Effects of a Novel Dental Gel on Enamel Surface Recovery from Acid Challenge.

BACKGROUND: Objective was to evaluate the in vivo effects of a novel dental gel (Livionex gelR) vs. a comparison dental gel on the surfaces of pre-eroded enamel chips. METHODS: On days 1-5, after toothbrushing with dentifrice, nine subjects each wore 8 enamel chips mounted on a palatal appliance for 4 h. Enamel blocks were pre-demineralized daily. After 2 day washout, subjects repeated the protocol using fresh chips and the second toothpaste on days 8-12. Samples were evaluated using electron microscopy. RESULTS: Ten standardized enamel surface photomicrographs/sample (total 1440 images) were evaluated for signs of erosion visually and on a scale of 0-3 by 1 evaluator. No significant differences were found between the 2 groups (p>0.32, 95% C.I.). Minimal surface erosion on approx. 15% of sample area was visible in both groups. CONCLUSION: The enamel surface appeared similar after usage of a test or control dentifrice. Based on this study, the test formulation did not affect enamel surface recovery from an erosive challenge. PRACTICAL IMPLICATIONS: Dentifrices can contribute to maintaining a healthy enamel surface. An all-natural dental gel formulation with novel anti-plaque mechanism achieved similar recovery from acid challenge to enamel as a control gel.

The effect of topical anesthetic hydration on the depth of thermal injury from the plasma skin regeneration device.

BACKGROUND AND OBJECTIVES: The plasma skin regeneration (PSR) device delivers thermal energy to the skin by converting nitrogen gas to plasma. Prior to treatment, hydration of the skin is recommended as it is thought to limit the zone of thermal damage. However, there is limited data on optimal hydration time. This pilot study aims to determine the effect of topical anesthetic application time on the depth of thermal injury from a PSR device using histology. STUDY DESIGN/MATERIALS AND METHODS: PSR (1.8 and 3.5 J) was performed after 0, 30, or 60 minutes of topical anesthetic application. Rhytidectomy was then performed and skin was fixed for histologic analysis. Four patients (two control and four treatment sites per patient) undergoing rhytidectomy were recruited for the study. Each patient served as his/her own control (no hydration). A scoring system for tissue injury was developed. Epidermal injury, the presence of vacuolization, blistering, damage to adnexal structures, and depth of dermal collagen changes were evaluated in over 1,400 high-power microscopy fields. RESULTS: There was a significant difference in the average thermal injury score, depth of thermal damage, and epidermal injury when comparing controls to 30 minutes of hydration (P = 0.012, 0.012, 0.017, respectively). There was no statistical difference between controls and 60 minutes of hydration or between 30 and 60 minutes of hydration. Epidermal vacuolization at low energy and patchy distribution of thermal injury was also observed. CONCLUSION: Topical hydration influences the amount of thermal damage when applied to skin for 30 minutes prior to treatment with the PSR device. There was a trend toward decreasing thermal damage at 60 minutes, and there was no difference between treatment for 30 or 60 minutes. The data suggest that application of topical anesthetic for a short period of time prior to treatment with the PSR device is cost-effective, safe, and may be clinically beneficial.

Spatial frequency domain imaging of burn wounds in a preclinical model of graded burn severity.

Frequent monitoring of early-stage burns is necessary for deciding optimal treatment and management. Both superficial and full thickness burns are relatively easy to diagnose based on clinical observation. In between these two extremes are superficial-partial thickness and deep-partial thickness burns. These burns, while visually similar, differ dramatically in terms of clinical treatment and are known to progress in severity over time. The objective of this study was to determine the potential of spatial frequency domain imaging (SFDI) for noninvasively mapping quantitative changes in chromophore and optical properties that may be an indicative of burn wound severity. A controlled protocol of graded burn severity was developed and applied to 17 rats. SFDI data was acquired at multiple near-infrared wavelengths over a course of 3 h. Burn severity was verified using hematoxylin and eosin histology. From this study, we found that changes in water concentration (edema), deoxygenated hemoglobin concentration, and optical scattering (tissue denaturation) to be statistically significant at differentiating superficial partial-thickness burns from deep-partial thickness burns.

The role of actin and myosin in PtK2 spindle length changes induced by laser microbeam irradiations across the spindle.

