O
RIGINALA
RTICLEW
ALDOL
INOJ
ÚNIOR1, W
ILLIAMD
IASB
ELANGERO2The effects of Holmium YAG laser (Ho:YAG) on the patellar
tendon of rats after 12 and 24 weeks of follow up
SUMMARY
The authors have studied the effects of the Holmium: Yttrium Aluminum Grenade (Ho:YAG) laser on tendon sizes (proximal and distal length and width) and on the cellularity and arrange-ment of collagen fibers in 20 Wistar variety, male, white, adult rats (Rattus Novergicus). The animals have been divided in two groups, according to the follow-up time (12 and 24 weeks) and pursuant to the for form of laser application (continuous or two-point). A Holmium laser (pulsed, solid state, 2.1 micron waves, 40 watts, OmniTip 30º tip) apparatus was used. After the ani-mals were sacrificed, the proximal and distal length and width of the operated side of such rats were compared to those of the non-operated side be means of non-parametric testing (consi-dering p=0,05). The length in the operated side was significan-tly bigger for both follow-up groups, when compared to the length of the non-operated side, however there was no significant diffe-rence in such measures in function of the type of laser applicati-on. In the same manner, the width, both in the proximal and
dis-tal regions, was significantly bigger in the operated side in both follow-up groups, without showing any significant difference whatsoever in function of the type of application. When the mea-sures in both follow-up groups were compared, distal length and width showed a trend to become bigger after 24 weeks, while width in the proximal region was significantly bigger in this group. As for the subjective microscopic evaluation, both in longitudi-nal and cross sections, it was possible to observe and increase in the number of fibroblasts, mainly in the 12-week group. The average fibroblast concentration in the tendon with 24-week fo-llow-up group was deemed to be intermediate between the 12-week group and non-operated tendon. The conjunctive tissue was exuberant in the region amidst the fasciculi for the 12-week group, with evident vascular neoformation. In the 24-week group, collagen fibers were regularly arranged, parallel to the tendon axle.
Keywords: Laser; Collagen; Tendon; Wound healing.
Study conducted at the Orthopaedics and Traumatology Department – FCM - UNICAMP
Correspondences to: Alameda Santos 211, conjunto 209 – Cerqueira César – 01419-002 – São Paulo/SP – e-mail: waldo@uol.com.br
1 - Master in Surgery by the Medical Sciences College (FCM) – UNICAMP – Head of the department of Orthopaedics at the Real e Benemérita Associação Portuguesa de Beneficência
2 - PhD in Surgery by the Medical Sciences College (FCM) - UNICAMP – Coordinator of the Traumatology and Pediatric Orthopaedics Groups – Coordinator of the Laboratory of Orthopaedic Materials Investigations, Orthopaedics and Traumatology Department – FCM-UNICAMP.
Received in: 10/14/04 approved in 11/30/04
INTRODUCTION
Collagen ability to suffer retraction through thermal and che-mical methods has been known since 1871, although publicati-ons have only been issued from 1900 on. Partridge* was the first
author to describe, in 1948, the shrinkage and laxity of rats’ tail tendon in distilled water at 60º C. From 1950’s on, such pheno-menon has been further studied, especially for industrial purpo-ses, since this technology was used in the leather industry.
Recently, the group leaded by Hayashi was responsible for many in vitro and in vivo studies aiming to analyze the effects of the Ho:YAG laser as a heat source for promoting the shrinkage of collagen-rich structures(1,2,3). Those authors concluded this
effect was energy-dependent and that laxity properties of such protein did not change after laser application.
After that, Vangsness et al.(4) evaluated the effect of the laser
on calcaneus tendons and patellar ligaments of human cada-vers and reported a reduction of the mechanical resistance of these structures in up to 70%, suggesting the need of further in
vivo studies on animal models in order to analyze whether such
resistance could be retrieved over time or not.
