Fluorescence spectroscopy of epithelial tissue throughout the dysplasia-carcinoma sequence in an animal model: spectroscopic changes precede morphologic changes

BACKGROUND AND OBJECTIVE: Recent studies have indicated that chondrocyte viability decreases with prolonged or repeated laser irradiation. To optimize laser-mediated cartilage reshaping, the heating process must be finely controlled. In this study, we use high-power Nd:YAG laser irradiation (lambda = 1.32 microm) combined with cryogen spray cooling (CSC) in an attempt to reshape porcine septal cartilage while enhancing chondrocyte viability. STUDY DESIGN/MATERIALS AND METHODS: Chondrocyte viability was determined after high-power (50 W/cm2) Nd:YAG-mediated cartilage reshaping with and without cryogen spray cooling (CSC) and correlated with dynamic measurements of tissue optical and thermal properties. RESULTS: After 1.5 to 2.0 seconds of laser exposure, characteristic changes in diffuse reflectance (indicating the onset of accelerated stress relaxation) was observed in both laser only and laser with CSC specimens. After 2 seconds of laser exposure, specimens in both groups retained the curved shape for up to 14 days. After one laser exposure, chondrocyte viability was 94.35 +/- 6.1% with CSC and 68.77 +/- 20.1% (P < 0.05) without CSC. After two laser exposures, a similar trend was observed with CSC (70.18 +/- 16.44%) opposed to without CSC (28 +/- 45%; P < 0.05). CONCLUSION: CSC during high-power laser irradiation allows rapid heating while minimizing extreme front surface temperature elevations and axial thermal gradients. Laser irradiation with CSC can be used to effectively reshape cartilage tissue with the additional advantage of increasing chondrocyte viability.     

Shape retention in porcine-septal cartilage following Nd:YAG (lambda = 1.32 microm) laser-mediated reshaping

BACKGROUND AND OBJECTIVE: Photothermal heating of mechanically deformed cartilage accelerates stress relaxation and results in sustained shape change. In this study, shape retention was measured in Nd:YAG laser reshaped porcine septal cartilage. MATERIALS AND METHODS: Specimens were laser reshaped either 4 (Group I) or 28 hours (Group II) following extraction from the crania. Specimens were bent into approximately semicircular shapes and irradiated half way between the endpoints of the semicircle. Resultant bend angle was calculated based on linear measurements. Shape retention was calculated by comparing resultant curvature with pre-irradiation measurements. RESULTS: Mechanical deformation alone resulted in initial bend angles varying from 188 degrees to 229 degrees. Resultant bend angles varied from 84 degrees to 194 degrees corresponding to shape retention varying from 58 to 75%. Non-irradiated cartilage retained less than 46% of the original bend. Shape retention was greater in Group II, compared to Group I. In Group I, no cephalocranial difference in shape retention was observed, though in Group II greater shape retention was observed in rostral specimens. CONCLUSION: While laser heating does significantly reshape cartilage, clinical use of this technology will require "overbending" of the cartilage graft to compensate for this memory effect. The degree of overbending is likely to vary with cartilage type and location.     

Laser solder welding of articular cartilage: tensile strength and chondrocyte viability.

BACKGROUND AND OBJECTIVE: The surgical treatment of full-thickness cartilage defects in the knee joint remains a therapeutic challenge. Recently, new techniques for articular cartilage transplantation, such as mosaicplasty, have become available for cartilage repair. The long-term success of these techniques, however, depends not only on the chondrocyte viability but also on a lateral integration of the implant. The goal of this study was to evaluate the feasibility of cartilage welding by using albumin solder that was dye-enhanced to allow coagulation with 808-nm laser diode irradiation. STUDY DESIGN/MATERIALS AND METHODS: Conventional histology of light microscopy was compared with a viability staining to precisely determine the extent of thermal damage after laser welding. Indocyanine green (ICG) enhanced albumin solder (25% albumin, 0.5% HA, 0.1% ICG) was used for articular cartilage welding. For coagulation, the solder was irradiated through the cartilage implant by 808-nm laser light and the tensile strength of the weld was measured. RESULTS: Viability staining revealed a thermal damage of typically 500 m in depth at an irradiance of approximately 10 W/cm(2) for 8 seconds, whereas conventional histologies showed only half of the extent found by the viability test. Heat-bath investigations revealed a threshold temperature of minimum 54 degrees C for thermal damage of chondrocytes. Efficient cartilage bonding was obtained by using bovine albumin solder as adhesive. Maximum tensile strength of more than 10 N/cm(2) was achieved. CONCLUSIONS: Viability tests revealed that the thermal damage is much greater (up to twice) than expected after light microscopic characterization. This study shows the feasibility to strongly laser weld cartilage on cartilage by use of a dye-enhanced albumin solder. Possibilities to reduce the range of damage are suggested.     

