Er: Yag laser promotes gingival wound vhealing by photodissociating water

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Er: Yag laser promotes gingival wound vhealing by photodissociating water
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  369 Photomedicine and LaserSurgeryVolume 23, Number4, 2005©Mary Ann Liebert, Inc.Pp. 369–372 Er:YAG Laser Promotes Gingival Wound Repair byPhoto-Dissociating Water Molecules RACHELLUBART, Ph.D., 1 GABI KESLER, D.M.D., 2 RONITLAVIE, Ph.D., 1 andHARRYFRIEDMANN, Ph.D. 1 ABSTRACT Objective: The aim of the present study was to show that rapid wound repairfollowing Er:YAG treatment andits bactericidal effect can also be related to reactive oxygen species (ROS) generation in irradiated tissue.  Background data: The Er:YAG laserwith a wavelength of 2,940 nm (corresponding to the vibrational OHstretch frequency of water) is of great value in dental medicine, owing to its dual ability to ablate soft and hardtissues with minimal damage to surrounding structures. The relatively rapid postoperative healing time seenafterablation of the gingiva is attributed to the very narrow zone of thermal disruption.  Methods: Waterwasirradiated with an Er:YAG laserat an energy of 100–130 mJ/cm 2 and 10–30-Hz pulse repetition rate The con-centration of OH radicals produced following irradiation was assessed by spin trapping coupled with electronparamagnetic resonance (EPR) spectroscopy.  Results: We found that the Er-YAG laserdissociates waterandgenerates OH radicals. The concentration of radicals produced was strongly dependent on the pulse repeti-tion rate and energy density perlaserpulse. Conclusions: The dissociation of waterneeded to generate OHradicals is possibly due to intermolecularvibrational (V-V) energy transferin water, competing with vibra-tional relaxation, thus leading to waterdissociation. High amounts of oxygen radicals (e.g., hydroxyl groups)have a sterilization effect, whereas low concentrations of ROS stimulate fibroblasts, causing collagen and ex-tracellularmatrix formation. ROS formation may explain the wound healing effect of the Er-YAG laserindentistry.INTRODUCTION A NERBIUM  /  YTTRIUMALUMINUMGARNET (Er:YAG) laser re-pairs soft tissue without surgery. In addition, there is typi-cally no scarring or gingival infections following Er:YAG lasergingival ablation. Keller et al. 1 demonstrated minimal damagezones, 20–40 microns in depth, of oral mucosa of pigs after ir-radiation with the Er:YAG laser. The laser has a therapeutic ef-fect on the gingiva due to its inherent ability to induce tissueshrinkage and enhance collagen remodeling. The relativelyspeedy postoperative healing time seen after ablation of thegingiva, based on collagen maturation, was attributed to thevery narrow zone of thermal disruption. 2 Also, skin ablativestudies have shown that Er:YAG laser irradiation produces aclean and precise lesion with only minimal adjacent injury athigh ablation efficiency. 3–5 In the present study, we attempt to explain the rapid gingivalwound repair following Er:YAG irradiation and the bacterici-dal effects of this treatment by proposing that Er:YAG treatmentmight generate reactive oxygen species (ROS) in irradiated tis-sue. This assumption is based on the fact that the Er:YAG laseroperates at 2,940 nm, which is the exact vibrational OH stretchfrequency of water. High amounts of ROS generated duringtreatment produce an important defense mechanism against in-fectious agents, 6–8 whereas low ROS levels have been shownto stimulate various cell activities. For example, minute con-centrations of ROS promote cell growth 9–12 and can function asan important regulator of cell survival by stimulating signaltransduction processes for transcription factor activation andgene expression. 13,14 ROS concentration changes and alterations in Ca 2+ homeosta-sisare closely linked. 15,16 Low-energy visible light has recently 1 Department of Physics and Chemistry, Bar-Ilan University, Ramat-Gan, Israel. 2 Dental Laser Clinic, Petach Tiqva, Israel.  370Lubart et al. been found to induce generation of ROS and stimulate an in-crease in intracellular calcium concentration in fibroblasts andcardiac cells. 16,17 This may also explain the ability of light topromote wound healing. 