From the theoretical point of view, thermal energy created by laser could be beneficial for the treatment of joint instabilities by promoting capsular shrinkage. However, it is unclear if joint sta-bility recovery is produced by a simple collagen retraction or by a disarranged fibroblast proliferation originated from a normal healing process, due to the necrosis produced by heat. In addi-tion, there are doubts regarding whether immobilization after heat application is necessary or not in order to maintain the results
Figure 2 - Two-point type laser application on patellar tendon Figure 1- Continuous-type laser
application on patellar tendon
*PARTRIDGE, apud BANGA et al. (5)
achieved. The purpose of this study is to analyze the medium and long-term effects of Ho:YAG laser in an experimental model, over a collagen-rich tissue in order to assess the effect of Ho:YAG on celullarity and on rats’ patellar tendon sizes, without immobi-lization, as a result of the type of laser application and of the follow-up time.
MATERIALS AND METHODS
Twenty adult, male, white, Wistar variety rats (Rattus
Novergi-cus) with average weight of 410 grams, provided by the Biotery
Center (CEMIB) from the State University of Campinas (UNI-CAMP) were used. ***
Rats were divided into 2 groups of 10 animals each, accor-ding to the follow-up time (12 and 24 weeks). Each group was then divided into 2 subgroups of five animals each, according to the type of laser application: continuous (CO) or two-point (TP) (Figures 1 and 2).
A Trimedyne Holmium (Ho:YAG) pulsed laser equipment was used, which is manufactured by Trimedyne®, Inc. 2801
Barran-ca, Irvine, CA 92714, adjusted to a frequency of 19Hz, 25W po-wer, with pulses of 0.5 seconds.
All animals were submitted to anesthesia with 50mg/ Kg sodium Pentobarbital, endovenously injected via tail. After trichotomy of the posterior legs and aseptic cleaning with iodine alcohol solution, a lateral parapatellar incision was performed and the patellar tendon was accessed. Laser shots were appli-ed with the knee joint immersappli-ed in a vat fillappli-ed with saline solution in order to mimic the arthroscopic procedure. On the left side, laser was applied in either a continuous (CO) or two-point (TP) fashion, randomly selecting the rat, with the
right side being used as control. All surgeri-es were performed by the same team and with the same equipment. Application was made on all tendons by the same surgeon intending to reduce variables regarding con-tact time with the laser and its pressure over the tendon. Laser exposure time in each pro-cedure (CO or TP) was approximately two seconds. Surgical wound was sutured with a nylon suture 000 and the animal remained warm until recovered from anesthesia. All animals have been left free, in plastic cages, with sterilized wood chips. The animals were fed with industrialized ration and filtered wa-ter “ad libitum”. Afwa-ter the follow-up time, the animals were sacrificed with a deeper anes-thesia with ethylic ether, and, following car-dio-respiratory failure, the ligaments on left and right side were exposed. Tendons leng-th and widleng-th were measured wileng-th a pachy-meter by Mitutoyo Corporation (Tokyo – Ja-pan) by the same individual and using the same technique. Measures were taken
simul-taneously at the right and left side of each animal. Length was measured from the lower pole of the patella up to its insertion on tibial anterior tuberosity, and width was measured in its proximal portion, just below the lower pole of the patella, and in its distal portion, just above the tibial anterior tuberosity, keeping the left knee joint (operated) in a flexed position, in the same position as the right knee (control). For evaluating the changes on the leng-ths and widleng-ths of the tendons submitted to the laser application, each operated side measure was subtracted from the measure of the control side, with such difference being named as delta, which would be stretching or enlargement if positive, and shor-tening or narrowing if negative.
Following, the femur was sectioned in its distal portion and the tibia, in its proximal portion, with shear. This segment was kept in a 10% formaldehyde solution for 24 hours and transfer-red to a 70% ethylic alcohol solution. Six tendons, three of them receiving the CO type of application and three receiving the TP type, have been randomly selected from each group (12 and 24 weeks). 4m -thick sections were obtained, which were stained by hematoxylin and eosin, Masson trichomic. With the purpose of rendering the study tendon regions uniform, the ten first sec-tions of each block have been discarded.
The evaluation was performed as a result of the comparative analysis on cells amount and on the arrangement and appea-rance of the collagen fibers – modified from Belangero et al.(6).
