Joachim Rychly

Calcium phosphate surfaces promote osteogenic differentiation of mesenchymal stem cells

Although studies in vivo revealed promising results in bone regeneration after implantation of scaffolds together with osteogenic progenitor cells, basic questions remain how material surfaces control the biology of mesenchymal stem cells (MSC). We used human MSC derived from bone marrow and studied the osteogenic differentiation on calcium phosphate surfaces. In osteogenic differentiation medium MSC differentiated to osteoblasts on hydroxyapatite and BONITmatrix®, a degradable xerogel composite, within 14 days. Cells revealed a higher alkaline phosphatase (ALP) activity and increased RNA expression of collagen I and osteocalcin using real‐time RTPCR compared with cells on tissue culture plastic. To test whether material surface characteristics alone are able to stimulate osteogenic differentiation, MSC were cultured on the materials in expansion medium without soluble additives for osteogenic differentiation. Indeed, cells on calcium phosphate without osteogenic differentiation additives developed to osteoblasts as shown by increased ALP activity and expression of osteogenic genes, which was not the case on tissue culture plastic. Because we reasoned that the stimulating effect on osteogenesis by calcium phosphate surfaces depends on an altered cell–extracellular matrix interaction we studied the dynamic behaviour of focal adhesions using cells transfected with GFP labelled vinculin. On BONITmatrix®, an increased mobility of focal adhesions was observed compared with cells on tissue culture plastic. In conclusion, calcium phosphate surfaces are able to drive MSC to osteoblasts in the absence of osteogenic differentiation supplements in the medium. An altered dynamic behaviour of focal adhesions on calcium phosphate surfaces might be involved in the molecular mechanisms which promote osteogenic differentiation.

Cell-extracellular matrix interaction and physico-chemical characteristics of titanium surfaces depend on the roughness of the material.

The interaction of cells with the extracellular matrix at the interface of an implant determines the biology of cells and tissues. We analysed components of cell adhesion and measured physico-chemical characteristics of structural modifications of titanium surfaces: polished, machined, glass particle-blasted, corundum-blasted, vacuum plasma-sprayed. Scanning electron microscopy and profilometry revealed a differentiated topography from smooth to rough surfaces, respectively. Osteoblastic MG-63 cells showed an increased spreading on surfaces with low roughness, although without a straight correlation with the surface topography. Integrin expression was increased on structured surfaces compared with polished material, and the organization of the actin cytoskeleton and fibronectin was impaired on extremely rough surfaces. Electrochemical methods, especially the electrochemical impedance spectroscopy (EIS) was used to evaluate physico-chemical characteristics, and the impedance curves revealed a dependence on the roughness of the material surfaces. Further analyses of the EIS results were performed using equivalent circuits which model the electrical flow through the interface. First indications for a correlation between parameters from the equivalent circuits with surface properties were obtained which promise a relevance for the biological response of the cells.

The mode of mechanical integrin stressing controls intracellular signaling in osteoblasts.

Following the idea that integrin receptors function as mechanotransducers, we applied defined physical forces to integrins in osteoblastic cells using a magnetic drag force device to show how cells sense different modes of physical forces. Application of mechanical stress to the β1‐integrin subunit revealed that cyclic forces of 1 Hz were more effective to stimulate the cellular calcium response than continuous load. Cyclic forces also induced an enhanced cytoskeletal anchorage of tyrosine‐phosphorylated proteins and increased activation of the focal adhesion kinase (FAK) and mitogen activated protein (MAP) kinase. These events were dependent on an intact cytoskeleton and the presence of intracellular calcium. Analyses of the intracellular spatial organization of the calcium responses revealed that calcium signals originate in a restricted region in the vicinity of the stressed receptors, which indicates that cells are able to sense locally applied stress on the cell surface via integrins. The calcium signals can spread throughout the cell including the nucleus, which shows that calcium also is a candidate to transmit mechanically induced information into different cellular compartments.

Structural alterations of adhesion mediating components in cells cultured on poly-β-hydroxy butyric acid

Polymers may serve as a biodegradable material in tissue engineering. To assess the biocompatibility of poly-beta-hydroxy butyric acid (PHB), we studied the structural organization of cellular molecules involved in adhesion using osteoblastic and epithelial cell lines. On PHB, both cell lines revealed a rounded cell shape due to reduced spreading. The filamentous organization of the actin cytoskeleton was impaired. In double immunofluorescence analyses we demostrated that the colocalization of the fibronectin fibrils with the actin filaments was lost in cultures on PHB. Similarly, collagen II distribution was altered, whereas the organization of collagen I was not obviously affected. Further evidence for impaired structural organization was obtained for the beta1-integrin receptor and vinculin which mediate the interaction of the cytoskeleton with the extracellular matrix. In confluent epithelial cells, the tight junction protein ZO-1 showed a larger lateral extension in the cell-cell contacts when cells were grown on PHB. Because structural organization of components which mediate cell-matrix and cell-cell adhesion controls cell physiology these parameters could be a sensitive indicator for the biocompatibility of implant materials.