18,19 In the present study, we irradiated samples of water with anEr:YAG laser and attempted to detect OH radicals generatedduring irradiation. The OH radicals were measured using theelectron paramagnetic resonance (EPR) spin trapping technique.These radicals were produced under conditions similar to thoseused in operative dentistry. MATERIALS AND METHODS Spin trap 5,5-dimethyl-1-pyrroline-  N  -oxide (DMPO), pur-chased from Sigma, coupled with EPR was used to detect oxy-gen radicals in water. DMPO is a common spin probe that cantrap •OH to yield DMPO-OH, possessing a quartet signal in itsEPR spectrum.  DMPO+•OH →  DMPO-OH  DMPO was purified in the dark, in a phosphate buffered sa-line (PBS), pH 7.4, with activated charcoal. After approxi-mately 30 min, the solution was filtered and its concentrationdetermined spectrophotometrically using  227nm = 8.0 mM  1  cm  1 . 20 The solution was stored at  20°C for no longer than 4weeks. Samples drawn by a syringe containing double distilledwater (DDW) and 0.02 M DMPO, were introduced into a gas-permeable Teflon capillary (Zeus Industries, Raritan, NJ) andinserted into a narrow quartz tube, open at both ends. The tubewas then placed into the EPR cavity and spectra were recordedon a Bruker EPR 100d X-band spectrometer after illuminatingthe samples in the EPR cavity. EPR measurement conditionswere as follows: frequency, 9.75 GHz; microwave power, 20mW; scan width, 85 G; resolution, 1024; receiver gain, 2  10 5 ; conversion time, 82 msec; time constant, 655 msec; sweeptime, 168 msec. For quantification of the spin, double integra-tion of the two central peaks of the quartet signal was per-formed using the Bruker WIN-EPR software, version 2.11.  Illumination Illumination was attained by a medical Er:YAG pulsed laser(DELight ™Er:YAG laser, HOYAConBio) tuned to producepulse repetition rates of 10–30 Hz, 200–220 µsec/pulse, withpulse energy density ranging from 100 to 130 mJ/cm 2 . Aplas-tic tube (500 µLin volume) containing DDWand DMPO (0.02M) was placed in an ice basket, and the laser beam was di-rected to the center of the tube. The EPR spectrum was imme-diately measured. RESULTS  Detection of DMPO-OH spin adduct signal To measure •OH formation in water, we used an Er:YAGbeam of 130 mJ/cm 2 pulse for 1 min, with increasing pulse re-pletion rates of 10–30 Hz. These laser parameters are used bydentists for periodontal therapy. 21 As can be seen in Figure 1,the DMPO-OH quartet signal (four peaks), which monitors OHradical formation, depended on the beam pulse repetition rate.While a frequency of 10 Hz did not induce a significant quartet(Fig. 1B), compared to the non-illuminated control (Fig. 1A),increasing the frequency to 20 or 25 Hz resulted in a detectablequartet signal (Fig. 1C,D). Apulse repetition rate of 30 Hz re-sulted in a massive DMPO-OH quartet, 10 times larger thanthat measured at 25 Hz (Fig. 1E). Generation of a DMPO-OH spin adduct as a function of the beam pulse energy density To study the concentration of •OH production as a functionof the beam pulse energy density, we calculated the area of theDMPO-OH quartet second low-field peak from samples illu-minated with the same pulse repetition rate, but with increasingpulse energy densities (100–130 mJ/cm 2 ). The OH radical con-centrations at 20 Hz and 25 Hz were quite low (Fig. 2), but asexpected (Fig. 1), there was an increase in the OH concentra-tion when the pulse repetition rate increased from 20 to 25 Hz.The radical concentration at 30 Hz at a beam pulse energy den-sity of 100–120 mJ/cm 2 was not different from that at 20 or 25Hz. Ahuge jump of OH concentration occurred when the beampulse energy density increased to >120 mJ/cm 2 . Thus, •OHproduction by an Er:YAG laser at 30 Hz was not linear with thebeam pulse energy density. DISCUSSION In the present study, we demonstrate that the Er:YAG lasercan produce OH radicals in water. The concentration of the OH FIG. 1. EPR spectra of DMPO-OH spin adduct (indicated by  ). The quartet signal (4 peaks), is attributed to •OH detectionin H 2 O illuminated for 1 min with a pulse energy of 130mJ/cm 2 and a pulse repetition rate of (A) control, (B) 10 Hz, (C) 20 Hz, (D) 25 Hz, (E) 30 Hz. The y-scale of graph E is 5times higher than A–D. ABCDE  Er:YAG Laserand Gingival Wound Repair371 radicals is non-linearly dependent on the pulse energy densityand pulse repetition rate (Figs. 1 and 2). Although the frequencyof the Er:YAG laser coincides with the vibrational OH stretchfrequency, it is difficult to generate OH radicals in irradiatedwater since the vibrational energy very rapidly decreases (life-time 740fs) to rotational (V-R energy transfer) and translational(V-Tenergy transfer) motion. The surprising effect in water isthat intermolecular vibrational (V-V) energy transfer may com-pete with vibrational relaxation. 