J. Hubacek, J. Pospisilova

ENT Clinic, Faculty Hospital Olomouc, CZ

 

Abstract

According to mitotic and synthetic activities of fibroblasts in the scar and in the granuloma the authors can form the following conclusions: (1) Laser irradiation influences fibroblasts and collagen directly in the irradiated area (number of fibroblasts increased by 62.5 per cent) and in the surrounding tissue (increased by 34.8 per cent), laser stimulates formation of IIIb type collagen typical for inflammatory reactions, increased synthesis and mitotic activity of fibroblasts were also monitored, (2) Irradiation in the third week of healing increases synthesis of I type collagen, (3) Laser irradiation is effective mainly in the event of poorly healing wounds.

 

Introduction

In clinical practice, wound healing is the decisive factor to ensure the outcome of a successfull operation. Poorly healing or even disintegrating wounds prolong the time of healing and they can be the cause of serious complications. On the other hand, they lead to undesirable presence of keloid scars, scarry stenosis and contractures.

In clinical practice, laser irradiation has proved to act as a stimulating agent in poorly healing wounds. Since no effect of therapeutic doses of laser irradiation on the growth of pathogenic bacteria [1] could be demonstrated, we believe laser being beneficial in the stimulating of immune responses [2, 3], or of initial regenerative processes.

Our previous studies followed the effect of laser irradiation upon the number of fibroblasts in early stages of wound healing [4] as well as on types of collagen depending on the time elapsed between the operation and irradiation [5]. These experiments were supplemented by studying mitotic and synthetic activity of fibroblasts with the use of radioisotopes.

 

Materials and methods

A group of 24 Wistar male rats (weighing 200 grams) had little 10 x 10 x 5 mm sponges of viscous cellulose implanted into subcutaneum from an incision on their back. Eight rats were left as a control group. The others had the incision and areas of both the implants exposed to daily irradiation with a 10 mW He-Ne laser for the period of 1 minute (i.e. 0.6 J), i.e. total dose of 1,8 J, from the 2nd to the 7th day after the surgery, 5 times in total. 

Half of the animals from both groups were decapitated on the 17th day, the other half on the 25th day after operation. Two hours before decapitation, radioisotopes of  ³H thymidine 4 MBq to follow synthesis of DNK, and  ¹⁴C proline 0.4 MBq to follow biosynthesis of collagen, were injected. 

The granuloma and scars were extirpated, mechanically homogenized and the fibroblast mitotic and metabolic activity was examined. 

DNA was determined after Niinikoski [6], the activity in the samples being measured  by scintillation with a two-channel spectrometer. The collagen proteine types were identified according to Ehrlich, the quantity and activity of hydroxyproline likewise by scintillation according to Kivirikko [7].

 

Results

a)    With regard to mitotic activity, no changes in granulomas were observed on the 17th day (see Table 1), neither in the number of cells, nor in their activity. 25 days after operation, the number of cells in granuloma decreased significantly, however the cells show high mitotic activity. The scars show these changes already on the 17th day. 

 

	Day 17		Day 25
	Cells	Specific activity	Cells	Specific activity
Control granuloma	8.5	6523.8	7.81	4180.9
Laser granuloma	6.5	6725.4	1.92	10176.0
Control scar	2.05	11658.5	0.84	50288.0
Laser scar	0.89	16539.3	0.85	42142.3

Table 1:     Mitotic activity of fibroblasts

 

b)     In order to be able to determine synthetic activity of fibroblasts - collagen production - only granulomas could be used, due to requirements on the volume of tissue in the chromatographic division (see Table 2). In control granulomas an increased amount of I type collagen and its activity can be seen, as well as a decrease of both the parameters of III type collagen on day 17 and day 25. In both the time intervals, after laser irradiation  metabolic activity of I type collagen was significantly increased whilst its total amount in tissue decreased. In III type collagen both the amount and its metabolic activity increased.

 

	Collagen Type	Day 17			Day 25
		Hydroxyproline	%	Activity	Hydroxyproline	%	Activity
Control Granuloma	I	27.9	85	7383	59.5	100	3807
	III	3.6	11	3478	0	0	0
Granulom (laser)	I	19.7	80	31208	25.6	88	10732
	III	4.7	20	20085	2.0	12	38000

Table 2:    Collagen formation

 

Discussion

Our observation has supplemented and confirmed the conclusions of our previous experiments in which we had followed:

1. Activity of fibroblasts in healing wound on both earlobes of rabbits when the wound on the right earlobe was irradiated only (4),
2. Types of collagen in the scar and in the experimental granuloma on the back of the rats after an early and a late irradiation (5). 

 

It has been proved that:

a)    early irradiation (day 2 to day 7 after the surgery) with a dosage of 1.5 J a day, five times in total, does not cause any differences in comparison with the control group on the third day, however, on the eighth day, the numbers of fibroblasts were found increased by 62.5 per cent in directly irradiated wound and by 34.8 per cent in unirradiated wound in animals with the second ear exposed to laser beam,

b)    early irradiation (day 2 to day 7 after the surgery) with a dosage of 1.8 J, five times in total, will increase glycoproteins and III type collagen, therefore it increases inflammatory reaction (see Table 3). Late irradiation (day 15 to day 21) applied in the same way increases the level of I type collagen significantly, therefore deposition of collagen in the tissue is increased substantially. Late irradiation suppresses inflammatory reaction of ligament (III type collagen decreased, missing in the scar). Material was processed on the 22nd day after the surgery.