Identification of chondrocyte proliferation following laser irradiation, thermal injury, and mechanical trauma

BACKGROUND AND OBJECTIVE: Cartilage has a limited regenerative capacity, and there are a lack of reliable techniques and methods to stimulate growth of new tissue to treat degenerative diseases and trauma. This study focused on identifying chondrocyte cell proliferation in ex vivo cartilage tissue following heating Nd:YAG laser using whole-mount analysis and flow cytometry, and compared findings with results produced by contact, and water bath heating methods, mechanical injury, and the addition of transforming growth factor-beta (TGF-beta). STUDY DESIGN/MATERIALS AND METHODS: Ex vivo rabbit nasal septal cartilages were either irradiated with an Nd:YAG laser (lambda = 1.32 microm, 2-16 seconds, 6 W/cm(2)), heated by immersion in a warm saline bath, heated by direct contact with a metal rod, or mechanically damaged by scoring with a scalpel or crushing. After treatment, specimens were incubated for 7 or 14 days in growth media containing 10 microM bromodeoxyuridine (BrdU). Additional specimens were cultured with both BrdU and TGF-beta. Both whole-mount BrdU-double-antibody detection techniques and flow cytometry were used to determine the presence of DNA replication as a marker of proliferation. RESULT: An annular region of regenerating chondrocytes was identified surrounding the laser irradiation zone in whole-mount tissue specimens, and the diameter of this region increased with irradiation time. Using whole-mount analysis, no evidence of chondrocyte DNA replication was observed in tissues heated using non-laser methods, grown in TGF-beta, or mechanically traumatized. In contrast, flow cytometry identified the presence of BrdU-positive cells in the S-phase of the cell cycle (synthesis of DNA) for all protocols, indicating chondrocyte proliferation. The percentage of cells that are in S-phase increased with irradiation time. CONCLUSION: These data provide evidence that laser irradiation, along with other thermal and mechanical treatments, causes a proliferative response in chondrocytes, and this is observed ex vivo in the absence of cellular and humoral repair mechanisms. The advantage of using optical methods to generate heat in cartilage is that microspot injuries could be created in tissue and scanned across surfaces in clinical applications.     

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.     

Viability and volume of in situ bovine articular chondrocytes-changes following a single impact and effects of medium osmolarity

OBJECTIVE: Mechanical stress above the physiological range can profoundly influence articular cartilage causing matrix damage, changes to chondrocyte metabolism and cell injury/death. It has also been implicated as a risk factor in the development of osteoarthritis (OA). The mechanism of cell damage is not understood, but chondrocyte volume could be a determinant of the sensitivity and subsequent response to load. For example, in OA, it is possible that the chondrocyte swelling that occurs renders the cells more sensitive to the damaging effects of mechanical stress. This study had two aims: (1) to investigate the changes to the volume and viability of in situ chondrocytes near an injury to cartilage resulting from a single blunt impact, and (2) to determine if alterations to chondrocyte volume at the time of impact influenced cell viability. METHODS: Explants of bovine articular cartilage were incubated with the fluorescent indicators calcein-AM and propidium iodide permitting the measurement of cell volume and viability, respectively, using confocal laser scanning microscopy (CLSM). Cartilage was then subjected to a single impact (optimally 100g from 10 cm) delivered from a drop tower which caused areas of chondrocyte injury/death within the superficial zone (SZ). The presence of lactate dehydrogenase (LDH; an enzyme released following cell injury) was used to determine the effects of medium osmolarity on the response of chondrocytes to a single impact. RESULTS: A single impact caused discrete areas of chondrocyte injury/death which were almost exclusively within the SZ of cartilage. There appeared to be two phases of cell death, a rapid phase lasting approximately 3 min, followed by a slower progressive 'wave of cell death' away from the initial area lasting for approximately 20 min. The volume of the majority (88.1+/-5.99% (n=7) of the viable chondrocytes in this region decreased significantly (P<0.006). By monitoring LDH release, a single impact 5 min after changing the culture medium to hyper-, or hypo-osmolarity, reduced or stimulated chondrocyte injury, respectively. CONCLUSIONS: A single impact caused temporal and spatial changes to in situ chondrocyte viability with cell shrinkage occurring in the majority of cells. However, chondrocyte shrinkage by raising medium osmolarity at the time of impact protected the cells from injury, whereas swollen chondrocytes were markedly more sensitive. These data showed that chondrocyte volume could be an important determinant of the sensitivity and response of in situ chondrocytes to mechanical stress.     

