Frank Lüthen

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.

Osteoblast response to biomimetically altered titanium surfaces

Bioinert titanium (Ti) materials are generally encapsulated by fibrous tissue after implantation into the living body. To improve the bone-bonding ability of Ti implants, we activated commercially pure titanium (cpTi) by a simple chemical pre-treatment in HCl and NaOH. Subsequently, we exposed the treated samples to simulated body fluid (SBF) for 2 (TiCT) and 14 days (TiHCA), respectively, to mimic the early stages of bone bonding and to investigate the in vitro response of osteoblasts on thus altered biomimetic surfaces. Sample surfaces were characterized by scanning electron microscopy, energy-dispersive X-ray analysis, cross-sectional transmission electron microscopy analyses, Fourier transform infrared and Raman spectroscopy. It was shown that the efflorescence consisting of sodium titanate that is present on pre-treated cpTi surfaces transformed to calcium titanate after 2 days in SBF. After 14 days in SBF a homogeneous biomimetic apatite layer precipitated. Human osteoblasts (MG-63) revealed a well spread morphology on both functionalized Ti surfaces. On TiCT, the gene expression of the differentiation proteins alkaline phosphatase (ALP) and bone sialo protein was increased after 2 days. On both TiCT and TiHCA, the collagen I and ALP expression on the protein level was enhanced at 7 and 14 days. The TiCT and the TiHCA surfaces reveal the tendency to increase the differentiated cell function of MG-63 osteoblasts. Thus, chemical pre-treatment of titanium seems to be a promising method to generate osteoconductive surfaces.

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.

Calpain-mediated breakdown of cytoskeletal proteins contributes to cholecystokinin-induced damage of rat pancreatic acini

The cytosolic cysteine protease calpain is implicated in a multitude of cellular functions but also plays a role in cell damage. Our previous results suggest that an activation of calpain accompanied by a decrease in its endogenous inhibitor calpastatin may contribute to pancreatic damage during cerulein‐induced acute pancreatitis. The present study aimed at the time course of secretagogue‐induced calpain activation and cellular substrates of the protease. Isolated rat pancreatic acini were incubated with a supramaximal concentration of cholecystokinin (0.1 μM CCK) for 30 min in the presence or absence of the calpain inhibitor Z‐Val‐Phe methyl ester (100 μM ZVP). The activation of calpain and the expression of calpastatin and the actin cytoskeleton‐associated proteins αII‐spectrin, E‐cadherin and vinculin were studied by immunoblotting. The cell damage was assessed by lactate dehydrogenase release and ultrastructural analysis including fluorescence‐labelled actin filaments. Immediately after administration, CCK led to activation of both calpain isoforms, μ‐ and m‐calpain. The protease activation was accompanied by a decrease in the E‐cadherin level and formation of calpain‐specific breakdown products of αII‐spectrin. A calpain‐specific cleavage product of vinculin appeared concomitantly with changes in the actin filament organization. No effect of CCK on calpastatin was found. Inhibition of calpain by ZVP reduced CCK‐induced damage of the actin‐associated proteins and the cellular ultrastructure including the actin cytoskeleton. The results suggest that CCK‐induced acinar cell damage requires activation of calpain and that the actin cytoskeleton belongs to the cellular targets of the protease.

Evaluation of Osseointegration of Titanium Alloyed Implants Modified by Plasma Polymerization

By means of plasma polymerization, positively charged, nanometre-thin coatings can be applied to implant surfaces. The aim of the present study was to quantify the adhesion of human bone cells in vitro and to evaluate the bone ongrowth in vivo, on titanium surfaces modified by plasma polymer coatings. Different implant surface configurations were examined: titanium alloy (Ti6Al4V) coated with plasma-polymerized allylamine (PPAAm) and plasma-polymerized ethylenediamine (PPEDA) versus uncoated. Shear stress on human osteoblast-like MG-63 cells was investigated in vitro using a spinning disc device. Furthermore, bone-to-implant contact (BIC) was evaluated in vivo. Custom-made conical titanium implants were inserted at the medial tibia of female Sprague-Dawley rats. After a follow-up of six weeks, the BIC was determined by means of histomorphometry. The quantification of cell adhesion showed a significantly higher shear stress for MG-63 cells on PPAAm and PPEDA compared to uncoated Ti6Al4V. Uncoated titanium alloyed implants showed the lowest BIC (40.4%). Implants with PPAAm coating revealed a clear but not significant increase of the BIC (58.5%) and implants with PPEDA a significantly increased BIC (63.7%). In conclusion, plasma polymer coatings demonstrate enhanced cell adhesion and bone ongrowth compared to uncoated titanium surfaces.

Morphometric immunohistochemical examination of the inflammatory tissue reaction after implantation of calcium phosphate-coated titanium plates in rats

Calcium phosphate (CaP) preparations are established coatings for titanium-based medical implants used for bone reconstruction. However, biodegradation of the coating can result in microparticles that subsequently cause inflammatory reactions. The present study was therefore aimed at investigating the inflammatory response to two series of CaP-coated titanium plates: Ti-brushite (Ti-B) and Ti-hydroxyapatite (Ti-H) implants. Fifteen male LEW.1A rats received one plate of each series and a pellet (5 x 2 mm) of sol-gel derived silica/CaP (SCP implants) implanted into the back musculature. After 7, 14 and 28 days, five rats were killed and the implants were removed with the surrounding tissue. Quantitative immunohistochemistry was performed on frozen sections. Total monocytes/macrophages, tissue macrophages, T-cells, MHC-class-II-positive cells and proliferating cells were counted. For the Ti-B implants, the number of monocytes/macrophages remained constant while the other cell populations increased. In contrast, for the Ti-H implants the number of monocytes/macrophages decreased while the other cell populations remained constant. The SCP implants demonstrated degradation and scattering into smaller particles with an increase for all cell populations except the proliferating cells. Human mesenchymal stem cells demonstrated adherence and a flat morphology on Ti-B and Ti-H implants and no remarkable difference between both implants. Taken together, the in vivo data demonstrate that the short-term inflammatory response against a hydroxyapatite coating is lower in comparison to a brushite coating, and that the morphology of cells growing in vitro is similar on both layers.