22 The rate of this V-Vprocessis inversely proportional to r 6 (where r is the intermoleculardistance), for dipole–dipole interactions according to the Foerstertransfer mechanism, and varies exponentially with r for repul-sive interactions. 23 Given that, at a very high local temperature,energetic collisions can sufficiently decrease r, V-Venergytransfer will compete with the V-R and V-Tprocesses, leadingto disequipartition among vibrational, rotational, and transla-tional energy modes, enabling the accumulation of internal en-ergy by V-Vup-pumping, resulting in dissociation when theinternal energy is sufficiently high. 23 The efficiency of the mol-ecular dissociation rate for a given energy dose increases withthe repetition rate of the laser pulses, since heat and vibra-tionally excited molecules escape from the region of high laserintensity when the pulse interval is too drawn out. Since disso-ciation of a water molecule requires at least 10 Er:YAG laserphotons, dependence of OH production on the pulse energydensity and the pulse repetition rate was expected to be non-linear, as observed. The fact that the Er:YAG delivers the exactresonance frequency of the OH fundamental vibrational fre-quency allows for a very efficient use of the laser energy due tothe strong absorption of resonant radiation, permitting the useof minimal laser power, and causing strong destructive heatingonly within the region of the laser beam. The strong local heat-ing leads to coagulation, thus sterilizing the wound.Afurther important consequence of the strong resonant ab-sorption is that the penetration depth in the tissues irradiated bythe laser beam, is limited to thin layers, allowing precise con-trol and ablation of a single thin tissue layer at each pass. Whenusing non-resonant laser radiation, dissociation is caused onlyby the heating of the tissues, with equipartition of the energyover all vibrational, rotational, and translational degrees of free-dom. Moreover, in order to deliver the amount of energy neededto destroy a layer of tissue, very high laser energy is needed,since in the absence of resonance, only that layer absorbs asmall proportion of this energy.The operator has reduced control due to the weak absorptionthat strongly increases penetration depth, accompanied by dif-fusion, thus causing the region of tissue destruction to enlarge.The risk of infection and large scar formation thereby increases.The present findings can explain the healing effect of theEr:YAG laser when used clinically due to the extreme limita-tion of the region where tissue destruction occurs. The OH rad-icals are extremely toxic, so that even in very small amountsthey may be expected to have a bactericidal effects. The reac-tion of the hydroxyl radicals (and of H atoms) with any organicmaterial present in the medium will probably dominate the re-combination of two OH radicals to form hydrogen peroxide (orof two H atoms to form a hydrogen molecule). Thus, even atthe higher pulse repetition rates, only small amounts of hydro-gen peroxide will be formed. These small amounts may have awound healing effect, especially since the wound is kept sterileby OH production. 24 REFERENCES 1.Keller, U., Hibst, R., and Mohr, W. (1995). Tierexperimentelle Un-tersuchungen zur Wündheilung der Mündschleimhaut nach Laser-behandlung. Dtsch. Zahnarztl. Z. 50:58–60.2.Kesler, G., Koren, R., Kesler, A., et al. (2000). Differences in histi-chemical characteristics of gingival collagen after Er:YAG laserperiodontal plastic surgery. J. Clin. Laser Med. Surg. 18:203–207.3.Kaufmann. R., and Hibst, R. (1989). Pulsed Er:YAG—and 308nm—excimer laser: an in vitro and in vivo study of skin ablativeeffects. Lasers Surg. Med. 9:132–140.4.Walsh, J.T., Jr., and Deutsch, T.F. (1989). Er:YAG laser ablationoftissue: measurement of ablation rates. Lasers Surg. Med. 9:327–337.5.Walsh, J.T., Flotte, T.J., and Deutsch, T.F. (1989). Er:YAG laserablation of tissue: effect of pulse duration and tissue type on ther-mal damage. Lasers Surg. Med. 9:314–326.6.Gordillo, G.M., and Sen, C.K. (2003). Revisiting the essential roleof oxygen in wound healing. Am. J. Surg. 186:259–263.7.Rojkind, M., Dominguez-Rosales, J.A., Nieto, N., et al. (2002).Role of hydrogen peroxide and oxidative stress in healing re-sponses. Cell. Mol. Life Sci. 59:1872–1891.8.Belotsky, S., Avtalion, R., Sinyakov, M., et