JBJS | Quantification of Laser-Induced Cartilage Injury by Confocal Microscopy in an ex Vivo Model

The Journal of Bone & Joint Surgery, Volume 83, Issue 4

Articles | April 01, 2001

Quantification of Laser-Induced Cartilage Injury by Confocal Microscopy in an ex Vivo Model

P. Mainil-Varlet, MDPhD; D. Monin, MD; C. Weiler, MD; S. Grogan, PhD; T. Schaffner, MD; B. Züger, MSc; M. Frenz, PhD

Investigation performed at the Institutes of Pathology and Applied Physics, University of Bern, Bern, Switzerland
P. Mainil-Varlet, MD, PhD D. Monin, MD S. Grogan, PhD T. Schaffner, MD B. Züger, MSc M. Frenz, PhD Institutes of Pathology (P.M.-V., D.M., S.G., and T.S.) and Applied Physics (B.Z. and M.F.), University of Bern, Murtenstrasse 31, CH-3010 Bern, Switzerland. E-mail address for P. Mainil-Varlet: mainil@patho.unibe.ch
C. Weiler, MD Institut für Pathologie, Institutsbereich Innenstadt, Thalkirchner Strasse 36, D-80337 Munich, Germany
In support of their research or preparation of this manuscript, one or more of the authors received grants or outside funding from the Swiss Commission for Technology and Innovation and Stiftung zur Förderung der wissenschaftlichen Forschung an der Universität Bern. None of the authors received payments or other benefits or a commitment or agreement to provide such benefits from a commercial entity. No commercial entity paid or directed, or agreed to pay or direct, any benefits to any research fund, foundation, educational institution, or other charitable or nonprofit organization with which the authors are affiliated or associated.

The Journal of Bone & Joint Surgery. 2001; 83:566-566

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Abstract

Background:

The application of lasers in orthopaedic surgery is increasing. However, some investigators have reported that osteonecrosis may occur after laser meniscectomy. The objective of the present study was to evaluate the effect of laser wavelength and energy on cartilage injury in an ex vivo model.

Methods:

Fresh bovine articular cartilage was exposed to either holmium:yttrium-aluminum-garnet (Ho:YAG) or erbium:YAG-laser (Er:YAG) irradiation. Both lasers were operated in a free-running mode and at a pulse-repetition rate of 8 Hz. The effect of laser treatment at several energy levels (Er:YAG at 100 and 150 mJ and Ho:YAG at 500 and 800 mJ) was examined. For each light source and energy level, ten cartilage samples were assessed by conventional histological analysis and by confocal microscopy. Thermal damage was assessed by determining cell viability.

Results:

The extent of thermal damage demonstrated by confocal microscopy was much greater than that demonstrated by histological analysis. The extent of thermal injury after Ho:YAG-laser irradiation was much greater than that after Er:YAG-laser irradiation, which was associated with almost no damage. In addition, the ablation depth was greater after treatment with the Er:YAG laser than after treatment with the Ho:YAG laser.

Conclusions:

In the present study, histological analysis underestimated thermal damage after laser irradiation. In addition, our findings highlighted problems associated with use of high-power settings of Ho:YAG lasers during arthroscopic surgery.

Clinical Relevance:

Débridement and smoothing of cartilage in patients with osteoarthritis or cartilage defects should cause minimal injury to the surrounding cartilage in order to avoid additional tissue destruction.

Figures in this Article

Introduction

Introduction | Materials and Methods | Results | Discussion | References

The ability to excise dense musculoskeletal tissue or abnormal articular cartilage with precision makes the laser a potentially valuable tool in orthopaedic surgery¹. The applications of lasers are increasing and currently include meniscectomy, débridement, chondroplasty, and synovectomy^1-4. The ultimate clinical acceptability of the applications is contingent on the ability of the laser to ablate tissue with minimal damage to the surrounding normal cartilage. The first clinical studies of laser-assisted arthroscopic surgery were performed in the 1970s with use of carbon-dioxide (CO2) lasers^2,5. The main disadvantage of those lasers was that they could not be transmitted through flexible optical fibers. As a consequence, CO2-laser radiation required the injection of nitrogen or air into the joint, a procedure that has been associated with complications⁶. In contrast, the neodymium:yttrium-aluminum-garnet (Nd:YAG) laser, which was first used in combination with intra-articularly administered fluid, is poorly absorbed by hyaline cartilage and fibrocartilage, resulting in considerable thermal damage to the collateral tissue⁶.

