Multiscale analysis of craniomaxillofacial bone repair: A preclinical mini-pig study.

BACKGROUND: The rate of reparative osteogenesis controls when an implant is sufficiently stable as to allow functional loading. Using a mini pig model, the rate of reparative osteogenesis in two types of implant sites for example, an osteotomy versus a fresh extraction socket were compared. METHODS: Eight adult mini pigs were used for the study. In phase I, three premolars were extracted on one side of the oral cavity; 12 weeks later, in phase II, osteotomies were produced in healed extraction sites, and contralateral premolars were extracted. Animals were sacrificed 1, 5, and 12 weeks after phase II. Bone repair and remodeling were evaluated using quantitative micro-computed tomographic imaging, histology, and histochemical assays coupled with quantitative dynamic histomorphometry. RESULTS: One week after surgery, extraction sockets and osteotomy sites exhibited similar patterns of new bone deposition. Five weeks after surgery, mineral apposition rates (MARs) were elevated at the injury sites relative to intact bone. Twelve weeks after surgery, the density of new bone in both injury sites was equivalent to intact bone but quantitative dynamic histomorphometry and cellular activity assays demonstrated bone remodeling was still underway. CONCLUSIONS: The mechanisms and rates of reparative osteogenesis were equivalent between fresh extraction sockets and osteotomies. The volume of new bone required to fill a socket, however, was significantly greater than the volume required to fill an osteotomy. These data provide a framework for estimating the rate of reparative osteogenesis and the time to loading of implants placed in healed sites versus fresh extraction sockets.

A novel cryo-embedding method for in-depth analysis of craniofacial mini pig bone specimens.

The disconnect between preclinical and clinical results underscores the imperative for establishing good animal models, then gleaning all available data on efficacy, safety, and potential toxicities associated with a device or drug. Mini pigs are a commonly used animal model for testing orthopedic and dental devices because their skeletons are large enough to accommodate human-sized implants. The challenge comes with the analyses of their hard tissues: current methods are time-consuming, destructive, and largely limited to histological observations made from the analysis of very few tissue sections. We developed and employed cryo-based methods that preserved the microarchitecture and the cellular/molecular integrity of mini pig hard tissues, then demonstrated that the results of these histological, histochemical, immunohistochemical, and dynamic histomorphometric analyses e.g., mineral apposition rates were comparable with similar data from preclinical rodent models. Thus, the ability to assess static and dynamic bone states increases the translational value of mini pig and other large animal model studies. In sum, this method represents logical means to minimize the number of animals in a study while simultaneously maximizing the amount of information collected from each specimen.

Interspecies comparison of alveolar bone biology: Tooth extraction socket healing in mini pigs and mice.

BACKGROUND: in an effort to identify and validate which animal models are best suited for dental implant research, we used multiscale analyses to examine tooth extraction wound healing in a well-accepted model, the Yucatan mini pig and a more controversial model, the laboratory mouse. METHODS: first molar extractions were performed in adult, skeletally mature mini pigs and mice. Alveolar bone repair was evaluated at early, intermediate and late timepoints using quantitative micro-computed tomographic (µCT) imaging, histology, molecular, and cellular assays. Vital dye labeling was employed to quantify mineral apposition rates (MAR) in both species. RESULTS: Despite a 3000-fold difference in weight, the relative proportions of the mini pig and murine maxillae and are equivalent. Quantitative µCT demonstrated that within the posterior alveolar bone, the volume of mineralized bone was lower in mini pig than in the mice; during healing, however, the bone volume fraction was equivalent. The histologic appearance of healing sites was also comparable, and alkaline phosphatase (ALP) and tartrate resistant acid phosphatase (TRAP) staining showed a similar temporal and spatial distribution of bone remodeling. Vital dye labeling indicated equivalent MAR between the species. The absolute duration of the healing period differed: in mice, complete healing was accomplished in ∼21 days. In mini pigs, the same process took four times longer. CONCLUSIONS: Extraction socket healing is histologically equivalent between mini pigs and mice, supporting the hypothesis that the underlying mechanisms of alveolar bone healing are conserved among species.

A combination of topical antiseptics for the treatment of sore throat blocks voltage-gated neuronal sodium channels.