al. (2004). Visible lightaffects chemiluminescence of carp (Cyprinus carpio) blood leuko-cytes. Photomed. Laser Surg. 22:255–2558.9.Burdon, R.H. (1995). Superoxide and hydrogen peroxide in rela-tion to mammalian cell proliferation. Free Radic. Biol. Med. 18:775–794.10.Gill, V. (1995). Hydrogen peroxide and the proliferation of BHK-21 cells. Free Radic. Res. 23:471–486.11.Murrell, G.A.C., Francis, M.J.O., and Bromley, L. (1990). Modu-lation of fibroblast proliferation by oxygen free radicals. Biochem.J. 265:659–665.12.Callaghan, G.A., Riordan, C., Gilmore, W.S., et al. (1996). Reac-tive oxygen species inducible by low-intensity laser irradiationalter DNAsynthesis in the haemopoietic cell line U937. LasersSurg. Med. 19:201–206. 0.000350.00030.000250.00020.000150.00015 10 -5 095 100 105 110 115 120 125 130 135 energy (mJ/cm 2 )    i  n   t  e  g  r  a   t   i  o  n  a  r  e  a  o   f  s  e  c  o  n   d   l  o  w    f   i  e   l   d  p  e  a   k   (  a .  u .   )  20Hz25Hz30Hz FIG. 2. Changes in the intensity of the DMPO-OH peaks as afunction of the laser parameters. The y-axis represents doubleintegration area for the second low field peak of the DMPO-OH quartet, and the x-axis represents different pulse energydensities of the beam with different pulse repetition rates, 20Hz (  ), 25 Hz (  ), and 30 Hz (  ).  372Lubart et al. 13.Suzuki, Y.J., and Ford, G.D. (1999). Redox regulation of signaltransduction in cardiac and smooth muscle. J. Mol. Cell. Cardiol.31:345–353.14.Rhee, S.G. (1999). Redox signaling: hydrogen peroxide as intra-cellular messenger. Exp. Mol. Med. 31:53–59.15.Goldman, R., Moshonov, S., and Zor, U. (1998). Generation of re-active oxygen species in a human keratinocyte cell line: role of calcium. Arch. Biochem. Biophys. 350:10–18.16.Lavi, R., Shainberg, A, Friedmann, H, et al. (2003). Low-energyvisible light induces reactive oxygen species generation and stimu-lates an increase of intracellular calcium concentration in cardiaccells. J. Biol. Chem. 278:40917–40922.17.Lubart, R., Friedmann, H., Sinyakov, M., et al. (1997). Low-energy doses of HeNe radiation change intracellular Ca 2+ concen-tration in fibroblasts. Laser Ther 9:115–120.18.Steinlechner, C.W.B., and Dyson, M. (1993). The effects of lowlevel laser therapy; on the proliferation of keratinocytes. LaserTher. 5:65–73.19.Conlan, M.J., Rapley, J.W., and Cobb, C.M. (1996). Biostimula-tion of wound healing by low-energy laser irradiation. Areview. J.Clin. Periodontol. 23:492–496.20.Kalyanaraman, B. (1982). Detection of toxic free radicals in biol-ogy and medicine. In: Hodgson, E., Bend, J.R., and Philpot, R.M.(eds.)  Reviews in Biochemical Toxicology, Vol. 4. New York:Else-vier, pp. 73–140.21.Aoki, I., and Takasaki, A.A. (2004), Potential applications of erbium:YAG laser in periodontics. J. Periodont. Res. 39:275–286.22.Nitzan, A. (1999). Ultrafast relaxation in water. Nature 402:473–475.23.Adamovich, I.V. (2001). Three-dimensional analytic model of vi-brational energy transfer in molecule–molecule collision. Am.Inst. Aeronaut. Astronaut. J. 39:1916–1925.24.Lubart, R., Sinyakov, M., Friedmann, H., et al. (1999). Photobio-stimulation by visible light: involvement of hydrogen peroxide.Trends Photochem. Photobiol. 6:169–174. Address reprint requests to:  Dr. Gabi Kesler 91 Rothschild St.Petach Tikva 49651, Israel E-mail: drkeslerg@012.net.il  This article has been cited by: 1.Gavriel Kesler, Dana Kesler Shvero, Yariv Siman Tov, George Romanos. 2011. Platelet Derived Growth Factor Secretionand Bone Healing After Er:YAG Laser Bone Irradiation.  Journal of Oral Implantology   37 :sp1, 195-204. [CrossRef ]2.Rachel Lubart, Harry Friedmann, Ronit Lavie, Abraham Baruchin. 2011. A novel explanation for the healing effect of theEr:YAG laser during skin rejuvenation.  Journal of Cosmetic and Laser Therapy   13 :1, 33-34. [CrossRef ]3.Rachel Lubart, Harry Friedmann, Ronit Lavie, Abraham M. Baruchin. 2010. A novel explanation for the healing effect of the Er:YAG laser during skin rejuvenation.  Journal of Cosmetic and Laser Therapy   12 :6, 256-257. [CrossRef ]4.Manal M. Azzeh. 2007. Treatment of Gingival Hyperpigmentation by Erbium-Doped:Yttrium, Aluminum, and Garnet Laserfor Esthetic Purposes.  Journal of Periodontology   78 :1, 177-184. [CrossRef ]5.2006. Laser Literature WatchLaser Literature Watch. Photomedicine and Laser Surgery   24 :1, 74-99. [Citation] [PDF] [PDF Plus]
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