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ORIGINAL ARTICLE
Year : 2012  |  Volume : 6  |  Issue : 2  |  Page : 40-45

Histopathological examination of oral mucosal incisions welded by 980 nm Diode Laser in vivo


Department of Physics, College of Education and Pure Science, University of Kirkuk, Ministry of Higher Education, Iraq

Date of Web Publication31-Jan-2013

Correspondence Address:
Balsam M Mirdan
Institute of Laser / University of Baghdad, Baghdad
Iraq
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0976-2868.106639

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  Abstract 

Continuous mode of operation (CW)980 nm Laser tissue welding has been studied for full thickness incisions on the oral cavity of the rabbit in vivo. Materials and Methods: Surgical incisions were done on the hard palate of the rabbits; the incisions were irradiated with 980 nm diode laser, 0.63 W as an output power and 27 s as an exposure time. Results: The immediate clinical results have shown a hemostasis of the bleeding, while the histopathological examination showed comparable results though it revealed a fastened healing pattern in comparison with the sutured incisions. Discussion: The immediately laser welded tissues was as a result of the denaturation and coagulation of the tissue proteins via the increase of the tissue temperature due to the laser-tissue photothermal interaction. Conclusion: The use of 980 nm diode laser in soft tissue welding is a successful method of wound closure in oral mucosa with the minimum side effects.

Keywords: 980 nm laser, photothermal interaction, laser tissue interaction, tissue welding


How to cite this article:
Mirdan BM. Histopathological examination of oral mucosal incisions welded by 980 nm Diode Laser in vivo. J Dent Lasers 2012;6:40-5

How to cite this URL:
Mirdan BM. Histopathological examination of oral mucosal incisions welded by 980 nm Diode Laser in vivo. J Dent Lasers [serial online] 2012 [cited 2017 Sep 20];6:40-5. Available from: http://www.jdentlasers.org/text.asp?2012/6/2/40/106639


  Introduction Top


In order to enhance the primary intention in wound healing process, the wound edges should be approximated adjacent to each other, [1] sutures are used to re approximate the wound edges, where the coagulation occurs uniformly. [2]

The suture may become a source of surgical wound contamination and infection due to the adherence and the colonization of bacteria, in addition; the suture - as a foreign body - can induce an inflammatory response in the living tissue, which may appear as granulomas or even a localized abscess formation. [3]

Currently; lasers have been used successfully in the coagulation, soft tissue welding, [4],[5] and soldering with the use of a filler material to restrict the thermal effect to the soldering region. [6] Laser tissue welding is utilized to join tissues together, through a local raising of the biological tissue temperature up to 60°C, which leads to the denaturation and coagulation of the proteins. [4],[5]

The laser soft tissue welding or soldering provides disinfection, water tightness, an early reepithelialization, maximal tensile strength during early healing, no foreign body reaction, and minimum scar formation. [7]

The first successful laser soft tissue welding in vitro using Nd: yag laser was performed in 1966 by Yahr and Strully. [8],[9] Tabakoğlu and Gülsoy[6] studied welding of the skin in Wistar rat with the use of 980 nm wavelength laser. Abbood [10] studied the application of 980 nm wavelength laser of welding the human skin in vitro experiment.

Diode laser emitting 980 nm wavelength was utilized in the present work to weld the oral soft tissue incisions and to discuss the influence of the used parameters on the results; via immediate clinical observation of the welded tissues and the histopathological examination.


  Materials and Methods Top


Thirty rabbits have been employed and then scarified in the present study; each rabbit was generally anesthetized with an intramuscular injection of (5 mg/kg) Zylazine in combination with (35 mg/kg) Ketamine. [11] Two full thickness incisions were done along the right and left sides of the hard palate of the rabbit using surgical blades; the incision length was about 1.5 cm. The right side incision has been sutured with one stitch of (3:0) silk suture to be the control for the comparison with the laser irradiated incision. The left side incision was irradiated with the 980 nm wavelength diode laser operated on continuous wave (CW) mode, 0.63 W output power and 0.6 mm spot size, so; the power density was 55.681 W/cm 2 for 27 s exposure time; laser parameters have been selected depending on a pilot study. The rabbits were divided into five groups; each group contains six rabbits, histopathological examination was done to the groups after the following periods postoperatively:

  • Group (a) rabbits were sacrificed immediately postoperatively.
  • Group (b) rabbits were sacrificed 24 hours postoperatively.
  • Group (c) rabbits were sacrificed 3 days postoperatively.
  • Group (d) rabbits were sacrificed 10 days postoperatively.
  • Group (e) rabbits were sacrificed 14 days postoperatively.

