Lasers in Surgery and Medicine 531-39 (1985)

The Biomedical Effects of Laser

Application
Endre Mester, MD, Andrew F. Mester, MD, and Adam Mester, MD
Postgraduate Medical School Laser Research Laboratory (f.M.), Second Department of
Pediatrics ENT Unit ( A X M.), and Department of Radiology (A.M.), Semrnelweis Medical
University, Budapest, Hungary.

This paper briefly reviews the authors experimental and clinical use of lasers
over a 20-year period, during which laser effects on 15 biological systems were
studied. Low-energy laser radiation was found to have a stimulating effect on
cells, and high-energy radiation had an inhibiting effect. The application of lasers
to stimulate wound healing in cases of nonhealing ulcers is recommended.

Key words: laser beam, laser biostimulation, application of soft laser, wound healing, non-
healing ulcers

INTRODUCTION
One of the most significant discoveries in the present century in the field of
medical science is the laser. It involves new, almost inconceivable perspectives in the
fields of biological research and application in medical practice. It is beyond the scope
of this paper to summarize the related works of professional literature; thus only the
results we obtained in the course of experimental and clinical activity are presented
here.

REVIEW OF RESEARCH
Beginning with a pulse-operated ruby laser device, later with continuous oper-
ating He-Ne and argon gas lasers, we studied laser effects in 15 various biological
systems.
The effect of ruby laser on the bacterium (Staphylococcuspyog.aureus) phago-
cytosis of leukocytes was investigated. Leukocytes were obtained by sedimentation in
part from circulating human blood containing heparin, and in part from the abdominal

Address reprint requests to Dr. Mester, Postgraduate Medical School Lasers Research Laboratory,
Budapest, XIII Szabolcs 35., Bp Pf.:112, Hungary 1389.
Accepted for publication October 31, 1984.

0 1985 Alan R. Liss. Inc.

