Changes in caveolin-1, caveolin-3 and vascular endothelial growth factor expression and protein content after botulinum toxin A injection in the right masseter muscle of dystrophin deficient (mdx-) mice.

Progressive muscle wasting, frequently associated with inflammation, muscle fibre degeneration and fibrosis, is a characteristic of DMD (Duchenne muscular dystrophy). Its most common used animal model, the mdx mouse, however can overcome muscle degeneration by regeneration processes and is for this reason not suitable to answer all scientific questions. The aim of this study was to evaluate the ability of botulinum toxin A (BTX-A) in breaking down muscle regeneration in mdx mice. For this purpose, the right masseter muscle of 100 days old mdx and healthy mice was paralyzed by a single specific intramuscular injection of BTX-A. After 21 days, right and left masseter and temporal muscles as well as tongue muscle were carefully dissected, and gene and protein expression of caveolin-1, caveolin-3 and vascular endothelial growth factor (VEGF) were determined using quantitative RT-PCR and Western blot technique. Statistics were performed using Student's t-test and Mann Whitney U-test (significance level: P ≤ 0.05). After BTX-A injection, in both mice strains and for all three studied genes, no significant differences in mRNA amount could be detected between treated and untreated masseter muscles. A significant increase in caveolin-1, caveolin-3 and VEGF mRNA expression could only be found in the right temporal muscle of control mice compared to the left side. All three investigated proteins were more frequent to be found in dystrophic masseter muscle samples compared to the corresponding control samples, whereas significant decreased caveolin-3 protein levels could only be detected in the treated masseter versus untreated masseter muscle of controls. In contrast to previous conclusions, with this study it was not possible to prove a BTX-A-induced dystrophic phenotype in control animals, in which only the known decreases of caveolin-3 protein expression could be verified due to denervation. At the same time, however, gene and protein expression in dystrophic mice was not changed after BTX-A injection.

Differential expression of genes involved in the calcium homeostasis in masticatory muscles of MDX mice.

Duchenne Muscular Dystrophy (DMD) and its murine model, mdx, are characterized by Ca(2+) induced muscle damage and muscle weakness followed by distorted dentofacial morphology. In both, DMD patients and in mdx mice, could be proven so far that only the extraocular muscles (EOM) are not affected by muscular dystrophy. The EOMs are protected against calcium overload by enhanced expression of genes involved in the Ca(2+) homeostasis. We could recently demonstrate that masticatory muscles of mdx mice are differentially affected by muscle dystrophy. The dystrophic masseter and temporalis shows muscle histology comparable to all other skeletal muscles in this animal model, whereas dystrophic tongue muscles seem to develop a milder phenotype. Due to this fact it is to hypothesize that an altered Ca(2+) homeostasis seems to underlie the mdx masticatory muscle pathology. Aim of this study was to examine the mRNA and protein levels of the sarcoplasmic reticulum Ca(2+) ATPases SERCA1 and SERCA2, the plasma membrane Ca(2+) ATPases Atp2b1 and Atp2b4, the sodium/calcium exchanger NCX1, the ryanodine receptor 1, parvalbumin, sarcolipin, phospholamban and the L-type Ca(2+) channel alpha-1 subunit (Cacna1s) in Musculus masseter, temporalis, and tongue of 100 day old control and mdx mice. In mdx masseter muscle significant increased mRNA levels of NCX1 and Cacna1s were found compared to control mice. In contrast, the mRNA amount of RYR1 was significant reduced in mdx temporalis muscle, whereas ATP2b4 was significant increased. In mdx tongue a down-regulation of the ATP2b1, sarcolipin and parvalbumin mRNA expression was found, whereas the phospholamban mRNA level was significantly increased compared to controls. These data were verified by western blot analyses. Our findings revealed that mdx masticatory muscles showed an unequally altered expression of genes involved in the Ca(2+) homeostasis that can support the differences in masticatory muscles response to dystrophin deficiency.

Histological changes and changes in the myosin mRNA content of the porcine masticatory muscles after masseter treatment with botulinum toxin A.

