Wound healing in rabbit corneas after flapless refractive lenticule extraction with a 345 nm ultraviolet femtosecond laser

Purpose To characterize corneal wound healing in a rabbit model after flapless refractive lenticule extraction with a 345 nm ultraviolet femtosecond laser. Setting Departments of Ophthalmology and Anatomy II, University of Erlangen-Nürnberg and Wavelight GmbH, Erlangen, Germany. Design Methods Flapless refractive lenticule extraction was performed in 1 eye each of 20 New Zealand white rabbits (−5.0 diopters). Groups of 4 animals were euthanized after 48 hours, 1 week, 2 weeks, 4 weeks, and 3 months, respectively. Corneal samples were prepared for histology and fluorescence microscopy. To assess corneal cell death, proliferation, and myofibroblastic transdifferentiation, terminal uridine deoxynucleotidyl nick end-labeling (TUNEL) assay as well as immunostaining for Ki67 and α-smooth muscle actin (αSMA) were performed on sagittal cryosections. Results Histology revealed a zone of keratocyte depletion with a thickness of approximately 50 μm around the extraction site. At 48 hours, pronounced TUNEL staining of keratocytes was detected around the interface (159.9 cells/mm ± 18.4 [SD]), which steadily decreased to 74.9 ± 19.8 cells/mm at 1 week and 5.7 ± 4.8 cells/mm at 2 weeks. Ki67 staining of keratocytes was evident at 48 hours (10.0 ± 3.8 cells/mm), which then decreased at 1 week (5.2 ± 1.7 cells/mm) and 2 weeks (0.4 ± 0.5 cells/mm). From 4 weeks onward, no TUNEL or Ki67 staining was detected. The corneal stroma was αSMA-negative at all timepoints. Conclusion Application of the 345 nm laser showed no signs of problematic repair processes in the cornea, which supports the initiation of the clinical phase

Entwicklung und Umsetzung von neuen Anwendungsmöglichkeiten an einem Femtosekundenlaser für die Hornhautchirurgie sowie deren experimentelle Verifizierung anhand von Gewebeproben

Das Hauptanwendungsgebiet für einen Femtosekundenlaser in der refraktiven Chirurgie ist der Flapschnitt. In dieser Arbeit wurde ein optimiertes Patienteninterface für ein Ultrakurzpulslasersystem getestet. Das Patienteninterface dient der mechanischen Adaption des Lasers an das Auge und besteht aus einem Saugring und einem Applanationskegel, an dem das Auge angesaugt und applaniert wird. Im Rahmen der Untersuchungen wurden größtmögliche Flaps an Gewebeproben mit dem Patienteninterface ohne Komplikationen erzeugt. DesWeiteren wurden Kunststoffvarianten des Interfaces getestet, da dieses in ein Einmalprodukt überführt werden soll. Weitere Untersuchungen befassten sich mit dem Augeninnendruck während einer OP und verschiedenen Pulsenergien desLasers bzw. Flapdicken. Femtosekundenlaser können neben Flapschnittgeometrien noch beliebig andere Schnittgeometrien generieren. Weitere denkbare Anwendungsmöglichkeiten in der Hornhautchirurgie sind: Schnitte für Keratoplastiken, Tunnelschnitte für intracorneale Ringsegmente und bogenförmige Schnitte in die Cornea zur Korrektur von hohen Astigmatismen (Astigmatische Keratotomie). Im Rahmen der Diplomarbeit wurden für Schnitte der perforierenden Keratoplastik, wobei die Hornhaut komplett transplantiert wird, und für Schnitte der lamellären Keratoplastik, wobei einzelne Schichten der Hornhaut transplantiert werden, experimentell optimale Schnittparameter ermittelt. Dabei liegen die Pulsenergien etwas höher als beim Flapschnitt, die Linien- und Punktabstände für den Schnitt sind ähnlich. In einem letzten Punkt folgt eine theoretische Betrachtung zur Optimierung des Flapschnittes, wobei dieser keilförmig nach einer Myopiebehandlung gekürzt werden kann, da der Flap nach dem Laserabtrag im Stroma nicht mehr genau in das stromale Bett passt

Clinical and biological behaviour of vestibular schwannomas: signalling cascades involved in vestibular schwannoma resemble molecular and cellular mechanisms of injury-induced Schwann cell dedifferentiation.

Klenke C, Widera D, Sepehrnia A, et al. Clinical and biological behaviour of vestibular schwannomas: signalling cascades involved in vestibular schwannoma resemble molecular and cellular mechanisms of injury-induced Schwann cell dedifferentiation. Head & Neck Oncology. 2013;5(2):20

Inhibition of endogenous antagonists with an engineered BMP-2 variant increases BMP-2 efficacy in rat femoral defect healing

Bone morphogenetic proteins (BMP) have been used successfully by orthopedic clinicians to augment bone healing. However, these osteoinductive proteins must be applied at high concentrations to induce bone formation. The limited therapeutic efficacy may be due to the local expression of BMP antagonists such as Noggin that neutralize exogenous and endogenous BMPs. If so, inhibiting BMP antagonists may provide an attractive option to augment BMP induced bone formation. The engineered BMP-2 variant L51P is deficient in BMP receptor type I binding, but maintains its affinity for BMP receptor type II and BMP antagonists including Noggin, Chordin and Gremlin. This modification makes L51P a BMP receptor-inactive inhibitor of BMP antagonists. We implanted β-tricalcium phosphate ceramics loaded with BMP-2 and/or L51P into a critical size defect model in the rat femur to investigate whether the inhibition of BMP antagonist with L51P enhances the therapeutic efficacy of exogenous BMP-2. Our study reveals that L51P reduces the demand of exogenous BMP-2 to induce bone healing markedly, without promoting bone formation directly when applied alone

