Walter Sutter

Initial effects of low-level laser therapy on growth and differentiation of human osteoblast-like cells.

ZusammenfassungDer Einsatz von Soft-Lasern im Rahmen einer Low level-Laser Therapie ist mittlerweile eine klinisch etablierte Behandlungsmethode. In vitro Studien haben gezeigt, dass Soft-Laser auch eine biostimulatorische Wirkung auf unterschiedlichste Zelltypen haben. Das Ziel dieser Untersuchung war die Effekte von Low level-Laser Therapie auf das initiale Wachstums- und Differenzierungsverhalten von in vitro kultivierten humanen osteoblastären Zellen zu untersuchen. SaOS-2 Zellen wurden mit Laser-Dosen von 1 J/cm2 und 2 J/cm2 mit einem Dioden Laser mit 670 nm Wellenlänge und einer Ausgangsleistung von 400 mW bestrahlt. Unbehandelte Zellen dienten als Kontrollgruppe. 24 h, 48 h und 72 h nach der Bestrahlung wurden die Zellen geerntet und ihre Vitalität bestimmt. Zusätzlich wurde die Aktivität der Alkalischen Phosphatase ermittelt und die Expression von Osteopontin und Collagen Typ I mittels semiquantitativer RT-PCR untersucht. Zellen, die mit 1 J/cm2 bestrahlt worden waren wiesen, sowohl eine höhere Vitalität als auch eine höhere Aktivität der Alkalischen Phosphatase gegenüber den Kontrollen auf. Auch die Expression von Osteopontin und Collagen Typ I mRNA war gegenüber der Kontrollgruppe erhöht. Hingegen führte eine Verdopplung der Laserleistung zu einer Abnahme der Zellviabilität in den ersten 48 h und zu einer konstant niedrigeren Alkalischen Phosphataseaktivität. Während die Expression von Collagen Typ I und Osteopontin mRNA in unbehandelten und mit 1 J/cm2 bestrahlten Zellen im Verlauf des Experiments leicht abnahm, konnte eine Zunahme ihrer Expression nach Bestrahlung mit 2 J/cm2 beobachtet werden. Unsere Beobachtungen deuten darauf hin, dass Low level-Laser Therapie eine biostimulatorische Wirkung auf SaOS-2 Zellen bereits in der inititalen Kulturphase hat. Diese Ergebnisse können dazu beitragen, neue Therapie-Konzepte in der Regeneration von Knochendefekten zu entwickeln. Weitere Untersuchungen über einen verlängerten Zeitraum wären hilfreich, dieses Potential genauer zu beurteilen.SummaryLow-level laser therapy is a clinically well established tool for enhancement of wound healing. In vitro studies have also shown that low level laser therapy has a biostimulatory effect on cells of different origin. The aim of this in vitro study was to investigate the initial effect of low-level laser therapy on growth and differentiation of human osteoblast-like cells. SaOS-2 cells were irradiated with laser doses of 1 J/cm2 and 2 J/cm2 using a diode laser with 670 nm wave length and an output power of 400 mW. Untreated cells were used as controls. At 24 h, 48 h and 72 h post irradiation, cells were collected and assayed for viability of attached cells and alkaline phosphatase specific activity. In addition, mRNA expression levels of osteopontin and collagen type I were assessed using semi-quantitative RT-PCR. Over the observation period, cell viability, alkaline phosphatase activity and the expression of osteopontin and collagen type I mRNA were slightly enhanced in cells irradiated with 1 J/cm2 compared with untreated control cells. Increasing the laser dose to 2 J/cm2 reduced cell viability during the first 48 h and resulted in persistently lower alkaline phosphatase activity compared with the other two groups. The expression of osteopontin and collagen type I mRNA slightly decreased with time in untreated controls and cells irradiated with 1 J/cm2, but their expression was increased by treatment with 2 J/cm2 after 72 h. These results indicate that low-level laser therapy has a biostimulatory effect on human osteoblast-like cells during the first 72 h after irradiation. Further studies are needed to determine the potential of low-level laser therapy as new treatment concept in bone regeneration.

Potential involvement of MYC- and p53-related pathways in tumorigenesis in human oral squamous cell carcinoma revealed by proteomic analysis.

Oral squamous cellular carcinoma is a malignant tumor with poor prognosis. Discovery of early markers to discriminate between malignant and normal cells is of high importance in clinical diagnosis. Subcellular fractions from 10 oral squamous cell carcinoma and corresponding control samples, enriched in mitochondrial and cytosolic proteins, as well as blood from the tumor were analyzed by proteomics, two-dimensional gel electrophoresis, followed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Three-hundred and fifty different gene products were identified. Twenty proteins showed deranged levels in oral squamous cell carcinoma in comparison with the control samples and are potentially involved in tumor growth and metastasis. Of these, 16 proteins were upregulated. By applying pathway analysis, we found 8 of the upregulated gene products to be linked to three main locus genes, p53, MYC, and MYCN, and could be candidate biomarkers for OSCC. The findings of this pilot study show that OSCC gene ontology combined with proteomic analysis is a powerful tool in systems biology for the elucidation of the complexity of expression profiles in cellular processes. Application of such pathway analysis has the potential to generate new insights into complex molecular mechanisms underlying disease related processes and could therefore significantly contribute to the efficient performance of the entire discovery process.