OBJECTIVES: Botulinum toxin A (Botox) is increasingly used for treatment of muscle hyperfunction. For a better understanding of the possible morphologic and chewing changes in patients induced by a therapy with Botox, muscle fiber and myosin heavy chain (MyHC) mRNA alterations were examined in this animal study. MATERIALS AND METHODS: The investigation was carried out on 14-week-old pigs (seven treated animals, eight controls; calculated animal size with a power of 0.5). To initialise the total immobilisation of the right masseter, the Botox injection was distributed into ten areas. After a 56-day period, muscle tissue was taken from the left and right side of the masseter (three regions), temporal (two regions), medial pterygoid and geniohyoid muscles using a standardized method. The muscle fiber cross sections were examined immunohistochemically. Fiber staining was accomplished with antibodies to specific MyHC isoforms. The MyHC mRNA changes were analysed using real-time RT-PCR. RESULTS: Muscles adapt to such stress by changing fiber types and MyHC mRNA content. Paralysed masseters display atrophic changes while other masticatory muscles show hypertrophic changes. The results indicated that the typical distributions of type IIa und IIb fiber types in masticatory muscles were increased in the masseter muscles due to Botox application. On the other hand, the masseters without Botox in the treated group showed a significant increase of type I MyHC. CONCLUSIONS: Application of Botox may lead to uncontrolled structural changes in affected and unaffected muscles. CLINICAL RELEVANCE: Treatment of muscle hypertrophy with Botox may cause muscle imbalance.

Comparison of reference points in different methods of temporomandibular joint imaging.

PURPOSE: Conventional radiography is a well-established method for imaging of the temporomandibular joint (TMJ) structures. However, the dental computer tomography becomes more important for the visualization of teeth in the jaw-bone. The applicability of dental computer tomography for the visualization of the TMJ it not yet been proven. The aim of the study was to identify TMJ structures using reference points with the magnetic resonance imaging (MRI) and the computed tomography (CT). METHODS: In order to compare the visualization and measurement of the TMJ a total of eight human cadaver heads was examined with CT and MRI and analysed using reference points. RESULTS: In both imaging techniques the selected reference points and distances are well definable and allow objective evaluation of anatomical structures. The CT images display a clearly better contrast to noise ratio than the MR images. The distance measurement of different width and length showed significant correlation of both images techniques. CONCLUSIONS: In TMJ diagnostics, maximum information could be obtained using both imaging techniques together due to synergistic effects.

Changes in insulin like growth factors, myostatin and vascular endothelial growth factor in rat musculus latissimus dorsi by poly-3-hydroxybutyrate implants.

The present study aimed at researching the synergistic effect between an ectopic bone substitute and surrounding muscle tissue. To describe this effect, changes of insulin like growth factors (IGF1, IGF2), myostatin (GDF8) and vascular endothelial growth factor (VEGF) mRNA content of 12 Wistar-King rats musculus latissimus dorsi with implanted poly-3-hydroxybutyrate (PHB) scaffold were examined after 6 and 12 weeks. At each time interval six rats were killed and implants and surrounding tissues prepared for genetic evaluation. Eight rats without any implants served as controls. RNA was extracted from homogenized muscle tissue and reverse transcribed. Changes in mRNA content were measured by Real-Time PCR using specific primers for IGF1, IGF2, GDF8 and VEGF. Comparing the level of VEGF mRNA in muscle after 6 and 12 weeks to the controls, we could assess a significant increase of VEGF gene expression (p<0.05) whereas the level of mRNA expression was higher after 6 than after 12 weeks of treatment. Expression of IGF1 gene was also significantly increased as compared to the controls over the observed period of time (p<0.05). In the case of the IGF2 gene, the expression was significantly elevated after 6 weeks (p<0.05), but not significantly increased after 12 weeks (p>0.05). We observed a significantly decreased GDF8 gene expression (p<0.05) both after retrieval of implants after 6 as well as after 12 weeks. Moreover, mRNA level of GDF8 after 6 and 12 weeks were comparable the same. Our results show that PHB implants in rat musculus latissimus dorsi interact with the surrounding muscle tissue. This interaction works itself on growth potential of the muscle.

The effect of treatment with fentanyl patches on pain relief and improvement in overall daily functioning in patients with postherpetic neuralgia.