Shape retention in porcine and rabbit nasal septal cartilage using saline bath immersion and Nd:YAG laser irradiation

BACKGROUND AND OBJECTIVES: The process of altering the shape of cartilage using heat has been referred to as thermoforming, and presents certain clinical benefits in reconstructive surgical procedures within the head and neck. Thermoforming allows cartilage in the upper airway and face to be reshaped without the use of classic surgical maneuvers such as carving, morselizing, or suturing. The goal of this study was to determine the dependence of cartilage shape change on both temperature and laser dosimetry using two thermoforming methods: saline bath immersion and laser irradiation. STUDY DESIGN/MATERIALS AND METHODS: Ex-vivo rabbit and porcine nasal septal cartilages were mechanically deformed and reshaped using the two thermoforming methods. With saline bath immersion using rabbit cartilage, each specimen was deformed by securing it to a small copper tube (outer diameter 8 mm) using dental bands. For porcine cartilage immersed in a saline bath, each sample was mechanically deformed between two pieces of wire mesh attached to a semicircular acrylic block. With both porcine and rabbit cartilage, the specimen and apparatus were then immersed in a hot saline bath for time intervals varying from 20 and 320 seconds and at constant temperatures between 62 and 74 degrees C. In laser reshaping, the cartilage specimens were mechanically deformed on a jig and consecutively irradiated with an Nd:YAG laser (lambda = 1.32 microm) in several spots for 6-16 seconds and irradiances of 10.2-40.7 W/cm2 per spot. After either saline bath heating or irradiation, cartilage specimens were immersed in room temperature saline for 15 minutes, then upon removal from the jig the length between the ends of each specimen was measured in order to calculate the resulting bend angle. RESULTS: The transition zone for cartilage reshaping was defined as where a significant increase in bend angle was observed between consecutive times of immersion/irradiation at the same temperature/irradiance. For the saline bath experiments, the transition zone was observed between 59-68 degrees C and 62-68 degrees C for porcine and rabbit cartilage, respectively. Similar transition zones occurred with laser irradiation below irradiances of 20.4 W/cm2 for both porcine and rabbit cartilage. In addition, the dosimetry pairs in the transition zones produce peak temperatures below the thresholds determined from the saline bath immersion studies. CONCLUSIONS: The critical transition temperature region was determined by the sharp increase in bend angle at consecutive times of immersion at the same temperature. This range was determined to be 59-68 degrees C and 62-68 degrees C for porcine and rabbit cartilage, respectively. Similar transition zones for dosimetry occurred below 20.4 W/cm2 during cartilage irradiation in both species.     

Temperature dependent change in equilibrium elastic modulus after thermally induced stress relaxation in porcine septal cartilage

BACKGROUND AND OBJECTIVES: Laser cartilage reshaping (LCR) is a promising method for the in situ treatment of structural deformities in the nasal septum, external ear and trachea. Laser heating leads to changes in cartilage mechanical properties and produces relaxation of internal stress allowing formation of a new stable shape. While some animal and preliminary human studies have demonstrated clinical feasibility of LCR, application of the method outside specialized centers requires a better understanding of the evolution of cartilage mechanical properties with temperature. The purpose of this study was to (1) develop a method for reliable evaluation of mechanical changes in the porcine septal cartilage undergoing stress relaxation during laser heating and (2) model the mechanical changes in cartilage at steady state following laser heating. STUDY DESIGN/MATERIALS AND METHODS: Rectangular cartilage specimens harvested from porcine septum were heated uniformly by a radio-frequency (RF) electric field (500 kHz) for 8 and 12 seconds to maximum temperatures from 50 to 90 degrees C. Cylindrical samples were fashioned from the heated specimens and their equilibrium elastic modulus was measured in a step unconfined compression experiment. Functional dependencies of the elastic modulus and maximum temperature were interpolated from the measurements. Profiles of the elastic modulus produced after 8 and 12 seconds of laser irradiation (Nd:YAG, lambda = 1.34 microm, spot diameter 4.8 mm, laser power 8 W) were calculated from interpolation functions and surface temperature histories measured with a thermal camera. The calculated elastic modulus profiles were incorporated into a numerical model of uniaxial unconfined compression of laser irradiated cylindrical samples. The reaction force to a 0.1 compressive strain was calculated and compared with the reaction force obtained in analogous mechanical measurements experiment. RESULTS: RF heating of rectangular cartilage sample produces a spatially uniform temperature field (temperature variations < or = 4 degrees C) in a central region of the sample which is also large enough for reliable mechanical testing. Output power adjustment of the RF generator allows production of temperature histories that are very similar to those produced by laser heating at temperatures above 60 degrees C. This allows creation of RF cartilage samples with mechanical properties similar to laser irradiated cartilage, however with a spatially uniform temperature field. Cartilage equilibrium elastic modulus as a function of peak temperature were obtained from the mechanical testing of RF heated samples. In the temperature interval from 60 to 80 degrees C, the equilibrium modulus decreased from 0.08+/- 0.01 MPa to 0.016+/-0.007 MPa, respectively. The results of the numerical simulation of uniaxial compression of laser heated samples demonstrate good correlation with experimentally obtained reaction force. CONCLUSIONS: The thermal history and corresponding thermally induced modification of mechanical properties of laser irradiated septal cartilage can be mimicked by heating tissue samples with RF electric current with the added advantage of a uniform temperature profile. The spatial distribution of the mechanical properties obtained in septal cartilage after laser irradiation could be computed from mechanical testing of RF heated samples and used for numerical simulation of LCR procedure. Generalization of this methodology to incorporate orthogonal mechanical properties may aid in optimizing clinical laser cartilage reshaping procedures.     