Modulating the Actin Cytoskeleton Affects Mechanically Induced Signal Transduction and Differentiation in Mesenchymal Stem Cells

Mechanical interactions of mesenchymal stem cells (MSC) with the environment play a significant role in controlling the diverse biological functions of these cells. Mechanical forces are transduced by integrins to the actin cytoskeleton that functions as a scaffold to switch mechanical signals into biochemical pathways. To explore the significance of cytoskeletal mechanisms in human MSC we modulated the actin cytoskeleton using the depolymerising drugs cytochalasin D (CytD) and latrunculin A (LatA), as well as the stabilizing drug jasplakinolide (Jasp) and examined the activation of the signalling molecules ERK and AKT during mechanical loading. All three drugs provoked significant changes in cell morphology and organisation of the cytoskeleton. Application of mechanical forces to β1-integrin receptors using magnetic beads without deformation of the cell shape induced a phosphorylation of ERK and AKT. Of the two drugs that inhibited the cytoskeletal polymerization, LatA completely blocked the activation of ERK and AKT due to mechanical forces, whereas CytD inhibited the activation of AKT but not of ERK. Activation of both signalling molecules by integrin loading was not affected due to cell treatment with the cytoskeleton stabilizing drug Jasp. To correlate the effects of the drugs on mechanically induced activation of AKT and ERK with parameters of MSC differentiation, we studied ALP activity as a marker for osteogenic differentiation and examined the uptake of fat droplets as marker for adipogenic differentiation in the presence of the drugs. All three drugs inhibited ALP activity of MSC in osteogenic differentiation medium. Adipogenic differentiation was enhanced by CytD and Jasp, but not by LatA. The results indicate that modulation of the cytoskeleton using perturbing drugs can differentially modify both mechanically induced signal transduction and MSC differentiation. In addition to activation of the signalling molecules ERK and AKT, other cytoskeletal mechanisms are involved in MSC differentiation.

Increased cytosolic Ca2+ amplifies oxygen radical-induced alterations of the ultrastructure and the energy metabolism of isolated rat pancreatic acinar cells

Background: Oxygen radicals have been implicated as important mediators in the early pathogenesis of acute pancreatitis, but the mechanism by which they produce pancreatic tissue injury remains unclear. We have, therefore, investigated the effects of oxygen radicals on isolated rat pancreatic acinar cells as to the ultrastructure, cytosolic Ca2+ concentration and energy metabolism. Methods: Acinar cells were exposed to an oxygen radical-generating system consisting of xanthine oxidase, hypoxanthine and chelated iron ions. Cell injury was assessed by LDH release and electron microscopy. Cytosolic Ca2+ levels and mitochondrial membrane potential were determined by flow cytometry; adenine nucleotide concentrations by HPLC. Mitochondrial dehydrogenase activity was measured by spectrophotometric assay. Results: Oxygen radicals damaged the plasma membrane as shown by a 6-fold LDH increase in the incubation medium within 180 min. At the ultrastructural level, mitochondria were the most susceptible to oxidative stress. In correlation to the pronounced mitochondrial damage, the mitochondrial dehydrogenase activity declined by 70%, whereas the mitochondrial membrane potential was enhanced by 27% after 120 min. Together this may cause the 85% decrease in the ATP concentration and the corresponding increase in ADP/AMP observed in parallel. In addition, an immediate 26% increase in cytosolic Ca2+ was found, a change which could be inhibited by BAPTA, reducing cellular damage. Conclusion: Cytosolic Ca2+ synergizes with oxygen radicals causing alterations of the ultrastructure and energy metabolism of acinar cells which might contribute to the cellular changes found in early stages of acute pancreatitis.