Postherpetic neuralgia often causes persistent pain, which can compromise patients' quality of life. The aim of the study was to examine the effect of pain reduction after the application of transdermal fentanyl on the quality of life of patients with postherpetic neuralgia. The study lasted 12 weeks. The study population included adult patients experiencing spontaneous pain, expressed by 4 points on the pain numeric rating scale (NRS). To assess quality of life questionnaire SF-36, the Zung depression scale and NRS were used. Pharmacotherapy included amitryptyline (Amitriptylinum VP) 10 mg orally once daily, gabapentine (Gabapentin TEVA 100 mg) 200-900 mg/24 hour orally twice daily and fentanyl skin patches (Durogesic) 12.5-100 microg/hour. Thirty four patients were qualified for the study. Comparison of pain intensity before the treatment and after 12 weeks showed that all the patients experienced relief. The treatment evidently reduced limitations in the physical and social domains. There was little improvement in the general physical capacity and mental condition. Analysis of correlation between changes in quality of life in the emotional, physical and social domains revealed that they were strongly interlinked. Increase in physical activity was accompanied by positive changes in the mental condition, better general wellbeing was correlated with increased social activity, and increased social activity was accompanied by improved physical condition. The more effective the treatment was, the more evident was improvement in the patients' quality of life in the physical, social and mental domains.

Morphological evaluation of bone defect regeneration after treatment with two different forms of bone substitution materials on the basis of BONITmatrix.

In the design of biomaterials for therapeutic application the evaluation of cellular/tissue responses play a key role. In this study, the in vivo bone-regenerative capacity and resorption of granular BONITmatrix and a paste-like bone substitution material on the basis of BONITmatrix were investigated in a rat cranial defect model. The results obtained with both biomaterials were compared to each other. For these, the paste-like composite and the granular BONITmatrix were implanted in adult male WOK-W rats, the skulls were harvested after eight weeks, and histopathological examinated. The comparison of the both tested biomaterials showed that the paste-like composite is much better to handle, the resorption of the material and the ossification process is much faster than those of granular BONITmatrix. The amount of newly formed bone was also measured and more bone formation was found in bone defects filled with the paste-like composite compared to those with granular BONITmatrix. The present study showed that both biomaterials could stimulate bone regeneration, but the paste-like composite leads in comparison to granular BONITmatrix to an accelerated more comprehensive bone regeneration.

Comparative animal study on hard tissue integration and bone formation of different Nobel Biocare implants.

Dental implantation aims at optimal and long-term hard tissue integration. Beside primary stability, loading time and other factors, e.g. the surface of the endosteal part of the implant, is a matter of special importance. In this animal trial, hard tissue integration of two different implant types was studied using radiological, histological and histomorphometric analysis. Two different implants with an oxidized surface (TiUnite; Nobel Biocare AB, Goteborg, Sweden, NobelReplace Tapered Groovy 4.3 x 10 mm and Replace Select Tapered 4.3 x 10 mm) were inserted into the right and left mandibles of 10 German domestic pigs between canine and premolar and immediately provided with a ceramic crown. The primary implant stability was determined using resonance frequency analysis. After 70 days, the test animals were killed and specimens were collected for histological and histomorphometric examination. All implants showed good primary stability after surgery. Histological and histomorphometrical analysis revealed no significant differences in the bone apposition. The immediate loading of the different implant types don't have any negative effects on the bone apposition in the period of 70 days. The long-term effects of immediate loading of these types of implant requires further studies.

Different bone sesitivity to malformations induced by procarbazine in fetal rats.

The study aimed at to induce cleft-lip-alveolus-palate (CLAP) applying procarbazine in rat fetuses at the 14(th) day of pregnancy, to supply thiocyanate and/or folic acid sufficient for preventive treatment and subsequently to investigate cleft extent in the palatal area as well as bone maturity. In this animal model, female primiparous inbred rats (LEW.1A) were used. The gravid animals were separated into treatment groups: group K (control), group P (procarbazine), group TP (thiocyanate and procarbazine) and group FTP (folic acid, thiocyanate, procarbazine). The results reveal that procarbazine may induce clefts in the palate area. Clefts occurred most frequently in group TP and mainly comprised subtotal clefts of the posterior secondary palate. As for palatal length, group FTP displayed the longest palate which was significantly different only from group K. A different picture was shown for the secondary palate with group TP displaying the shortest values which were significantly different from those in groups K, P, and FTP. Thus, group TP showed the most marked negative changes both for cleft frequency and palatal length as compared to group K and the other groups. The preventive application of either thiocyanate (TP) or thiocaynate and folic acid combined (group FTP) failed to completely prevent cleft formation in the palate area. In conclusion, a preventive effect on palatal clefts and growth inhibition could not be proved for the vitaminoid thiocyanate.

