Accuracy of cone-beam computed tomography imaging of the temporomandibular joint: comparisons with panoramic radiology and linear tomography.

INTRODUCTION: Cone-beam computed tomography (CBCT) is increasingly being used as an imaging modality, particularly in the assessment of the temporomandibular joint (TMJ). A blinded observational cross-sectional in-vitro study was conducted to compare the diagnostic accuracy of observers viewing images made with CBCT, panoramic radiography, and linear tomography. The task was to detect cortical erosions affecting the mandibular condylar head. METHODS: The sample consisted of 37 TMJ articulations from 30 skulls with either normal condylar morphology (n = 19) or erosion of the lateral pole (n = 18). The articulations were imaged by using corrected angle linear tomography (TOMO), normal (Pan-N) and TMJ-specific (Pan-TM) panoramic radiography, and CBCT. Digital images were obtained with photostimulable phosphor plates for all modalities except CBCT. The CBCT detector used an amorphous silicon flat-panel array combined with cesium iodide. Images and 10 rereads were presented to 10 observers on a flat-panel display at a pixel-to-monitor ratio of 1:1. CBCT multi-planar images were presented both statically (CBCT-S) and interactively (CBCT-I). The observers were permitted to scroll through axial (0.4 mm) and para-sagittal (1 mm) sections and then independently rate their confidence about the presence or absence of cortical erosion. Intraobserver reliability was determined by weighted kappa and diagnostic accuracy by the fitted area under the ROC curve. Means were compared by using ANOVA (P < or =.05). RESULTS: Intraobserver reliability was moderate (0.57 +/- 0.22; range, 0.34-0.78). Pan-N (0.72 +/- 0.15), CBCT-I (0.65 +/- 0.21), and CBCT-S (0.65 +/- 0.17) reliability was significantly greater than TOMO (0.44 +/- 0.25). The diagnostic accuracy of CBCT-I (0.95 +/- 0.05) and CBCT-S (0.77 +/- 0.17) was significantly greater than all other modalities (Pan-N [0.64 +/- 0.11], Pan-TM [0.55 +/- 0.11], TOMO [0.58 +/- 0.15]). CBCT-I was also more accurate than CBCT-S, and Pan-N was more accurate than Pan-TM and TOMO. CONCLUSIONS: CBCT images provide superior reliability and greater accuracy than TOMO and TMJ panoramic projections in the detection of condylar cortical erosion.

Human adult olfactory neural progenitors promote axotomized rubrospinal tract axonal reinnervation and locomotor recovery.

We investigated the effects of engrafted human adult olfactory neuroepithelial neurosphere forming cells (NSFCs) on regeneration and reinnervation of rubrospinal tract (RST) axons and locomotor recovery following partial cervical hemisection that completely ablated the RST. Weekly behavioral testing showed greater functional recovery of forelimb use during the 12 weeks after NSFCs engraftment than in the control rats. Anterograde tracing with biotinylated dextran amine (BDA) confirmed the presence of RST axons within the white matter 4-8 segments caudal to the grafts. Both immunofluorescence and immunoelectron microscopy revealed the BDA-labeled RST axonal terminals reestablished synaptic connections with motoneurons in the ventral horn of the distal cervical spinal cord. Further study of forelimb functional recovery in NSFCs-engrafted subgroups considered the effects of a second dorsolateral funiculotomy, irreversibly destroying the recovery, and the injection of muscimol, blocking RST neuronal activity reversibly. These results highlight the unique potential of human olfactory neuroepithelial-derived progenitors as an autologous cell source for spinal cord repair.

Induction of neuronal differentiation of adult human olfactory neuroepithelial-derived progenitors.

Neurosphere forming cells (NSFCs) have been established from cultures of adult olfactory neuroepithelium obtained from patients and cadavers as described previously. They remained undifferentiated in serum or defined media with or without neurotrophic factors. Many factors affect the differentiation of stem cells along a neuronal pathway. Retinoic acid (RA), forskolin (FN), and sonic hedgehog (Shh) have been reported to act as growth promoters during neurogenesis of embryonic CNS in vivo. The effect of RA, FN, and Shh on NSFCs' neuronal lineage restriction has not been described. The application of RA, FN, and Shh to NSFCs induced the expression of motoneuronal transcription factors, tyrosine hydroxylase, an indicator of dopamine production, and neurite formation. These studies further heighten the potential for using olfactory neuroepithelial progenitors for future autologous cell replacement strategies in neurodegenerative conditions and trauma as well as for use in diagnostic evaluation.

Role of transcription factors in motoneuron differentiation of adult human olfactory neuroepithelial-derived progenitors.

