Temperature-Controlled CO2 Laser Tissue Welding of Ocular Tissues

Temperature-Controlled CO2 Laser Tissue Welding of Ocular Tissues

SURVEY OF OPHTHALMOLOGY VOLUME 42· SUPPLEMENT 1 • NOVEMBER 1997 Temperature-Controlled CO 2 Laser Tissue Welding of Ocular Tissues ADIEL BARAK, MD, ...

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SURVEY OF OPHTHALMOLOGY VOLUME 42· SUPPLEMENT 1 • NOVEMBER

1997

Temperature-Controlled CO 2 Laser Tissue Welding of Ocular Tissues ADIEL BARAK, MD, l OPHIR EYAL,2 MORDECHAI ROSNER, MD, l EDUWARD BELOTSERKOUSKY, MA, 2ARIEH SOLOMON, MD, l MICHAEL BELKIN, MA, MD, l AND PROF. AVRAHAM KATZIR2

IThe Goldschleger Eye Institute, Sheba Medical Center, Tel Hashomer, and 2 The Department for Applied Physics, Tel Aviv University, Tel Aviv, Israel

Abstract. Lasers can be used for binding tissues by welding, but the clinical application of this method has been limited by the difficulties in defining and maintaining the optimal conditions. Fiberoptic radiometry allows accurate remote temperature measurements for control oflaser tissue welding. We evaluated the use of a temperature-controlled tissue welding system to close corneal and corneoscleral wounds. Eighty ex vivo bovine eyes were used for the determination of welding parameters optimal for corneal wound closure. A 4 mm central corneal cut was closed with use of a CO~ laser (600 mw, 0.9 mm spot size), with tissue temperatures ranging from 45-70°C and welding time ranging from 1-30 seconds. Wound strength was measured as burst pressure of the sealed wound. The welding parameters found to cause the strongest wound binding were used to weld a limbal incision of 4 mm in 10 adult albino rabbits. The fellow eye of each animal was used as a control, and the same wound was closed with one 10/0 mersilen suture. Two animals were killed immediately after the procedure, and the eyes were sent for histologic examination. Eight rabbits were followed for 1 month. Clinical examination and refraction were done 1 day, 1 week, 2 weeks, and 1 month after the procedure. Corneal topographic evaluations were done 1 week after the procedure. After I month the animals were killed and the eyes were examined histologically. The optimal results of wound binding by laser welding in the enucleated bovine eyes were achieved with 55-60°C and at a welding time of 12-20 seconds. At these parameters the burst pressure of corneal wounds was 70 mm Hg. All laser-welded limbal wounds in the rabbits were tightly closed at the end of procedure and during the follow-up period. The refractive results after laser welding were equal to those of the controlled suture-closed wound. Laser tissue welding combined with tissue temperature monitoring can be used to close corneal wounds. (Surv Ophtbalmol 42 [Suppl 1]:S77-S81, 1997. © 1997 by Elsevier Science Inc. All rights reserved.) Key words.

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corneal surgery



laser

Lasers have been used experimentally for bonding tissues. The probable advantages of this method are that it is noncontact, does not introduce foreign materials, and is fast. Previous studies have shown laser welding to be an effective way of connecting blood vessels,1tJ peripheral nerves,s bowl segments,4 and many other biological tissues. In ophthalmology, previous attempts to weld corneal tissue with CO 2 lasers have failed. fi,7



welding

The mechanism for producing tissue welding is poorly understood. The biochemical basis is believed to be a change in the extracellular matrix, especially in the collagen proteins. Collagen molecule, in its tertiary strncture, is a stable molecule with a melting temperature of 60-70°C in mammals. Extensive crosslinkage between collagen fibrils increases the melting temperature to over lOO°e. The structural alteration following laser welding consisted of hoS77

© 1997 by Elsevier Science Inc.

