Klaus Liefeith

Two-photon techniques in tissue engineering

PURPOSE NIR radiation in the range of about 800 nm is less absorbed by biological tissues and is suited for triggering photonic effects using femtosecond pulsed Ti:Sa lasers. Especially in the life sciences, two-photon techniques are gaining greater importance. We introduce two laser applications for tissue engineering: the autofluorescent visualization of cells seeded on 3D scaffolds after two-photon excitation; and the manufacturing of 3D-structured hydrogel-like scaffolds by triggering free-radical polymerization processes within polymerizable precursors. METHODS Primary bovine chondrocytes were cultivated on collagen I/III scaffolds using a flow chamber system coupled with a two-photon laser scanning microscope (2PLSM). During the incubation the cell population was hydrostatically stimulated. The selective visualization of unlabeled cells and scaffolds was achieved by spectral autofluorescence imaging. To gain some insight into scaffold-mediated effects on cell growth and cell differentiation, hydrogel-like scaffolds with well defined 3D structures were generated by two-photon polymerization (2PP) using methacrylated urethane and polyethyleneglycol diacrylate. RESULTS We were able to show that spectral autofluorescence imaging provides spatially resolved data for the non-invasive online control of the tissue engineering process as well as the quantification of cell distribution within the scaffold. The fabrication of 3D 2PP scaffolds made from hydrogel-forming monomers and their effect on cell attachment and cell growth were also shown. CONCLUSIONS Two-photon techniques provide powerful tools for both the non-invasive online visualization of 3D cell-scaffold constructs and the structuring of 3D cultivation environments. The application of these techniques is also suitable for integration into micro-systems technology (e.g. BioMEMS, Cells-on-Chip, Lab-on-a Chip).

In vitro degradation and mechanical properties of PLA-PCL copolymer unit cell scaffolds generated by two-photon polymerization.

The manufacture of 3D scaffolds with specific controlled porous architecture, defined microstructure and an adjustable degradation profile was achieved using two-photon polymerization (TPP) with a size of 2  ×  4  ×  2 mm(3). Scaffolds made from poly(D,L-lactide-co-ɛ-caprolactone) copolymer with varying lactic acid (LA) and ɛ -caprolactone (CL) ratios (LC16:4, 18:2 and 9:1) were generated via ring-opening-polymerization and photoactivation. The reactivity was quantified using photo-DSC, yielding a double bond conversion ranging from 70% to 90%. The pore sizes for all LC scaffolds were see 300 μm and throat sizes varied from 152 to 177 μm. In vitro degradation was conducted at different temperatures; 37, 50 and 65 °C. Change in compressive properties immersed at 37 °C over time was also measured. Variations in thermal, degradation and mechanical properties of the LC scaffolds were related to the LA/CL ratio. Scaffold LC16:4 showed significantly lower glass transition temperature (T g) (4.8 °C) in comparison with the LC 18:2 and 9:1 (see 32 °C). Rates of mass loss for the LC16:4 scaffolds at all temperatures were significantly lower than that for LC18:2 and 9:1. The degradation activation energies for scaffold materials ranged from 82.7 to 94.9 kJ mol(-1). A prediction for degradation time was applied through a correlation between long-term degradation studies at 37 °C and short-term studies at elevated temperatures (50 and 65 °C) using the half-life of mass loss (Time (M1/2)) parameter. However, the initial compressive moduli for LC18:2 and 9:1 scaffolds were 7 to 14 times higher than LC16:4 (see 0.27) which was suggested to be due to its higher CL content (20%). All scaffolds showed a gradual loss in their compressive strength and modulus over time as a result of progressive mass loss over time. The manufacturing process utilized and the scaffolds produced have potential for use in tissue engineering and regenerative medicine applications.