L51P - A BMP2 variant with osteoinductive activity via inhibition of Noggin

Bone morphogenetic proteins (BMP) have to be applied at high concentrations to stimulate bone healing. The limited therapeutic efficacy may be due to the local presence of BMP antagonists such as Noggin. Thus, inhibiting BMP antagonists is an attractive therapeutic option. We hypothesized that the engineered BMP2 variant L51P stimulates osteoinduction by antagonizing Noggin-mediated inhibition of BMP2. Primary murine osteoblasts (OB) were treated with L51P, BMP2, and Noggin. OB proliferation and differentiation were quantified with XTT and alkaline phosphatase (ALP) assays. BMP receptor dependent intracellular signaling in OB was evaluated with Smad and p38 MAPK phosphorylation assays. BMP2, Noggin, BMP receptor Ia/Ib/II, osteocalcin, and ALP mRNA expressions were analyzed with real-time PCR. L51P stimulated OB differentiation by blocking Noggin mediated inhibition of BMP2. L51P did not induce OB differentiation directly and did not activate BMP receptor dependent intracellular signaling via the Smad pathway. Treatment of OB cultures with BMP2 but not with L51P resulted in an increased expression of ALP, BMP2, and Noggin mRNA. By inhibiting the BMP antagonist Noggin, L51P enhances BMP2 activity and stimulates osteoinduction without exhibiting direct osteoinductive function. Indirect osteoinduction with L51P seems to be advantageous to osteoinduction with BMP2 as BMP2 stimulates the expression of Noggin thereby self-limiting its own osteoinductive activity. Treatment with L51P is the first protein-based approach available to augment BMP2 induced bone regeneration through inhibition of BMP antagonists. The described strategy may help to decrease the amounts of exogenous BMPs currently required to stimulate bone healing

1,8-Cineol Reduces Mucus-Production in a Novel Human <i>Ex Vivo</i> Model of Late Rhinosinusitis

<div><p>Inflammatory diseases of the respiratory system such as rhinosinusitis, chronic obstructive pulmonary disease, or bronchial asthma are strongly associated with overproduction and hypersecretion of mucus lining the epithelial airway surface. 1,8-cineol, the active ingredient of the pharmaceutical drug Soledum, is commonly applied for treating such inflammatory airway diseases. However, its potential effects on mucus overproduction still remain unclear.In the present study, we successfully established <i>ex vivo</i> cultures of human nasal turbinate slices to investigate the effects of 1,8-cineol on mucus hypersecretion in experimentally induced rhinosinusitis. The presence of acetyl-α-tubulin-positive cilia confirmed the integrity of the <i>ex vivo</i> cultured epithelium. Mucin-filled goblet cells were also detectable in nasal slice cultures, as revealed by Alcian Blue and Periodic acid-Schiff stainings. Treatment of nasal slice cultures with lipopolysaccharides mimicking bacterial infection as observed during late rhinosinusitis led to a significantly increased number of mucin-filled goblet cells. Notably, the number of mucin-filled goblet cells was found to be significantly decreased after co-treatment with 1,8-cineol. On a molecular level, real time PCR-analysis further showed 1,8-cineol to significantly reduce the expression levels of the mucin genes MUC2 and MUC19 in close association with significantly attenuated NF-κB-activity. In conclusion, we demonstrate for the first time a 1,8-cineol-dependent reduction of mucin-filled goblet cells and MUC2-gene expression associated with an attenuated NF-κB-activity in human nasal slice cultures. Our findings suggest that these effects partially account for the clinical benefits of 1,8-cineol-based therapy during rhinosinusitis. Therefore, topical application of 1,8-cineol may offer a novel therapeutic approach to reduce bacteria-induced mucus hypersecretion.</p></div

Cultured human nasal slices show unimpaired epithelium containing mucus-filled goblet cells.

<p><b>A,B</b>: Overview images of the established nasal slice culture system showing sliced nasal tissue cultured in culture plate inserts within a 12-well-plate. <b>C,D</b>: Immunohistochemical staining revealed the presence of acetyl-α-tubulin-positive cilia in nasal slice cultures. <b>E</b>: Hematoxylin and eosin-staining displayed the integrity of the <i>ex vivo</i> cultured epithelium containing ciliated epithelial cells (arrowheads), goblet cells (arrows) and a basal membrane (BM). Scale Bar: 20 μm. <b>F, G</b>: Mucin-filled goblet cells (arrows) were detected in cultivated nasal slices by Alcian Blue-staining and Periodic acid-Schiff stain. Scale Bar: 20 μm.</p

Schematic view on nasal epithelium containing mucus-filled goblet cells during LPS-induced rhinosinusitis and after co-treatment with 1,8-cineol.

<p>Co-treatment with LPS and 1,8-cineol leads to reduced production of mucin and decreased expression levels of mucin genes closely associated with attenuated NF-κB-activity.</p