32 Mester, Mester, and Mester

cavity of rats after a quantity of bouillon was injected in the rats. For the purpose of
experimentation, sedimented native cells were applied. Phagocytosis was determined
in accordance with the method of Ludany and Vajda [9]. We found that the bacterium
phagocytosis of leukocytes was considerably increased by an incident energy density
(IED) of 0.05 J/cm2 and inhibited by IED values of 2-4 Jicm. Further, we proved
that radiation sensitivity of leukocytes was increased by the application of methylene
blue and Janus B green staining solutions, and was suspended when acridinorange
solution was used. Small IEDs applied in series to native leukocytes cumulated and
acted to inhibit large energies, in conformity with the Arndt-Schulz law.
The above effect appeared to be mainly a ferment one. The results obtained by
others and the changes in catalase activity of the leukocytes pointed to that. In the
course of investigations, properly prepared samples of leukocytes of rats were sub-
jected to laser radiation with various IED values. Subsequently, catalase activity was
measured in accordance with Frenycis method [9], and it was seen that-when
compared to untreated control samples-activity in the experimental samples was
increased by a value of 0.05 J/cm2, particularly by one of 5 J/cm2 and the same was
decreased by 50 J/cm2.
The in vitro effect of the ruby laser on the growth of Erlichs ascites tumors
was also investigated [8]. Growth of the tumor, mitosis-index of the tumor cells,
electronmicroscopic picture of the tumor cells, and survival of test animals were
compared to corresponding features of a control group inoculated with tumor cells
from the same donor that were not, however, treated with laser rays. In six series of
experiments, body weight values of animals inoculated with cells treated with laser
rays surpassed that of animals in the control group by 10-16%, and the number of
tumor cells was 19-30% higher in the experimental group than in the control group.
The differences proved to be highly significant in three series. In an experimental
group of ten mice, survival time following inoculation with cells treated with laser
rays was shorter than in the control group.
The stimulating effect of low-power radiation and the inhibiting effect of high-
power radiation was also observed in hair growth of C57B1 and white mice [9].
Dosages of 1 J/cm2 IED lrubylweek applied for 3 to 5 weeks involved increased hair
growth, but following the tenth to eleventh radiation, inhibition occurred. In this
experiment, cumulative small doses in repeated application also resulted in an inhib-
iting effect. The biological effects of radiation applied fractionally were cumulative
even when fractionation was applied with time delays. Following treatment of longer
duration, histologically inflammatory, later atrophic, changes occurred on the hair
follicles. After 15 treatments, some parts of the epithelium became larger because of
the proliferation of basal cells. This may, perhaps, be interpreted as a preblastomatosis
phenomenon; however, after the thirtieth irradiation, atrophy, induced by the total
sum of effects, occurred.
During this period of time, changes also occurred in the internal organs of some
animals; for example, subcapsular coagulation-type cellular necroses extended over
some sheets in the liver area. In three mice, necroses occurred in the wall of the small
intestine on the anterior abdominal wall side, together with consequential peritonitis.
It is well known from the investigations of Fine et al [3] and others that the liver and
the intestines are responsive to the effects of a direct high-power laser; however, it is
possible that necroses may come into existence from the cumulative effect of such
low-power pulses even through the intact shn. This possibility requires a thorough
consideration.
 Biomedical Effects of Laser 33
The healing of 10-mm-diameter round-shaped total skin defects artificially
created on the back of white mice was significantly accelerated by the effect of ruby
laser rays [7]. The laser rays most probably represented a stimulus causing a higher
activity of the cells at the lips of the wound and at the wound basis, increasing the
number of divisions and accelerating the formation of granulation tissue. Microscopic
investigation revealed a larger number of dividing cell forms in the area of the treated
wound; and the wound closed more quickly. The stimulating effect was greatest when
the wounds were treated twice a week, 4 times altogether, with 1 . 1 J/cm2 IED being
applied for each irradiation.
Since the intestinal mucosa is highly responsive to radiation, in situ experiments
were carried out on the effect of ruby laser rays on the micromotility of the intestinal
mucosa, as well as on the movement of intestinal villi [9]. We found that smaller
volumes of energy (1-3 J/cm2) increased the automatic movement of intestinal villi,
and larger volumes inhibited or even suppressed automatic movement. Following the
application of a ray of approximately 7 J/cm2 IED, the signs of destruction were
already visible. The increasing effects of radiation on automatic movement are
thought to take place through the mediation of the ganglions.
We also examined the effects of ruby laser on the corneas of 18 rabbits treated
with vessel formation stimulating total extract of the adrenal glands of rats [9].
Observations were made on 2 weeks' experiment material by means of loupe magni-
fication on macroscopic specimens and on tissue sections. In the range of smaller
IED values (1-3 J/cm2) the angiogenesis-promoting degenerative effect of the total
extract of the adrenal gland was decreased qualitatively, as well as quantitatively, by
the laser treatment. In the range of higher IED values (5 J/cm2) the above effect was
quantitatively increased with accompanying inflammation, ulceration, and perfora-
tion. Destructive doses of laser radiation also had remote effects, as the vascular area
was increased also in the cornea of the opposite-side control eyes treated only with
total extract of the adrenal gland.
Within the range of IED values of 0.05 to 26 J/cm2, the effects of ruby laser
beams on hemoglobin synthesis were observed on short-term rat bone marrow
cultures irradiated in vitro. Parallel development of heme and globin synthesis was
followed by measuring the incorporation of 59-FE and C-14 glycine (Ammersham)
in other cultures. In the range of low IED values heme synthesis was significantly
increased, tending toward inhibition as IED values rose; in the globin synthesis
observation, a small decrease occurred, followed by an increase. The latter remained
moderate and appeared primarily with IED values that already involved inhibition in
heme synthesis. The differences between the degrees of the two components of
hemoglobin were assumed to be related to the different degrees of power absorption
by the two kinds of proteins [9].
In Escherichiu coli CR 54 cultures, in the presence of an exogenic light stabilizer
(methylene blue), the relative cell count rose significantly under the effect of 1 J/cm2
IED, when compared to the control cell count. In bacterial cultures treated with high
IEDs (120 J/cm2) the count of surviving cells decreased parallel with the IED value
applied. The bacterial cells that survived formed much smaller colonies than the
control cells, and the mean sizes of the former decreased as IED values rose. Clearly
these colonies were formed by sublethally injured bacterial cells [ 111.
When experiments were repeated under identical conditions, the quantitative
changes of DNA were measured on the basis of the incorporation of I4C-marked
thimidine, By means of radioactive ribonucleic acid-precursor incorporation measure-
34 Mester, Mester, and Mester