An optical microscope was used (model Leica DML DC 300 F) with an objective lens 20x and with the Program “Imaging Pro-cessing Analising System (Leica QWin)” for capturing images. From each slide, five fields were selected, which were grouped in an image database in order to facilitate the comparison to each other. This comparison was performed among tendons of each subgroup (CO and TP) within the same group (12 and 24 we-eks), and then among tendons of each group (12 and 24 weeks), via ordinary optical mi-croscopy. Two tendons from each of the subgroups – totaling four tendons by group -have been randomly selected for controls, which did not receive laser application (right side).
The results analysis for the measurements was performed by using non-parametric tes-ts. In order to evaluate the significance of the laser application effect on tendon length and width measurements (proximal and distal re-gions) a Mann-Whitney’s U-test was used. For comparing the effects of the kind of laser application, the Wilcoxon test has been used. On the evaluation of the follow-up time effect the Mann-Whitney test has been used as well. For all tests, p = 0.05 was established. Sli-des evaluation was performed in a subjecti-ve fashion, disregarding statistical tests.
Table 1 - Mean, median and standard deviation for length, proximal region width and distal region width of rat tendons submitted to a laser application in a continuous fashion (L-CO) or in a two-point fashion (L-TP) and of tendons not submitted to the laser, assessed after 12 weeks of follow-up.
Legend: n = number of tendons; CO = Continuous laser; TP = Two-point laser
RESULTS
I – Effect of the Kind of Laser Application on T I – Effect of the Kind of Laser Application on T I – Effect of the Kind of Laser Application on T I – Effect of the Kind of Laser Application on T I – Effect of the Kind of Laser Application on Ten-en-en-en- en-dons Sizes
dons Sizes dons Sizes dons Sizes dons Sizes
In this phase, difference values (deltas) between each mea-surement on the side submitted to laser and the control side are considered according to the type of laser application. After 12 and 24 weeks of follow-up, no significant differences were noti-ced between the continuous (CO) or the two-point (TP) applica-tions, both in length measurements and in proximal or distal wi-dth measurements (Tables 1 and 2).
Because there were no significant differences between the types of laser application, this variable was not considered for analysis of the laser effects and of the follow-up time on tendons sizes.
II – Effect of the laser on tendons sizes II – Effect of the laser on tendons sizes II – Effect of the laser on tendons sizes II – Effect of the laser on tendons sizes II – Effect of the laser on tendons sizes
As shown in Tables 1 and 2, there was a significant stretching of the tendons in animals submitted to laser application and with a follow-up time of 24 weeks (p = 0.01). A significant increase was also noticed on proximal and distal width on tendons sub-mitted to laser application with 12 and 24-week follow-up time.
III – Effect of the laser and follow-up time on III – Effect of the laser and follow-up time on III – Effect of the laser and follow-up time on III – Effect of the laser and follow-up time on III – Effect of the laser and follow-up time on ten-dons sizes
dons sizes dons sizes dons sizes dons sizes
From Figures 3, 4 and 5 it can be seen that length and distal width measurements of tendons submitted to laser application tended to be greater in the 24-week follow-up group (p = 0.07 and p = 0.06, respectively), while proximal width measurement was significantly higher after 24 weeks (p = 0.01).
IV IV IV IV
IV- Macroscopic evaluation- Macroscopic evaluation- Macroscopic evaluation- Macroscopic evaluation- Macroscopic evaluation
After 12 weeks of follow-up, tendon appearance was typical, exhibiting a loss in brightness, a subtle yellowish color and volu-me increase. In the 24-week group, although voluvolu-me was lower and brightness was more evident, the tendon appearance was notably different from the non-operated side.
V V V V
V- Microscopic eva-- Microscopic eva-- Microscopic eva-- Microscopic eva-- Microscopic eva-luation luation luation luation luation Microscopic evaluation was performed by analy-zing six tendons (three re-ceiving CO laser and three receiving TP laser), ran-domly selected from each of both groups. Histologi-cal sections showed a very regular and typical pattern. However, there were no d i f f e r e n c e s b e t w e e n subgroups.
