Evaluation of Femtosecond Laser-Induced Breakdown Spectroscopy for Analysis of Animal Tissues

The aim of this work was to evaluate the performance of femtosecond laser-induced breakdown spectroscopy (fs-LIBS) for the determination of elements in animal tissues. Sample pellets were prepared from certified reference materials, such as liver, kidney, muscle, hepatopancreas, and oyster, after cryogenic grinding assisted homogenization. Individual samples were placed in a two-axis computer-controlled translation stage that moved in the plane orthogonal to a beam originating from a Ti:Sapphire chirped-pulse amplification (CPA) laser system operating at 800 mu and producing a train of 840 mu J and 40 fs pulses at 90 Hz. The plasma emission was coupled into the optical fiber of a high-resolution intensified charge-coupled device (ICCD)-echelle spectrometer. Time-resolved characteristics of the laser-produced plasmas showed that the best results were obtained with delay times between 80 and 120 ns. Data obtained indicate both that it is a matrix-independent sampling process and that fs-LIBS can be used for the determination of Ca, Cu, Fe, K, Mg, Na, and P, but efforts must be made to obtain more appropriate detection limits for Al, Sr, and Zn.

Combined Retrograde Flexible Ureteroscopic Lithotripsy with Holmium YAG Laser for Renal Calculi Associated with Ipsilateral Ureteral Stones

Purpose: The purpose of this study was to evaluate the effectiveness of combined ureteroscopic holmium YAG lithotripsy for renal calculi associated with ipsilateral ureteral stones. Materials and Methods: Between August 2002 and March 2007, retrograde flexible ureteroscopic stone treatment was attempted in 351 cases. Indication for treatment was concurrent symptomatic ureteral stones in 63 patients (group I). Additional operative time and perioperative complication rates were compared to a group of 39 patients submitted to ureteroscopic treatment for ureteral calculi exclusively (group II). Results: Mean ureteral stone size was 8.0 +/- 2.6 mm and 8.1 +/- 3.4 mm for groups I and II, respectively. Mean operative time for group I was 67.9 +/- 29.5 minutes and for group 2 was 49.3 +/- 13.2 minutes (p < 0.001). Flexible ureteroscopic therapy for renal calculi increased 18 minutes in the mean operative time. The overall complication rate was 3.1% and 2.5% for groups I and II, respectively (p = 0.87). Mean renal stone size was 10.7 +/- 6.4 mm, overall stone free rate in group I was 81%. However, considering only patients with renal stones smaller than 15 mm, the stone free rate was 88%. Successful treatment occurred in 81% of patients presenting lower pole stones, but only 76% of patients with multiple renal stones became stone free. As expected, stone free rate showed a significant negative correlation with renal stone size (p = 0.03; r = -0.36). Logistic regression model indicated an independent association of renal stones smaller than 15 mm and stone free rate (OR = 13.5; p = 0.01). Conclusion: Combined ureteroscopic treatment for ureteral and ipsilateral renal calculi is a safe and attractive option for patients presenting for symptomatic ureteral stone and ipsilateral renal calculi smaller than 15 mm.

Comparison of the Effects of Electrical Field Stimulation and Low-Level Laser Therapy on Bone Loss in Spinal Cord-Injured Rats

Objective: This study investigated the effects of low-level laser therapy (LLLT) and electrical stimulation (ES) on bone loss in spinal cord-injured rats. Materials and Methods: Thirty-seven male Wistar rats were divided into four groups: standard control group (CG); spinal cord-injured control (SC); spinal cord-injured treated with laser (SCL; GaAlAs, 830 nm, CW, 30mW/cm, 250 J/cm(2)); and spinal cord-injured treated with electrical field stimulation (SCE; 1.5 MHz, 1: 4 duty cycles, 30 mW, 20 min). Biomechanical, densitometric, and morphometric analyses were performed. Results: SC rats showed a significant decrease in bone mass, biomechanical properties, and morphometric parameters (versus CG). SCE rats showed significantly higher values of inner diameter and internal and external areas of tibia diaphyses; and the SCL group showed a trend toward the same result (versus SC). No increase was found in either mechanical or densitometric parameters. Conclusion: We conclude that the mentioned treatments were able to initiate a positive bone-tissue response, maybe through stimulation of osteoblasts, which was able to determine the observed morphometric modifications. However, the evoked tissue response could not determine either biomechanical or densitometric modifications.