ments, it was observed that owing to the effect of low IED values, marked uridine
was incorporated by E coli cells to an increased extent, reflecting the stimulative
effect on the synthesis of the RNA.
In order to study the biological effect of lasers, two test objects specifically
susceptible to radiations were selected. In dog experiments the micromotility of
intestinal mucosa was increased by small IED radiation (2 J/cm2). This effect was
suspended, or considerably weakened, when amino-ethylthiouronium (AET) was
previously administered parenterally. AET also weakened both the stimulating and
the inhibiting effects of laser beams in the case of bacteriophagocytosis of leukocytes.
AET protected the SA radicals of thiolenzymes from destructive radiation effects
[ 101.
According to recent investigations, the application of ruby laser to the Auer-
bachs plexus of guinea pigs increased acetylcholine release. We concluded the
aforesaid to be a stimulative effect, too [25].
The investigation of the effects of laser irradiation and antithymocyte serum
treatment on the survival of mouse-skin homotransplants was, in effect, a model
experiment [21]. In connection with extensive burn injuries, when free graft of foreign
skm becomes indispensable, the investigations clinical significance is accentuated by
the fact that it is by the length of time the transplant remains viable, or how long it
will be capable of providing protection and inhibiting the fluid and ion losses of
persons who suffered serious burn injuries is by no means unimportant.
As transplant, the tail skin of male GBA mice originating from an inbred tribe
was used. The recipients were random male Swiss albino mice of 20-25 g body
weight. Laser irradiation was performed with a continuously operating 20 mW output
He-Ne gas laser, with an unfocused, perpendicularly incident ray of identical power
density.
Antithymocyte treatment was performed on the second and fifth day following
the transplantation with an i.p. 0.5-0.5 ml volume of antithymocyte serum (ATS).
The ATS was produced in rabbits with thymocytes of male Swiss mice of 10-11 days
of age. The efficiency of the ATS was measured with the increase in survival rate.
Survival rate of the transplant was increased only to a small extent by the laser
beam applied alone. The survival time in animals treated with ATS rose only by
56.2% compared with that of control animals. Treatment with laser increased this
effect by 28.5%, i.e., the mean survival time (MST) was 84.7% higher in animals
treated with ATS and laser irradiation than in the control animals.
As reported in the professional literature, recently, a combination of immuno-
suppression and allograft, as well as xenograft, transplants has been used in the
treatment of burn injuries. While highly effective, immunosupressive substances
induced deterioration of protection against infections of the organism. This is espe-
cially hazardous for persons who have suffered burn injuires, even when treated with
antibiotics.
However, the joined application of low-power level laser irradiation and im-
munosuppressive treatment may offer new perspectives in cases of allograft, as well
as xenograft, transplantations. In our transplantation interventions carried out on
mice, the joined application of antithymocyte serum treatment and laser irradiation
allowed a longer survival time of the allotransplant than it was possible to achieve
when ATS or laser irradiation were applied alone.
We also studied the effect of laser irradiation on changes in lymphocyte trans-
 Biomedical Effects of Laser 35

formation [ 141. The lymphocyte transformation test is a specific method of examinat-