  Results Top


Clinical examination

The clinical observation of the incisions was done immediately after the procedure:

Sutured incisions

There was a normal closure of the incision wound edges with a hemostasis of the bleeding [Figure 1].
Figure 1: Photograph showing the sutured incision on the right side ( ) along the hard palate of rabbit immediately

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Laser welded incisions

The edges of wound were closed along the incision with an evident hemostasis. The welding line was obvious with a blanching in the tissues adjacent to the welding line [Figure 2].
Figure 2: Photograph showing the laser welded incision on the left side ( ) along the hard palate of the rabbit immediately

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Histopathological examination

Immediately examined Sutured incisions

The sutured tissues have been excluded from the histopathological examination; they were torn and separated from the incision line, due to the weak attachment after removing the suture for the samples preparation.

Immediately examined laser welded incisions

The histopathological examination showed a well-defined blood clot in the welding area [Figure 3]. The welding area was characterized by denaturation and coagulation of the tissue proteins and collagen fibers at the deep layers of the incision [Figure 4] and [Figure 5].
Figure 3: Optical micrograph showing oral mucosa of the hard palate of rabbit immediately after laser welding showing the performed incision ( ) and the blood clot ( ) (H and E ×100)

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Figure 4: Optical micrograph showing oral mucosa of the hard palate of rabbit immediately after laser welding showing coagulation and denaturation of the tissue collagen and proteins ( ) at the deep layers of the incision (H and E ×100)

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Figure 5: Optical micrograph showing oral mucosa of the hard palate of rabbit immediately after laser welding showing coagulation and denaturation of the tissue proteins and collagen fibers ( ) with a higher magnification of Figure 4 (H and E ×400)

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Sutured incisions at day 1

At the first day postoperatively; tissue sections showed infiltration of large numbers of dead and living neutrophils at the site of incision [Figure 6]. The submucosal connective tissue showed severe hemorrhage with infiltration of neutrophils [Figure 7]. Furthermore there were congested blood capillaries containing neutrophils in their lumina [Figure 8]. Interstitial edema was also seen [Figure 9]. Serum protein was noticed in the dilated blood vessels of submucosal connective tissue. The epithelial edges were still separated over the incision surface.
Figure 6: Optical micrograph showing oral mucosa of the hard palate of rabbit at the first day of suturing, showing infiltration of large numbers of neutrophils at the site of incision (H and E ×100)

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Figure 7: Optical micrograph showing oral mucosa of the hard palate of rabbit at the first day of suturing, showing severe hemorrhage, with infiltration of neutrophils (H and E 400×)

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Figure 8: Optical micrograph showing oral mucosa of the hard palate of rabbit at the first day of suturing, showing congested blood capillaries containing neutrophils in their lumina (H and E ×400)

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Figure 9: Optical micrograph showing oral mucosa of the hard palate of rabbit at the first day of suturing, showing interstitial edema (H and E ×400)

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Laser welded incisions at day 1

After 24 hours, laser welded incisions have been examined microscopically; the acute inflammatory response was more prominent in comparison with sutured ones as it was characterized by a profuse infiltration of the neutrophils more than the associated with the suture side, hemorrhage and congestion of the blood vessels [Figure 10] and [Figure 11], it was characterized by the formation of fibrinous exudate covering the area of the incision. The fiberinous exudate consists of fibrin network with neutrophils and few red blood cells (RBCs) [Figure 12].
Figure 10: Optical micrograph of oral mucosa of the hard palate of rabbit at the first day after laser welding showing the profuse infiltration of the neutrophils to the welding line (H and E ×100)

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Figure 11: Optical micrograph of oral mucosa of the hard palate of rabbit at the first day after laser welding showing the severe infiltration of the neutrophils to the welding line in a higher magnification of Figure 10 (H and E ×400)

