The importance of stem cell engineering in head and neck oncology.

Head and neck squamous cell carcinoma is the sixth leading cause of cancer worldwide. The most common risk factors are carcinogens (tobacco, alcohol), and infection of the human papilloma virus. Surgery is still considered as the treatment of choice in case of head and neck cancer, followed by a reconstructive surgery to enhance the quality of life in the patients. However, the widespread use of artificial implants does not provide appropriate physiological activities and often cannot act as a long-term solution for the patients. Here we review the applicability of multiple stem cell types for tissue engineering of cartilage, trachea, vocal folds and nerves for head and neck injuries. The ability of the cells to self-renew and maintain their pluripotency state makes them an attractive tool in tissue engineering.

Evaluation of Cartilage Repair by Mesenchymal Stem Cells Seeded on a PEOT/PBT Scaffold in an Osteochondral Defect.

The main objective of this study was to evaluate the effectiveness of a mesenchymal stem cell (MSC)-seeded polyethylene-oxide-terephthalate/polybutylene-terephthalate (PEOT/PBT) scaffold for cartilage tissue repair in an osteochondral defect using a rabbit model. Material characterisation using scanning electron microscopy indicated that the scaffold had a 3D architecture characteristic of the additive manufacturing fabrication method, with a strut diameter of 296 ± 52 µm and a pore size of 512 ± 22 µm × 476 ± 25 µm × 180 ± 30 µm. In vitro optimisation revealed that the scaffold did not generate an adverse cell response, optimal cell loading conditions were achieved using 50 µg/ml fibronectin and a cell seeding density of 25 × 10(6) cells/ml and glycosaminoglycan (GAG) accumulation after 28 days culture in the presence of TGFß3 indicated positive chondrogenesis. Cell-seeded scaffolds were implanted in osteochondral defects for 12 weeks, with cell-free scaffolds and empty defects employed as controls. On examination of toluidine blue staining for chondrogenesis and GAG accumulation, both the empty defect and the cell-seeded scaffold appeared to promote repair. However, the empty defect and the cell-free scaffold stained positive for collagen type I or fibrocartilage, while the cell-seeded scaffold stained positive for collagen type II indicative of hyaline cartilage and was statistically better than the cell-free scaffold in the blinded histological evaluation. In summary, MSCs in combination with a 3D PEOT/PBT scaffold created a reparative environment for cartilage repair.

[Investigation of simple and complex prelamination of vascularized flaps in experiment].

Prelamination, permitting improvement not esthetics only, but also function of the damaged region, to create necessary, by contents and configuration, vascularized complexes of tissues, is applied for effective treatment of complex defects in tissues. In experiment on laboratory animals for prelamination of vascularized flaps were applied separate and combined placement of mucosal and cartilaginous autotransplants. Results of the investigation witness possibility and efficacy of creation of content--component complexes of tissues, owing necessary characteristics for their further application in the clinic during performance of reconstructive interventions.

The majority of myelinated and unmyelinated sensory nerve fibers that innervate bone express the tropomyosin receptor kinase A.

Although skeletal pain is a leading cause of chronic pain and disability, relatively little is known about the specific populations of nerve fibers that innervate the skeleton. Recent studies have reported that therapies blocking nerve growth factor (NGF) or its cognate receptor, tropomyosin receptor kinase A (TrkA) are efficacious in attenuating skeletal pain. A potential factor to consider when assessing the analgesic efficacy of targeting NGF-TrkA signaling in a pain state is the fraction of NGF-responsive TrkA+ nociceptors that innervate the tissue from which the pain is arising, as this innervation and the analgesic efficacy of targeting NGF-TrkA signaling may vary considerably from tissue to tissue. To explore this in the skeleton, tissue slices and whole mount preparations of the normal, adult mouse femur were analyzed using immunohistochemistry and confocal microscopy. Analysis of these preparations revealed that 80% of the unmyelinated/thinly myelinated sensory nerve fibers that express calcitonin gene-related peptide (CGRP) and innervate the periosteum, mineralized bone and bone marrow also express TrkA. Similarly, the majority of myelinated sensory nerve fibers that express neurofilament 200 kDa (NF200) which innervate the periosteum, mineralized bone and bone marrow also co-express TrkA. In the normal femur, the relative density of CGRP+, NF200+ and TrkA+ sensory nerve fibers per unit volume is: periosteum>bone marrow>mineralized bone>cartilage with the respective relative densities being 100:2:0.1:0. The observation that the majority of sensory nerve fibers innervating the skeleton express TrkA+, may in part explain why therapies that block NGF/TrkA pathway are highly efficacious in attenuating skeletal pain.