ing cellular immunoreaction to detect sensibilization in vitro. Some mitogenic
substances are capable of transforming lymphocytes to large-size, blast-type cells in
a nonspecific way, too. When this transformation takes place, together with the
changes in cell morphology, changes in cellular metabolism also occur with respect
to the increase of synthesis of DNA and RNA. In human lymphocytes the de novo
synthesis of nucleic acids due to stimulation by phytohemagglutinine (PHA) starts,
and the aforesaid transformation can easily be measured on the basis of radioactive
purines and pyridines, as these values reflect the quantity of DNA-synthetizing cells.
In this work the effects of laser radiation on human lymphocytes stimulated with
PHA were examined by measuring the incorporation of radioactive timidine into
DNA. From microscopic measurements, as well as from fluid scintillation ones, it
became clear that no cell propagation or blast formation could be registered in either
the control cultures or the lymphocyte cultures treated with laser, when they were not
stimulated. In lymphocyte cultures stimulated with PHA, blast formation could be
detected microscopically to 60-80%, thus conforming with reports in the literature.
In cultures stimulated with PHA and treated with laser, a 20% increase in blast
formation could be observed, when compared to untreated but stimulated cells. With
1 J/cm2 ruby laser energy being applied, the rate of DNA synthesis of lymphocytes
stimulated with PHA made a multiple of that in untreated ones. With unstimulated
lymphocytes the laser treatment alone proved to be inefficient.
Considering that laser treatment proved to have no effect at all on unstimulated
lymphocyte cultures, the impression was obtained that under the condition given, the
laser beam did not directly stimulate the lymphocyte metabolism but had an effect on
one or more of the factors that induce stimulation.
This experiment and the preceding experiment on mouse skin transplants start
from the aspect of the investigation of the presumable immunological effects of lasers.
With the purpose of clarifying the immunosuppressive effects of lasers, we
observed their direct influence on T and B lymphocytes. Ruby, He-Ne, and argon
lasers were applied, and it was established that the phenomenon depended on the
volume of incident energy, on the laser output, and on the wavelength. In the range
of low IED values (0.5-1 J/cm2) most favorable results were obtained with a
combination of pulse-type and continuous operation lasers (argon, 488 nm wave-
length; He-Ne, 632 nm wavelength) [20].
Similarly, the effects of monochromatic polarized and nonpolarized normal light
were compared with those of laser light with respect to the immunosuppressive effect
of human lymphocytes [20]. The He-Ne gas laser had an output of 632 nm, linearly
polarized 50 mW power. The incoherent light source consisted of a halogen lamp,
with fluorescent light mirror and corresponding filters , with identical power, wave-
length, and IED values being applied. We found that the effect of incoherent light
was 0.74% when compared to that of the laser. With plano-polarization of corre-
sponding plane, however, an efficiency of 80% could be achieved. To explain the
20% difference between the efficiency of the two respective light sources requires
further investigations.
CLINICAL RESULTS
Because of the positive results of our experiments, in 1971 we started to deal
with the treatment of human wounds, ulcers difficult to heal, or not healing at all,
36 Mester, Mester, and Mester

and with the problem of responsiveness of wound healing to laser treatment. The
investigation of the process of wound healing at the molecular level has become
practical as a result of the development, during the last two decades, of biochemical,
autoradiographic, enzyme-histochemical, and electron-microscopic examination
methods. At the microscopic level, inflammation, proliferation, and reorganization
occur. These represent respective phases of wound healing. The first two phases, ie,
inflammation and proliferation, involve the development of granulated tissues, and
the third phase is characterized by contraction, on the one hand, and by ripening
of the scar tissue, on the other. Following the characteristic changes occurring during
the early postinjury phase, fibroblasts appear at a later date. The high tensile strength
of collagen produced by fibroblasts serves to provide and-in case given-to replace
the tissue defects that occur from injuries.
Our experiments involved ulcers, as well as nonhealing wounds that did not
positively respond to the usual therapeutic procedures, including plastic surgery.
Treatment was first performed with an He-Ne laser (50 mW) and, later, with an argon
laser (wavelength 488 nm, 100 mW power), inasmuch 4 J/cm2 twice a week on the
total wound surface. Results are shown in Table I, grouped from an etiological point
of view. The average duration of healing was 12-16 weeks.
Definite healing can usually be expected in clinical cases where no general
circulatory disturbances are present, or where a general circulatory disturbance can
be corrected. In accordance with our experience, laser irradiation can also be applied
as preparative, eventually complementary, treatment for dermatoplastic interventions.