Cell Therapy Attenuates Cardiac Dysfunction Post Myocardial Infarction: Effect of Timing, Routes of Injection and a Fibrin Scaffold

Background: Cell therapy approaches for biologic cardiac repair hold great promises, although basic fundamental issues remain poorly understood. In the present study we examined the effects of timing and routes of administration of bone marrow cells (BMC) post-myocardial infarction (MI) and the efficacy of an injectable biopolymer scaffold to improve cardiac cell retention and function. Methodology/Principal Findings: (99m)Tc-labeled BMC (6x10(6) cells) were injected by 4 different routes in adult rats: intravenous (IV), left ventricular cavity (LV), left ventricular cavity with temporal aorta occlusion (LV(+)) to mimic coronary injection, and intramyocardial (IM). The injections were performed 1, 2, 3, or 7 days post-MI and cell retention was estimated by gamma-emission counting of the organs excised 24 hs after cell injection. IM injection improved cell retention and attenuated cardiac dysfunction, whereas IV, LV or LV* routes were somewhat inefficient (< 1%). Cardiac BMC retention was not influenced by timing except for the IM injection that showed greater cell retention at 7 (16%) vs. 1, 2 or 3 (average of 7%) days post-MI. Cardiac cell retention was further improved by an injectable fibrin scaffold at day 3 post-MI (17 vs. 7%), even though morphometric and function parameters evaluated 4 weeks later displayed similar improvements. Conclusions/Significance: These results show that cells injected post-MI display comparable tissue distribution profile regardless of the route of injection and that there is no time effect for cardiac cell accumulation for injections performed 1 to 3 days post-MI. As expected the IM injection is the most efficient for cardiac cell retention, it can be further improved by co-injection with a fibrin scaffold and it significantly attenuates cardiac dysfunction evaluated 4 weeks post myocardial infarction. These pharmacokinetic data obtained under similar experimental conditions are essential for further development of these novel approaches.

Effect of GaAlAs Laser Irradiation on the Epiphyseal Cartilage of Rats

Objective: To study the effect of an 830-nm gallium-aluminum-arsenic (GaAlAs) diode laser at two different energy densities (5 and 15 J/cm(2)) on the epiphyseal cartilage of rats by evaluating bone length and the number of chondrocytes and thickness of each zone of the epiphyseal cartilage. Background Data: Few studies have been conducted on the effects of low-level laser therapy on the epiphyseal cartilage at different irradiation doses. Materials and Methods: A total of 30 male Wistar rats with 23 days of age and weighing 90 g on average were randomly divided into 3 groups: control group (CG, no stimulation), G5 group (energy density, 5 J/cm(2)), and G15 group (energy density, 15 J/cm(2)). Laser treatment sessions were administered every other day for a total of 10 sessions. The animals were killed 24 h after the last treatment session. Histological slides of the epiphyseal cartilage were stained with hematoxylin-eosin (HE), photographed with a Zeiss photomicroscope, and subjected to histometric and histological analyses. Statistical analysis was performed using one-way analysis of variance followed by Tukey's post hoc test. All statistical tests were performed at a significance level of 0.05. Results: Histological analysis and x-ray radiographs revealed an increase in thickness of the epiphyseal cartilage and in the number of chondrocytes in the G5 and G15 groups. Conclusion: The 830-nm GaAlAs diode laser, within the parameters used in this study, induced changes in the thickness of the epiphyseal cartilage and increased the number of chondrocytes, but this was not sufficient to induce changes in bone length.

Effects of Low-Level Laser Therapy on Pain and Scar Formation After Inguinal Herniation Surgery: A Randomized Controlled Single-Blind Study

Objective: The aim of this study was to investigate the efficacy of an infrared GaAlAs laser operating with a wavelength of 830 nm in the postsurgical scarring process after inguinal-hernia surgery. Background: Low-level laser therapy (LLLT) has been shown to be beneficial in the tissue-repair process, as previously demonstrated in tissue culture and animal experiments. However, there is lack of studies on the effects of LLLT on postsurgical scarring of incisions in humans using an infrared 830-nm GaAlAs laser. Method: Twenty-eight patients who underwent surgery for inguinal hernias were randomly divided into an experimental group (G1) and a control group (G2). G1 received LLLT, with the first application performed 24 h after surgery and then on days 3, 5, and 7. The incisions were irradiated with an 830-nm diode laser operating with a continuous power output of 40 mW, a spot-size aperture of 0.08 cm(2) for 26 s, energy per point of 1.04 J, and an energy density of 13 J/cm(2). Ten points per scar were irradiated. Six months after surgery, both groups were reevaluated using the Vancouver Scar Scale (VSS), the Visual Analog Scale, and measurement of the scar thickness. Results: G1 showed significantly better results in the VSS totals (2.14 +/- 1.51) compared with G2 (4.85 +/- 1.87); in the thickness measurements (0.11 cm) compared with G2 (0.19 cm); and in the malleability (0.14) compared with G2 (1.07). The pain score was also around 50% higher in G2. Conclusion: Infra-red LLLT (830 nm) applied after inguinal-hernia surgery was effective in preventing the formation of keloids. In addition, LLLT resulted in better scar appearance and quality 6 mo postsurgery.