Investigations on the Secondary Structure of Polypeptide Chains in Polyelectrolyte Multilayers and their Effect on the Adhesion and Spreading of Osteoblasts

Inspired by the composition of the native extracellular matrix, biomimetic polyelectrolyte multilayers were assembled from polypeptides and the glycosaminoglycan chondroitin sulfate (CS). To investigate whether peptide conformation imposes an effect on the cell biological functions of osteoblasts, the secondary structure was analyzed by in situ infra-red and circular dichroism spectroscopy. Multilayers composed of polypeptides and CS reveal a predominantly random coiled conformation and impede osteoblast spreading. On the contrary, polypeptide chains in assemblies of poly-l-lysine and poly-l-glutamic acid (PGA) primarily adopt an intermolecular β sheet structure and reveal an increased area of spread, which consequently supports the proliferation of osteoblasts. When CS is replaced by PGA in mixed multilayers, we observe a structural rearrangement from random coils to β sheets with a concomitant improved cell response. We conclude that polypeptide conformation in biomimetic multilayer assemblies affects osteoblast response by altering the stiffness of the multilayer.

Two-photon laser scanning microscopy on native cartilage and collagen membranes for tissue engineering

In our experiments 2-Photon laser scanning microscopy (2PLSM) has been used to acquire 3-dimensional structural information on native unstained biological samples for tissue engineering purposes. Using near infrared (NIR) femtosecond laser pulses for 2-photon excitation and second harmonic generation (SHG) it was possible to achieve microscopic images at great depths in strongly (light) scattering collagen membranes (depth up to 300 μm) and cartilage samples (depth up to 460 μm). With the objective of optimizing the process of chondrocyte growth on collagen scaffolding materials for implantation into human knee joints, two types of samples have been investigated. (1) Both arthritic and non-arthritic bovine and human cartilage samples were examined in order to differentiate between these states and to estimate the density of chondrocytes. In particular, imaging depth, fluorescence intensity and surface topology appear promising as key information for discriminating between the non-arthritic and arthritic states. Human chondrocyte densities between 2-106/cm3 and 20-106/cm3, depending on the relative position of the sample under investigation within the cartilage, were measured using an automated procedure. (2) Chondrocytes which had been sown out on different types of I/III-collagen membranes, were discriminated from the scaffolding membranes on the basis of their native fluorescence emission spectra. With respect to the different membranes, either SHG signals from the collagen fibers of the membranes or differences in the emission spectra of the chondrocytes and the scaffolding collagenes were used to identify chondrocytes and membranes.

Biomimetic assembly of polyelectrolyte multilayers containing phosvitin monitored with reflectometric interference spectroscopy.

Coatings of biomaterials or implants that facilitate biomineralization possess a great potential for applications focused to the replacement, augmentation, and regeneration of bone tissue. Biomimetic approaches utilize biomolecules for either templating or supporting the crystallization process. One of these promising biomolecules is phosvitin (PV), an egg yolk protein known to transport and store inorganic phosphates and calcium ions. The incorporation of PV into polyelectrolyte multilayers is favorable due to PVs high degree of phosphorylation and thus a high acidity. Utilizing the reflectometric interference spectroscopy, the adsorption kinetics of this novel polyelectrolyte system composed of poly-L-lysine and the heavily phosphorylated phosvitin were monitored. The results demonstrate an unexpected nonregular growth regime called overshoot. Effective measures of shifting this irregular polyelectrolyte adsorption process back to a regular multilayer growth regime are reported in this paper.

Biomimetic organic-inorganic nanocomposite coatings for titanium implants. In vitro and in vivo biological testing

Recently described organic-inorganic nanocomposite coatings of the chemical composition: (PLL/PGA)(10)CaP[(PLL/PGA)(5)CaP](4) (coating A) and (PLL/PGA)(10)CaP[(PLL/PGA)(5)CaP](4)(PLL/PGA)(5) (coating B), applied to chemically etched titanium plates, have been tested by extensive cell culture tests and in vivo biological experiments, with uncoated titanium plates serving as controls. Before testing, coated samples were stored for extended periods of time (from 2 weeks to 8 months) under dry, sterile conditions. Cells of the cell-lines MC3T3-E1 and/or SAOS-2 were used for the following cell culture tests: initial adhesion (4 h) and proliferation (up to 21 days), cell activity (XTT test), morphology, synthesis of collagen type I and alkaline phosphatase activity (all incubation up to 21 days). In addition, coating B was tested against uncoated control in a validated in vivo pull-out model in rabbit tibia. The results of both in vitro and in vivo experiments show excellent biological properties of chemically etched titanium which are even surpassed by surfaces covered with coating B. Thus, after 8 weeks of healing the implants coated with B were significantly better attached to the cortical bone of rabbit thibiae than uncoated titanium controls with more than twice the force needed to detach coated implants. However, coating A (top crystal layer) had an adverse effect on both cell proliferation and activity, which is explained by morphological observations, showing inhibited spreading of the cells on its rough surfaces. The results also show the remarkable stability of the coatings when shelved under dry and sterile conditions.

