Visualization of bone formation in sheep's middle ear by using fluorochrome sequential labelling (FSL).

One factor for the lacking integration of the middle ear stapes footplate prosthesis or the missing healing of stapes footplate fractures could be the known osteogenic inactivity. In contrast, it was recently demonstrated that titanium prostheses with an applied collagen matrix and immobilised growth factors stimulate osteoblastic activation and differentiation on the stapes footplate. Regarding those findings, the aim of this study was to evaluate the potential of bone regeneration including bone remodeling in the middle ear. Ten one-year-old female merino sheep underwent a middle ear surgery without implantation of middle ear prostheses or any other component for activating bone formation. Post-operatively, four fluorochromes (tetracycline, alizarin complexion, calcein green and xylenol orange) were administered by subcutaneous injection at different time points after surgery (1 day: tetracycline, 7 days: alizarin, 14 days: calcein, 28 days: xylenol). After 12 weeks, the temporal bones including the lateral skull base were extracted and histologically analyzed. Fluorescence microscopy analysis of the entire stapes with the oval niche, but in particular stapes footplate and the Crura stapedis revealed evidence of new bone formation. Calcein was detected in all and xylenol in 60% of the animals. In contrast, tetracycline and alizarin could only be verified in two animals. The authors were able to demonstrate the osseoregenerative potential of the middle ear, in particular of the stapes footplate, using fluorescence sequence labelling.

Development of electrospun, biomimetic tympanic membrane implants with tunable mechanical and oscillatory properties for myringoplasty.

Most commonly, autologous grafts are used in tympanic membrane (TM) reconstruction. However, apart from the limited availability and the increased surgical risk, they cannot replicate the full functionality of the human TM properly. Hence, biomimetic synthetic TM implants have been developed in our project to overcome these drawbacks. These innovative TM implants are made from synthetic biopolymer polycaprolactone (PCL) and silk fibroin (SF) by electrospinning technology. Static and dynamic experiments have shown that the mechanical and oscillatory behavior of the TM implants can be tuned by adjusting the solution concentration, the SF and PCL mixing ratio and the electrospinning parameters. In addition, candidates for TM implants could have comparable acousto-mechanical properties to human TMs. Finally, these candidates were further validated in in vitro experiments by performing TM reconstruction in human cadaver temporal bones. The reconstructed TM with SF-PCL blend membranes fully recovered the acoustic vibration when the perforation was smaller than 50%. Furthermore, the handling, medium adhesion and transparency of the developed TM implants were similar to those of human TMs.

Histological evaluation of extraction sites grafted with Bio-Oss Collagen: Randomized controlled trial.

The combination of bovine bone matrix with collagen shows good results in bone and volume preservation after tooth extraction. To determine the ideal time to apply an implant after augmentation with Bio-Oss Collagen and to observe if there are differences in the age of the patients and the sex, the aim of this randomized controlled clinical trial was to compare the post-extraction changes in angiogenic and osteogenic aspects during spontaneous bone regeneration with those during socket preservation using Bio-Oss Collagen. Sixty-six patients were included in this study. After 8-12 weeks, bone biopsies were embedded in paraffin and histological and immune-histological investigated. Using qRT-PCR bone (Alpl, Bglap, Runx2) and angiogenic markers (VEGF, caveolin-1) were identified. The histomorphometric analysis of all examined samples showed no differences between treated and untreated sockets, but a tissue compression. After classification in bone regeneration stages, more samples with woven bone were present in treated sockets than in controls. The Alpl expression correlates with increase in mature bone tissue. In treated sockets a significant decrease in CD34 and caveolin-1 protein expression was found. Additionally, a significant increase of Runx2 and VEGF mRNA was detected in patients younger than 50 years. Thus, all specimens showed ossification in different stages after eight weeks of healing. The treated group gives an earlier stage of ossification than controls, but produces densified tissue with greater volume fraction. It can be assumed that successful implant placement in Bio-Oss Collagen augmented extraction sockets is possible after eight weeks of bone healing.

Influence of botulinum toxin A on craniofacial morphology after injection into the right masseter muscle of dystrophin deficient (mdx-) mice.