Involvement of calcitonin gene-related peptide innervation in the promoting effect of low-intensity pulsed ultrasound on spinal fusion without decortication.

STUDY DESIGN: A prospective left-right comparison designed experiment using a rabbit posterolateral intertransverse process fusion model. OBJECTIVE: To investigate the involvement of calcitonin gene-related peptide (CGRP) innervation in the promoting effect of low intensity pulsed ultrasound stimulation (LIPUS) on spinal fusion without decortication. SUMMARY OF BACKGROUND DATA: Sensory neuropeptide CGRP is involved in bone repair and ectopic ossification. Comparison of CGRP innervations in ectopic bone between sham LIPUS and LIPUS sides can help us to understand the relationship between sensory nerve innervation and LIPUS. METHODS: A total of 27 New Zealand white rabbits underwent bilateral posterolateral intertransverse process fusion with implantation of porous poly-D,L-lactic acid blocks loaded with 1.25 µg recombinant human bone morphogenetic protein-4 solution. One side was provided LIPUS daily whereas the other side served as control. Animals were killed and the operated lumbar vertebrae were harvested for histomorphologic evaluation at 3 days (n = 3), 1 week (n = 6), 3 weeks (n = 6), 7 weeks (n = 6), and 12 weeks (n = 6) following surgery, respectively. RESULTS: LIPUS accelerated the invasion of CGRP-positive nerve fibers during ectopic ossification spatially and temporally. Spatially, CGRP-positive nerve fibers were also observed in the new formed cartilage and bone tissues on LIPUS side, whereas they were only detected in the fibrous tissue and bone marrow on sham LIPUS side. Temporally, the density of CGRP-positive nerve fibers was significantly higher on the LIPUS side when compared with the sham LIPUS side. CONCLUSION: LIPUS promoted the invasion of CGRP sensory nerve in ectopic bone, which may in turn contribute to the promoting effect of LIPUS on ectopic ossification.

Case reports with special reference to the innervation, development and clinical significance of the subclavius posticus and the termination of the facial vein in South Indian cadavers

During the routine dissection of the head andneck, a variation in the termination of thecommon facial vein was noticed in two (a maleaged 93 years and a female aged 53 years)South Indian cadavers. In the male, an accessorymuscle, the subclavius posticus was alsoobserved in the left posterior triangle. Themuscle was attached posteriorly to the superiorangle and the adjoining part of the superiorborder of the scapula along with theattachment of the inferior belly of the omohyoidmuscle. Anteriorly, the muscle wasattached to the first costal cartilage. On itscourse, this muscle crossed the brachial plexusand subclavian vessels anteriorly and wasinnervated by the suprascapular nerve. Therelationship of this muscle to the neurovascularstructures in the neck should be borne inmind while accessing the thoracic outlet syndrome.In addition to this, the common facialvein joined the external jugular vein on theleft side.In the female cadaver the termination ofthe common facial vein was also into theexternal jugular vein on the left side, but noaccessory muscle was found. Knowledge of thevariation of the vein is important for the intravenouscanulation, for therapeutic or diagnosticpurposes, and for the surgeons performingreconstructive surgery (AU)

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Laryngeal muscle responses to mechanical displacement of the thyroid cartilage in humans.