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BARAKETAL

mogenizing changes in the collagen fibrils with interdigitations of altered individual fibrils.ll The cause for the structural changes is believed to be a thermal effect, yet little is known about the tissue temperature during laser irradiation. 8 Because of the specific temperature at which the transaction occurs, and because fibril denaturation occurs at higher temperatures, controlling the temperature of the welded tissue is crucial for achieving reliable welding. We combined a laser, whose energy is delivered via an optical fiber, and a monitoring thermometer system that controls the incident laser power to produce a desirable tissue temperature. This system was tested by welding a hole in a rat's bladder. 12 We used this system for welding of linear cuts in rabbit corneas.

proximal face to the thermal detector. A "lock-in" amplifier filtered and amplified the detector signals, which were sampled and processed by a personal computer with an analog-to-digital adapter. The laser radiometer system was calibrated and used to determine the optimal welding parameters with use of ex vivo bovine corneas. The fibers of the radiometer and the laser were held 1 mm above the corneal surface. For calibrations, the tissue was heated by CO 2 laser irradiation. The radiometer readings were compared to those made with a thin thermocouple (chrome alume) inserted under the upper surface of the cornea. Calibration curves were plotted and later used for temperature control of welding.

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Materials and Methods A pulsed CO 2 laser (Synrad Inc., Bothell, WA) was used to irradiate tissue. The laser radiation was transmitted to the tissue by silver halide optical fibers. The laser output at the fiber tip was set to 600 m W, at 0.9 mm caliber spot size on exit from fiber. The laser power could be turned on and off by a computer program that utilized a radiometer output signal in a proportional feedback control algorithm. The fibers used in the experiments were silver halide (AgClxBr l _x ). These fibers are polycrystalline, multimode, unclad fibers of 0.9 mm caliber and are transparent in the mid and far infrared (2-20 /-Lm). One fiber carried the laser radiation from the laser source to the tissue which was welded, and another fiber guided the infrared radiation from the heated corneal surface to the thermal detector in the radiometer. A fiberoptic radiometer measured the tissue temperature of the welded tissue (Fig. 1). An optical system focused the infrared radiation from the fiber's

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1. Diagram of the laser and temperature-control system used to irradiate the tissue.

Fig.

Laser Welding To determine the optimal laser setting for wound closure 80 ex vivo bovine eyes were used. A full-thickness 4-mm linear cut was made in the central portion of the cornea with a No. 15 Beaver blade (Beaver, Waltham, MA) The anterior chambers were reformed by continuous saline flow with use of an anterior chamber maintainer that was inserted at the limbus. The laser and the radiometer fibers were held 1 mm in front of the cut; the linear wounds were sealed by applying laser irradiation to the cuts. In all wounds six continuous applications of laser were performed side by side so that the whole length of the wound was irradiated. This was repeated for combinations of welding temperature ranging from 45-65°C and time ranging from 1-30 seconds for each application. In the first 20 eyes the wounds were irradiated without any approximation of the edges, and in the other 60 eyes the wound edges were approximated during the lasing with use of a forceps. A slit-lamp was used to monitor the wounds during the welding procedure. After completion of the welding, the area of surrounding thermal effects were recorded with a caliber. Depth of the wound closure was evaluated as percent oftotal corneal thickness. To test the strength of the wounds' closure achieved, the eyes were continuously inflated with saline through an anterior chamber maintainer with a syringe-driven infusion pump. The intraocular pressures at which the wounds started to leak were recorded with a pressure transducer and monitor (Cobe, Stanford, CT [Fig. 2]). This was tested immediately after the completion of the welding.