The holmium:YAG (Ho:YAG) and erbium:YAG (Er:YAG) lasers emitting at 2.12 and 2.94 m, respectively, are better suited for tissue treatment because their wavelengths are strongly absorbed in water and can be transmitted through flexible optical fibers to allow for tissue treatment even in a wet environment^7,8.

The Er:YAG laser is known for very shallow optical penetration, in the range of a few micrometers, and for precision of ablation with minimal thermal damage to the surrounding tissue².

The Ho:YAG laser, which is currently in clinical use, is employed to gain access to small articular compartments with minimal scuffing of the chondral surface while providing excellent hemostatic control. Smooth articulation surfaces can be created with the Ho:YAG laser when it is used for chondroplasty in patients with chondral fractures or chondromalacia^2,4,9-14.

The results of in vitro studies have been promising^15,16; however, several investigators have reported that osteonecrosis may occur after contact laser meniscectomy^15,16. Raunest and Derra also showed that the degree of reactive synovitis was greater and the degenerative changes were more rapidly progressive after laser surgery than after conventional procedures¹⁷. A similar observation was made recently in a review of eighty meniscectomies¹⁸.

Despite case reports and experimental investigations, little is known about the extent of acute cartilage damage induced by different laser systems and energies. The goal of the present study was to evaluate the effect of laser wavelength and energy on tissue removal, short-term viability of cartilage, and necrosis in an ex vivo model.

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Fig. 1:: Experimental design setup for laser treatment and the control of distance from the optical fiber to the tissue. Er:YAG = erbium:yttrium-aluminum-garnet laser, Ho:YAG = holmium:YAG, PBS = phosphate-buffered saline solution, ZrF4 = zirconium fluoride, and CCD = charge coupled device.

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Fig. 2:: Representative histological findings after laser irradiation. A: Er:YAG laser (150 mJ, single pulse) (safranin O). B: Er:YAG laser (150 mJ, single pulse) (alcian blue). C: Ho:YAG laser (800 mJ, single pulse) (safranin O). D: Ho:YAG laser (800 mJ, single pulse) (alcian blue).

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Fig. 3:: Detailed light microscopy view revealing three zones of tissue damage after irradiation with a single-pulse Ho:YAG laser at 800 mJ. Zone 1 is characterized by tissue vacuolization and loss of cell structure, disappearance of nuclei and other organelles, and marginalization of nuclei; zone 2, by small damaged cells containing condensed or marginalized nuclei; and zone 3, by proteoglycan loss without major cell alteration.

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Fig. 4:: Typical findings seen with the cell viability test after laser irradiation. Living cells yield a green fluorescent product (calcein), which can be demonstrated intracellularly by fluorescence microscopy or confocal microscopy. Ethidium homodimer-1 is a red fluorescent nucleic acid stain that is able to pass only through the damaged plasma membranes of injured or dead cells. A: Er:YAG laser (150 mJ, single pulse). B: Er:YAG laser (150 mJ, double pulse). C: Ho:YAG laser (800 mJ, single pulse). D: Ho:YAG laser (800 mJ, double pulse).

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TABLE I: Effect of Laser Type and Energy Level on Tissue Removal and Tissue Damage

*The values are given as the mean and standard deviation of eight to ten measurements, and they represent the distance between the deepest location of the ablated tissue and the lowest point of visible damage. †The standard deviation is not given because the mean was calculated from the results of three experiments (217, 222, and 236 m). ‡The standard deviation is not given because the mean was calculated from the results of three experiments (1345, 1439, and 1482 m).

	Laser Type and Energy Level
Er:YAG*				Ho:YAG*
100 mJ		150 mJ		500 mJ		800 mJ
1 Pulse	2 Pulses	1 Pulse	2 Pulses	1 Pulse	2 Pulses	1 Pulse	2 Pulses
Ablation depth (m)
Confocal microscopy	?75 ± 10	122 ± 9	174 ± 13	485 ± 24	35 ± 4	120 ± 4	?175 ± 15	225†
Histological analysis	90 ± 9	111 ± 5	180 ± 22	500 ± 30	40 ± 3	?130 ± 12	170 ± 9	230 ± 11
Damage depth (m)
Confocal microscopy	<50	<50	<50	<50	1090 ± 110	1072 ± 83	1240 58	1422‡
Histological analysis	<10	<10	<10	<10	351 ± 22	?412 ± 18	?553 43	580 ± 22