Speakers may use laryngeal sensory feedback to adjust vocal fold tension and length before initiating voice. The mechanism for accurately initiating voice at an intended pitch is unknown, given the absence of laryngeal muscle spindles in animals and conflicting findings regarding their existence in humans. Previous reports of rapid changes in voice fundamental frequency following thyroid cartilage displacement suggest that changes in vocal fold length modulate laryngeal muscle contraction in humans. We tested the hypothesis that voice changes resulting from mechanical perturbation are due to rapid responses in the intrinsic laryngeal muscles. Hooked wire electrodes were used to record from the thyroarytenoid, cricothyroid, and sternothyroid muscles along with surface electrodes on the skin overlying the thyroid cartilage in 10 normal adults. Servomotor displacements produced consistent changes in the subjects' vocal fundamental frequency at 70-80 ms, demonstrating changes in vocal fold length and tension. No simultaneous electromyographic responses occurred in the thyroarytenoid or cricothyroid muscles in any subjects. Instead, short-latency responses at 25-40 ms following stimulus onset occurred in the sternothyroid muscles, simultaneous with responses in the surface recordings. The sternothyroid responses may modulate long-latency changes in voice fundamental frequency (approximately 150 ms). The absence of intrinsic laryngeal muscle responses is consistent with a lack of spindles in these muscles. Our results suggest that other sensory receptors, such as mucosal mechanoreceptors, provide feedback for voice control.

Neuropeptide effects on rat chondrocytes and perichondrial cells in vitro.

This study examines if cultured chondrocytes and perichondrial cells change the level of cAMP and/or cGMP in response to application of the neuropeptide calcitonin gene-related peptide (CGRP). Cells collected from the knee region of 4-8 days old rat pups were cultured in vitro. Cultures were exposed to 10(-10)-10(-6) M CGRP during 10 minutes. The levels of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) in the cultures and in controls were determined by radioimmunoassay. The results show that application of CGRP causes a distinctly increased level of cAMP, that was absent when CGRP was applied together with the CGRP(1) receptor antagonist. The level of cGMP was not obviously altered. Hence, it is possible that terminals of primary sensory neurones present in developing cartilage influence chondrocytes and perichondrial cells via local release of CGRP.

Neuropeptide content and physiological properties of rat cartilage-projecting sensory neurones co-cultured with perichondrial cells.

In young rats the cartilaginous epiphyses forming the knee joint are supplied with blood vessels and peptidergic sensory nerve fibres through the perichondrium and cartilage canals. In the present study we show that cartilage-related dorsal root ganglion neurones co-cultured with perichondrial cells develop extensive neurite trees and express calcitonin gene-related peptide (CGRP) and substance P (SP) in in vivo-like proportions using retrograde tracing and immunohistochemistry. Moreover, whole cell patch clamp recordings from these cells showed that the majority is depolarised by application of H(+)-ions. These results are compatible with the hypothesis that a local imbalance of blood flow and metabolism during normal skeletal maturation may cause tissue acidosis eliciting release of CGRP/SP from sensory nerve endings.

Bifid ribs observed in the third and the fourth ribs.

Three cases of bifid ribs were found in two cadavers during routine dissections at the Iwate Medical University School of Dentistry. All of the cases were found in the third or the fourth rib. The distal parts of the osseous rib bifurcated with an angle of 60 degrees and both of the branches had their own costal cartilage. The costal cartilage fused again to form the trunk which was connected to the sternum. The space between the two branches was filled with presumably normal intercostal muscles. Blood supply was maintained by a small branch from the interthoracic artery to the upper branches. However, the intercostal nerves did not branch toward the upper branch but only ran along the lower margins of the lower branches of the bifid ribs.

Sensory and sympathetic innervation of the vertebral endplate in patients with degenerative disc disease.