Amylmetacresol and dichloro-benzylalcohol are ingredients of lozenges used for the treatment of sore throat. In a former in vitro study, a local anaesthetic-like effect of these substances has been described. Since amylmetacresol and dichloro-benzylalcohol are co-administered in over-the-counter lozenges, the intention of this study is to evaluate the in vitro effects of the combination of these compounds on the voltage-gated sodium channel. We analysed the block of inward sodium currents induced by the combination of amylmetacresol, dichloro-benzylalcohol and the local anaesthetic lidocaine. Tonic and use-dependent block and effects on the inactivated channel state of the neuronal sodium channel were examined. Therefore, the α-subunit of the voltage-gated NaV1.2 sodium channel was heterologously expressed in HEK 293 cells in vitro. Inward sodium currents were investigated in the whole-cell configuration of the patch-clamp technique. The combination of amylmetacresol and dichloro-benzylalcohol and the combination of amylmetacresol and lidocaine induced a block of resting and inactivated sodium channels both displaying a pronounced block at the inactivated channel state. In addition, the combination of all three compounds also resulted in a voltage-dependent block of inward sodium currents. While use-dependent block by co-application of amylmetacresol and dichloro-benzylalcohol was moderate (<20 %), lidocaine and amylmetacresol induced a robust use-dependent block (up to 50 %). This study demonstrates local anaesthetic-like effects of a combination of amylmetacresol and dichloro-benzylalcohol as established ingredients of lozenges. In the presence of amylmetacresol, dichloro-benzylalcohol and lidocaine, a prominent block of inward sodium currents is apparent.

Inhibition of voltage-gated Na⁺ channels by the synthetic cannabinoid ajulemic acid.

BACKGROUND: The synthetic cannabinoid ajulemic acid has been demonstrated to alleviate pain in patients suffering from chronic neuropathic pain. Cannabinoids interact with several molecules within the pain circuit, including a potent inhibition of voltage-gated sodium channels. In this study, we closely characterized this property on neuronal and nonneuronal sodium channels. METHODS: The inhibition of sodium inward currents by ajulemic acid was studied in vitro. Human embryonic kidney 293t cells were used as the expression system for Nav1.2, 1.3, 1.4, 1.5, 1.5N406K, 1.5F1760A, and 1.7; Nav1.8 was transiently expressed in ND7/23 cells. Nav1.2, Nav1.3, and Nav 1.8 were from rats, and Nav1.4, Nav1.5, and Nav1.7 were of human origin. Sodium currents were analyzed by means of the whole cell patch-clamp technique. The investigated concentrations of ajulemic acid were 0.1, 0.3, 1, 3, 10, and 30 µmol/L. RESULTS: Ajulemic acid reversibly and concentration-dependently inhibited all voltage-gated sodium channel (Nav) isoforms investigated in this study, including Nav1.2, 1.3, 1.4, 1.5, 1.7, and 1.8. Tonic block of resting channels yielded half-maximal inhibitory concentration values between 2 and 9 µmol/L and was strongly enhanced on inactivated channels, suggesting state-dependent inhibition by ajulemic acid. Tonic block did not differ significantly when comparing Nav1.2 and Nav1.3, Nav1.4 and Nav1.5, and Nav1.7 and Nav1.8. Statistical analysis of other combinations of subunits (e.g., Nav1.2 and Nav1.4) by analysis of variance yielded a significant difference in block. Although we did not observe any relevant use-dependent block, ajulemic acid induced a strong hyperpolarizing shift of the voltage dependency of fast inactivation and modest shift of slow inactivation. The local anesthetic-insensitive Nav1.5 constructs N406K and F1760A displayed a preserved sensitivity to block by ajulemic acid. Finally, we found that low concentrations of ajulemic acid efficiently inhibited Navß4 peptide-mediated resurgent currents in Nav1.5. CONCLUSIONS: Our data suggest that block of sodium channels can be a relevant mechanism by which ajulemic acid alleviates neuropathic pain. The potent inhibition of resurgent currents and the preserved block on local anesthetic-insensitive channels indicates that ajulemic acid interacts with a conserved but yet unknown site of sodium channels.

Interaction of alfaxalone with the neuronal and the skeletal muscle sodium channel.

The neurosteroid alfaxalone exerts potent anesthetic activity in humans and animals. In former studies on myelinated axons, alfaxalone was assumed to produce a local anesthetic-like effect on the peripheral nervous system. Therefore,the present in vitro study aimed to characterize possible modulatory actions of alfaxalone on voltage-gated sodium channels. -Subunits of voltage-gated neuronal (Nav1.2)and skeletal muscle (Nav1.4) sodium channels were stably expressed in human embryonic kidney cells, and in vitro effects of alfaxalone were compared with lidocaine by means of the patch clamp technique. Alfaxalone preferentially blocked slow inactivated channels and therefore could provide membrane-stabilizing effects in ischemic/hypoxic tissues where slow inactivation is regarded to play a crucial role.