TABLE I. Distribution of Cases by Their Etiology

Healed Improved Not healed

I Skin ulcers of mechanical origin 48 9 6

2 Skin ulcers not healing after burn 40 8 5
3 Radionecrosis caused by 35 10 6
electrocautery and X-ray
treatment of tumors
4 Diabetic lipodystrophy 12 2 3
5 Trophic wound healing troubles 80 6 3
6 Long-existing varicose crural ulcer 130 5
7 Obstinate ulcers following a 15 5
recurrent erysipelas
8 Postthrombotic ulcers 216 90 50
9 Uecubitus 33 10
10 Postoperative wound-healing 160 5 6
troubles
1 1 Skin necroses due to coumarin 18
treatment
12 Skin necroses due to infection 18 5 3
13 Erosio portionis uteri 8 2 2
14 Ulcus in urinary bladder (with 8 3 1
fiber optics)
15 Allergic vasculitis 8
16 Ulcus oris 5
17 Ulcus ventriculi (with fiber optics) 40
18 Sturge-Weber syndrome 1
Totals 875 160 85
 Biomedical Effects of Laser 37

It will be the task of further experiments and clinical observations to state the
place of lasers among the therapeutic means used to stimulate wound healing. A
summary of our experiments and investigations follows.
The healing process was followed by means of a series of electron microscopic
examinations [15]. In the sample taken prior to irradiation, quite a number of cells
corresponding mostly to fibroblasts and collagenous fibres were observed. Following
the first irradiation the number of collagenous fibres increased and the cellular
substance diminished. In addition, vesicles having electron-dense nuclei were found
in both intracytoplasmatic and intercellular material. Following the second laser
treatment, the number of lysosoma-type bodies was increased in the intracellular area,
mitochondria became swollen, and further multiplication of collagen and of the
vesicles was observed in the intercellular space. Similarly, a further multiplication of
the collagen and the vesicles followed the third irradiation. No changes occurred in
the clinical picture. In the samples excised from the nonirradiated areas of the wound
surface, similar, but less expressed, changes were observable at various dates.
On the basis of activity measurement results obtained after *C-glycine and 3H-
proline incorporation [ll], we felt the point of attack to be at the synthesis of
collagene, and we assumed that it was primarily the collagenous phase of wound
healing that was affected by laser stimulation. This assumption was confirmed by our
electron-microscopic examinations. In our opinion, the structural basis of healing
consists of the activation of collagene production, and a certain role should also be
attributed to the vesicles with central nuclei. These are assumed to contain bioactive
substances that also act as catalysts of healing in the nonirradiated areas. As further
evidence for this assumption, we know that it is not necessary to fully irradiate the
entire ulcer in order to achieve the stimulating effect. The possibility that the remote
effect was induced by a humoral substance created in the irradiated area was raised.
To investigate this, we returned to a method applied earlier, ie to stimulating the
bacterium-phagocytosis of leukocytes with laser rays.
In the course of this experimental series [16] sedimented leukocytes from the
abdominal cavity were selected as experimental subjects. In order to clarify the effect
of time, irradiations were performed with identical mean energy levels, in normal
and Q-switching, with ruby laser (treatment durations of 1 ms and 1 ns, respectively).
Sedimented leukocytes were suspended in Ringers solution containing a homologous
serum immediately after the irradiation, and the solution was distributed to five
aliquots. The control samples were treated identically with the experimental ones-
except that they received no laser rays at all. One control and one irradiated sample
were centrifuged immediately after the irradiation and then 1, 2, 3, and 4 hours later.
The supernatants of the cells were mixed with fresh normal leukocyte populations,
and the effects of the supernatants of the phagocytosis were studied.
The results confirmed that from the irradiated cells a substance capable of
increasing the phagocyte function in a normal leukocyte population was released. In
addition, the concentration of stimulating substance altered as a function of time
elapsed since that of irradiation in the surrounding medium, and at the end of the 4
hours experimental time, the supernatants effects became inhibitive. Following
irradiations performed in Q-switching mode, both the stimulating and inhibiting
effects of the medium occurred in a more explicit way and at an earlier time.
As a result of these findings we assumed that laser beams influenced enzymatic
events in an early stage of wound healing. Therefore, we investigated the effects of
38 Mester, Mester, and Mester