Physiological functions of the human finger.

Using morphological data describing the physiological curvature morphology of the corresponding articulating surfaces in each finger joint, it is shown that a) the flexion of each finger joint is described by two angles of flexion; b) in each finger joint, a "pump mechanism" for synovial fluid is present whose function is to lubricate and nourish the joint cartilage and c) finger posture has six kinematic degrees of freedom (DOF). Since six muscle forces control finger posture, the relationship between the muscle forces and finger posture is unambiguously described. The states of flexion of the interphalangeal joints restrict possible flexions in the metacarpophalangeal joint. Since the muscle forces act simultaneously on all three finger joints, the interdependence of the flexional states in the three finger joints can be attributed to the alignment of the lines of force and their sites of insertion, as a function of the corresponding flexion in the joints.

Evaluation of shape and size changes of bone and remodelled bone substitute after different fixation methods.

Suitable tissue fixation is indispensable to histological analysis. This investigation, therefore, sought to evaluate changes of shape and size of bone specimens and remodelled bone substitute material following different fixation methods. Mandibular bones of 9 pigs (Sus scrofa domesticus) served as specimens. Two mandibular premolars were extracted respectively and the extraction alveoli were filled with synthetic bone substitute material. The samples were collected after 70 days. Fixation of 6 specimens respectively was done for 7 days in 4% formalin (formaldehyde), 70% ethanol and glycerol at 18 degrees C room temperature. The samples were radiographically examined before and after fixation using a reference specimen and subsequently underwent histological analysis. After fixation in formalin, the samples showed no size changes. After fixation in glycerol, morphological analysis revealed minor shape changes. Fixation in ethanol causes shrinking of the tissue specimens. Histological inspection of the tissues shows no morphological changes except slight shrinking. In conclusion there is no universal fixative that could met all requirements and permited proper examination without affecting tissues or bone specimens.

A novel post-and-core restoration system--results of three years of clinical application of the "Wuerzburg Post".

The Wuerzburg Post is a new post-and-core restoration system designed to eliminate the weak parts of post-and-core restorations and the associated problems, respectively. In contrast to conventional posts, the Wuerzburg Post is a short and thick post, which no longer relies on cementation or luting for retention in the root, but on stress-free positive locking, which it achieves by means of a post which can be spread into a predefined and form-congruent undercut cavity. The second key feature is an annular groove which runs in the dentin, girded by a corresponding structure, ensuring regular force transmission and stress dissipation, as opposed to the classic ferrule design. There are two versions: one with a machined core which can be prepared like a classic build-up to support crowns and bridges, and another one with a 2.25 mm ball end to connect to common dies which can be integrated into removable prostheses. As the system utilizes prefabricated parts made from Titanium, a precise fit is ensured, enabling the user to restore teeth quickly and easily. Over the course of the past three years, 129 posts were inserted, most commonly on upper and lower incisors and canines. The main application was restoration of fractured telescopes. During the observation period, five failures were observed. Two of the failiures did not cause significant damage to the tooth, and were subsequently immediately repairable. The survival rate amounts to over 95% after three years under risk.

Adaptation of myosin heavy chain mRNA expression after implantation of poly(3)hydroxybutyrate scaffolds in rat m. latissimus dorsi.

The aim of this study is to identify the synergistic effect between an ectopic bone substitute and surrounding tissues, in this case muscle tissue, which is known to have a considerable potential for adaptation. To describe this effect, changes of myosin heavy chain (MyHC) isoform mRNA content of 12 Wistar-King rats m. latissimus dorsi with implanted poly(3)hydroxybutyrate (PHB) scaffolds were examined after six and 12 weeks. At each time interval six rats were killed and implants and surrounding tissues prepared for genetic evaluation. Eight rats without any implants served as controls. After homogenisation of muscle tissue, RNA was extracted and reverse transcribed. Changes in mRNA content were measured by Real-Time PCR using specific primers for type I MyHC, IIa, IIb and IIx isoforms. The mRNA level of myosin isoform type I of the muscles surrounding the implant was significantly increased (p<0.02) compared to the control group. Further, the studied muscle tissue showed a significant decrease in MyHC isoform IIx mRNA compared to the controls (p<0.02). Implantation of PHB scaffolds into rat m. latissimus dorsi causes an increase of its' content of slow myosin isoforms indicating a synergistic effect between the PHB scaffold and the surrounding muscle tissue.