Neurosphereforming cell (NSFC) lines have been established from cultures of human adult olfactory neuroepithelium. Few of these cells ever express mature neuronal or glial markers in minimal essential medium supplemented with 10% fetal bovine serum or defined medium. However, these neural progenitors have the potential to differentiate along glial or neuronal lineages. To evaluate the potential of NSFCs to form motoneurons, transcription factors Olig2, Ngn2, and HB9 were introduced into NSFCs to determine if their expression is sufficient for motoneuron specification and differentiation, as has been shown in the early development of the avian and murine central nervous systems in vivo. NSFCs transfected with Olig2, Ngn2, and HB9 alone exhibited no phenotypic lineage restriction. In contrast, simultaneous transfection of Ngn2 and HB9 cDNA increased the expression of Isl1/2, a motoneuron marker, when the cells were maintained in medium supplemented with retinoic acid, forskolin, and sonic hedgehog. Furthermore, a population of Olig2-expressing NSFCs also expressed Ngn2. Cotransfection of NSFCs with Olig2 and HB9, but not Olig2 and Ngn2, increased Isl1/2 expression. Coculture of NSFCs trans-fected with Ngn2-HB92 or Olig2 and HB9 with purified chicken skeletal muscle demonstrated frequent contacts that resembled neuromuscular junctions. These studies demonstrate that transcription factors governing the early development of chick and mouse motoneuron formation are able to drive human adult olfactory neuroepithelial progenitors to differentiate into motoneurons in vitro. Our long-term goal is to develop cell populations for future studies of the therapeutic utility of these olfactory-derived NSFCs for autologous cell replacement strategies for central nervous system trauma and neurodegenerative diseases.

Human adult olfactory neuroepithelial derived progenitors retain telomerase activity and lack apoptotic activity.

Olfactory epithelium (OE) contains a population of progenitors responsible for its life-long regenerative capacity. Procedures for the isolation of these progenitors have been established [F.J. Roisen, K.M. Klueber, C.L. Lu, L.M. Hatcher, A. Dozier, C.B. Shields, Adult human olfactory stem cells, Brain Res., 890 (2001) 11-12.] and over 40 patient-specific cell lines from adult postmortem OE and endoscopic biopsy from patients undergoing nasal sinus surgery have been obtained. As these cells emerged in primary cultures, they formed neurospheres (NSFCs). The purpose of the present study was to further characterize these adult human olfactory-derived progenitors. Subcultures of the NSFCs have been passaged nearly 200 times, with a mitotic cycle of 18-20 h. Telomerase activity remains in stem cells; therefore, ELISA was employed to determine the telomerase activity of different lines and passages. Since progenitors undergo low levels of apoptosis, the levels of apoptosis were also examined in these populations. The levels of telomerase and apoptotic activity in 12 NSFC lines remained relatively constant irrespective of donor age, culture duration, or sex. To further study the apoptotic characteristics of the NSFCs, nine different caspases (cysteine proteases) known to be critical in apoptosis were evaluated using gene-microarrays comparing cells from a single line at passages 14, 88, and 183. No increases were found in caspase activity in all passages studied. ELISA confirmed the absence of caspase activity over the entire range of passages. This study further suggests that NSFCs can be obtained and used from patients, irrespective of age, sex, or time in culture without altered viability expanding the potential utility of these cells for autologous transplantation and possible diagnostic testing.

Human adult olfactory neural progenitors rescue axotomized rodent rubrospinal neurons and promote functional recovery.

Previously, our lab reported the isolation of patient-specific neurosphere-forming progenitor lines from human adult olfactory epithelium from cadavers as well as patients undergoing nasal sinus surgery. RT-PCR and ELISA demonstrated that the neurosphere-forming cells (NSFCs) produced BDNF. Since rubrospinal tract (RST) neurons have been shown to respond to exogenous BNDF, it was hypothesized that if the NSFCs remained viable following engraftment into traumatized spinal cord, they would rescue axotomized RS neurons from retrograde cell atrophy and promote functional recovery. One week after a partial cervical hemisection, GFP-labeled NSFCs suspended in Matrigel matrix or Matrigel matrix alone was injected into the lesion site. GFP-labeled cells survived up to 12 weeks in the lesion cavity or migrated within the ipsilateral white matter; the apparent number and mean somal area of fluorogold (FG)-labeled axotomized RST neurons were greater in the NSFC-engrafted rats than in lesion controls. Twelve weeks after engraftment, retrograde tracing with FG revealed that some RST neurons regenerated axons 4-5 segments caudal to the engraftment site; anterograde tracing with biotinylated dextran amine confirmed regeneration of RST axons through the transplants within the white matter for 3-6 segments caudal to the grafts. A few RST axons terminated in gray matter close to motoneurons. Matrix alone did not elicit regeneration. Behavioral analysis revealed that NSFC-engrafted rats displayed better performance during spontaneous vertical exploration and horizontal rope walking than lesion Matrigel only controls 11 weeks post transplantation. These results emphasize the unique potential of human olfactory neuroepithelial-derived progenitors as an autologous source of stem cells for spinal cord repair.

Induction of oligodendrocytes from adult human olfactory epithelial-derived progenitors by transcription factors.