Laser application pro-duced collagen hyaliniza-tion areas and fibroblasts proliferation.
Hypercellu-larity and an increase of the number of collagen fibers at the periphery of the tendon (paratendon) were well-defined in the 12-week group (Figure 6). Both in longitudinal and transversal sections, an increase of fibroblasts was seen, which was more evident in the 12-week group. In the 24-week group, average concentration of fibroblasts was considered as intermediate be-tween the 12-week group and the non-operated group (Figure 7). In the region between fascicles, the connective tissue was more exuberant in the 12-week groups and with a more intense vascular neoformation. Calcification areas were seen on longitu-dinal sections of the 12-week and 24-week groups submitted to both types of laser application (CO and TP). Hyalinized collagen fibers were seen in the 12-week group, mixed with areas where fibers were randomly arranged, in a non-parallel orientation in relation to the tendon long axis. After 24 weeks, the collagen fibers arrangement in the groups showed a similar appearance to the non-operated side (Figure 8).
DISCUSSION
Many studies have been performed, both in vitro and in vivo, in different animal species (cattle, dogs, rats, rabbits, sheep) with the purpose of understanding the shrinkage-and-recovery process occurring subsequently to heat application(7,1,8,9,10,11).
Nevertheless, there are still just a few studies in vivo evalua-ting the biological effects of heat on collagen-rich tissues, espe-cially in medium and long term(12).
When temperatures ranging 65oC to 75oC are reached,
co-llagen suffers a structural disarrangement resulting in cell com-ponents’ death, which resist to up to 40o C. Recovery process is
then initiated, characterized by neovascularization and fibroblasts proliferation(8).
By the moment this experimental model has been proposed, the main purpose was to study the effects of laser on rats’ pate-llar tendon recovery process when animals are kept free of im-mobilization for a period of 12 and 24 weeks.
Immediate postoperative mobilization was important for eva-luating tendon’s ability to remodel after thermal in-jury. With the shortening, the patellar tendon beca-me more required than the one of the normal side, sin-ce in the case of rats, knee extended position is not physiological for ambulati-on. Tendon stretching may have occurred on the dead portion of the tendon du-ring recovery process, es-pecially because of the well-known fact that the greater the shortening, the lower the tissue resistance to mechanical tracti-on(13,3,9,11). As the average
Table 2 - Mean, median and standard deviation for length, proximal region width and distal region width of rat tendons submitted to a laser application in a continuous fashion (L-CO) or in a two-point fashion (L-TP) and of tendons not submitted to the laser, assessed after 24 weeks of follow-up.
Legend: n = number of tendons; CO = Continuous laser; TP = Two-point laser
Figure 3 - Values of the differences between tendons lengths as a result of follow-up time
Figure 4 - Values of the proximal width differences on tendons as a result of follow-up time
Figure 5 - Values of the distal width differences on tendons as a result of follow-up time.
mm, its deepest portion may be intact, since Ho:YAG laser penetration within the conditions used here ranges from 0.3 mm to 0.5 mm, maintaining jo-int function in the early re-covery phase, as well as the intact portion serving as a model for recovery.
With the unaided eye, clear differences could be seen both in the length and in the distal and proximal width of tendons between the normal side and the operated side in both fo-llow-up groups (12 and 24 weeks). On the other hand,
all animals from those groups had an apparently normal and similar function, both on the operated side and on the control side. Usually, recovery and collagen fibers neoformation proces-ses take 12 weeks in average to be completed(11). After this
peri-od, neoformed tissue is considered as physiologically mature, meaning that it is able to perform its mechanical functions. The fact that a greater length was found on the operated side after 12 weeks indicates the need of immobilizing at the immediate postoperative period, in order to keep the shrinkage resulting from heat, which will serve as a model for vascular neoformati-on, and, mainly, the fibroblast neoformation. Even when compa-ring tendons with initially smaller lengths (rats with 10 weeks), we could see that the operated side had become significantly longer after 24 weeks, inclusively in comparison to the 12-week group.