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TABLE I: Effect of Laser Type and Energy Level on Tissue Removal and Tissue Damage

	Laser Type and Energy Level
Er:YAG*				Ho:YAG*
100 mJ		150 mJ		500 mJ		800 mJ
1 Pulse	2 Pulses	1 Pulse	2 Pulses	1 Pulse	2 Pulses	1 Pulse	2 Pulses
Ablation depth (m)
Confocal microscopy	?75 ± 10	122 ± 9	174 ± 13	485 ± 24	35 ± 4	120 ± 4	?175 ± 15	225†
Histological analysis	90 ± 9	111 ± 5	180 ± 22	500 ± 30	40 ± 3	?130 ± 12	170 ± 9	230 ± 11
Damage depth (m)
Confocal microscopy	<50	<50	<50	<50	1090 ± 110	1072 ± 83	1240 58	1422‡
Histological analysis	<10	<10	<10	<10	351 ± 22	?412 ± 18	?553 43	580 ± 22

Materials and Methods

Introduction | Materials and Methods | Results | Discussion | References

Bovine hyaline cartilage was obtained from a local slaughterhouse six to twelve hours post mortem. Knee-joint cartilage was removed from the bone and was trimmed to pieces measuring 3 ¥ 2 cm in size. Prior to use, the samples were kept in gauze that had been soaked in phosphate-buffered saline solution (pH 7.4). For laser treatment, the cartilage samples were mounted in a container filled with phosphate-buffered saline solution at room temperature.

An optical-fiber-coupled multiwavelength laser system, consisting of an Er:YAG laser and a Ho:YAG laser, was used as the light source¹⁹. Both lasers were operated in the free-running mode at a pulse-repetition rate of 8 Hz. The pulse duration was t = 400 s FWHM (full width at half maximum). The laser radiation was coupled into a zirconium fluoride (ZrF4) optical fiber (core diameter, 600 m), which was guarded at its distal end by a short piece of a low-OH quartz fiber (core diameter, 1000 m). The laser energies measured at the distal fiber tip were 100 and 150 mJ for the Er:YAG laser and 500 and 800 mJ for the Ho:YAG laser, corresponding to radiant exposures of 12.7, 19.1, 63.7, and 101.9 J/cm², respectively. These Ho:YAG-laser energies are at the low end of the energy range used in clinical practice. The delivery fiber was positioned at a 30° angle of incidence to the tissue surface, allowing for the limited space in the joint, and at a constant distance of 500 m between the optical fiber tip and the tissue surface, which was measured under video control. The cartilage samples were exposed to two laser translation protocols. In the first protocol, the irradiated field consisted of a single line measuring 4 mm in length. In the second protocol, the irradiated field consisted of an area measuring 4 ¥ 4 mm to mimic clinical smoothing of cartilage. The fiber was moved by a computerized translation stage such that a 50% overlap of the laser spot was obtained between consecutive laser pulses (Fig. 1). Furthermore, for each irradiation protocol, single and double laser pulses were applied. For each light source and energy level, ten cartilage samples were analyzed in each of two groups (described below).

Group I

One part of each treated sample was fixed in 4% buffered formalin and embedded in paraffin. Serial sections (4 m thick) were cut perpendicular to the laser incision and stained with Masson trichrome, safranin O, or alcian blue. Measurements of the depth of the incision as well as the extent of the thermal damage were then made with use of a light microscope (Leica Leitz, Bensheim, Germany) coupled to an analog camera (Kappa CF 20/4 DX; Kappa Messtechnik, Gleichen, Germany). For morphometric analysis, Image-Pro Plus software (Media Cybernetics, Silver Spring, Maryland) was used.

Group II

At no more than two hours after laser treatment, the other part of the sample was embedded in agar (Fluka Chemical, Buchs, Switzerland; gelling temperature, 35°C) and sectioned with a tissue slicer (TC-Z; Sorvall, Newtown, Connecticut) into 200-m-thick slices. The slices were then incubated for one hour in calcein AM and ethidium homodimer-1 (Molecular Probes, Eugene, Oregon) and analyzed by confocal microscopy (model 400; Carl Zeiss, Göttingen, Germany). Calcein AM is a fluorogenic esterase substrate that is hydrolyzed by living cells to yield a green fluorescent product (calcein), which can be demonstrated intracellularly by fluorescence microscopy or confocal microscopy; thus, green fluorescence is an indicator of viable (living) cells that have esterase activity and an intact membrane that retains esterase products. Ethidium homodimer-1 is a high-affinity, red, fluorescent nucleic acid stain that is able to pass through only the damaged membranes of dead cells²⁰. For each analyzed knee, a piece of cartilage that had not been exposed to laser irradiation was also processed with use of standard histological analysis and confocal microscopy to serve as a control. Laser-treated samples with corresponding controls having more than 2% to 3% dead cells were excluded from the experiment. The results of confocal microscopy and histological analysis were compared with use of the Wilcoxon ranked-sign test.

