Elisabeth Stein

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.

Particle size of hydroxyapatite granules calcified from red algae affects the osteogenic potential of human mesenchymal stem cells in vitro.

Hydroxyapatite (HA) microparticles as a carrier in an injectable tissue-engineered bone filler are considered promising candidates for the treatment of small bone defects in the craniomaxillofacial region. HA granules calcified from red algae, varying in size, were evaluated in vitro for their suitability to be used as a carrier for human mesenchymal stem cells (hMSCs). Three groups of granules were produced in grain sizes of 10–100, 200–500 and 600–1,000 µm. After seeding and culturing hMSCs under osteogenic differentiation conditions onto HA particles for 3, 6 and 9 days, cellular proliferation (tetrazolium salt, XTT), alkaline phosphatase (ALP)-specific activity and total protein synthesis were investigated. The osteoblastic phenotype of the cells was evaluated by assaying the bone-specific genes osteocalcin, osteopontin and collagen type I. XTT assay revealed significantly higher (p < 0.01) proliferation of cells grown on the smallest grain size after 9 days of culture. Regarding ALP-specific activity, significantly higher levels of activity were detected in cells grown on the smallest grain size. Different grain sizes had no significant effects on the secretion of osteocalcin and osteopontin. Collagen type I production was significantly higher (p < 0.05) in cells grown on the biggest grain size in comparison with the two other grain sizes. These results show that the particle size of HA microparticles affects the osteogenic potential of cultured hMSCs and lead to the conclusion that particle size has differential effects on ALP-specific activity and collagen type I production.

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.

In Vitro and In Vivo Uptake Study of Escherichia coli Nissle 1917 Bacterial Ghosts: Cell-Based Delivery System to Target Ocular Surface Diseases

PURPOSE For the successful topical administration of drugs or vaccines to treat ocular surface diseases, efficient and well-tolerated delivery systems/carriers for conjunctival delivery are crucial in the development of new treatment strategies. The present study investigated the efficiency of internalization of bacterial ghosts (BGs) produced from probiotic Escherichia coli Nissle 1917 (EcN) by human conjunctival epithelial (HCjE) cell line, the EcN BGs cytotoxicity for HCjE cells, and in vivo uptake of EcN BGs by conjunctival guinea pig epithelial cells. METHODS The uptake of EcN BGs by HCjE cells was analyzed by laser scanning microscopy and flow cytometry. Immunohistochemistry was used to localize the EcN BGs in the guinea pig conjunctival tissue. Cytotoxicity of EcN BGs on HCjE cells was evaluated by measurement of LDH. RESULTS Laser scanning microscopy and flow cytometry revealed that EcN BGs internalization by HCjE cells was time- and dose dependent. No cytotoxic effect on HCjE cells was observed after EcN BGs inoculation for 30 and 120 minutes, as well as 24 hours. In addition, the uptake of EcN BGs was detected in the conjunctival cells after in vivo administration of EcN BGs into the eye of the guinea pig. CONCLUSIONS The findings that EcN BGs are nontoxic and effectively internalized in vitro by human and in vivo by guinea pig conjunctival cells comprise an important contribution to the future use of BGs as a system for conjunctival delivery of drugs and vaccines, either to treat or prevent ocular surface diseases.

Escherichia coli Nissle 1917 bacterial ghosts retain crucial surface properties and express chlamydial antigen: an imaging study of a delivery system for the ocular surface

To target chronic inflammatory ocular surface diseases, a drug delivery platform is needed that is safe, possesses immunomodulatory properties, and can be used either for drug delivery, or as a foreign antigen carrier. A new therapeutic approach that we have previously proposed uses nonliving bacterial ghosts (BGs) as a carrier-delivery system which can be engineered to carry foreign antigens and/or be loaded with therapeutic drugs. The parent strain chosen for development of our BG delivery system is the probiotic Escherichia coli strain Nissle 1917 (EcN), whose intrinsic properties trigger the innate immune system with the flagella and fimbriae used to attach and stimulate epithelial cells. In previous studies, we have shown that EcN BGs are safe for the ocular surface route, but evidence that EcN BGs retain flagella and fimbriae after transformation, has never been visually confirmed. In this study, we used different visualization techniques to determine whether flagella and fimbriae are retained on EcN BGs engineered either for drug delivery or as a foreign antigen carrier. We have also shown by immunoelectron microscopy that EcN retains two foreign antigens after processing to become EcN BGs. Furthermore, we demonstrated that BGs derived from EcN and expressing a foreign antigen attachment to conjunctival epithelial cells in vitro without causing reduced cell viability. These results are an important step in constructing a delivery system based on a nonliving probiotic that is suitable for use in ocular surface diseases pairing immunomodulation and targeted delivery.