Analysis of the influence of the macro- and microstructure of dental zirconium implants on osseointegration: a minipig study

OBJECTIVES It was the aim of this study to analyze the influence of implant design and surface topography on the osseointegration of dental zirconium implants. STUDY DESIGN Six different implant designs were tested in the study. Nine or 10 test implants were inserted in the frontal skull in each of 10 miniature pigs. Biopsies were harvested after 2 and 4 months and subjected to microradiography. RESULTS No significant differences between titanium and zirconium were found regarding the microradiographically detected bone-implant contact (BIC). Cylindric zirconium implants showed a higher BIC at the 2-month follow-up than conic zirconium implants. Among zirconium implants, those with an intermediate Ra value showed a significantly higher BIC compared with low and high Ra implants 4 months after surgery. CONCLUSIONS Regarding osseointegration, titanium and zirconium showed equal properties. Cylindric implant design and intermediate surface roughness seemed to enhance osseointegration.

ortho-Nitrobenzyl alcohol based two- photon excitation controlled drug release system

Controlled drug delivery is highly desired for many medicinal cases where time and spatial dependency is required. The present paper provides a promising but also simple molecular architecture for photoinduced release of chemical compounds with primary amines based on one-and two-photon absorption. Silane coupling to the surface and biotin/streptavidin chemistry ensure the stability of the system.

Dependence of the initial adhesion of biofilm forming Pseudomonas putida mt2 on physico-chemical material properties

Bacterial adhesion is strongly dependent on the physico-chemical properties of materials and plays a fundamental role in the development of a growing biofilm. Selected materials were characterized with respect to their physico-chemical surface properties. The different materials, glass and several polymer foils, showed a stepwise range of surface tensions (γs) between 10.3 and 44.7 mN m−1. Measured zeta potential values were in the range between −74.8 and −28.3 mV. The initial bacterial adhesion parameter q max was found to vary between 6.6 × 106 and 28.1 × 106 cm−2. By correlation of the initial adhesions kinetic parameters with the surface tension data, the optimal conditions for the immobilization of Pseudomonas putida mt2 were found to be at a surface tension of 24.7 mN m−1. Both higher and lower surface tensions lead to a smaller number of adherent cells per unit surface area. Higher energy surfaces, commonly termed hydrophilic, could constrain bacterial adhesion because of their more highly ordered water structure (exclusion zone) close to the surface. At low energy surfaces, commonly referred to as hydrophobic, cell adhesion is inhibited due to a thin, less dense zone (depletion layer or clathrate structure) close to the surface. Correlation of q max with zeta potential results in a linear relationship. Since P. putida carries weak negative charges, a measurable repulsive effect can be assumed on negative surfaces.

Antibacterial and anti-encrustation biodegradable polymer coating for urinary catheter

Bacterial biofilm and crystalline deposits are the common causes of failure of long-term indwelling urinary catheter. Bacteria colonise the catheter surface causing serious infections in the urinary tract and encrustations that can block the catheter and induce trauma in patients. In this study, the strategy used to resist bacterial adhesion and encrustation represents a combination of the antibacterial effects of norfloxacin and silver nanoparticles and the PLGA-based neutralisation of alkali products of urea hydrolysis gained through the degradation of the polymer in an aqueous milieu. Silver nanoparticles were coated with tetraether lipids (TEL) to avoid aggregation when dispersed in acetone and during the film formation. The polymer films loaded with the two antibacterial agents were applied on Polyurethane (PUR) and Silicon sheets. We demonstrated the antibacterial and anti-adhesion effectiveness of the coatings whereby commercially available biocompatible polymers PUR and Silicon were used as controls. Using artificial urine and an in vitro encrustation model, it was shown that the coatings resist the encrustation for at least 2 weeks. This combination of a biodegradable polymer and wide-range antibacterial agents represents a potentially attractive biocompatible coating for urinary catheters.