Analysis of Nd:YAG laser-mediated thermal damage in rabbit nasal septal cartilage

BACKGROUND AND OBJECTIVES: Laser cartilage reshaping (LCR) involves the use of photo-thermal heating to reshape cartilage. Its clinical relevance depends on the ability to minimize thermal injury in irradiated regions. The present study seeks to understand the safety of LCR by determining shape change and resultant tissue viability as a function of laser dosimetry. STUDY DESIGN/MATERIALS AND METHODS: Rabbit nasal septal cartilage were irradiated using a Nd:YAG laser (lambda = 1.32 microm, 5.4 mm spot diameter) with different exposure times of 4, 6, 8, 10, 12, and 16 seconds and powers of 4, 6, and 8 W. Temperature on the cartilage surface in the laser-irradiated region was collected using infrared thermography, this data was then used to predict tissue damage via a rate process model. A Live/Dead viability assay combined with fluorescent confocal microscopy was used to measure the amount of thermal damage generated in the irradiated specimens. RESULTS: Considerable thermal injury occurred at and below the laser-reshaping parameters that produced clinically relevant shape change using the present Nd:YAG laser. Confocal microscopy identified dead cells spanning the entire cross-sectional thickness of the cartilage specimen (about 500 microm thick) at laser power density and exposure times above 4 W and 6 seconds; damage increased with time and irradiance. The damage predictions made by the rate process model compared favorably with measured data. CONCLUSIONS: These results demonstrate that significant thermal damage is concurrent with clinically relevant shape change. This contradicts previous notions that there is a privileged laser dosimetry parameter where clinically relevant shape change and tissue viability coexist.     

Measurement of the elastic modulus of rabbit nasal septal cartilage during Nd:YAG (lambda = 1.32 microm) laser irradiation

BACKGROUND AND OBJECTIVES: The objective of this study was to quantitatively measure changes in the elastic moduli of rabbit nasal septal cartilage during laser heating. While the efficacy of laser cartilage reshaping has been established for use in nasal surgery, few studies have investigated the temperature-dependent viscoelastic behavior of cartilage. STUDY DESIGN/MATERIALS AND METHODS: Cyclic force versus displacement curves were generated during the Nd:YAG laser (lambda = 1.32 microm, 10 second exposure time, 21.22 W/cm2) irradiation of cartilage specimens secured in cantilevered geometry. Samples were irradiated three times with 30 second cooling intervals between each laser exposure. Measurements were recorded before, during, and after laser irradiation, and then following complete rehydration in normal saline (NS) for 1 hour at 25 degrees C. Elastic modulus was calculated assuming linear viscoelastic behavior. RESULTS: The elastic modulus in native tissue decreased during and after successive laser exposures from about 6 to 3.5 MPa. After rehydration, the modulus returned to near-baseline value. Surface temperature reached a maximum of 65 degrees C. CONCLUSIONS: The laser irradiation of cartilage using parameters similar to those used in reshaping does not produce significant irreversible changes in the mechanical properties of the tissue. Measurement of the elastic modulus is an effective means of characterizing alterations in cartilage mechanical behavior during and after laser heating.     