In Vivo Experiment Tissue welding was evaluated in 10 adult albino female rabbits, weighing 2.5 kg each. The animals were anesthetized with 0.75 mL ketamine and 0.75 mL xylazine hydrochloride. Full-thickness linear wounds, mimicking cataract wounds, of 4 mm were made in the upper limbal portion of the cornea with

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a Beaver knife. The anterior chambers were reformed by continuous saline flow with use of an anterior chamber maintainer, which was inserted at the limbus. In one eye of each animal the wounds were sealed with laser irradiation. The edges of the wound were joined by a forceps while the CO 2 laser was applied. In all applications tissue temperature rose to 55°C, and the duration of each application was 12 seconds. In all wounds six continuous applications of laser were performed side by side so that the entire length of the wound was irradiated. The wounds in the fellow eye of each animal were closed with single mersilen 10/0 suture. All animals were clinically evaluated prior to the procedure. Two animals were killed and the eyes enucleated immediately after the procedure to evaluate the immediate welding effects. The other animals were examined every day in the first week after the procedure and every week for 4 weeks after the operation. The examination included biomicroscopic examination (Haag-Streit 9000 slit-lamp, Haag-Streit, Bern, Switzerland) of the wound, Seidel testing with fluorescein dye, and evaluation of corneal clarity around the wound and the anterior segment for inflammation. Intraocular pressure and refraction were recorded at 1 day, 1 week, and every week for 4 weeks. Intraocular pressure was recorded with a Schiotz tonometer. Refraction was performed with a handheld schiascope. Corneal topography with a Topcon (Tokyo, Japan) corneal topographer was done 1 week after the procedure. Histological evaluation was performed in eyes of two rabbits that were immediately killed after the welding procedure and in eight eyes of rabbits that were sacrificed 4 weeks after the laser welding. The eyes were sectioned through the area of welding and submitted for paraffin embedding. Sections 6 f.Lm in length through the welding site were stained by PERSONAL COMPllTER

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hematoxylin and eosin and evaluated by light microscope. Microsoft Excel (Microsoft, Seattle, WA) software was used for the statistical analysis. Maximal burst pressure in bovine eyes of wounds whose edges were joined by forceps and wounds whose edges were not joined by forceps was compared using the Student's t-test. In the in vivo experiment of the intraocular pressure measurements, the amount of inflammation, and amount of postoperative astigmatism were compared in the eyes whose wounds were closed by laser welding to those in the eyes whose wounds were closed by sutures. A Student's (-test was used for the statistical evaluation.

Results Calibration curve of temperature versus radiometer signal (Fig. 3) showed that the radiometer response was near linear in the temperature range of interest, which was 40-60 c C. By using the radiometer signal to control the laser irradiation, a temperature control of only ± 1.5°C was achieved at the corneal surface (Fig. 4). The temperature was measured independently by thermocouple and radiometric measurements. Effective welding was achieved by the laser irradiation of central corneal cuts in the ex vivo bovine eyes. In the 60 eyes in which the wound edges were approximated with use of forceps at the time of the laser application, the outer stromal lamellae fused, as demonstrated by a thin, white line on the surface of the wound, which developed immediately after the procedure. The irradiated tissue around the wound edges whitened and showed minimal or no charring. When the laser was applied while the wound outer edges were not approximated by forceps, the heating of the tissue caused shrinkage of the outer corneal lamellae, and on gross inspection, the outer lamellae in all wounds were separated ex-

Surv Ophthalmol42 (Suppl1) November 1997

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ternally. However, the continuous heating also caused shrinkage of the inner corneal lamellae, so the wounds were watertight. In the wounds that were held together by a forceps, bonding strength rose with the higher tissue temperature and longer time of irradiation to a near plateau at 55-60°C and 12-15 seconds irradiation time. At these parameters the maximal burst pressure of the bovine corneas was 70 ± 5.0 mm Hg (Fig. 5). When the laser was used at higher settings that produced higher tissue temperature or longer exposure time, charring of the tissue appeared and wound bonding strength declined. In wounds in which edges were not approximated by a forceps, the maximal burst pressure was 42 ± 8.2 mm Hg, significantly lower than the burst pressure achieved when the wound edges were approximated during welding (P < 0.01). In wounds in which suture was used to close the cut, the maximal burst pressure achieved was 62 ± 4.8 mm Hg. This burst pressure was also significantly lower than the burst pressure achieved in the joined tissue laser welding (P < 0.05). The results of the in vivo experiment in rabbits' eyes with corneoscleral wounds showed tight wound closures with no postoperative leak at the end of the laser irradiation. Mean intraocular pressure prior to the procedure was 22.1 ± 3.1 mm Hg in laser-welded eyes and 22.2 ± 2.8 mm Hg in suture-closed eyes. One day after the procedure the intraocular pressure was 23.2 ± 4.3 mm Hg in the laser welded eyes and 22.9 ± 3.9 mm Hg in the suture-closed wounds (P = nonsignificant). No significant difference in intraocular pressure was seen at 1 week and 1 month after the procedure. Immediately after the procedure there was marked inflammatory reaction with fibrin formation in the anterior chamber in both the laser- and suture-closed wounds. The inflammatory

