Enhanced cellular migration and prolonged chondrogenic differentiation in decellularized cartilage scaffolds under dynamic culture conditions.

Lesions of aural, nasal and tracheal cartilage are frequently reconstructed by complex surgeries which are based on harvesting autologous cartilage from other locations such as the rib. Cartilage tissue engineering (CTE) is regarded as a promising alternative to attain vital cartilage. Nevertheless, CTE with nearly natural properties poses a significant challenge to research due to the complex reciprocal interactions between cells and extracellular matrix which have to be imitated and which are still not fully understood. Thus, we used a custom-made glass bioreactor to enhance cell migration into decellularized porcine cartilage scaffolds (DECM) and mimic physiological conditions. The DECM seeded with human nasal chondrocytes (HPCH) were cultured in the glass reactor for 6 weeks and examined by histological and immunohistochemical staining, biochemical analyses and real time-PCR at 14, 28 and 42 days. The migration depth and the number of migrated cells were quantified by computational analysis. Compared to the static cultivation, the dynamic culture (DC) fostered migration of HPCH into deeper tissue layers. Furthermore, cultivation in the bioreactor enhanced differentiation of the cells during the first 14 days, but differentiation diminished in the course of further cultivation. We consider the DC in the presented bioreactor as a promising tool to facilitate CTE and to help to better understand the complex physiological processes during cartilage regeneration. Maintaining differentiation of chondrocytes and improving cellular migration by further optimizing culture conditions is an important prerequisite for future clinical application.

Photoinactivation results of Enterococcus moraviensis with blue and violet light suggest the involvement of an unconsidered photosensitizer.

Microorganisms can be photoinactivated with 405 and 450 nm irradiation, due to endogenous photosensitizers, which absorb light of these wavelengths and generate reactive oxygen species that destroy the cells from within. The photosensitizers assumed to be responsible are porphyrins in the spectral region around 405 nm and flavins at about 450 nm. The aim of this study was to investigate this hypothesis on enterococci, considering that they do not contain porphyrins. In photoinactivation experiments with Enterococcus moraviensis, 405 nm and 450 nm irradiation both led to a reduction of the bacterial concentration by several orders of magnitude with 405 nm irradiation being much more efficient. The measurement and analysis of the fluorescence spectra revealed no signs of porphyrins whereas flavins seemed to be rapidly converted to lumichrome by 405 nm radiation. Therefore, probably none of the usual suspects, porphyrins and flavins, was responsible for the photoinactivation of Enterococcus moraviensis during 405 nm irradiation. Fluorescence experiments revealed the spectra of lumichrome and NADH, which are both known photosensitizers. Presumably, one of them or both were actually involved here. As NADH and flavins (and therefore their photodegradation product lumichrome) are abundant in all microorganisms, they are probably also involved in 405 nm photoinactivation processes of other species.

Photoinactivation Sensitivity of Staphylococcus carnosus to Visible-light Irradiation as a Function of Wavelength.

Inactivation properties of visible light are of increasing interest due to multiple possible fields of application concerning antibacterial treatment. For violet wavelengths, the generation of reactive oxygen species by porphyrins is accepted as underlying mechanism. However, there is still little knowledge about photosensitizers at blue wavelengths. While flavins were named as possible candidates, there is still no experimental evidence. This study investigates the photoinactivation sensitivity of Staphylococcus carnosus to selected wavelengths between 390 and 500 nm in 10- to 25-nm intervals. Absorption and fluorescence measurements in bacterial lysates confirmed inactivation findings. By means of a mathematical calculation in MATLAB® , a fit of different photosensitizer absorption spectra to the measured action spectrum was determined to gain knowledge about the extent to which specific photosensitizers are involved. The most effective wavelength for S. carnosus at 415 nm could be explained by the involvement of zinc protoporphyrin IX. Between 450 and 470 nm, inactivation results indicated a broad plateau, statistically distinguishable from 440 and 480 nm. This observation points to flavins as responsible photosensitizers, which furthermore seem to be involved at violet wavelengths. A spectral scan of sensitivities might generally be an advantageous approach for examining irradiation impact.

Realisation and assessment of a low-cost LED device for contact lens disinfection by visible violet light.

This study presents a device for efficient, low-cost and eye-friendly overnight disinfection of contact lenses by visible violet light as an alternative to disinfection with biocide-containing solutions. Bacterial solutions with one Pseudomonas and one Staphylococcus strain each were irradiated for up to 8 h in commercial transparent contact lens cases by the presented light-emitting diode (LED) device. Samples were taken at different intervals and distributed on agar plates. The surviving bacteria were determined by counting of colony-forming units and compared to the specific requirements of the stand-alone test for contact lens disinfection of the hygiene standard ISO 14729. The concentration of both microorganisms was reduced by three orders of magnitude after less than 4 h of irradiation. The LED current and intensity have not yet been at maximum and could be further increased if necessary for other microorganisms. The presented device fulfils the requirement of the stand-alone test of the contact lens hygienic standard ISO 14729 for the tested Pseudomonas and Staphylococcus strains. According to literature data, the inactivation of Serratia marcescens, Candida albicans and Fusarium solani seems also possible, but may require increased LED current and intensity.

Automated bioreactor system for cartilage tissue engineering of human primary nasal septal chondrocytes.

An automated bioreactor system for three-dimensional (3D) cultivation of facial cartilage replacement matrices (e.g. whole human auricles) with automatised medium exchange, gas flow and temperature control was developed. The measurement of O2 saturation and pH value in the medium was performed with a non-invasive optical method. The whole system can be observed via remote monitoring worldwide. First results demonstrated that the complete system remained sterile throughout a period of 42 days. Human chondrocytes migrated into the employed cartilage replacement matrix consisting of decellularised porcine nasoseptal cartilage (pNSC). Furthermore, an improved migration and new synthesis of aggrecan was detected. A first evaluation of the system was conducted by comparison of the results from laboratory analysis with computational fluid dynamics (CFD).