This study investigates spindle biomechanical properties to better understand how spindles function. In this report, laser microbeam cutting across mitotic spindles resulted in movement of spindle poles toward the spindle equator. The pole on the cut side moved first, the other pole moved later, resulting in a shorter but symmetric spindle. Intervening spindle microtubules bent and buckled during the equatorial movement of the poles. Because of this and because there were no detectable microtubules within the ablation zone, other cytoskeletal elements would seem to be involved in the equatorial movement of the poles. One possibility is actin and myosin since pharmacological poisoning of the actin-myosin system altered the equatorial movements of both irradiated and unirradiated poles. Immunofluorescence microscopy confirmed that actin, myosin and monophosphorylated myosin are associated with spindle fibers and showed that some actin and monophosphorylated myosin remained in the irradiated regions. Overall, our experiments suggest that actin, myosin and microtubules interact to control spindle length. We suggest that actin and myosin, possibly in conjunction with the spindle matrix, cause the irradiated pole to move toward the equator and that cross-talk between the two half spindles causes the unirradiated pole to move toward the equator until a balanced length is obtained.

Neuronal growth cones respond to laser-induced axonal damage.

Although it is well known that damage to neurons results in release of substances that inhibit axonal growth, release of chemical signals from damaged axons that attract axon growth cones has not been observed. In this study, a 532 nm 12 ns laser was focused to a diffraction-limited spot to produce site-specific damage to single goldfish axons in vitro. The axons underwent a localized decrease in thickness ('thinning') within seconds. Analysis by fluorescence and transmission electron microscopy indicated that there was no gross rupture of the cell membrane. Mitochondrial transport along the axonal cytoskeleton immediately stopped at the damage site, but recovered over several minutes. Within seconds of damage nearby growth cones extended filopodia towards the injury and were often observed to contact the damaged site. Turning of the growth cone towards the injured axon also was observed. Repair of the laser-induced damage was evidenced by recovery of the axon thickness as well as restoration of mitochondrial movement. We describe a new process of growth cone response to damaged axons. This has been possible through the interface of optics (laser subcellular surgery), fluorescence and electron microscopy, and a goldfish retinal ganglion cell culture model.

Analysis of DNA double-strand break response and chromatin structure in mitosis using laser microirradiation.

In this study the femtosecond near-IR and nanosecond green lasers are used to induce alterations in mitotic chromosomes. The subsequent double-strand break responses are studied. We show that both lasers are capable of creating comparable chromosomal alterations and that a phase paling observed within 1-2 s of laser exposure is associated with an alteration of chromatin as confirmed by serial section electron microscopy, DAPI, γH2AX and phospho-H3 staining. Additionally, the accumulation of dark material observed using phase contrast light microscopy (indicative of a change in refractive index of the chromatin) ∼ 34 s post-laser exposure corresponds spatially to the accumulation of Nbs1, Ku and ubiquitin. This study demonstrates that chromosomes selectively altered in mitosis initiate the DNA damage response within 30 s and that the accumulation of proteins are visually represented by phase-dark material at the irradiation site, allowing us to determine the fate of the damage as cells enter G1. These results occur with two widely different laser systems, making this approach to study DNA damage responses in the mitotic phase generally available to many different labs. Additionally, we present a summary of most of the published laser studies on chromosomes in order to provide a general guide of the lasers and operating parameters used by other laboratories.

An instruction on the in vivo shell-less chorioallantoic membrane 3-dimensional tumor spheroid model.

The traditional shell chicken chorioallantoic membrane (CAM) model has been used extensively in cancer research to study tumor growth and angiogenesis. Here we present a combined in vivo tumor spheroid and shell-less CAM three-dimensional model for use in quantitative and qualitative analysis. With this model, the angiogenic and tumorigenic environments can be generated locally without exogenous growth factors. This physiological model offers a stable, static and flat environment that features a large working area and wider field of view useful for imaging and biomedical engineering applications. The short experimental time frame allows for rapid data acquisition, screening and validation of biomedical devices. The method and application of this shell-less model are discussed in detail, providing a useful tool for biomedical engineering research.

Preliminary investigation on use of high-resolution optical coherence tomography to monitor injury and repair in the rat sciatic nerve.