The Effects of a 785-nm AlGaInP Laser on the Regeneration of Rat Anterior Tibialis Muscle After Surgically-Induced Injury

Objective: This study aims to investigate the effects of low-level laser therapy (LLLT) on muscle regeneration. For this purpose, the anterior tibialis muscle of 48 male Wistar rats received AlGaInP laser treatment (785 nm) after surgically-induced injury. Background Data: Few studies have been conducted on the effects of LLLT on muscle regeneration at different irradiation doses. Materials and Methods: The animals were randomized into four groups: uninjured rats (UN); uninjured and laser-irradiated rats (ULI); injured rats (IN); and injured and laser-irradiated rats (ILI). The direct contact laser treatment was started 24 h after surgery. An AlGaInP diode laser emitting 75 mW of continuous power at 785 nm was used for irradiation. The laser probe was placed at three treatment points to deliver 0.9 J per point, for a total dose of 2.7 J per treatment session. The animals were euthanized after treatment sessions 1, 2, and 4. Mounted sections were stained with hematoxylin and eosin and used for quantitative morphological analysis, in which the number of leukocytes and fibroblasts were counted over an area of 4480 mu m(2). The data were statistically analyzed by analysis of variance (ANOVA) and the Bonferroni t-test. Results: Quantitative data showed that the number of both polymorphonuclear and mononuclear leukocytes in the inflammatory infiltrate at the injury site was smaller in the ILI(1), ILI(2), and ILI(4) subgroups compared with their respective control subgroups (IN(1), IN(2), and IN(4)) for sessions 1, 2, and 4, respectively (p < 0.05). On the other hand, the number of fibroblasts increased after the fourth treatment session (p < 0.05). With regard to the regeneration of muscle fibers following injury, only after the fourth treatment session was it possible to find muscle precursor cells such as myoblasts and some myotubes in the ILI(4) subgroup. Conclusion: During the acute inflammatory phase, the AlGaInP laser treatment was found to have anti-inflammatory effects, reducing the number of leukocytes at the injury site and accelerating the regeneration of connective tissue.

Effect of Laser (670 nm) on Healing of Wounds Covered with Occlusive Dressing: A Histologic and Biomechanical Analysis

Objectives: To analyze the effects of low-level laser therapy (LLLT), 670 nm, with doses of 4 and 7 J/cm(2), on the repair of surgical wounds covered by occlusive dressings. Background Data: The effect of LLLT on the healing process of covered wounds is not well defined. Materials and Methods: For the histologic analysis with HE staining, 50 male Wistar rats were submitted to surgical incisions and divided into 10 groups (n=5): control; stimulated with 4 and 7 J/cm(2) daily, for 7 and 14 days, with or without occlusion. Reepithelization and the number of leukocytes, fibroblasts, and fibrocytes were obtained with an image processor. For the biomechanical analysis, 25 rats were submitted to a surgical incision and divided into five groups (n=5): treated for 14 days with and without occlusive dressing, and the sham group. Samples of the lesions were collected and submitted to the tensile test. One-way analysis of variance was performed, followed by post hoc analysis. A Tukey test was used on the biomechanical data, and the Tamhane test on the histologic data. A significance level of 5% was chosen (p <= 0.05). Results: The 4 and 7J/cm(2) laser with and without occlusive dressing did not alter significantly the reepithelization rate of the wounds. The 7 J/cm(2) laser reduced the number of leukocytes significantly. The number of fibroblasts was higher in the groups treated with laser for 7 days, and was significant in the covered 4 J/cm(2) laser group. Conclusions: Greater interference of the laser-treatment procedure was noted with 7 days of stimulation, and the occlusive dressing did not alter its biostimulatory effects.