Neurosphere-forming cell (NSFC) lines have been derived from cultures of adult olfactory neuroepithelium obtained from patients and cadavers. These progenitors remain undifferentiated when maintained in minimal essential medium with 10% fetal bovine serum, but have the potential to differentiate along glial or neuronal lineages. However, few of these cells ever express mature neuronal or glial markers in defined medium. To evaluate the potential of NSFCs to form oligodendrocytes, two transcription factors, Olig2 and Nkx2.2, were introduced into NSFCs to determine whether their expression is sufficient for oligodendrocyte differentiation, as has been shown in the embryonic avian and murine central nervous systems in vivo. NSFCs transfected with Olig2 or Nkx2.2 alone exhibited no phenotypic lineage restriction. In contrast, simultaneous transfection of Olig2 and Nkx2.2 cDNA produced characteristic oligodendrocyte morphology and antigenicity, including myelin basic protein (MBP). Furthermore, a population of Olig2-expressing NSFCs also expressed Sox10. Cotransfection of NSFCs with Nkx2.2 and Sox10, but not Olig2 and Sox10, produced a MBP(+) oligodendrocytic phenotype. Coculture of NSFCs transfected with Olig2 and Nkx2.2 or Nkx2.2 and Sox10 with purified sensory neurons, demonstrated frequent contacts between NSFC processes and axons, including the early stages of ensheathment. These studies demonstrate transcription factors governing early development of chick and mouse oligodendrocyte formation, also apply to human progenitors isolated from adult olfactory neuroepithelium. Our long-term goal is to develop cell populations for future studies used to determine the therapeutic utility of these olfactory-derived NSFCs for autologous transplantation into donors with central nervous system trauma or neurodegenerative diseases.

Endoscopic biopsy of human olfactory epithelium as a source of progenitor cells.

BACKGROUND: The adult central nervous system contains progenitor cells; however, invasive surgery is required for their harvest. Olfactory neuroepithelium (ONe) has attracted attention because it is extracranial and contains progenitor cells that account for its regenerative capacity. Olfactory progenitor cells have been cultured from postmortem ONe. Our aim was to determine if olfactory progenitors could be obtained via biopsy from patients in a feasible, effective, and safe manner. METHODS: Endoscopic biopsy was performed on individuals undergoing sinus surgery (n = 42). Olfactory function was assessed pre- and postoperatively. Specimens were cultured under conditions for olfactory progenitor cell development. RESULTS: Progenitor cells emerged in cultures from 50% of our patients. The superior turbinate, biopsied with cutting punch forceps, gave the highest yield. No adverse impact on olfaction or complications with the biopsy were observed. CONCLUSION: Endoscopic biopsy of ONe for obtaining olfactory progenitor cells from living donors is feasible, effective, and safe.

Adult human olfactory neural progenitors cultured in defined medium.

Neurosphere-forming cells (NSFCs) derived from primary cultures of adult human olfactory epithelium were established in minimum essential medium (MEM) with Hanks balanced salts and 10% heat-inactivated fetal bovine serum (FBS). A totally defined medium (DM) was employed to examine their proliferation, lineage restriction and differentiation. DMEM/F12 (DF) was found to support NSFCs and served as the base medium for this study. NSFCs were adapted to the DM through serial serum reductions at successive feedings. NSFCs in DF supplemented with N2, B27 or insulin attained saturation density and formed extensive processes. Immunolocalization of lineage specific markers [i.e., nestin, beta-tubulin III, peripherin, neural cell adhesion molecule, A2B5, O4, microtubule-associated-protein-2 (MAP2) and glial fibrillary acidic protein], as well as 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide and ornithine decarboxylase assays were employed to characterize the NSFCs. The effects of trophic factors including epidermal growth factor (EGF), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), neurotrophic factors (NT-3), and basic fibroblast growth factor (bFGF) were evaluated. With the reduction of serum and the addition N2, B27, and other nutrients, there was a change in lineage restriction including an increase the expression of A2B5 and other glial markers as well as the expression of mature neuronal markers with a simultaneous reduction of nestin reactivity. NSFCs proliferated and maintained their pluripotency for over a year in the DM. Further studies will determine the utility of NSFCs for cell replacement therapy.

Neuroanatomy for the dentist in the twenty-first century.

Both the anatomy and physiology parts of national boards have questions on neuroscience. Currently, there are course guidelines established for dental neuroanatomy but not for dental neuroscience. As a result, there is great variability in what and how neurosciences are taught to dental students. At first glance, it is difficult to determine where neurosciences fit in the dental curriculum. One area where there is a close tie between basic science and clinical care is the realm of pain control. Since the Institute of Medicine study recommended that basic and clinical sciences curricula provide clinically relevant education, a neuroscience curriculum can integrate basic understanding of how the nervous system works in the care and management of dental pain. This paper describes the integrated approach to teaching neuroanatomy as a component of the head and neck gross anatomy course at the University of Louisville. This integrated strategy provides dental students with the basic concepts of neuroscience, pain pathways, autonomic nervous system, and detailed information on the cranial nerves.