BACKGROUND AND OBJECTIVE: Optical coherence tomography (OCT) has been used in limited settings to study peripheral nerve injury. The purpose of the study is to determine whether high-resolution OCT can be used to monitor nerve injury and regeneration in the rat sciatic nerve following crush injury, ligation, and transection with microsurgical repair. STUDY DESIGN/MATERIALS AND METHODS: Forty-five rats were segregated into three groups. The right sciatic nerve was suture ligated (n = 15), cut then microsurgically repaired (n = 15), or crushed (n = 15). The left sciatic nerve served as the control; only surgical exposure and skin closure were performed. Each group was further divided into three subgroups where they were assigned survival durations of 4, 15, or 24 weeks. Following euthanasia, nerves were harvested, fixed in formalin, and imaged at the injury site, as well as proximal and distal ends. The OCT system resolution was approximately 7 microm in tissue with a 1,060 nm central wavelength. RESULTS: Control (uninjured) nerve tissue showed homogenous signal distribution to a relatively uniform depth; in contrast, damaged nerves showed irregular signal distribution and intensity. Changes in signal distribution were most significant at the injury site and distal regions. Increases in signal irregularity were evident during longer recovery times. Histological analysis determined that OCT imaging was limited to the surrounding perineurium and scar tissue. CONCLUSION: OCT has the potential to be a valuable tool for monitoring nerve injury and repair, and the changes that accompany wound healing, providing clinicians with a non-invasive tool to treat nerve injuries.

Applications of a new In vivo tumor spheroid based shell-less chorioallantoic membrane 3-D model in bioengineering research.

The chicken chorioallantoic membrane (CAM) is a classical in vivo biological model in studies of angiogenesis. Combined with the right tumor system and experimental configuration this classical model can offer new approaches to investigating tumor processes. The increase in development of biotechnological devices for cancer diagnosis and treatment, calls for more sophisticated tumor models that can easily adapt to the technology, and provide a more accurate, stable and consistent platform for rapid quantitative and qualitative analysis. As we discuss a variety of applications of this novel in vivo tumor spheroid based shell-less CAM model in biomedical engineering research, we will show that it is extremely versatile and easily adaptable to an array of biomedical applications. The model is particularly useful in quantitative studies of the progression of avascular tumors into vascularized tumors in the CAM. Its environment is more stable, flat and has a large working area and wider field of view excellent for imaging and longitudinal studies. Finally, rapid data acquisition, screening and validation of biomedical devices and therapeutics are possible with the short experimental window.

Enhanced detection of early-stage oral cancer in vivo by optical coherence tomography using multimodal delivery of gold nanoparticles.

Contrast in optical coherence tomography (OCT) images can be enhanced by utilizing surface plasmon resonant gold nanoparticles. To improve the poor in vivo transport of gold nanoparticles through biological barriers, an efficient delivery strategy is needed. In this study, the improved penetration and distribution of gold nanoparticles were achieved by microneedle and ultrasound, respectively, and it was demonstrated that this multimodal delivery of antibody-conjugated PEGylated gold nanoparticles enhanced the contrast in in vivo OCT images of oral dysplasia in a hamster model.

Cartilage regeneration in the rabbit nasal septum.

OBJECTIVE: Rhinoplasty frequently includes harvesting of nasal septal cartilage. The objective of this prospective basic investigation is to determine whether cartilage can regenerate after submucosal resection (SMR) of the nasal septum in the rabbit. Neocartilage formation has not heretofore been described in this model. METHODS: By lateral rhinotomy, SMR was performed on 17 rabbits followed by reapproximation of the perichondrium. After 7 months, septi were fixed, sectioned, and examined histologically. Findings were photographed and data tabulated according to location and extent. RESULTS: Sites of matrix-secreting isogenous chondrocyte islands were identified between the perichondrial flaps of every animal, principally in the anterior inferior septum. The width of the islands averaged 190 microm, and the mean neocartilage height was found to be 840 microm. The newly formed cartilage consisted of chondrocytes within chondrons and was comparable in shape and structure to native septal cartilage. CONCLUSIONS: After SMR, rabbit cartilage tissue can regenerate and form matrix within the potential space created by surgery. The surrounding stem cell-rich perichondrium may be the site of origin for these chondrocytes. These findings suggest that after SMR of the human nasal septum, it may be possible for new cartilage tissue to develop provided the mucosa is well approximated. This biologic effect may be enhanced by insertion of cytokine-rich tissue scaffolds that exploit the native ability of septal perichondrium to regenerate and repair cartilage tissue.