Long-term effect of pulsed Nd:YAG laser irradiation on cultured human periodontal fibroblasts

BACKGROUND AND OBJECTIVES: The purpose of this study was to investigate the long-term effect of Nd:YAG laser irradiation on cultured human periodontal fibroblasts (hPF). STUDY DESIGN/MATERIALS AND METHODS: The cultured hPF were irradiated by pulsed Nd:YAG laser. The power delivery was 50 mJ x 10 pps (pulse per second) with irradiation duration 60, 120, 180, or 240 seconds. The viability and collagen content of laser-irradiated hPF were assessed on day 5 after laser treatment. Light microscope and transmission electron microscope (TEM) were used to observe cytomorphological change. The irradiated hPF cultured in mineralizing medium for 28 days were examined by alizarin red S and Von Kossa stain. RESULTS: The cellular viability and collagen content of hPF decreased after Nd:YAG laser irradiation. Cell damage was noted with retraction of cellular processes, loss of normal architecture, and lysis of some cells. However, survived hPF proliferated and migrated to the cell-debris-associated deposits. The electron-dense cytoplasm and amorphous organelles in laser-damaged cells was revealed by TEM. In vitro mineralization was demonstrated in the long-term laser-irradiated hPF cultured in mineralizing medium. CONCLUSION: Nd:YAG laser irradiation induced partial loss of cellular viability and collagen content. The co-existence of viable cells and progressive degeneration of laser-damaged cells was associated with the in vitro mineralization of hPF.     

Laser-assisted straightening of deformed cartilage: numerical model

BACKGROUND AND OBJECTIVES: The potential application of laser cartilage reshaping (LCR) for correction of septal deviations has generated increasing clinical interest, because septoplasty is among the top five most common operations performed. However, few studies have investigated stress fields existing in the nasal septal cartilage during LCR of septal deviations. The objectives of this study were to: (1) formulate a finite-element model describing stress fields in mechanically straightened septal cartilage, (2) calculate stress fields in the septum after a given pattern of laser irradiation produced thermally induced stress relaxation in selected sites, and (3) investigate the dependence of the overall stress relaxation in a straightened septum as a function of the number, location, and size of laser irradiation sites. STUDY DESIGN/MATERIALS AND METHODS: The cartilagenous nasal septum was modeled as 24 x 24 x 2-mm slab. The deviation was represented as a bulge running along the center of the septum with a maximum elevation of 2 mm above the surface. A straightening deformation was represented in form of displacement boundary condition applied to the bulge convex surface with maximum displacement amplitude at center of the septum. Laser irradiation applied in a pattern of one, two, and three lines parallel to the bulge was assumed. The effect of thermally induced stress relaxation was modeled as a simultaneous change in the cartilage mechanical properties and reduction of strain to zero occurring inside the laser heated zone. The finite-element method was used to calculate stress fields within cross-section of the straightened septum and the force of reaction to the straightening deformation before and after laser irradiation. Calculations were performed for the width and depth of thermally modified zones varying from 0.5 to 3 mm and from 0.5 to 2 mm, respectively. Irradiation of convex and concave sides of the deviation was studied. RESULTS: The straightening deformation produced a stress field with both regions of tension and compression present. Maximum stress values were obtained on the surface where the straightening deformation was applied. Reaction force decreased with increasing width and depth of the relaxation zones and depends on location and number of these zones. The maximum reduction of reaction force obtained with three zones (3 mm wide and 2 mm deep) optimally placed in regions of stress concentration was 98%. However, using the same pattern of stress relaxation zones but with a depth of only 1 mm produces a reaction force reaction of 91%. Irradiation of convex side of the deviation reduced reaction force approximately twice as much as irradiation of the concave side. CONCLUSIONS: The present numerical simulation of the stress field in laser-reshaped deviated septum shows highly non-homogeneous stress distributions before and after laser treatment. Using reasonable assumptions on how the mechanical behavior of cartilage changes after heating, the model allows estimation reaction force and its reduction following localized laser irradiation as a function of size and location of laser heated zones.     

冷冻保存对软骨细胞存活率及代谢活性的影响

目的了解各种冷冻保存方法对软骨细胞的存活率和代谢活性的影响，寻找满意的软骨组织冻存方法。方法采用梯度慢速降温法和连续慢速降温法对兔关节软骨进行低温冷冻保存处理，通过荧光染色及^35SO4摄入率了解冻存后软骨细胞的存活率和代谢活性。结果采用梯度降温法的软骨细胞存活率为61％，显著高于连续降温法；冷冻保存对软骨细胞的代谢活性有一定的影响．但与对照组的差异并无统计学意义。结论梯度降温法较传统保存方法能显著提高冷冻保存后软骨细胞的存活率，并能维持软骨细胞的代谢活性，是理想的关节软骨冷冻保存方法。     

The effect of monopolar radiofrequency energy on partial-thickness defects of articular cartilage.