Effect of Low-Level Laser Therapy on Malondialdehyde Concentration in Random Cutaneous Flap Viability

Objective: The aim of this study was to assess the effects of 830 and 670 nm laser on malondialdehyde (MDA) concentration in random skin-flap survival. Background Data: Low-level laser therapy (LLLT) has been reported to be successful in stimulating the formation of new blood vessels and activating superoxide-dismutase delivery, thus helping the inhibition of free-radical action and consequently reducing necrosis. Materials and Methods: Thirty Wistar rats were used and divided into three groups, with 10 rats in each one. A random skin flap was raised on the dorsum of each animal. Group 1 was the control group; group 2 received 830 nm laser radiation; and group 3 was submitted to 670 nm laser radiation. The animals underwent laser therapy with 36 J/cm(2) energy density immediately after surgery and on the 4 days subsequent to surgery. The application site of the laser radiation was 1 point, 2.5 cm from the flap's cranial base. The percentage of the skin-flap necrosis area was calculated 7 days postoperative using the paper-template method, and a skin sample was collected immediately after as a way of determining the MDA concentration. Results: Statistically significant differences were found between the necrosis percentages, with higher values seen in group 1 compared with groups 2 and 3. Groups 2 and 3 did not present statistically significant differences (p > 0.05). Group 3 had a lower concentration of MDA values compared to the control group (p < 0.05). Conclusion: LLLT was effective in increasing the random skin-flap viability in rats, and the 670 nm laser was efficient in reducing the MDA concentration.

Effects of Er:YAG Laser Irradiation on the Microtensile Bond Strength to Bleached Enamel

Objective: The objective of this study was to evaluate the influence of different Er:YAG laser (lambda = 2.94 mu m) energy parameters on the microtensile bond strength (mu TBS) and superficial morphology of bovine enamel bleached with 16% carbamide peroxide. Background: Laser irradiation could improve adhesion to bleached enamel surfaces. Methods: Sixty bovine enamel blocks (7x3x3 mm(3)) were randomly assigned to six groups according to enamel preparation procedures (n = 10): G1-bleaching and Er:YAG laser irradiation with 25.52 J/cm(2) (laser A, LA); G2-bleaching and Er:YAG laser irradiation with 4.42J/cm(2) (laser B, LB); G3-bleaching; G4-Er:YAG laser irradiation with 25.52 J/cm(2); G5-Er:YAG laser irradiation with 4.42J/cm(2); G6-control, no treatment. G1 to G3 were bleached for 6 h during 21 days. Afterwards, enamel surfaces in all groups were slightly abraded with 600-grit SiC papers and G1, G2, G4 and G5 were irradiated according to each protocol. Enamel blocks were then restored with an etch-and-rinse adhesive system and a 4-mm thick composite buildup was made in two increments (n = 9). After 24 h, restored blocks were serially sectioned with a cross-section area of similar to 1 mm(2) at the bonded interface and tested in tension in a universal testing machine (1 mm/min). Failure mode was determined at a magnification of x100 using a stereomicroscope. One treated block of each group was selected for scanning electron microscopy (SEM) analysis. mu TBS data were analyzed by two-way ANOVA and no statistical differences were observed among groups. Results: Mean bond strengths (SD) in MPa were: G1-30.4(6.2); G2-27.9(8.5); G3-32.3(3.9); G4-23.7(5.8); G5-29.3(6.0); G6-29.1(6.1). A large number of adhesive failures was recorded for bleached and irradiated enamel surfaces. Conclusions: Bleached enamel surfaces mu TBS values were not significantly different from those of unbleached enamel. Even though Er:YAG laser irradiation with both parameters had no influence on mu TBS for bleached and unbleached enamel, SEM analysis revealed that Er:YAG laser irradiation with 25.52J/cm(2) should not be recommended, as enamel ablation was observed, whereas irradiation with 4.42J/cm(2) did not promote any remarkable changes on enamel surface.

Nd:YAG Laser Improves Biocompatibility of Human Dental Root Surfaces

Objective: Our goal was to compare the in vivo biocompatibility of dental root surfaces submitted to four different treatments after tooth avulsion followed by implantation into rat subcutaneous tissue. Background Data: Dental root surface preparation prior to replanting teeth remains a challenge for endodontists. Root surface changes made by Nd:YAG irradiation could be an alternative preparation. Methods: Forty-eight freshly extracted human dental roots were randomly divided into four treatment groups prior to implantation into rat subcutaneous tissue: G1, dry root, left in the environment up to 3 h; G2, the same treatment as G1, followed by a soaking treatment in a 2.4% sodium fluoride solution (pH 5.5); G3, root soaked in physiologic saline after avulsion for 72 h; G4, the same treatment as G1, followed by Nd:YAG laser irradiation (2.0 W, 20 Hz, 100 mJ, and 124.34 J/cm(2)). The animals were sacrificed 1, 7, and 45 d later. Histological and scanning electron microscopy analyses were done. Results: All dental roots were involved and in intimate contact with connective tissue capsules of variable thicknesses. Differences were observed in the degree of inflammation and in connective tissue maturation. In G3 the inflammatory infiltrate was maintained for 45 d, whereas the Nd:YAG laser irradiation (G4) led to milder responses. The overall aspects of the root surfaces were similar, except by the irradiated roots, where fusion and resolidification of the root surface covering the dentinal tubules were observed. Conclusion: Nd:YAG laser irradiation improves the biocompatibility of dental root and thus could be an alternative treatment of dental root prior to replantation.