Results

Introduction | Materials and Methods | Results | Discussion | References

Histological Analysis

Histological examination of the Er:YAG-laser-treated samples stained with alcian blue or safranin O-fast green revealed effective tissue ablation and little or no visible thermal damage at any of the energy levels (Fig. 2, A and B). In contrast, the Ho:YAG-laser-treated samples showed an extensive zone of thermal damage with a depth of almost 600 m (Fig. 2, C and D). Closer examination revealed three different zones of thermal damage, which demonstrated a decrease in proteoglycan content on alcian blue or safranin O-fast green staining (Fig. 3). Zone 1, directly adjacent to the ablated region, showed vacuolization of the tissue, empty chondrocyte lacunae, or lacunae containing necrotic cells with no identifiable nuclei or other organelles. Zone 2 demonstrated small damaged cells containing contracted or marginalized nuclei with no visible or identifiable cytoplasmic organelles. The cells in zone 3 showed no clear-cut abnormalities.

Confocal Microscopy

The extent of thermal damage demonstrated by the cell viability test (Fig. 4) was found to be significantly (p < 0.05) greater than that demonstrated by conventional histological analysis, even with the zone of proteoglycan depletion taken into account. The cell viability test showed a clear line of demarcation between dead cells (red fluorescence) and living cells (green fluorescence). According to this technique, the extent of thermal injury was as deep as 1400 m after two 800-mJ pulses of Ho:YAG radiation. Only limited, superficial tissue ablation with a depth of about 150 m was obtained with this type of laser, and the created surface was rough on macroscopic observation.

In contrast, the ablation depth after two 150-mJ pulses of Er:YAG-laser irradiation was around 500 m and the thermal damage zone was less than 50 m on both morphological analysis and cell viability testing; thus, the findings of the two methods were not significantly different. In fact, exact measurement of thermal damage in this limited zone adjacent to the region of tissue ablation was difficult. The ablated surface also appeared to be smoother than that of the Ho:YAG-irradiated cartilage. Tissue damage and ablation after the various forms of laser treatment, as evaluated by standard histological and histomorphometric methods and by the cell viability test with confocal microscopy, are summarized in Table I. No additional tissue damage was observed in the samples treated with the irradiation protocol.

Discussion

Introduction | Materials and Methods | Results | Discussion | References

The different effects of the Er:YAG and Ho:YAG lasers were due to the lasers’ different absorption coefficients in water^21,22. The Er:YAG laser demonstrated a high ablation efficiency and precision, which, by virtue of the high absorption coefficient, led to thermally damaged zones that were restricted to a depth of no more than 50 m. The Ho:YAG laser demonstrated poor ablation efficiency because its water absorption coefficient is more than 300 times lower than that of the Er:YAG laser. Most of the energy from the pulse of the Ho:YAG laser was used to heat a relatively large volume of tissue, and this heat was not efficiently removed. The deposited heat slowly diffused out of the irradiated volume and left a zone of thermal damage around the crater as demonstrated by the cell viability test (Fig. 4, C and D). The experimental results of the present study qualitatively confirmed the thermal effects that have been generated with a Ho:YAG laser in polyacrylamide gels and visualized by a color Schlieren technique²².

Conventional histological evaluation revealed three zones of thermal damage following Ho:YAG-laser irradiation. The first zone was largely characterized by vaporized tissue that had been directly exposed to the laser beam and partially ablated, leaving an empty crater. The second zone, located adjacent to and beneath this evaporated crater, was characterized by mechanically ruptured tissue that may have been damaged by the explosive expansion and subsequent collapse of a water vapor bubble⁸. The depth of this second zone was approximately twice the depth of the vaporized tissue. The third zone, although sharply demarcated by proteoglycan depletion, contained a large number of cells that appeared to be unchanged as judged by light microscopy.