PURPOSE: To evaluate the effect of monopolar radiofrequency (RF) energy on partial-thickness defects of articular cartilage, comparing the outcome of partial-thickness defects treated with monopolar RF energy with that of treatment by conversion of partial-thickness defects to full-thickness defects by curettage and microfracture. TYPE OF STUDY: Randomized trial using adult female sheep. MATERIALS AND METHODS: Thirty-six sheep were used in this study. Both stifles in each animal were randomly assigned to 1 of the following 3 procedures: (1) partial-thickness defect without any treatment to serve as a sham-operated control, (2) partial-thickness defect with RF energy treatment, and (3) partial-thickness defect treated by conversion of the defect to a full-thickness defect by curettage and microfracture. Nine sheep were euthanized at 0, 2, 12, and 24 weeks after surgery (n = 6 per group). After euthanasia, cartilage samples were harvested from the defect sites, and chondrocyte viability was analyzed by confocal laser microscopy using a triple-labeling technique. Cartilage samples also were decalcified and stained with hematoxylin and eosin and safranin-O for histologic analysis. Surface properties of cartilage samples were analyzed using scanning electron microscopy. RESULTS: The analysis of chondrocyte viability showed that RF treatment caused death of almost all chondrocytes in the defect. Histologic analysis showed that RF treatment caused detrimental effects to chondrocytes and proteoglycan concentration that progressed over time, and that full-thickness defects were repaired by fibrocartilage by 24 weeks after surgery. Scanning electron microscopy analysis indicated that RF-treated groups were significantly smoother and less irregular than control groups at 2, 12, and 24 weeks after surgery. CONCLUSIONS: This study showed that monopolar RF energy caused long-term damage to cartilage in this sheep model and did not appear to have the beneficial effects reported in a previous study that evaluated application of this technique using a bipolar RF probe.     

Morphologic changes and removal of debris on apical dentin surfaces after Nd:YAG laser and diode laser irradiation

OBJECTIVE: To verify the effects of laser energy on intracanal dentin surfaces, by analyzing the morphologic changes and removal of debris in the apical third of 30 extracted human teeth, prepared and irradiated with the Nd:YAG laser and diode laser. BACKGROUND DATA: Lasers have been widely used in endodontics. The morphologic changes in dentin walls caused by Nd:YAG and diode laser irradiation could improve apical seals and cleanliness. MATERIALS AND METHODS: The protocol used for Nd:YAG laser irradiation was 1.5 W, 100 mJ, and 15 Hz, in pulsed mode, and for diode laser was 2.5 W in continuous mode. Each specimen was irradiated four times at a speed of 2 mm/sec with a 20-sec interval between applications. Five calibrated examiners scored the morphologic changes and debris removal on a 4-point scale. RESULTS: In analyzing the scores, there were no statistically significant differences between the two types of laser for either parameter, according to Kruskal-Wallis testing at p = 0.05. The SEM images showed fusion and resolidification of the dentin surface, with partial removal of debris on the specimens irradiated with the Nd:YAG laser and the diode laser, compared with controls. CONCLUSION: Both lasers promote morphologic changes and debris removal. These alterations of the dentin surface appeared to be more evident in the Nd:YAG laser group, but the diode laser group showed more uniform changes.     

Viability of cultured human nasal septum chondrocytes after crushing

OBJECTIVE: To investigate how different degrees of crushing affect the viability of human nasal septum chondrocytes in adherent cell cultures. METHODS: Cartilage grafts were harvested from the nasal septa of 15 patients who underwent submucosal resection. Five cartilage pieces were prepared from each specimen as follows: the cartilage was left intact, slightly crushed, moderately crushed, significantly crushed, or severely crushed. Chondrocytes were isolated for trypan blue dye exclusion testing, and the numbers of viable cells were determined at 1, 2, 3, and 10 days after culturing. Comparisons were made among the groups. RESULTS: The day 1 viability rates for the intact, slightly crushed, moderately crushed, significantly crushed, and severely crushed cartilage preparations were 96%, 92%, 82%, 72%, and 54%, respectively. The corresponding rates on day 10 were 93%, 90%, 84%, 75%, and 68%. CONCLUSIONS: The viability and proliferative capacity of crushed human septal cartilage depend on the degree of crushing sustained. Slightly or moderately crushed cartilage grafts show good chondrocyte viability and proliferation and are valuable for fashioning soft nasal contours, filling defects, and concealing dorsal irregularities. However, significant or severe crushing reduces chondrocyte viability and proliferation and may result in unpredictable degrees of graft volume loss.     