In Vitro Adhesion of Streptococcus sanguinis to Dentine Root Surface After Treatment with Er:Yag Laser, Ultrasonic System, or Manual Curette

Objective: The purpose of this in vitro study was to evaluate the dentine root surface roughness and the adherence of Streptococcus sanguinis (ATCC 10556) after treatment with an ultrasonic system, Er:YAG laser, or manual curette. Background Data: Bacterial adhesion and formation of dental biofilm after scaling and root planing may be a challenge to the long-term stability of periodontal therapy. Materials and Methods: Forty flattened bovine roots were randomly assigned to one of the following groups: ultrasonic system (n = 10); Er:YAG laser (n = 10); manual curette (n = 10); or control untreated roots (n = 10). The mean surface roughness (Ra, mu m) of the specimens before and after exposure to each treatment was determined using a surface profilometer. In addition, S. sanguinis was grown on the treated and untreated specimens and the amounts of retained bacteria on the surfaces were measured by culture method. Results: All treatments increased the Ra; however, the roughest surface was produced by the curettes. In addition, the specimens treated with curettes showed the highest S. sanguinis adhesion. There was a significant positive correlation between roughness values and bacterial cells counts. Conclusion: S. sanguinis adhesion was the highest on the curette-treated dentine root surfaces, which also presented the greatest surface roughness.

Effect of Laser Phototherapy on the Release of TNF-alpha and MMP-1 by Endodontic Sealer-Stimulated Macrophages

Objective: Our aim was to analyze the effect of laser phototherapy on the secretory activity of macrophages activated by interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS), and stimulated by substances leached from an epoxy resin-based sealer (AH-Plus) and a calcium hydroxide-based sealer (Sealapex). Background Data: Laser phototherapy can modulate the inflammatory process, improving wound healing. This type of therapy could be useful for modulating postoperative symptoms seen after endodontic treatment. Materials and Methods: Cytotoxicity was indirectly assessed by measuring mitochondrial activity. Macrophages were stimulated by the leached substances or not (controls), and the groups were then irradiated or not. The secretion of pro-inflammatory cytokines (TNF-alpha and MMP-1) was analyzed using ELISA. Two irradiations at 6-h intervals were done with an As-Ga-Al diode laser (780 nm, 70 mW, spot size 4.0 mm(2), 3 J/cm(2), for 1.5 sec) in contact mode. Results: The sealers were non-cytotoxic to macrophages. The production of TNF-alpha was significantly decreased by laser phototherapy, regardless of experimental group. The level of secretion of MMP-1 was similar in all groups. Conclusion: Based on the conditions of this study we concluded that in activated macrophages, laser phototherapy impairs the secretion of the pro-inflammatory cytokine TNF-alpha, but has no influence on MMP-1 secretion.

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.

Er : YAG laser, ultrasonic system, and curette produce different profiles on dentine root surfaces: An in vitro study

Objective: The aim of the present study was to compare the in vitro effects of the Er:YAG laser, an ultrasonic system, and manual curette on dentine root surface by roughness and micro-morphological analysis. Materials and Methods: Thirty-six flattened bovine roots were randomly assigned to one of the following groups: group 1 (n = 12): Er: YAG laser ( 2940 nm), 120 mJ/pulse, 10 Hz, 8.4 J/cm(2); group 2 ( n = 12): ultrasonic system; and group 3 ( n = 12): manual curette. The mean surface roughness (Ra) of each sample was measured using a profilometer before and after the treatments. The micro-morphology of the treated and untreated ( control) root surfaces was evaluated with scanning electron microscopy (SEM) at 50 x and 1000 x magnification. Results: Analysis with the profilometer showed that for equal times of instrumentation, the smoothest surfaces were produced by the Er: YAG laser and the ultrasonic system, followed by the curette ( p < 0.05). Morphological analyses demonstrated that treatment with the Er: YAG laser produced some areas with an irregular surface, craters, and ablation of the intertubular dentin. The smear layer was removed and dentine tubules were opened by both curettes and the ultrasonic system. The micro-morphology of the dentine root surface after ultrasonic treatment, however, demonstrated randomly distributed areas cratering. Conclusion: All instruments increased the roughness of the dentine root surface after treatment; however, the curette produced rougher surfaces than the other devices. SEM analysis revealed distinct root surface profiles produced by the three devices.