The Ocular Conjunctiva as a Mucosal Immunization Route: A Profile of the Immune Response to the Model Antigen Tetanus Toxoid

Background In a quest for a needle-free vaccine administration strategy, we evaluated the ocular conjunctiva as an alternative mucosal immunization route by profiling and comparing the local and systemic immune responses to the subcutaneous or conjunctival administration of tetanus toxoid (TTd), a model antigen. Materials and methods BALB/c and C57BL/6 mice were immunized either subcutaneously with TTd alone or via the conjunctiva with TTd alone, TTd mixed with 2% glycerol or TTd with merthiolate-inactivated whole-cell B. pertussis (wBP) as adjuvants. Mice were immunized on days 0, 7 and 14 via both routes, and an evaluation of the local and systemic immune responses was performed two weeks after the last immunization. Four weeks after the last immunization, the mice were challenged with a lethal dose (2 × LD50) of tetanus toxin. Results The conjunctival application of TTd in BALB/c mice induced TTd-specific secretory IgA production and skewed the TTd-specific immune response toward a Th1/Th17 profile, as determined by the stimulation of IFNγ and IL-17A secretion and/or the concurrent pronounced reduction of IL-4 secretion, irrespective of the adjuvant. In conjunctivaly immunized C57BL/6 mice, only TTd administered with wBP promoted the establishment of a mixed Th1/Th17 TTd-specific immune response, whereas TTd alone or TTd in conjunction with glycerol initiated a dominant Th1 response against TTd. Immunization via the conjunctiva with TTd plus wBP adjuvant resulted in a 33% survival rate of challenged mice compared to a 0% survival rate in non-immunized animals (p<0.05). Conclusion Conjunctival immunization with TTd alone or with various adjuvants induced TTd-specific local and systemic immune responses, predominantly of the Th1 type. The strongest immune responses developed in mice that received TTd together with wBP, which implies that this alternative route might tailor the immune response to fight intracellular bacteria or viruses more effectively.

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.

Delivery of a Chlamydial Adhesin N-PmpC Subunit Vaccine to the Ocular Mucosa Using Particulate Carriers.

Trachoma, caused by the intracellular bacterium Chlamydia trachomatis (Ct), remains the world’s leading preventable infectious cause of blindness. Recent attempts to develop effective vaccines rely on modified chlamydial antigen delivery platforms. As the mechanisms engaged in the pathology of the disease are not fully understood, designing a subunit vaccine specific to chlamydial antigens could improve safety for human use. We propose the delivery of chlamydia-specific antigens to the ocular mucosa using particulate carriers, bacterial ghosts (BGs). We therefore characterized humoral and cellular immune responses after conjunctival and subcutaneous immunization with a N-terminal portion (amino acid 1–893) of the chlamydial polymorphic membrane protein C (PmpC) of Ct serovar B, expressed in probiotic Escherichia coli Nissle 1917 bacterial ghosts (EcN BGs) in BALB/c mice. Three immunizations were performed at two-week intervals, and the immune responses were evaluated two weeks after the final immunization in mice. In a guinea pig model of ocular infection animals were immunized in the same manner as the mice, and protection against challenge was assessed two weeks after the last immunization. N-PmpC was successfully expressed within BGs and delivery to the ocular mucosa was well tolerated without signs of inflammation. N-PmpC-specific mucosal IgA levels in tears yielded significantly increased levels in the group immunized via the conjunctiva compared with the subcutaneously immunized mice. Immunization with N-PmpC EcN BGs via both immunization routes prompted the establishment of an N-PmpC-specific IFNγ immune response. Immunization via the conjunctiva resulted in a decrease in intensity of the transitional inflammatory reaction in conjunctiva of challenged guinea pigs compared with subcutaneously and non-immunized animals. The delivery of the chlamydial subunit vaccine to the ocular mucosa using a particulate carrier, such as BGs, induced both humoral and cellular immune responses. Further investigations are needed to improve the immunization scheme and dosage.

A Probiotic Adjuvant Lactobacillus rhamnosus Enhances Specific Immune Responses after Ocular Mucosal Immunization with Chlamydial Polymorphic Membrane Protein C

Recent advances in the development of chlamydia vaccines, using live-attenuated or ultraviolet light-inactivated chlamydia, are paving the way for new possibilities to oppose the societal challenges posed by chlamydia-related diseases, such as blinding trachoma. An effective subunit vaccine would mitigate the risks associated with the use of a whole-cell vaccine. Our rationale for the design of an efficient subunit vaccine against Chlamydia trachomatis (Ct) is based on the membrane proteins involved in the initial Ct-host cell contact and on the route of immunization that mimics the natural infection process (i.e., via the ocular mucosa). The first aim of our study was to characterize the specific conjunctival and vaginal immune responses following eye drop immunization in BALB/c mice, using the N-terminal portion of the Ct serovar E polymorphic membrane protein C (N-PmpC) as the subunit vaccine antigen. Second, we aimed to examine the adjuvant properties of the probiotic Lactobacillus rhamnosus (LB) when formulated with N-PmpC. N-PmpC applied alone stimulated the production of N-PmpC- and Ct serovar B-specific antibodies in serum, tears and vaginal washes, whereas the combination with LB significantly enhanced these responses. The N-PmpC/LB combination initiated a T cell response characterized by an elevated percentage of CD25+ T cells and CD8+ effector T cells, enhanced CD4+ T-helper 1 skewing, and increased regulatory T cell responses. Together, these results show that eye drop vaccination with combined use of N-PmpC and a live probiotic LB stimulates specific cellular and humoral immune responses, not only locally in the conjunctiva but also in the vaginal mucosa, which could be a promising approach in Ct vaccine development.

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.