Long-term viability and mechanical behavior following laser cartilage reshaping.

OBJECTIVE: To investigate the long-term in vivo effect of laser dosimetry on rabbit septal cartilage integrity, viability, and mechanical behavior. METHODS: Nasal septal cartilage specimens (control and irradiated pairs) were harvested from 18 rabbits. Specimens were mechanically deformed and irradiated with an Nd:YAG laser across a broad dosimetry range (4-8 W and 6-16 seconds). Treated specimens and controls were autologously implanted into a subperichondrial auricular pocket. Specimens were harvested an average +/- SD of 208 +/- 35 days later. Tissue integrity, histology, chondrocyte viability, and mechanical property evaluations were performed. Tissue damage results were compared with Monte Carlo simulation models. RESULTS: All laser-irradiated specimens demonstrated variable tissue resorption and calcification, which increased with increased dosimetry. Elastic moduli of the specimens were significantly either lower or higher than controls (all P<.05). Viability assays illustrated a total loss of viable chondrocytes within the laser-irradiated zones in all treated specimens. Histologic examination confirmed these findings. Experimental results were consistent with damage profiles determined using numerical simulations. CONCLUSION: The loss of structural integrity and chondrocyte viability observed across a broad dosimetry range underscores the importance of spatially selective heating methods prior to initiating application in human subjects.

Noninvasive measurement of ablation crater size and thermal injury after CO2 laser in the vocal cord with optical coherence tomography.

OBJECTIVE: To characterize tissue destruction after CO(2) laser-ablation of the vocal cords with the use of optical coherence tomography (OCT). STUDY DESIGN AND SETTING: OCT was used to image fresh porcine vocal cords after laser ablation. OCT and histology estimates of the ablation crater dimensions and the depth of thermal injury were obtained. RESULTS: The vocal cord substructures up to 2.29 mm in depth at 10 microm resolution, and the thermal disruption after laser ablation were identified by OCT. OCT and histology estimates of the lesion dimensions showed no significant differences. Crater depth is directly proportional to laser power, whereas crater width and the zone of thermal injury appear to be unrelated to laser power. CONCLUSIONS: OCT may be used to accurately characterize the native states and the laser-induced thermal injury of laryngeal mucosa, within the inherent limitation in its depth of penetration. OCT may be a useful diagnostic and monitoring tool in an otolaryngology practice.

Characterization of submucosal lesions using optical coherence tomography in the rabbit subglottis.

OBJECTIVE: To evaluate the efficacy of optical coherence tomography in differentiating between several simulated subglottic lesions, using an ex vivo rabbit laryngotracheal model. DESIGN: Laryngotracheal complexes were harvested from euthanized rabbits and divided into the following 4 groups: (1) control, (2) submucosal collagen injection (simulating scar formation), (3) dehydration and rehydration (simulating edema), and (4) repeated intubation trauma. The subglottic region was imaged using optical coherence tomography. Images were later correlated with conventional histologic findings. RESULTS: The epithelium, basement membrane, lamina propria, perichondrium, and cartilage (cricoid and tracheal) were clearly imaged. In group 2, an increase in the thickness of the lamina propria was observed, in addition to a characteristic optical pattern of the injected collagen. Dehydration (in group 3) produced a visible reduction in the thickness of the lamina propria, while rehydration of the same specimen with distilled water revealed a significant increase in submucosal swelling. Repeated intubation (in group 4) resulted in tissue edema that was seen as wavy heterogeneous thickening of the lamina propria. Edema produced by repeated intubation or distilled water immersion was easily differentiated from native and collagen-injected tissues. CONCLUSION: Optical coherence tomography successfully identifies the microstructure layers of the subglottis and can differentiate between edema and increased collagen deposition in the rabbit model.

Spatial distribution and function of sterol regulatory element-binding protein 1a and 2 homo- and heterodimers by in vivo two-photon imaging and spectroscopy fluorescence resonance energy transfer.