The cell viability test unambiguously revealed the extent of laser damage more accurately than conventional histological analysis did. Specifically, confocal microscopy revealed the extent of tissue damage to be approximately twice that demonstrated by conventional histological analysis (Table I). Although cell morphology appeared normal when viewed by light microscopy (Fig. 3, zone 3), it was apparent that cellular membranes were injured and compromised, allowing ethidium homodimer-1 to stain the nuclei.

The experimental results highlighted the problems associated with use of high-power Ho:YAG-laser settings during arthroscopic surgery. The cell viability test on the sites treated with Ho:YAG radiation showed an almost full-thickness cartilage defect. Our findings explain some of the adverse effects that have been observed after clinical use of the Ho:YAG laser. Lane et al., in 1997, found that energy levels of more than 1000 mJ caused full-thickness damage extending down to the subchondral bone¹⁰. Möller et al., in 1994, compared conventional mechanical cartilage abrasion with Ho:YAG laser débridement (energy level, 450 to 1440 mJ; pulse frequency, 1 to 15 Hz)²³. At one day following the procedure, the laser-treated surfaces were much smoother than the mechanically treated surfaces. However, at one and three months following treatment, the laser-treated surfaces were observed to be rougher than the mechanically treated surfaces²³. In view of our observations in the present study, the laser-induced chondrocyte death in the cartilage surface appeared to lead to matrix collapse in vivo and, thus, may have contributed to the rough cartilage surface observed by Möller et al.

The ex vivo model used in the present study is simple, reproducible, and, importantly, could eliminate the need for extensive experimentation on live animals. This ex vivo model, however, does not predict the long-term behavior of the cells after laser exposure. The advantage of this model is that the chondrocytes are exposed to laser irradiation in their native surroundings rather than in monolayer cell-culture systems, as previously described^5,24,25. The normal bovine cartilage used in the present study has different properties than those of osteoarthritic tissue cartilage, which has partial loss of matrix integrity because of the presence of cracks and has activated chondrocytes that may secrete various cytokines leading to an intensive matrix remodeling. However, this model may be employed to study cartilage in vitro under a range of experimental conditions.

The findings of the present study highlight the problems associated with use of high-power settings of Ho:YAG lasers during arthroscopic surgery. The thermal damage caused by this system, as assessed in vitro by confocal microscopy, is important. The Er:YAG laser demonstrated superior ablation effectiveness while causing less acute collateral tissue damage, and its use in orthopaedic surgery therefore warrants additional investigation. Standard histological analysis clearly underestimated the acute tissue damage after laser irradiation compared with that demonstrated by vital staining. The ex vivo model employed in the present study reduced the need for extensive experimentation on live animals and yielded rapid results—that is, a rough assessment of tissue survival without the need to wait for the outcome in vivo.

References

Introduction | Materials and Methods | Results | Discussion | References

Trauner KB, Nishioka NS, Flotte T, Patel D.: Acute and chronic response of articular cartilage to holmium:YAG laser irradiation. Clin Orthop.,1995.310: 52-7, 31052 1995 [PubMed]

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Atik OS, Tali T.: Does the Holmium:Yag laser cause osteonecrosis?. Bull Hosp Jt Dis.,1999.58: 111-3, 58111 1999 [PubMed]

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Abergel RP, Lyons RF, Castel JC, Dwyer RM, Uitto J.: Biostimulation of wound healing by lasers: experimental approaches in animal models and in fibroblast cultures. J Dermatol Surg Oncol.,1987.13: 127-33, 13127 1987 [PubMed]

Vangsness CT, Ghaderi B.: A literature review of lasers and articular cartilage. Orthopedics,1993.16: 593-8, 16593 1993 [PubMed]

Föhn O, Pratisto HS, Könz F, Ith M, Altermatt HJ, Frenz M, Weber HP.: Side-firing fiber device for underwater tissue ablation with Ho:YAG and Er:YAG laser radiation. J Biomed Optics.,1998.3: 112-22, 3112 1998

Pratisto H, Frenz M, Ith M, Altermatt HJ, Jansen ED, Weber HP.: Combination of fiber-guided pulsed erbium and holmium laser radiation for tissue ablation under water. Applied Optics.,1996.35: 3328-36, 353328 1996 [PubMed]

Janecki CJ, Perry MW, Bonati AO, Bendel M.: Safe parameters for laser chondroplasty of the knee. Lasers Surg Med.,1998.23: 141-50, 23141 1998 [PubMed]