Characterization of temperature dependent mechanical behavior of cartilage

BACKGROUND AND OBJECTIVES: Few quantitative studies have investigated the temperature dependent viscoelastic properties of cartilage tissue. Cartilage softens and can be reshaped when heated using laser, RF, or contact heating sources. The objectives of this study were to: (1) measure temperature dependent flexural storage moduli and mechanical relaxation in cartilage, (2) determine the impact of tissue water content and orientation on these mechanical properties, and (3) use these measurements to estimate the activation energy associated with the mechanical relaxation process. STUDY DESIGN/MATERIALS AND METHODS: Porcine nasal septal cartilage specimens (30 x 10 x 2 mm) were deformed using a single cantilever arrangement in a dynamic thermomechanical analyzer. Stress relaxation measurements were made at discrete temperatures ranging from 25 to 70 degrees C in response to cyclic deformation (within the linear viscoelastic region). The time and temperature dependent behavior of cartilage was measured using frequency multiplexing techniques (10-64 Hz), and these results were used to estimate the activation energy for the phase change using the Williams-Landel-Ferry (WLF) equation and the Arrhenius kinetic equation. In addition, the effect of tissue orientation was examined with specimens oriented in both transverse and longitudinal directions at room temperature. RESULTS: The storage moduli of porcine cartilage decreased with increasing temperature, and a critical change in mechanical properties was observed between 58 and 60 degrees C with a reduction in the storage modulus by 85-90%. The shift of the stress relaxation behavior from viscoelastic solid to viscoelastic liquid was observed between 50 and 57 degrees C and likely corresponds to the transition temperature region in which structural changes in the tissue occur. The storage moduli for transverse and longitudinally oriented specimens were 19-22 and 14-16 MPa, respectively at ambient temperature. Reducing the water content (<10% mass loss) by allowing it to dry under ambient conditions resulted in reduction in the storage modulus by 31-36%. The activation energy associated with the mechanical relaxation of cartilage was 147 kJ/mole at 60 degrees C. This value was calculated by measuring stress-strain relationship under conditions where linear viscoelastic behavior was observed (0.09-0.15% of strain) within the transition temperature region (58-60 degrees C). CONCLUSIONS: The anisotropic mechanical behavior of cartilage was quantitatively analyzed in the transversely and longitudinally oriented specimens. Viscoelastic behavior appeared to be strongly dependent on the water content. Using empirically determined estimates of the transition zone temperature range accompanying stress relaxation, the activation energy for stress relaxation was calculated using time and temperature superposition theory and WLF equation. Further investigation of the molecular changes, which occur during laser irradiation, may assist in understanding the thermal and mechanical behavior of cartilage and how the reshaping process might to be optimized.     

Laser cartilage reshaping in an in vivo rabbit model using a 1.54 microm Er:Glass laser

BACKGROUND AND OBJECTIVES: The potential applications for facial laser cartilage reshaping (LCR) have generated increasing clinical interest. This study aimed to evaluate in vivo LCR of the rabbit ear using a 1.54 micro m Er:Glass laser in combination with contact cooling. STUDY DESIGN/MATERIALS AND METHODS: LCR was performed in vivo on 12 rabbit ears using a 1.54 micro m Er:Glass laser (Aramis, Quantel Medical, Clermont Ferrand, France) connected to a 4 mm chilled (+5 degrees C) handpiece placed in contact to the skin. Ear curvature was predetermined using a perforated cylindrical guide also used to standardize laser beam delivery. The treatment consisted of 15 spots (3 millisecond, 7 pulses, 12 J/cm(2), 2 Hz, 84/cm(2) cumulative fluence) applied on 10 contiguous parallel rows along the ear. After irradiation, the aluminum jig was replaced by a holder (10 mm diameter plastic tube) maintaining the curvature. This holder was secured with sutures and covered by an adhesive gauze bandage dressing to keep new form during 7 days. In order to assess thermal damage, biopsies were taken on irradiated areas and 1 week, 3 weeks and 6 weeks and studied using haematoxylin-erythrosin-safran (HES) and orcein staining and PCNA to detect cells in cycle. RESULTS: Using the laser with the parameters given above, no immediate visible effects were observed on the skin (no swelling, no bleaching). There were also no late visible side effects like crusting, or blistering. The laser treatment produced changes in the shape of every ear after the dressing was removed. A slight tendency to recover its initial shape was observed for each ear. However, the curvature was stabilized after 10 days and the average shape retention was 64+/-4% at 6 weeks, with a curvature radius of 7.25+/-0.75 mm, instead of 5 mm initially. Histological examination of the laser irradiated side at 1 week showed an intact epidermis. A reduced inflammation process was seen in the dermis. A modification of half of the layer of cartilage was observed at the opposite side where the laser irradiation was applied and proliferative cells were detected inside. At 3 weeks, an important chondroblastic proliferation was observed around the area of contracted cartilage. At 6 weeks, significant thickening of the cartilage layer was observed (from 300 to 490 micro m) and new chondrocytes were clearly seen. CONCLUSIONS: Rabbit ear cartilage can be reshaped with an Er:Glass laser. This technique could offer exciting possibilities that may help patients whose cartilage-lined joints have been affected by disease or trauma. This technique could be certainly utilized to correct alar cartilage deformities and septum deviation of cleft lips.     