Continuous and two-point types of laser application were compared because literature shows evidences of point applica-tion producing the same amount of shrinkage as continuous application, but allowing tissue recovery to be faster due to the preservation of intact areas between heat application points(9).
According to the previous arguments, the final stretching in the
group submitted to the two-point application could be expected to be smaller. However, results did not corroborate such a hypothesis, because no significant differences were found regarding leng-th and distal and proximal width between both appli-cation types. The smaller size of the preserved cen-tral area as a result of the size of tips used can be a potential reason for the fact. The other theory could be related to the reduction of blood intake to the cen-tral portion of the patellar tendons, coming mainly from the proximal and distal regions, hit by heat in both groups. Such reduction may have decreased, at least transitorily, the recovery ability of such region. This result and theories previously raised can impose a doubt regarding the two-point application of the laser in this model.
Alternatively, in future studies the laser application in two points and continuous could eventually be compared by applying the laser on the central portion of tendon. In those conditions, proximal and distal portions would be preserved and the remai-ning viable area would be bigger than the area hit by laser. At first, this option was not chosen during project elaboration be-cause it was believed that central application could weaken the tendon, resulting in a rupture during the immediate postoperati-ve period, caused by the early joint mobilization. Corroborating to this theory, we can mention the in vitro study by Vangsness et al.(4), in which the authors analyzed the mechanical resistance of a human patellar tendon after Ho:YAG laser application on its central portion. During traction assays, they noticed the occur-rence of a rupture exactly at the region hit by laser.
Regarding proximal and distal width portions measurements of tendons in both groups (12 and 24 weeks), they were found
Figure 7 -H i s t o l o g i c a l appearance of the tendon, in a transversal section, where enlarged res-traints of the connective tis-sue (arrows) are noticed, with abundant presence of vessels splitting colla-gen fibers (Hematoxylin eosin, 12-week group, laser applied using the continuous type, objecti-ve lens 10x). Figure 8 -H i s t o l o g i c a l appearance of a 2 4 - w e e k group tendon, where oriented fibers and cells presenting a si-milar appearan-ce as the non-operated side are seen, both in quantity and morphology (Hemato-xylin eosin, 24-week group, laser applied in two-po-ints, objective lens 20x).
REFERENCES * Figure 6 - His-tological appe-arance of the p a r a t e n d o n (left), where an increase of ce-lls counts and vascular neo-formation is ob-served. On the right, heat-hya-linized collagen fibers are noticed (*). (Hemato-xylin eosin, 12-week group, laser applied in two-points, objective lens 20x).
1. Hayashi K, Markel MD, Thabit G II, Bogdanske JJ, Thielke RJ. The effect of nonablative laser energy on joint capsular properties. Am J Sports Med 1995; 23:482-487.
2. Hayashi K, Thabit G III, Vailas AC, Bogdanske JJ, Cooley AJ, Markel MD. The effect of nonablative laser energy on joint capsular properties. An in vitro his-tologic and biochemical study using a rabbit model. Am J Sports Med 1996a; 24:640-646.
3. Hayashi K, Thabit G III, Bogdannske JJ, Mascio LN, Markel MD. The effect of nonablative laser energy on the ultra structure of joint capsular collagen. Ar-throscopy 1996b; 12:474-81.
4. Vangsness CT, Mitchell W III, Nimni M, Erlich M, Saadat V, Schmotzer, H. Co-llagen shortening. Clin Orthopedic 1997, 337:267-271.
5. Banga I, Baló F, Szabo D. Contraction and relation of collagen. Nature 1954; 174:788.
6. Belangero WD, Köberle G, Hadler WA. Inflammatory reaction of rat striated muscle to particles of carbon fiber reinforced carbon. Braz J Med Biol Res 1993; 26:819-826.
7. Abelow SP. Use of lasers in orthopedic surgery: current concepts. Orthope-dics 1993; 16:551-556.
8. Hayashi K, Nieckarz JA, Thabit G III, Bogdanske JJ, Cooley AJ, Markel MD. Effect of nonablative laser energy on the joint capsule. An in vivo rabbit study using a holmium:yag laser. Lasers Surg Med 1997; 10:164-171.