Lane JG, Amiel ME, Monosov AZ, Amiel D.: Matrix assessment of the articular cartilage surface after chondroplasty with the holmium:YAG laser. Am J Sports Med.,1997.25: 560-9, 25560 1997 [PubMed]

Lubbers C, Siebert WE.: Arthroscopic holmium-YAG laser compared to conventional procedures on the knee joint. 2-year results of a randomized prospective study. Orthopäde.,1996.25: 64-72, German2564 1996 [PubMed]

Vangsness CT, Akl Y, Nelson SJ, Liaw LH, Smith CF, Marshall GJ.: In vitro analysis of laser meniscectomy. Clin Orthop.,1995.310: 21-6, 31021 1995 [PubMed]

Vangsness CT, Watson T, Saadatmanesh V, Moran K.: Pulsed Ho:YAG laser meniscectomy: effect of pulsewidth on tissue penetration rate and lateral thermal damage. Lasers Surg Med.,1995.16: 61-5, 1661 1995 [PubMed]

Wakitani S, Imoto K, Kimura T, Ochi T, Matsumoto K, Nakamura T.: Hepatocyte growth factor facilitates cartilage repair. Full thickness articular cartilage defect studied in rabbit knees. Acta Orthop Scand.,1997.68: 474-80, 68474 1997 [PubMed]

Rozbruch SR, Wickiewicz TL, DiCarlo EF, Potter HG.: Osteonecrosis of the knee following arthroscopic laser meniscectomy. Arthroscopy.,1996.12: 245-50, 12245 1996 [PubMed]

Thal R, Danziger MB, Kelly A.: Delayed articular cartilage slough: two cases resulting from holmium:YAG laser damage to normal articular cartilage and a review of the literature. Arthroscopy.,1996.12: 92-4, 1292 1996 [PubMed]

Raunest J, Derra E.: Laser-assisted induction of arthrosis. Orthopäde.,1996.25: 10-6., German2510 1996 [PubMed]

Tabib W, Beaufils P, Blin JL, Tremoulet J, Hardy P.: Arthroscopic meniscectomy with Ho-Yag laser versus mechanical mensicectomy. Mid-term results of a randomized prospective study of 80 meniscectomies. Rev Chir Orthop Réparatrice Appar Mot.,1999.85: 713-21, French85713 1999 [PubMed]

Pratisto H, Ith M, Frenz M, Weber HP.: Infrared multiwavelength laser system for establishing a surgical delivery path through water. Appl Phys Lett.,1995.67: 1963-5, 671963 1995

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TABLE I: Effect of Laser Type and Energy Level on Tissue Removal and Tissue Damage

	Laser Type and Energy Level
Er:YAG*				Ho:YAG*
100 mJ		150 mJ		500 mJ		800 mJ
1 Pulse	2 Pulses	1 Pulse	2 Pulses	1 Pulse	2 Pulses	1 Pulse	2 Pulses
Ablation depth (m)
Confocal microscopy	?75 ± 10	122 ± 9	174 ± 13	485 ± 24	35 ± 4	120 ± 4	?175 ± 15	225†
Histological analysis	90 ± 9	111 ± 5	180 ± 22	500 ± 30	40 ± 3	?130 ± 12	170 ± 9	230 ± 11
Damage depth (m)
Confocal microscopy	<50	<50	<50	<50	1090 ± 110	1072 ± 83	1240 58	1422‡
Histological analysis	<10	<10	<10	<10	351 ± 22	?412 ± 18	?553 43	580 ± 22

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TABLE I: Effect of Laser Type and Energy Level on Tissue Removal and Tissue Damage

	Laser Type and Energy Level
Er:YAG*				Ho:YAG*
100 mJ		150 mJ		500 mJ		800 mJ
1 Pulse	2 Pulses	1 Pulse	2 Pulses	1 Pulse	2 Pulses	1 Pulse	2 Pulses
Ablation depth (m)
Confocal microscopy	?75 ± 10	122 ± 9	174 ± 13	485 ± 24	35 ± 4	120 ± 4	?175 ± 15	225†
Histological analysis	90 ± 9	111 ± 5	180 ± 22	500 ± 30	40 ± 3	?130 ± 12	170 ± 9	230 ± 11
Damage depth (m)
Confocal microscopy	<50	<50	<50	<50	1090 ± 110	1072 ± 83	1240 58	1422‡
Histological analysis	<10	<10	<10	<10	351 ± 22	?412 ± 18	?553 43	580 ± 22