BACKGROUND: Severe craniofacial and dental abnormalities, typical for patients with progressive Duchenne muscular dystrophy (DMD), are an exellcent demonstration of Melvin L. Moss "functional matrix theory", highlighting the influence of muscle tissue on craniofacial growth and morphology. However, the currently best approved animal model for investigation of this interplay is the mdx-mouse, which offers only a limited time window for research, due to the ability of muscle regeneration, in contrast to the human course of the disease. The aim of this study was to evaluate craniofacial morphology after BTX-A induced muscle paralysis in C57Bl- and mdx-mice, to prove the suitability of BTX-A intervention to inhibit muscle regeneration in mdx-mice and thus, mimicking the human course of the DMD disease. METHODS: Paralysis of the right masseter muscle was induced in 100 days old C57Bl- and mdx-mice by a single specific intramuscular BTX-A injection. Mice skulls were obtained at 21 days and 42 days after BTX-A injection and 3D radiological evaluation was performed in order to measure various craniofacial dimensions in the sagittal, transversal and vertical plane. Statstical analysis were performed using SigmaStat®Version 3.5. In case of normal distribution, unpaired t-test and otherwise the Mann-Whitney-U test was applied. A statistical significance was given in case of p ≤ 0.05. RESULTS: In contrast to C57Bl-mice, in mdx-mice, three weeks after BTX-A treatment a significant decrease of skull dimensions was noted in most of the measurements followed by a significant increase at the second investigation period. CONCLUSIONS: BTX-A can induce changes in craniofacial morphology and presumably partially inhibit muscle regeneration in mdx-mice, but cannot completely intensify craniofacial effects elicited by dystrophy. Further research is necessary in order to fully understand muscle-bone interplay after BTX-A injection into dystrophic muscles.

Comparative effects of neurally adjusted ventilatory assist and variable pressure support on lung and diaphragmatic function in a model of acute respiratory distress syndrome: A randomised animal study.

BACKGROUND: Variable assisted mechanical ventilation has been shown to improve lung function and reduce lung injury. However, differences between extrinsic and intrinsic variability are unknown. OBJECTIVE: To investigate the effects of neurally adjusted ventilatory assist (NAVA, intrinsic variability), variable pressure support ventilation (Noisy PSV, extrinsic variability) and conventional pressure-controlled ventilation (PCV) on lung and diaphragmatic function and damage in experimental acute respiratory distress syndrome (ARDS). DESIGN: Randomised controlled animal study. SETTING: University Hospital Research Facility. SUBJECTS: A total of 24 juvenile female pigs. INTERVENTIONS: ARDS was induced by repetitive lung lavage and injurious ventilation. Animals were randomly assigned to 24 h of either: 1) NAVA, 2) Noisy PSV or 3) PCV (n=8 per group). Mechanical ventilation settings followed the ARDS Network recommendations. MEASUREMENTS: The primary outcome was histological lung damage. Secondary outcomes were respiratory variables and patterns, subject-ventilator asynchrony (SVA), pulmonary and diaphragmatic biomarkers, as well as diaphragmatic muscle atrophy and myosin isotypes. RESULTS: Global alveolar damage did not differ between groups, but NAVA resulted in less interstitial oedema in dorsal lung regions than Noisy PSV. Gas exchange and SVA incidence did not differ between groups. Compared with Noisy PSV, NAVA generated higher coefficients of variation of tidal volume and respiratory rate. During NAVA, only 40.4% of breaths were triggered by the electrical diaphragm signal. The IL-8 concentration in lung tissue was lower after NAVA compared with PCV and Noisy PSV, whereas Noisy PSV yielded lower type III procollagen mRNA expression than NAVA and PCV. Diaphragmatic muscle fibre diameters were smaller after PCV compared with assisted modes, whereas expression of myosin isotypes did not differ between groups. CONCLUSION: Noisy PSV and NAVA did not reduce global lung injury compared with PCV but affected different biomarkers and attenuated diaphragmatic atrophy. NAVA increased the respiratory variability; however, NAVA yielded a similar SVA incidence as Noisy PSV. TRIAL REGISTRATION: This trial was registered and approved by the Landesdirektion Dresden, Germany (AZ 24-9168.11-1/2012-2).

Effects of variable versus nonvariable controlled mechanical ventilation on pulmonary inflammation in experimental acute respiratory distress syndrome in pigs.