9. Medvecky MJ, Ong BC, Rokito AS, Sherman OH. Thermal capsular shrinka-ge: Basic science and clinical applications. Arthroscopy 2001; 17:624-635. 10. Schaefer SL, Ciarelli MJ, Arnoczky SP, Ross HE. Tissue shrinkage with the
holmium: yttrium aluminum garnet laser. A postoperative assessment of tis-sue length, stiffness and structure. Am J Sports Med 1997; 25:841-848. 11. Schulz MM, Lee TQ, Sandusky MD, Tibone J.E.; McMahon P. The healing effects
on the biomechanical properties of joint capsular tissue treated with Ho: YAG laser. An in vivo rabbit study. Arthroscopy 2001; 17:342-347.
12. Hayashi K, Hecht P, Thabit G III, Peters DM, Vanderby R Jr., Cooley AJ et al. The biologic response to laser thermal modification in an in vivo sheep model. Clin Orthopedic 2000; 373:265-276.
13. Arnoczky SP, Aksan A. Thermal modification of connective tissues: Basic sci-ence considerations and clinical implications. J Am Acad Orthopedic Surg 2000; 8:305-313.
14. Wong KL, Williams G. Complications of thermal capsulorrhaphy of the shoul-der. J Bone Joint Surg Am 2001; ; ; ; ; 83(Suppl. 2, Pt2):151-155.
significantly greater at the operated side and, as occurred with the final length, there was no significant difference in width as a result of the type of application. This finding is consistent to the
in vivo assays performed on rabbits’ patellar tendon(10). Laser is
known to stimulate fibroblasts neoformation and, consequently, collagen fibers deposing(2,11). Optical microscope analysis of the
slides showed that in such regions, even after 12 weeks, there still was an intense fibroblastic proliferation and cartilaginous neoformation areas, presenting calcification as well. The progres-sion to calcification indicates that thermal injury exceeded beyond necessary limits and may have caused extensive tissue necro-sis. In the 24-week follow-up groups, the histological appearan-ce was close to normal, with the presenappearan-ce of fibers directed to-wards the tendon long axis and with a decreased number of cells compared to the 12-week group. However, cartilaginous and calcified cartilage areas have also been seen in this follow-up grofollow-up (24 weeks). On the other hand, the exuberant fibro-blastic response, particularly present in 12-week animals, sho-ws that an important injury occurred on the tendon and by sim-ply performing a clinical control, these lesions are difficult to re-produce safely. Calcification areas were not expected or desi-red, especially because it is not possible to establish which func-tional progression would be with those tendons in case the ani-mals were kept alive. As there was an evident change in the mi-croscopic appearance between both groups, perhaps longer follow-up periods could provide additional information on the natural and continuous remodeling process occurring on ten-dons when submitted to physiological efforts.
Somehow confirming those findings, literature shows that laser is being replaced by radiofrequency equipment, which is cheaper, more user-friendly and easy to transport. But the most important reason for the replacement is that such equipment allo-ws surgeons to have, during the procedure, a touch sensation with the tip on the tissue, thus enhancing his/her tactile sensati-on and, csensati-onsequently, providing better csensati-ontrol over the proce-dure(14).
From the practical point of view, all those conclusions are relevant, although they cannot be directly transferred to the prac-tice. However, they may serve as a warning for keeping a judg-ment spirit for surgeons in searching for better outcomes based on these findings. Anyway, the issues raised here can only be answered upon new experimental and clinical studies, with well-prepared protocols.
CONCLUSIONS
Regarding the effect of Holmium Trimedyne laser applicati-on applicati-on rats’ patellar tendapplicati-on after 12 and 24 weeks, which were kept free of immobilization, we can conclude that:
There was a significant stretching of tendons, being more intense in the 24-week follow-up group. There was also a signifi-cant enlargement of the proximal and distal regions of tendons in the 12- and 24-week follow-up groups.
Histologically, the collagen fibers arrangement and the cellu-larity on tendons submitted to laser returned at their normal le-vels only after 24 weeks, although calcification areas are still found.