Sterol regulatory element-binding proteins (SREBPs) are a subfamily of basic helix-loop-helix-leucine zipper proteins that regulate lipid metabolism. We show novel evidence of the in vivo occurrence and subnuclear spatial localization of both exogenously expressed SREBP-1a and -2 homodimers and heterodimers obtained by two-photon imaging and spectroscopy fluorescence resonance energy transfer. SREBP-1a homodimers localize diffusely in the nucleus, whereas SREBP-2 homodimers and the SREBP-1a/SREBP-2 heterodimer localize predominantly to nuclear speckles or foci, with some cells showing a diffuse pattern. We also used tethered SREBP dimers to demonstrate that both homo- and heterodimeric SREBPs activate transcription in vivo. Ultrastructural analysis revealed that the punctate foci containing SREBP-2 are electron-dense nuclear bodies, similar or identical to structures containing the promyelocyte (PML) protein. Immunofluorescence studies suggest that a dynamic interplay exists between PML, as well as another component of the PML-containing nuclear body, SUMO-1, and SREBP-2 within these nuclear structures. These findings provide new insight into the overall process of transcriptional activation mediated by the SREBP family.

Detection and diagnosis of oral cancer by light-induced fluorescence.

BACKGROUND AND OBJECTIVE: New techniques for non-invasive early detection and diagnosis of oral dysplasia and carcinoma are required. Our objective was to determine in the hamster cheek pouch model whether differentiation between the healthy tissue and the different stages of oral premalignancy and malignancy is possible using laser-induced fluorescence after tissue exposure to 5-Aminolevulinic acid (ALA). STUDY DESIGN/MATERIALS AND METHODS: DMBA carcinogenesis was applied to one cheek pouch in 18 hamsters for 0-20 weeks. Prior to sacrifice, 20% ALA was applied to the cheek tissues. Excised cheek tissues were cryosectioned and imaged using fluorescence microscopy with excitation at 405 nm, detection at 635 nm. After fluorescence measurement, H&E staining and histopathological evaluation were performed. RESULTS: Fluorescence intensity was significantly lower in healthy tissue than in pathological tissues. Significantly higher intensities and more "fluorescence hot spots" occurred in severe dysplasia and carcinoma than in healthy tissue, hyperkeratosis, mild and moderate dysplasia. CONCLUSIONS: Light-induced fluorescence after ALA exposure can differentiate between the different stages of premalignancy and malignancy. Its ability to differentiate between healthy tissue and early pathology is particularly interesting

Laparoscopic photodynamic diagnosis of ovarian cancer using 5-aminolevulinic acid in a rat model.

OBJECTIVE: The objective of this study was to determine the efficacy and sensitivity of laparoscopic photodynamic diagnosis to detect 5-aminolevulinic acid (ALA)-induced fluorescent tumors in an animal model. METHODS: Cancer cells were injected into the peritoneum of rats to induce peritoneal carcinomatosis. After 3-4 weeks, ALA was administered to establish fluorescence in tumor nodules. All intraperitoneal surfaces were inspected using fluorescence and white light laparoscopy. Suspicious lesions were then biopsied in vivo under either fluorescence or white light laparoscopic guidance. Fluorescence intensities of the cancerous lesions compared to normal tissues were determined. A pathologist blinded to our clinical impression analyzed all biopsied specimens. We compared the sensitivity of fluorescence and white light laparoscopic-guided detection of cancerous lesions and determined the clinical utility of fluorescent photodynamic diagnosis in detecting metastatic ovarian cancer. RESULTS: Forty-three biopsies were performed in vivo under laparoscopic fluorescent guidance and 42 biopsies were taken using white light in various regions of the peritoneal surface from nine rats. Ten biopsies were also removed from nonfluorescent regions as nontumor controls. Cancerous lesions showed significantly higher fluorescent intensity compared to noncancerous lesions. Cancerous lesions that were difficult to differentiate from normal surrounding tissue under white light conditions were clearly detected by ALA-induced fluorescence. The average size of these metastatic lesions biopsied under fluorescent light was 1.0 mm (range: 0.3-2.5) compared to 1.5 mm (range: 0.5-2.9) with white light illumination (P < 0.05). CONCLUSIONS: Fluorescent laparoscopic detection of micrometastatic ovarian cancer using ALA is significantly more sensitive than white-light laparoscopy in detecting smaller cancerous lesions in an ovarian cancer rat model. Human trials are indicated.