We obtained intervertebral discs with cartilage endplates and underlying cancellous bone at operation from patients with degenerative disc disease and then used immunohistochemical techniques to localise the nerves and nerve endings in the specimens. We used antibodies for the ubiquitous neuronal protein gene product 9.5 (PGP 9.5). Immunoreactivity to neuropeptide Y was used to identify autonomic nerves and calcitonin gene-related peptide (CGRP) and substance P to identify sensory nerves. Blood vessels were identified by immunoreactivity with platelet-endothelial cell-adhesion molecule (CD31; PECAM). In a control group with no known history of chronic back pain, nerve fibres immunoreactive to PGP 9.5 and neuropeptide Y were most closely related to blood vessels, with occasional substance P and CGRP immunoreactivity. In patients with severe back pain and markedly reduced disc height, proliferation of blood vessels and accompanying nerve fibres was observed in the endplate region and underlying vertebral bodies. Many of these nerves were immunoreactive to substance P or CGRP, and in addition, substance P- and CGRP-immunoreactive nociceptors were seen unrelated to blood vessels. Quantification by image analysis showed a marked increase in CGRP-containing sensory nerve fibres compared with normal control subjects. We speculate that a chemotactic response to products of disc breakdown is responsible for the proliferation of vascularity and CGRP-containing sensory nerves found in the endplate region and vertebral body adjacent to degenerate discs. The neuropeptides substance P and CGRP have potent vasodilatory as well as pain-transmitting effects. The increase in sensory nerve endings suggests increase in blood flow, perhaps as an attempt to augment the nutrition of the degenerate disc. The increase in the density of sensory nerves, and the presence of endplate cartilage defects, strongly suggest that the endplates and vertebral bodies are sources of pain; this may explain the severe pain on movement experienced by some patients with degenerative disc disease.

Discrimination between the different compartments in sciatic nerve by 2H double-quantum-filtered NMR.

The 2H double-quantum-filtered (DQF) NMR spectrum of isolated rat sciatic nerve, equilibrated with deuterated saline, is composed of three quadrupolar-split water signals. On the basis of the time course of their shift by Co-EDTA2- and CoCl2, the signals with quadrupolar splittings of about 120, 470, and 9 Hz were assigned to water in the epineurium, endoneurium, and intra-axonal compartments, respectively. The signal of the bulk water, which experiences isotropic motion, was eliminated by the DQF pulse sequence. As the maximum intensities of the water signals in the three anisotropic compartments occur at different creation times, in the DQF pulse sequence, it is possible to resolve the signals and measure their properties, such as relaxation times, independently, without perturbing the system with shift reagents.

Tooth agenesis in Down syndrome.

We studied the frequency and pattern of tooth agenesis in a Danish population with Down syndrome, trisomy 21 (46 females and 54 males). The control group consisted of a normal Danish population (2424 females and 2431 males) [Rølling, 1980: Scand J Dent Res 88:365-369; Ravn and Nielsen, 1973: Tandlaaegebladet 77:12-22]. We found that individuals with Down syndrome have an occurrence of agenesis that is some 10 times greater that in the general population with a higher frequency in males than in females. Agenesis occurred more frequently in the mandible than in the maxilla and most often on the left side. The highly significant differences were primarily found in the occurrence of agenesis of the mandibular central incisors, followed by the maxillary lateral incisors and second premolars and the mandibular second premolars. The main components in the pattern of agenesis observed in Down syndrome are supposed to be related to the peripheral nervous system and abnormal cartilagenous tissue. The present study on Down syndrome suggests that the dentition, with its many different anomalies, from agenesis to malformation, can be used as an indicator in evaluating different aspects in the patheogenetic of aneuploidy conditions.

Neurohistological and histochemical observations on the lung of Francolinus pondicerianus (gray partridge or safed teeter) as revealed by the cholinesterase technique.

An investigation was undertaken to demonstrate the neural elements of the lung of Francolinus pondicerianus, from the point of view of neurohistology and histochemistry. The staining of the neural elements was done by the cholinesterase technique with a maintained pH of 5.2, temperature 40 degrees C and incubation period of 19 h. Distribution of nerves in association with bronchial cartilage, pulmonary vessels and bronchi has been described and discussed. The distribution of the ganglia in association with blood vessels, bronchi, cartilage, various plexuses and the neural terminal terminal network has also been described. The innervation of the bronchi and their branches, and formation of the neural terminal network has been studied, as well as the distribution of cholinesterase in bronchi, blood vessels, muscles, ganglia, and nerve fibres.