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Figure 12: Oral mucosa of the hard palate of rabbit at the first day after laser welding, showing formation of fibrinous exudate covering the area of incision consisting mainly of fibrin network with neutrophils and erythrocytes (H and E ×400)

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Sutured incisions at day 3

The microscopic picture of the sutured incisions has been examined 3 days postoperatively; it was characterized by the granulation tissue formation. An evidence of multiple abscesses containing liquefactive necrosis surrounded by numbers of neutrophils in most of the examined cases [Figure 13] and [Figure 14].
Figure 13: Optical micrograph showing oral mucosa of the hard palate of rabbit at the third day of suturing, showing the formation of abscesses characterized by liquefactive necrosis () containing numbers of neutrophils (H and E ×100)

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Figure 14: Optical micrograph showing higher magnification of Figure 13 (H and E ×400)

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Laser welded incisions at day 3

After 3 days of welding the incisions; the main microscopic features are characterized by the formation of granulation tissue consisting of newly formed congested blood capillaries and immature fibroblasts without any signs of suppuration [Figure 15] and [Figure 16].
Figure 15: Optical micrograph showing oral mucosa of the hard palate of rabbit at the third day of laser welding, showing the formation of granulation tissue, consisting of newly formed congested blood capillaries and immature fibroblasts (H and E ×100)

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Figure 16: Optical micrograph showing higher magnification of Figure 15. (H and E ×400)

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Sutured incisions at day 10

The microscopic picture was characterized by the reepithelialization in a cleft-like depression pattern in the mucosa [Figure 17], and the presence of granulation tissue at the suture site. Proliferation of the fibrous connective tissue is illustrated by the deposition of collagen fibers [Figure 18] and [Figure 19].
Figure 17: Optical micrograph of oral mucosa of the hard palate of rabbit at day 10 of suturing, showing the reepithelialization in a cleft-like pattern (H and E ×100)

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Figure 18: Optical micrograph of oral mucosa of the hard palate of rabbit at day 10 of suturing, showing the granulation tissue and the collagen fibers deposition (H and E ×100)

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Figure 19: Optical micrograph of oral mucosa of the hard palate of rabbit at day 10 of suturing, showing a higher magnification of Figure 18 (H and E ×400)

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Laser welded incisions at day 10

After 10 days; the fibrous connective tissue was wide, epithelial proliferation was obvious in a more flat pattern at the welding site rather than the appearance of the cleft-like depression in the sutured incisions [Figure 20], [Figure 21], [Figure 22].
Figure 20: Optical micrograph of oral mucosa of the hard palate of rabbit at day 10 of laser welding, showing the flat pattern of the epithelial proliferation (H and E ×100)

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Figure 21: Optical micrograph of oral mucosa of the hard palate of rabbit at day 10 of laser welding, showing the granulation tissue and the collagen fibers deposition (H and E ×100)

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Figure 22: Optical micrograph of oral mucosa of the hard palate of rabbit at day 10 of laser welding, showing a higher magnification of Figure 21 (H and E ×400)

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Sutured incisions at day 14

Histopathological examination of the suture site revealed an appearance of a wide area of fibrous connective tissue and collagen deposition [Figure 23]. Thin layers of keratinized stratified squamous epithelium with adjacent empty spaces which represent the abscess remnants were also shown [Figure 24].
Figure 23: Optical micrograph of oral mucosa of the hard palate of rabbit, 14 days postoperatively of suturing the incision, showing the wide area of fibrous connective tissue and collagen deposition (H and E ×400)

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Figure 24: Optical micrograph of oral mucosa of the hard palate of rabbit, 14 days postoperatively of suturing the incision, showing the empty spaces of the remnants of the abscess. (H and E ×400)