Changes in Human Endothelial Cell Energy Metabolic Capacities during in vitro Cultivation. The Role of "Aerobic Glycolysis" and Proliferation

Background: In this study the influence of cultivation and proliferation on energy metabolic characteristics of human umbilical vein endothelial cells (HUVEC) has been examined. The energy metabolic capacities of human endothelial cells freshly isolated from the umbilical vein were compared with those after cultivation for three passages and as subconfluent and confluent cultures. Methods: Expression of cell type-specific differentiation markers and proliferative activity were studied in dependency on cultivation characteristics. Furthermore, the energy metabolic characteristics of HUVEC were analyzed by measurement of the maximum catalytic activities of marker enzymes of various metabolic pathways. Results: Examination of a typical marker of proliferation, Ki67, confirmed that HUVEC changed in culture from a non-proliferative to a proliferative state. Compared to other cell types, the enzyme pattern of HUVEC showed a high glycolytic and a high NADPH regenerating capacity. These capacities increased by cultivation nearly to the same degree as marker enzymes of other metabolic pathways (e.g. citric acid cycle). Conclusion: Our data support the theory that metabolism of EC is primarily by “aerobic glycolysis”, i.e. the conversion of glucose to lactate in the presence of oxygen. These characteristics were independent of whether the cells are freshly isolated/non-proliferating or cell culture-adapted/proliferating.

A dual function of copper in designing regenerative implants.

The supply of titanium implants which are widely used in orthopaedics with both regenerative and anti-microbial properties will achieve a great progress in bone regeneration. We asked, whether by appropriate concentrations of copper ions it will be possible both to inhibit growth of bacteria and stimulate biological responses in mesenchymal stem cells (MSC). Using titanium material which released galvanically deposited copper at concentrations from 0.3 to 1.75 mM, growth of planktonic Staphylococcus aureus was blocked and more importantly adherent bacteria were cleared from the material surface within 24 h. To test biological responses of human bone marrow derived MSC due to copper ions, we found that copper stimulated the proliferation of MSC in a narrow concentration range around 0.1 mM. Similar copper concentrations enhanced osteogenic differentiation of MSC when cells were cultured in osteogenic differentiation medium. We observed increased activity of alkaline phosphatase (ALP), higher expression of collagen I, osteoprotegerin, osteopontin and finally mineralization of the cells. We conclude that titanium implants that release copper ions can be effective against bacterial infections at higher concentrations of copper near the implant surface and can promote bone regeneration when its concentration becomes lower due to diffusion.

Cellular investigations on electrochemically deposited calcium phosphate composites

Electrochemically deposited calcium phosphate (CaP) coatings are fast resorbable and existent only during the first period of osseointegration. In the present study, composite coatings with varying solubility (hydroxyapatite (HA), brushite with less HA and monetite (M) with less HA) were prepared and the influence of the degradation and the reprecipitation of CaP on osteoblastic cells were investigated. On the brushite composite coating a new precipitated, finely structured CaP phase was observed during immersion in cell culture medium with or without osteoblastic cells. The surface morphology of monetite and HA coatings were entirely unmodified under the same conditions. So it could be assumed that electrochemically deposited brushite with less HA acts as a precursor for new precipitated CaP. On this surface osteoblastic cells revealed a well-spread morphology with pronounced actin cytoskeleton and demonstrated good proliferation behaviour. Thus we suggest that brushite seems to be especially suitable for coating of implants as a matrix for nucleation and growth of new bone.

Capability of Differently Charged Plasma Polymer Coatings for Control of Tissue Interactions with Titanium Surfaces

Titanium surfaces were equipped with positively and negatively charged chemical functional groups by plasma polymerization. Their capability to influence the adhesion of human mesenchymal stem cells (hMSCs) and inflammation processes was investigated on titanium substrates, which are representative of real implant surfaces. For these purposes, titanium samples were coated with plasma polymers from allylamine (PPAAm) and acrylic acid (PPAAc). The process development was accompanied by physicochemical surface analysis using XPS, FT-IR and contact angle measurements. Very thin plasma polymer coatings were created, which are resistant to hydrolysis and delamination. Positively charged amino groups improve considerably the initial adhesion and spreading steps of hMSCs. PPAAm and PPAAc surfaces have an effect on the differentiation of hMSCs, e.g., the expression of osteogenic markers in dependence on culturing conditions. Acrylic acid groups appear to stimulate early mRNA differentiation markers (ALP, COL, Runx2) under basal conditions, whereas positively and negatively charged groups both stimulate late differentiation markers, like BSP and OCN, after 3 days of osteogenic stimulation. Long-term intramuscular implantation in rats revealed that PPAAc surfaces caused significantly stronger reactions by macrophages and antigen-presenting cells compared to untreated control (polished titanium) samples, while PPAAm films did not show a negative influence on the inflammatory reaction after implantation.