procedures to treat patients with damaged articular cartilage is to reconstruct the integrity of the articular cartilage surface in order to enable them to live an unrestricted painless professional and private life. This article gives an overview of the clinically established procedures, their indications and the present long-term results, as well as a crucial look on the limitations of each approach. Novel therapies, which integrate molecular biology techniques and tissue engineering into transplantation surgery, are introduced and analyzed in terms of their capability and future potential.

Subcellular distribution of mono(ADP-ribose) protein conjugates in rat liver

Summary Rat liver nuclei isolated by neutral sucrose or citric acid procedures contain 2 OH-resistant mono(ADP-ribosyl) protein conjugates were associated with the mitochondrial fraction, and, to a small degree, with the plasma membranes. The NH 2 OH-sensitive conjugates were primarily found in the fractions containing the endoplasmic reticulum. This distribution is in accordance with multiple and independent functions of mono ADP-ribosylation and poly ADP-ribosylation reactions.

Rapid determination of chain length pattern in poly (ADP-ribose) samples.

(3H)poly(ADP-ribose) synthesized from nuclei by incubation with (3H)NAD was released from protein by alkaline treatment and electrophoresed in dodecyl sulfate gels. Individual polymers up to at least 33 units were completely separated according to their chain length. Size distribution was visualized by fluorography of the gels, and quantified by radioactivity determination of sliced gels The method could be applied to crude nuclear extracts. It showed that nuclei of Ehrlich ascites tumor cells produced a poly(ADP-ribose) pattern distinctly different from that of rat liver nuclei.

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.

Mono ADP-ribosylation and poly ADP-ribosylation of proteins in normal and malignant tissues.

Three subclasses of (ADPR)n protein conjugates were quantified from intact tissue; proteins carrying poly(ADPR) and two types of mono(ADPR) protein conjugates, one susceptible, the other resistant to neutral hydroxylamine. Mono(ADPR) conjugates were found in all major compartments of the liver cell although the two subfractions were unevenly distributed. Poly(ADPR) protein conjugates appear to be restricted to the nucleus. Independent changes of the subclasses in normal and malignant tissues associated with cell growth and differentiation also point to independent functions. Hydroxylamine-resistant mono(ADPR) protein conjugates of various tissues changed with the degree of terminal differentiation. Formation of poly(ADPR) proteins, on the other hand, was stimulated by treatment of cells with alkylating agents which lead to DNA-fragmentation. This points to an involvement of polyADP-ribosylation in DNA excision repair.

ADP-ribosylation of nuclear proteins.

Covalent modification of nuclear proteins by mono ADP-ribosylation and poly ADP-ribosylation was studied in various tissues and under various growth conditions with the aid of a newly developed radioimmunoassay. Two types of (ADPR)n protein conjugates were found in vitro and in intact tissues which could be differentiated by their sensitivity towards neutral NH2OH. Analysis of the cell cycle of Physarum polycephalum showed independent synthesis of these two fractions. Widely differing ratios of the two types of conjugates were also found in different hepatic tissues. Yoshida hepatoma cells had very low levels of both types of protein bound ADPR residues (1 ADPR residue per 2,000–3,000 DNA bases), while neonatal liver was characterized by the (near) absence of NH2OH resistant ADPR protein conjugates. Conjugates carrying poly(ADPR) residues exhibited independent variations. Proliferating tissues consistently had somewhat lower levels of mono(ADPR) protein conjugates than the same tissues in resting conditions. The independent changes of mono and poly(ADPR) protein conjugates under various conditions, and the existence of multiple acceptor proteins in the nucleus point to multiple functions of ADP ribosylation rather than to a single role of this covalent modification reaction. Analysis of (ADPR)n histone H1 conjugates isolated from (3H) adenosine labeled HeLa cultures by a new procedure indicated modification of < 1% of total histone H1 by ADP-ribosylation. Most of the conjugates carried single ADPR residues, while (ADPR)n histone H1 conjugates formed by incubation of isolated nuclei with NAD contained mainly poly(ADPR) residues. There were additional basic differences in the degree of ADP- and polyADP-ribosylation as well as in the types of bonds linking ADPR to the histone which seriously limit the conclusions drawn from experiments with isolated nuclei or chromatin. The low levels of ADPR conjugates in vivo, together with the high enzymic capacity for ADPR transfer observed in many cell types, point to a rapid turnover of protein bound ADPR residues being consistent with the occurrence of rapid transient modifications of protein in localized regions of chromatin.

Nuclear poly (ADPR) and mono (ADPR) residues in tissues with different growth rates

Poly(ADPR) is formed in eukaryotes by a nuclear enzyme with NAD as a substrate [l-3] . Its localization, its association with nucelar proteins [4], its influence on DNA polymerase activity [S-7] , the stimulation of poly(ADPR) formation in vitro by exogenous DNA [ 1,8] , and the inhibition of poly(ADPR) degradation in vitro by DNA [9,10] are consistent with the postulate that the polymer might be involved in the regulation of DNA synthesis and cell proliferation [