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  Discussion Top


The evident hemostasis in the immediate clinical examination of sutured incisions was due to the uniform clot formation along the incision line; the clot formation was facilitated by the wound edges approximation to enhance the primary intention healing, this process is in a good agreement with Fragiskos, Ethicon, and Alexander. [2],[12],[13] While the clinical picture of the immediately laser welded tissues was as a result of the denaturation and coagulation of the tissue proteins via the increase of the tissue temperature due to the laser-tissue photothermal interaction, that observation is in a good agreement with Niemz, Li et al., and Tabakoğlu and Gülsoy.[4],[5],[6] The use of the 980 nm wavelength laser in such procedures was because it is within what is called the "therapeutic window", which is delineated between roughly 600 and 1200 nm; in this spectral range radiation penetrates biological tissues at a lower loss; as it is very poorly absorbed by hemoglobin, water, and other body pigments. This type of laser energy relies on a multiple pigment effect to produce a general tissue interaction depending on the representative quantities of each pigment in a given tissue; the previous hypothesis is in agreement with Niemz, Tabakoğlu and Gülsoy, and Breger and Eeg. [4],[6],[14] Using the (CW) mode in the tissue welding is preferred to achieve deep heat diffusion within the tissue to overcome the thermal relaxation time of the tissue at the applied wavelength; the effect that is in agreement with Niemz and Nathaniel et al. [4],[15]

The histopathological picture after 24 hours of the sutured incisions was in agreement with Schultz, Whaley and Burt; [16],[17] it was consistent with the normal inflammatory response that is associated with the healing process; characterized by the hemorrhage, presence of a large number of the inflammatory cells migration to the incision site, vasodilatation, digestion of the formed clot by the lysosomal enzymes, congestion of the blood vessels and edema. The multiple micro abscesses and the suppurative exudate formation have begun due to the degeneration of the dead tissues in sutured incisions in addition to the possibility of the infection due to the adherence and colonization of the microorganisms close to the suture tissue interface; the latter findings were also in agreement with Proksch et al., Cotran et al., and Rubin et al. [3],[18],[19]

While examining the laser welded incisions microscopically 24 hours postoperatively showed a difference in the picture, though they showed a comparable inflammatory response form with the sutured ones; the fibrinous exudate was a characteristic feature in the incision site, it occurred as a result to the inflammatory response, that finding is in agreement with Robert and Barnhart, [20] the stimulating inflammatory response could be due in part to the photothermal effect of the laser tissue welding; although there was no evidence of the thermal damage in the examined samples as agreed with Tabakoğlu and Gülsoy.[6] The fibrinous exudate is assumed to act as a matrix for the fibroblasts to build up the fibrous tissue in the area which in turn enhances the wound healing.

After three days of suturing the incisions; the granulation tissue appeared which was rich of RBCs, immature fibroblasts with inflammatory cells and newly formed capillaries. Micro abscesses became more obvious than they did at day one due to the progressed degeneration of the inflammatory process, eventually a delayed healing process is expected as a consequence; the resulted histopathological pattern was also in agreement with Cotran et al. and Rubin et al. [18],[19]

The granulation tissue of laser welded incisions at the third day was more uniform than that in the sutured ones; as there was no sign of suppuration, instead; the beginning of fibrous tissue formation was prominent microscopically due to the activity of the fibroblasts which were incorporated with the fibrinous exudate, the newly formed blood vessels were more obvious in comparison with those in sutured incisions for the same period; the recent histopathological picture was in a disagreement with Tabakoğlu and Gülsoy,[6] who reported the granulation tissue formation at the fourth day with a necrotic detachment of the epithelium; the disagreement is attributed to the variation in the laser parameters and the difference in the targeted tissues.

Ten days and two weeks postoperatively, the histopathological picture showed contraction of the wound which might have been occurred due to the effect of the myofibroblasts in the granulation tissue and the fibrous connective tissue. The observed features in the microscopic examination were consistent with the normal sequel of the primary intention healing process. That was in a good agreement with Rubin et al. [19]

Though laser welded tissues showed a comparable picture with the sutured ones in the groups that have been examined ten days and two weeks postoperatively, there were indications of fastened healing process; that was characterized by the more flat reepithelialization without the cleft-like formation that was observed in the sutured incisions for the same period; that finding could be attributed to the well approximated edges of the incisions that were welded by laser when compared with sutured ones. Heavy fibroblastic proliferation with marked collagen deposition without signs of infection was reported in the histopathological examination of the laser welding groups during the mentioned periods.


  Conclusion Top


The enhanced healing process, disinfection, and minimum side effects in comparison with the suturing were significant. The use of 980 nm diode laser operated on the (CW) mode in soft tissue welding is a promising modality of wound closure in oral mucosa.

