The effects of Holmium YAG laser (Ho:YAG) on the patellar tendon of rats after 12 and 24 weeks of follow up

Abstracts

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 arrangement 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 animals were sacrificed, the proximal and distal length and width of the operated size of such rats were compared to those of the non-operated size be means of non-parametric testing (considering p=0,05). The length in the operated size was significantly bigger for both follow-up groups, when compared to the length of the non-operated size, however there was no significant difference in such measures in function of the type of laser application. In the same manner, the width, both in the proximal and distal regions, was significantly bigger in the operated size in both follow-up groups, without showing any significant difference whatsoever in function of the type of application. When the measures 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 longitudinal 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 follow-up was group 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.

Laser; Collagen; Tendon; Wound healing

Os autores estudaram os efeitos do laser Hólmio:Ítrio-Alumínio-Granada (Ho: YAG) sobre as dimensões do tendão (comprimento e largura proximal e distal) e sobre a celularidade e arranjo das fibras de colágeno em 20 ratos adultos, machos, brancos (Ratus Novergicus) da varidade Wistar. Os animais foram divididos em dois grupos de acordo com o tempo de seguimento (12 e 24 semanas) e de acordo com a forma de aplicação do laser (contínua e em dois pontos). Utilizou-se aparelho de laser de Hólmio (pulsátil, estado sólido, com ondas de 2,1 microns, com potência de 40 watts, ponteira OmniTip de 30º) . Após o sacrifício, foram comparadas por meio de testes não paramétricos (considerando p = 0,05) as medidas de comprimento e da largura (proximal e distal) dos tendões do lado operado e do lado não operado. A medida do comprimento do lado operado foi significativamente maior nos dois grupos de seguimento, quando comparado com o lado não operado, porém, não houve diferença significativa dessas medidas em função do tipo de aplicação do laser. Do mesmo modo, a medida da largura, tanto na região proximal quanto distal, foram significativamente maiores no lado operado nos dois grupos de seguimento, sem apresentar diferença significativa em função do tipo de aplicação. Quando se compararam as medidas nos dois grupos de seguimento, o comprimento e a largura distal tenderam a ser maiores após 24 semanas, enquanto que a largura, na região proximal, foi significativamente maior nesse grupo. Quanto à avaliação microscópica subjetiva, tanto nos cortes longitudinais como transversais, pôde-se verificar aumento do número de fibroblastos, principalmente no grupo de 12 semanas. A concentração média de fibroblastos nos tendões com 24 semanas de seguimento foi considerada como intermediário entre o grupo de 12 semanas e os tendões não operados. Na região entre os fascículos, o tecido conjuntivo era exuberante no grupo com 12 semanas, com neoformação vascular evidente. No grupo de 24 semanas as fibras de colágeno se apresentavam com disposição regular e paralela ao longo eixo do tendão.

Laser; Colágeno; Tendões; Cicatrização de Feridas

ORIGINAL ARTICLE

The effects of Holmium YAG laser (Ho:YAG) on the patellar tendon of rats after 12 and 24 weeks of follow up

Waldo Lino Júnior^I; William Dias Belangero^II

^IMaster in Surgery by the Medical Sciences College (FCM) — UNICAMP — Head of the department of Orthopaedics at the Real e Benemérita Associação Portuguesa de Beneficência

^IIPhD 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

^{Correspondence}

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 arrangement 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 animals 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 (considering p=0,05). The length in the operated side was significantly bigger for both follow-up groups, when compared to the length of the non-operated side, however there was no significant difference in such measures in function of the type of laser application. In the same manner, the width, both in the proximal and distal 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 measures 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 longitudinal 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 follow-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.

INTRODUCTION

Collagen ability to suffer retraction through thermal and chemical methods has been known since 1871, although publications 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 phenomenon has been further studied, especially for industrial purposes, 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 cadavers 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 stability 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 addition, there are doubts regarding whether immobilization after heat application is necessary or not in order to maintain the results 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 immobilization, 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 Novergicus) with average weight of 410 grams, provided by the Biotery Center (CEMIB) from the State University of Campinas (UNICAMP) were used. ^**

Rats were divided into 2 groups of 10 animals each, according 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 Barranca, Irvine, CA 92714, adjusted to a frequency of 19Hz, 25W power, 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 applied with the knee joint immersed in a vat filled 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 surgeries 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 contact time with the laser and its pressure over the tendon. Laser exposure time in each procedure (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 water "ad libitum". After the follow-up time, the animals were sacrificed with a deeper anesthesia with ethylic ether, and, following cardio-respiratory failure, the ligaments on left and right side were exposed. Tendons length and width were measured with a pachymeter by Mitutoyo Corporation (Tokyo — Japan) by the same individual and using the same technique. Measures were taken simultaneously 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 lengths and widths 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 shortening 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 transferred 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 sections 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 appearance 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 Processing 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 weeks), and then among tendons of each group (12 and 24 weeks), via ordinary optical microscopy. 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 tests. In order to evaluate the significance of the laser application effect on tendon length and width measurements (proximal and distal regions) 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. Slides evaluation was performed in a subjective fashion, disregarding statistical tests.