reaction cleared in both groups by 7 days, and there was no significant difference in the amount of inflammation or in the rate of its clearance. In both groups, the corneas remained clear during the entire follow-up period. In the laser-welded wounds, the irradiated tissue whitened during the procedure, but showed no charring. The sealed wounds were seen as a thin, white line of approximately 100 /-Lm in width. By 1 week the wounds had sealed completely and were hardly seen by slit-lamp examination, as was the case with the sutured wounds. During I-month follow-up, no wound dehiscence or any other postoperative complications were observed in any of the animals. Mean refraction prior to the procedure was + 3.00 ± 0.21 diopters (D) in the laser-welded eyes and +3.15 ± 0.24 D in the sutured-closed eyes. One week after the procedure mean refraction of the laser-welded eye was +3.25 ± 0.5 X 85 D. In the suture-closed eyes mean refraction was + 3.50 ± 0.75 X 95 D (P = nonsignificant). Simulated keratometry in the laser-welded wounds was 48.00 X 47.25 D and 47.75 X 47.25 D in the suture-closed wound (P = nonsignificant). Histologic study of the welding site of the rabbits' eyes that were enucleated immediately after tissue welding showed perforating wounds with disorganization of the tissue surrounding the wound, caused by thermal effect. Tissue disorganization extended

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4. Temperature control at the corneal surface. A temperature control of ± 1.5°C was achieved at the corneal surface. The temperature was independently measured by thermocouple and radiometric measurements.

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5. Bonding strength achieved at different application times and temperatures.

Fig.

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to one third of corneal depth, with swelling of corneal lamellae surrounding the disorganized tissue, extending down to Descemet's membrane. Fusion of corneal tissue (seen as disorganized tissue extending over the corneal wound) was found in the external one third of the corneal stroma, surrounded by tissue swelling. The internal two thirds of the corneal stroma remained open with a clean cut and no signs of tissue fusion between the two sides of the wound. Descemet's membrane and endothelium at the sides of the wound appeared normal. In the rabbits' eyes that were enucleated 4 weeks after the procedure, histological evaluation showed healed perforating wounds with scarring consisting of hypercellular, vascularized, subepithelial scar and hypercellular, vascularized, deep stromal scar. The epithelium continuously covered the scarred area with minimal change in thickness. The stromal lamellae arrangement at the scarred area was disrupted and extensively vascularized, while the rest of the stroma was unremarkable. The endothelium was unremarkable and covered the inner surface of the healed wound.

high energy (600 mW) we used, as compared to only 300 m W used in previous studies, 6 and to the temperature-control which prevented tissue charring at such a high temperature. We chose to use a CO 2 laser for several reasons. First, the laser emission is mainly absorbed by water molecules. Because of the high water content of the cornea and the sclera, most of the emission is absorbed near the surface, and no damage is expected in the delicate intraocular tissues. Another reason for selecting the CO 2 laser is because of its mechanical advantages, It is cheap, easy-to-handle, and stable It is extensively used in medicine and many physicians are familiar with it. Our work has demonstrated the effectiveness of laser welding. However, more experience is needed before the system can be applied to clinical use. The rabbit model for wound closure that we used differs from human wound closure, and primate experiments are needed.