 
  References Top

1.Mostafa G., Cathey L. , Frederick LG. Review of Surgery Basic Science and Clinical Topics for ABSITE, Springer.1 st edition. 2006. p. 17.  Back to cited text no. 1
    
2.Fragiskos DF. Oral Surgery, Springer. 2007. p. 282.  Back to cited text no. 2
    
3.Proksch E, Brandner JM, Jensen JM. The skin: An indispensable barrier. Exp Dermatol 2008;17:1063-72.  Back to cited text no. 3
    
4.Niemz MH. Laser-Tissue Interactions Fundamentals and Applications, Third Enlarged Edition, Springer; 2007. p. 1-79.  Back to cited text no. 4
    
5.Li ZR, Chi YL, Ke RC. Sutureless end-to-end bowel anastomosis in rabbit using low power CO2 laser. World J Gastroenterol 2000;6:557-60.  Back to cited text no. 5
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6.Tabakoðlu HO, Gülsoy M. In vivo comparison of near infrared lasers for skin welding. Lasers Med Sci 2010;25:411-21.  Back to cited text no. 6
    
7.Suh DD, Schwartz IP, Canning DA, Snyder HM, Zderic SA, Kirsch AJ. Comparison of Dermal and Epithelial Approaches to Laser Tissue Soldering for skin flap closure. Lasers Surg Med 1998;22:268-74.  Back to cited text no. 7
    
8.Poppas DP, Choma TJ, Rooke CT, Klioze SD, Schlossberg SM. Preparation of human albumin solder for laser tissue welding. Lasers Surg Med 1993;13:577-80.  Back to cited text no. 8
    
9.Devoisselle JM, Soulie-Begu S, Mordon S, Desmettre T, Maillols H. A Preliminary Study of the In Vivo Behavior of an Emulsion Formulation of Indocyanine Green. Lasers Med Sci 1998;13:279-82.  Back to cited text no. 9
    
10.Abbood Ahmed Abdulrazzaq. Laser Assisted Human Skin Wound Closure Using 980 nm Diode Laser: An In Vitro Experimental Study. 2011.  Back to cited text no. 10
    
11.Plumb's Veterinary Drug Handbook: Desk Edition,6 th ed. 6, John Wiley & Sons, 2008.p.804.  Back to cited text no. 11
    
12.Ethicon. Wound closure manual. Johnson and Johnson company, 2007. p. 10-35.  Back to cited text no. 12
    
13.Trott AT. Wounds and lacerations: Emergency care and closure. 3 rd ed. Elsevier, Mosby; printed in USA.2005.  Back to cited text no. 13
    
14.Berger N, Eeg PH. Veterinary laser surgery - A practical guide. Ames, Iowa, USA: Blackwell publishing professional; 2006. p. 41.  Back to cited text no. 14
    
15.Fried NM, Hung VC, Walsh JT. Laser Tissue Welding: Laser Spot Size and Beam Profile Studies, IEEE journal of selected topics in quantum electronics, vol. 5. no. 4, July/August 1999.  Back to cited text no. 15
    
16.Gregory SS. Surgical wound healing and management. In: Granick MS, Gamelli RL, editors. Informa Healthcare USA, Inc. The Physiology of Wound Bed Preparation; 2007. p. 1-14.  Back to cited text no. 16
    
17.Whaley K. Burt AD Inflammation, healing and repair. Cited by Muirs textbook of pathology. 13 th ed. Arnold international student edition; 1998.  Back to cited text no. 17
    
18.Cotran RS, Kumer V, Collins T. Robbins pathologic basis of disease. 6 th ed. W. B. Saunders Company; 1999. p. 89-112.  Back to cited text no. 18
    
19.Rubin ER, Howard M, Gregory C, Sephel S, Woodward C. Essentials of Rubin's Pathology. 5 th ed. Lippincott Williams and Wilkins; 2009. p. 37-52.  Back to cited text no. 19
    
20.Barnhart RK, editor. Chambers Dictionary of Etymology. New York: Chambers Harrap Publishers; 1998. p. 363.  Back to cited text no. 20
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11], [Figure 12], [Figure 13], [Figure 14], [Figure 15], [Figure 16], [Figure 17], [Figure 18], [Figure 19], [Figure 20], [Figure 21], [Figure 22], [Figure 23], [Figure 24]



 

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