Effects of 810 nm laser irradiation on in vitro growth of bacteria: comparison of continuous wave and frequency modulated light

BACKGROUND AND OBJECTIVES: Low intensity laser therapy may modify growth of wound bacteria, which could affect wound healing. This study compares the effects on bacteria of 810 nm laser using various delivery modes (continuous wave or frequency modulated light at 26, 292, 1000, or 3800 Hz). STUDY DESIGN/MATERIALS AND METHODS: Staphylococcus (S.) aureus, Escherichia (E.) coli, and Pseudomonas (P.) aeruginosa were plated on agar and then irradiated (0.015 W/cm(2); 1-50 J/cm(2)) or used as controls (sham irradiated); growth was examined after 20 hours of incubation post exposure. RESULTS: There were interactions of species and modulation frequency in the overall effects of irradiation (P = 0.0001), and in the radiant exposure mediated effects (P = 0.0001); thus individual frequencies and each bacterium were analysed separately. Bacteria increased following 3800 Hz (P = 0.0001) and 1000 Hz (P = 0.0001) pulsed irradiation; at particular radiant exposures P. aeruginosa proliferated significantly more than other bacteria. Pulsed laser at 292 and 26 Hz also produced species-dependent effects (P = 0.0001; P = 0.0005); however, the effects for different radiant exposures were not significant. Bacterial growth increased overall, independent of species, using continuous mode laser, significantly so at 1 J/cm(2) (P = 0.02). Analysis of individual species demonstrated that laser-mediated growth of S. aureus and E. coli was dependent on pulse frequency; for S. aureus, however, there was no effect for different radiant exposures. Further tests to examine the radiant exposure effects on E. coli showed that growth increased at a frequency of 1000 Hz (2 J/cm(2); P = 0.03). P. aeruginosa growth increased up to 192% using pulsed irradiation at 1000-3800 Hz; whereas 26-292 Hz laser produced only a growth trend. CONCLUSIONS: The findings of this study point to the need for wound cultures prior to laser irradiation of infected wounds. Similar investigations using other common therapeutic wavelengths are recommended.     

The viability and proliferation of human chondrocytes following cryopreservation

Human articular cartilage samples were retrieved from the resected material of patients undergoing total knee replacement. Samples underwent automated controlled freezing at various stages of preparation: as intact articular cartilage discs, as minced articular cartilage, and as chondrocytes immediately after enzymatic isolation from fresh articular cartilage. Cell viability was examined using a LIVE/DEAD assay which provided fluorescent staining. Isolated chondrocytes were then cultured and Alamar blue assay was used for estimation of cell proliferation at days zero, four, seven, 14, 21 and 28 after seeding. The mean percentage viabilities of chondrocytes isolated from group A (fresh, intact articular cartilage disc samples), group B (following cryopreservation and then thawing, after initial isolation from articular cartilage), group C (from minced cryopreserved articular cartilage samples), and group D (from cryopreserved intact articular cartilage disc samples) were 74.7% (95% confidence interval (CI) 73.1 to 76.3), 47.0% (95% CI 43 to 51), 32.0% (95% CI 30.3 to 33.7) and 23.3% (95% CI 22.1 to 24.5), respectively. Isolated chondrocytes from all groups were expanded by the following mean proportions after 28 days of culturing: group A ten times, group B 18 times, group C 106 times, and group D 154 times. This experiment demonstrated that it is possible to isolate viable chondrocytes from cryopreserved intact human articular cartilage which can then be successfully cultured.