Discussion

1. Badeau AF, Lee CE, Morris JR. et al: Temperature response during microvascular anastomosis using milliwatt CO 2 laser. La5er Surg Med 6:179-185, 1986 2. Bass LS, Moazami N, Pocsidio J, et al: Changes in type I collagen following laser welding. Laser Surg Med 12:500-505, 1992 3. Burstein NL, WilliamsJM, Nowicki MJ. et al: Corneal welding using hydrogen fluoride lasers. Arch OphthalmolllO:12-13, 1992 4. Costello AJ,Johnson DE, Cromeens DM, et al: Sutureless end to end bowl ansastomosis using Nd: YAG and water soluble intraluminal stent. Laser Surg Med 10:179-184, 1990 5. Eaton AM, Bass LS, Libutti SI{, Schubert HD, Treat MR: Sutureless cataract incision closure using laser-activated tissue glues. Proc SPIE 1423:52-57, 1991 6. Galitis RP, Thompson KP, Ren Q, et al: Laser welding of synthetic epikeratoplasty lenticulus to the cornea.J Refract Corneal Surg 6:430-436, 1990 7. Keates RH, Fried S, Levy SN, MorrisJR: Carbon dioxide laser use in wound sealing and epikeratophakia. J Cat Refract Surg 13:290-295,1992 8. Maragh H, Hawn RS, Gould JD, Terzis JK: Is laser nerve repair comparable to microsuture coaptation? J Reconstruct Microsurg 4:189-195,1988 9. Murray LW, Su L, Kopchok GE, White RE: Cross linkage of extracellular matrix proteins: A preliminary report on a possible mechanism of argon laser welding. Laser Surg Med 9:490-496,1989 10. Neblett G, Morris JR, Thomsen S: A study of CO 2 laser assisted microsurgical anastomosis of small vessels. American V. Mueller, American Hospital Supply Corporation. 11. Schober R, Ulrich F, Sanders T, et al: Laser induced alteration of collagen substructure allows microsurgical tissue welding. Science 232:1421-1422,1986 12. Shenfeld 0, Eyal 0, Goldwasser B, Kaztir A: Temperature monitoring and control of CO 2 laser tissue welding using a silver halide fiber optic radiometer. Proc SPIE 1876:203-216, 1994

This study showed that laser treatment can close corneal and corneoscleral wounds with significant mechanical strength. The results of laser tissue welding to date have not been consistent and the clinical use of the method has not gained widespread use. Different groups have reported a wide range of conditions under which welding was performed. I - 3 The end point for the welding process, which is a crucial point in the procedure, was decided in most cases only by subjective changes, such as bleaching, browning, or shrinking. Most studies reported the laser energy used for welding, but failed to report the more relevant data of the temperatures in the laser-irradiated tissue. Only two groups reported the tissue temperature during irradiation, and it ranged from 45-1 00°c.1,g We believe that a temperature-control system is necessary for the successful laser welding of wounds. To our knowledge, the only previous reports of successful welding in ophthalmological tissues are a study by Burstein et al describing the welding procedure in corneal tissue with use of a hydrogen fluoride laser,3 and a study by Eaton,5 which used laser-activated tissue glues. The hydrogen fluoride laser is expensive, difficult to maintain and not widely used, and the report that presented the method did not include significant survival data, Eatonam's method of using laser-activated tissue glues is an important report, but the method can hardly be described as "welding." Prior to our study, attempts to weld corneal tissues with the commonly used and inexpensive CO 2 lasers failed. 5 ,7 We attribute our success in achieving effective welding using CO 2 laser to the

References

This work was sponsored by the Shauder Foundation for Experimental Surgery. Reprint address: Adiel Barak, MD, The Goldschleger Eye Institute, Sheba Medical Center, Tel Hashomer, 52621, Israel.