RESULTS

I — Effect of the Kind of Laser Application on Tendons Sizes

In this phase, difference values (deltas) between each measurement 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 noticed between the continuous (CO) or the two-point (TP) applications, both in length measurements and in proximal or distal width 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

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 submitted to laser application with 12 and 24-week follow-up time.

III — Effect of the laser and follow-up time on tendons 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- Macroscopic evaluation

After 12 weeks of follow-up, tendon appearance was typical, exhibiting a loss in brightness, a subtle yellowish color and volume increase. In the 24-week group, although volume was lower and brightness was more evident, the tendon appearance was notably different from the non-operated side.

V- Microscopic evaluation

Microscopic evaluation was performed by analyzing six tendons (three receiving CO laser and three receiving TP laser), randomly selected from each of both groups. Histological sections showed a very regular and typical pattern. However, there were no differencesbetween subgroups.

Laser application produced collagen hyalinization areas and fibroblasts proliferation. Hypercellularity 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 between 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 longitudinal 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 evaluating the biological effects of heat on collagen-rich tissues, especially in medium and long term⁽¹²⁾.

When temperatures ranging 65ºC to 75ºC are reached, collagen suffers a structural disarrangement resulting in cell components' 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' patellar tendon recovery process when animals are kept free of immobilization for a period of 12 and 24 weeks.

Immediate postoperative mobilization was important for evaluating tendon's ability to remodel after thermal injury. With the shortening, the patellar tendon became more required than the one of the normal side, since in the case of rats, knee extended position is not physiological for ambulation. Tendon stretching may have occurred on the dead portion of the tendon during recovery process, especially because of the well-known fact that the greater the shortening, the lower the tissue resistance to mechanical traction^(13,3,9,11). As the average width of this tendon is 0.7 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 joint function in the early recovery 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 follow-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 processes take 12 weeks in average to be completed⁽¹¹⁾. After this period, 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 neoformation, and, mainly, the fibroblast neoformation. Even when comparing 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 application 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 length and distal and proximal width between both application types. The smaller size of the preserved central 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 central 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 remaining viable area would be bigger than the area hit by laser. At first, this option was not chosen during project elaboration because it was believed that central application could weaken the tendon, resulting in a rupture during the immediate postoperative 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 occurrence 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 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 progression to calcification indicates that thermal injury exceeded beyond necessary limits and may have caused extensive tissue necrosis. In the 24-week follow-up groups, the histological appearance was close to normal, with the presence of fibers directed towards 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 group (24 weeks). On the other hand, the exuberant fibroblastic response, particularly present in 12-week animals, shows that an important injury occurred on the tendon and by simply performing a clinical control, these lesions are difficult to reproduce safely. Calcification areas were not expected or desired, especially because it is not possible to establish which functional progression would be with those tendons in case the animals were kept alive. As there was an evident change in the microscopic appearance between both groups, perhaps longer follow-up periods could provide additional information on the natural and continuous remodeling process occurring on tendons 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 allows surgeons to have, during the procedure, a touch sensation with the tip on the tissue, thus enhancing his/her tactile sensation and, consequently, providing better control over the procedure⁽¹⁴⁾.

From the practical point of view, all those conclusions are relevant, although they cannot be directly transferred to the practice. However, they may serve as a warning for keeping a judgment 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 application on rats' patellar tendon 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 significant 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 cellularity on tendons submitted to laser returned at their normal levels only after 24 weeks, although calcification areas are still found.

REFERENCES

Received in: 10/14/04 approved in 11/30/04

Study conducted at the Orthopaedics and Traumatology Department — FCM - UNICAMP

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• 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.
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*

PARTRIDGE, apud BANGA et al. (5)

**

The research project was duly approved by the Committee of Ethics in Animal Experimentation (CEEA) of the Biology Institute, UNICAMP, protocol # 194-1, of 06/30/2000.

Publication Dates

• Publication in this collection
  27 July 2005
• Date of issue
  2005

History

• Received
  14 Oct 2004
• Accepted
  30 Nov 2004