 

	Glycoproteins	Peptides	Type Ia	Type Ib	Type IIIa	Type IIIb
GRANULOMA - control	6.2	16.4	2.5	94.3	1.9	1.3
-   early irradiation	11.0	9.4	4.5	89.5	4.0	2.0
-   late irradiation	7.9	15.6	11.8	86.4	0.8	1.0
SCAR - control	13.1	8.3	0	98.0	1.2	0.8
-   early irradiation	15.6	33.4	0	95.9	2.8	1.3
-   late irradiation	22.2	7.0	9.6	90.4	0	0

Table 3:    Genetic type of collagen following laser irradiation (relative proportion in %)

 

Monitoring of mitotic and synthetic activity of fibroblasts with the use of radioisotopes, with the samples taken on day 17 and day 25, was evaluated in the second stage of wound healing when the number of fibroblasts, as well as their mitotic activity, decrease in the course of the healing, but, on the contrary, synthetic activity of fibroblasts in this stage of healing increases in terms of synthesis of I type collagen, typical for tissue. It can also be noticed in values of the control group (see Table 2). Monitored decrease of the number of fibroblasts after irradiation on days 17 and 25 (see Table 1) can be explained by accerelated healing process and by shortened regeneration processes thanks to their activation. It is also important to mention that after irradiation a lower number of cells holds high mitotic activity. Another finding, i.e. that on days 17 and 25 an increased amount of III type collagen as well as its increased metabolic activity (see Table 2) were monitored, confirms our previous findings related to different effects of early and late irradiation. Irradiation in the first week activated also in this case formation of III type collagen. 

A number of authors have been dealing with the effect of laser irradiation on fibroblasts. Mester et al. (8, 9) have documented increasing number of cells after irradiation of cultures of human fybrocytes with a He-Ne laser, furthermore, production of collagen being stimulated even after the first irradiation, the volume of collagen increasing after successive irradiations, laser beam increasing the activity of non-specific esterasis of fibroblasts, after skin incision of rats they monitored increased tensile firmness only after five days after the incision, whilst a significant difference could be noticed on the eighth day after the surgery. Lam et al. (10) monitored increased production of procollagen after irradiation of cultures. Stimulation of the growth of fibroblasts after laser irradiation has been described by Soundry et al. (11), significant stimulation of collagen production in granulation tissue after laser irradiation was mentioned by Bosatra et al. (12) and in fibroblast cultures by Lyons et al. (13). Ditrichova (14) irradiated fibroblast cultures in human embryonic lungs with laser with a dosage of 4 J/cm² repeatedly in four days, or with a flat dosage of 50 J/cm².  Electronic microscope revealed changes after repeated dosage of 4 J/cm² - there was Golgi`s apparatus noticeably developed, membranes of endoplasmatic reticulum with folds and sacs dilatations, numerous mitochondria the size and contrast of which were close to those of mitochondria of unirradiated fibroblasts. After irradiation with a flat dosage of 50 J/cm<sup>2</sup> significant dilatations of the sacs od Golgi`s apparatus as well as of granular endoplasmatic reticulum, and mitochondrias of oblong shape with a sharp contrast as well as with disintagrated mitochondrial crists were observed. The study has revealed that even if it is necessary to take into account that the experiments were performed with embryonal cells, therapeutic utilization of He-Ne laser to stimulate fibroblasts is useful with repeated lower dosages, since one-shot massive irradiation of fibroblasts led to disintegrative changes in mitochondrias.

Our results correspond with observations of the above mentioned, as well as of other authors on fibroblast stimulation and collagen formation by means of laser irradiation, adding up findings on different effects of laser in the course of different phases of wound healing, and knowledge about synthetic and mitotic activity of fibroblasts after irradiation with He-Ne laser.

Experimental results vindicate our clinical experience. In clinical practice laser beam accelerates wound healing of worsened healing processes, especially in oncological patients after operations in irradiated areas, and is also a suitable stimulus supporting tissue substitution in cases of cells deficiency (cartilage, bone). Thanks to impoved deposition of collagen, laser irradiation applied in the third week of healing could have positive influence on healing in areas where keloid scars formation could be anticipated.

 

Literature

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2. Hubacek, J., Matouskova, I., Grivnova, J., Polach, A.: Changes in Phagocytic Activity of Neutrophil Leukocytes after Irradiation with He-Ne Laser, Cs. Otolaryng., 34, 1985, s. 141-146
3. Hubacek, J., Krc, I., Matouskova, I.: Reaction of Lymphocytes of Palatal Tonsils after Application of He-Ne Laser, Cs. Gastroent. Vyz., 37, 1983, No. 8, s. 457-466
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8. Mester, E., Nagylucskay, S., Tisza, S., Mester, A.: Neuere Untersuchungen uber die Wirkung der Laserstrahlen auf die Wundheilung, Z. exper, Chirurg, 10, 1977, No. 5, s. 301-306
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10. Lam, T. S., Abertel, R. P., Dwyer, R. M., Nitto, J.: Biological Effects of Laser, Laser in Surgery and Medicine, 3, 1983, s. 189-190
11. Soundry, M., Franquin, J. C., Pourreau-Schneider, N., Martin, P. M.: Action d`un laser He-Ne sur la croissance cellulaire: étude in vitro sur fibroblastes gingivaux humains, J. Biol. Buccale, 16, 1988, s. 129-135
12. Bosatra, M., Jucci, A., Olliaro, P.: In Vitro Fibroblast and Dermis Fibroblast Activation by Laser Irradiation at Low Energy, An Electron Microscopis Study, Dermatologica (Basel), 168, 1984, s. 157-162
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