Phenothiazine Chloride and Soft Laser Light Have a Biostimulatory Effect on Human Osteoblastic Cells

OBJECTIVE Low-level laser therapy (LLLT) is a well accepted tool to accelerate wound healing and to reduce inflammation after oral implant insertion. Since there are no in vitro data on a combination of LLLT with prior photosensitization, it was the aim of this study to investigate if photosensitization with phenothiazine chloride results in an alteration of the biostimulatory effect of low-level laser irradiation. BACKGROUND DATA LLLT and antimicrobial photodynamic therapy are well established for the treatment of peri-implantitis. In vitro studies have shown a biostimulatory effect of LLLT on various cell types, including osteogenic cells. MATERIALS AND METHODS SaOS-2 cells were treated with the photosensitizer phenothiazine chloride before irradiation with matched laser light. At 24-h intervals the viability and differentiation were analyzed in treated and untreated cells. RESULTS While the biostimulatory effect of the LLLT could be observed for the lower irradiation dose, the pretreatment with phenothiazine chloride did not significantly affect the growth and differentiation of the SaOS-2 cells. CONCLUSION It can thus be concluded that combined treatment with phenothiazine chloride and LLLT does not result in a synergistic enhancement of the biostimulatory effect of LLLT, but there was also no evidence for antagonizing effects on growth and differentiation of human osteoblasts.

Effect of different biomaterials on the expression pattern of the transcription factor Ets2 in bone-like constructs

Tissue engineering offers an exciting prospect for reconstructive surgery by replacing missing natural scaffolds with artificial ones. For optimal success the artificial scaffold should provide an environment closely resembling the natural tissue. Little is known about the direct influence of the scaffold on the expression of regulators of bone development, such as transcription factors. The aim of this study was to investigate the influence of the scaffold material on the expression of V-ets erythroblastosis virus E26 oncogene homolog 2 (avian) (Ets2), a key transcription factor in bone biology. Human primary bone precursor cells were seeded in three-dimensional constructs consisting of hydroxyapatite (HA) or poly (lactic-co-glycolic acid) (PLGA). Cells grown on tissue culture polystyrene dishes served as controls. After cultivation for up to 21 days the expression of Ets2 and other important bone-specific genes was assessed by reverse transcription polymerase chain reaction (RT-PCR) and Western Blotting. Ets2 mRNA showed significantly higher expression in controls than in bone-like constructs, and more Ets2 mRNA was expressed in cells grown in HA than in PLGA constructs. At protein level however, Ets2 expression was higher in constructs than in controls after prolonged culture. Our study showed for the first time a differential expression of Ets2 in tissue engineered bone constructs in vitro, demonstrating that scaffold chemistry has an influence on the expression of genes regulating osteogenesis.

Evaluation of Maxillary Sinus Floor Augmentation on Digital Panoramic Radiographs with an Image Manipulation Programme after Simultaneous or Staged Dental Implant Insertion

Sinus floor augmentation is an effective method to regain bone height for the successful insertion of dental implants into the posterior maxilla. The aim of the study was to evaluate the behaviour of augmentation material following simultaneous or staged dental implant insertion, as visible on digital panoramic radiographs using the GNU Image Manipulation Program (GIMP). We evaluated one-stage (group 1) or two-stage (group 2) maxillary sinus floor augmentation procedures in 19 patients, using a high temperature-treated bovine porous hydroxyapatite material. Digital panoramic radiographic measurements were captured pre-operatively in both groups, pre-implant insertion in group 2, and immediately postoperatively and 6 months postoperatively in both groups. Forty parallel-walled bone-level implants were placed in a one-stage (n=18) or two-stage (n=22) protocol, with a mean residual bone height of 4.9±1.8 mm and survival rate of 100%. Mean bone height increased by 8.6±1.6 mm immediately post-implantation and by 7.9±1.7 mm after 6 months. Mean distal and mesial bone losses after abutment connection were 0.42±0.24 mm and 0.34±0.27 mm, respectively. No significant intergroup or intragroup differences between simultaneous and staged dental implant procedures were found. Our results show that the histogram tool in GIMP is useful for documentation of the area of the augmentation material used in maxillary sinus floor augmentation.

Expression patterns of Ets2 protein correlate with bone-specific proteins in cell-seeded three-dimensional bone constructs.

The transcription factor Ets2 and its transcriptional targets osteopontin (OPN) and osteocalcin (OC) are expressed in tissue-engineered bone constructs in vitro. Up to now little is known about the role of Ets2 in tissue-engineering applications. This study was intended to investigate the hypothesis that protein expression of Ets2 is correlated with the expression of bone-specific proteinsin tissue-engineeredbone constructs. Cell-seeded three-dimensional bone constructs manufactured with osteoblastic cells and poly(lactic-co-glycolic acid) polymer fleeces over a period of 21 days were analyzed by SDS-PAGE and Western blotting. The protein expression of OPN, OC, osteonectin and collagen type I was analyzed. Cellularity, alkaline phosphatase-specific activity and histology confirmed the osteoblastic phenotype of the constructs. Correlations between Ets2 expression and OPN and Ets2 and collagen type I expression could be detected during the phase of late osteoblastic differentiation between days 9 and 21. The correlation between OC and collagen type I was significant in this late stage of osteoblastic differentiation. These results suggest that there is a strong interplay of Ets2 with bone-specific proteins in cell-seeded three-dimensional bone constructs. This study is a crucial step to elucidate the complex interplay of bone-related proteins in the application of bone tissue engineering.