BACKGROUND: Mechanical ventilation with variable tidal volumes (VT) may improve lung function and reduce ventilator-induced lung injury in experimental acute respiratory distress syndrome (ARDS). However, previous investigations were limited to less than 6 h, and control groups did not follow clinical standards. We hypothesised that 24 h of mechanical ventilation with variable VT reduces pulmonary inflammation (as reflected by neutrophil infiltration), compared with standard protective, nonvariable ventilation. METHODS: Experimental ARDS was induced in 14 anaesthetised pigs with saline lung lavage followed by injurious mechanical ventilation. Pigs (n=7 per group) were randomly assigned to using variable VT or nonvariable VT modes of mechanical ventilation for 24 h. In both groups, ventilator settings including positive end-expiratory pressure and oxygen inspiratory fraction were adjusted according to the ARDS Network protocol. Pulmonary inflammation (primary endpoint) and perfusion were assessed by positron emission tomography using 2-deoxy-2-[18F]fluoro-d-glucose and 68Gallium (68Ga)-labelled microspheres, respectively. Gas exchange, respiratory mechanics, and haemodynamics were quantified. Lung aeration was determined using CT. RESULTS: The specific global uptake rate of 18F-FDG increased to a similar extent regardless of mode of mechanical ventilation (median uptake for variable VT=0.016 min-1 [inter-quartile range, 0.012-0.029] compared with median uptake for nonvariable VT=0.037 min-1 [0.008-0.053]; P=0.406). Gas exchange, respiratory mechanics, haemodynamics, and lung aeration and perfusion were similar in both variable and nonvariable VT ventilatory modes. CONCLUSION: In a porcine model of ARDS, 24 h of mechanical ventilation with variable VT did not attenuate pulmonary inflammation compared with standard protective mechanical ventilation with nonvariable VT.

Histological features of masticatory muscles after botulinum toxin A injection into the right masseter muscle of dystrophin deficient (mdx-) mice.

OBJECTIVE/BACKGROUND: The most frequently used animal model for human DMD (Duchenne muscular dystrophy) research is the mdx mouse. In both species, characteristic histological changes like inflammation, muscle fiber degeneration and fibrosis are the same, but in contrast to humans, in mdx mice, phases of muscle fiber degeneration are compensated by regeneration processes. AIM: Therefore, the interest of this study was to evaluate histological features in masticatory muscles after BTX-A injection into the right masseter muscle of wild type and dystrophic (mdx) mice, illustrating de- and regeneration processes induced by this substance. MATERIAL AND METHODS: The right masseter muscle of 100 days old healthy and mdx mice were selectively paralyzed by a single intramuscular BTX-A injection. Masseter as well as temporal muscle of injection and non-injection side were carefully dissected 21 days and 42 days after injection, respectively, and fiber diameter, cell nuclei position, necrosis and collagen content were analyzed histomorphologically in order to evaluate de- and regeneration processes in these muscles. Statistical analysis was performed using SigmaStat Software and Mann Whitney U-test (significance level: p < 0.05). RESULTS: At both investigation periods and in both mouse strains fiber diameter was significantly reduced and collagen content was significantly increased in the right injected masseter muscle whereas fiber diameters in mdx mice were much smaller, and these differences were even more apparent at the second investigation period. Necrosis and central located nuclei could generally be found in all mdx mice muscles investigated with an amount of centronucleation exceeding 60%, and a significant increase of necrosis six weeks after injection. In wild type mice central located nuclei could primarily be found in the treated masseter muscle with a portion of 2.7%, and this portion decreased after six weeks, whereas in mdx mice a decrease could also be seen in the non-injected muscles. In contrast, in wild type mice necrosis was not apparent at any time and in all muscles investigated. CONCLUSION: From our results it can be concluded that in mdx mice masticatory muscles de- and regeneration processes were extended, triggered by a selective BTX-A injection, or mdx mice at this age, independently of BTX-A treatment, went through another cycle of de- and regeneration as a characteristic of this disease.

Histological comparison between laser microtome sections and ground specimens of implant-containing tissues.

Evaluation of bone regeneration and peri-implant bone apposition can only be accomplished using laboratory techniques that allow assessment of decalcified hard tissue. It is known that 5-15µm thick sections can be prepared with the cutting-grinding technique, but their production causes a high material loss (≥0.5mm) between two sections and requires years of training and experience. With the development of the laser microtome it has become possible to cut decalcified bone without high sample material loss. Many scientific publications deal with the application possibilities of the individual methods So far, there is no comparison work between the cutting-grinding technique and laser microtomy. For this reason, new tissue sections were prepared by laser microtome and analyzed histologically from samples that had been previously been prepared by the cutting-grinding technique. Using both methods, it could be demonstrated that the different implants were completely surrounded by a connective tissue layer. In sections (50-100µm) produced by the routine cutting-grinding technique, magnifications up to 20× revealed no detailed histological information because cell structures could not be clearly identified. By contrast, laser microtome sections (10µm) revealed these information as e.g. osteocytes are already clearly visible at 10× magnification. Furthermore, the interface between implant and the surrounding bone could be clearly demonstrated due to visible demarcation between a capsule and connective tissue. At the histological level, laser microtome sections were clearly superior at thicknesses ≥30µm compared to sections produced by the cutting-grinding technique. In addition, laser microtomy has the advantages of time saving and markedly reduced sample loss, especially in cases of the production of serial sections.