laser irradiation applied at a stimulative IED value on skin wounds of rats, with
respect to succinic acid-dehydrogenase, lactic acid-dehydrogenase, acid phosphatase,
and nonspecific esterase activity localizable histochemically in accordance with Rae-
kallios method [22], in the early stage of wound healing [ 151. We found that 8 to 48
hours after wounding, in the basal cells of the epithelium within the sound tissue zone
next to the wound lips, activity of succinic acid-dehydrogenase, and in the fibroblasts,
that of lactic-acid-dehydrogenase and of nonspecific esterase increased when com-
pared to the control cells. Consequently, the preparation for increased functioning of
fibroblasts may be detected with enzyme-histochemical methods as early as 8 hours
after wounding takes place, and this activity will be increased by laser treatment.
The effect of laser beams on another decisive phase of wound healing, ie the
development of new blood vessels, was studied [ 2 3 ] . Our results showed that the
development of blood circulation of regenerating tissues was significantly influenced
by laser rays. In the course of experiments, Sanders ear chamber techniques were
applied and He-Ne radiation treatment was performed daily in the usual way. The
significant increase in recapillarization, which we attribute to the effects of the laser,
was best shown when comparing the photos taken the eighteenth post-implantation
day with those of the control animals.
The changes in tensile strength values due to laser radiation were followed in
an experimental series in rats, in which the healing of cut wounds closed with clamps
was studied. It was observed that tensile strength increased most dramatically on the
eighth postoperative day, in the proliferative-vascular phase of wound healing, giving
a good congruence with the results obtained earlier in the course of collagen produc-
tion revascularization experiments.
The effects of ruby laser on RNA and DNA protein synthesis in the macromol-
ecules of human fibroblast cultures were also studied [24]. We measured to what
extent the specific, radioactively marked mother substances of DNA and RNA
syntheses of proteins were incorporated by the cells of previously laser-treated and
untreated control cultures. The effects were examined in four series. In each series
three laser-treated and three untreated cultures were incubated separately with [3H]-
uridine, [3H]-timidine, and I4C-valine.
The incorporation of marked uridine and valine was increased to a small extent
by irradiation. The stimulating effect on the incorporation of timidine, however, was
much more dramatic. Since the extent of the incorporation of timidine commonly
supplies information about the number of cells in phase S of the cellular cycle, it may
be assumed that laser irradiation involved an increase in the number of cells in
reproducton.
We investigated the changes in E and F types of prostaglandin (PG) contents
from the effect of a 50-mW-output He-Ne laser at 1 J/cm2 IED values as a function
of time elapsed since the wounding of the dorsal skin of rats [2]. In 4 days, both kinds
of PG significantly accumulated when compared to the control animals. After 8 days,
however, the PG E2 content value dropped below the level observed with the control
group, while that of PG F2a continued to rise.
We concluded that the stimulating effect of lasers on wound healing was related
to the increase in PG formation, and that the changes in the levels of various
prostaglandins contributed to the acceleration of a partial process of wound healing-
to that of the inflammation phase.
 Biomedical Effects of Laser 39

CONCLUSIONS
Our experience with 875 clinical cases, that healed, together with the results of
the experiments carried out for the purpose of elucidating the bioregulative process
of wound healing, has convinced us to recommend the use of lasers to stimulate
wound healing.

ACKNOWLEDGMENTS
This work was supported by the Central Research Institute of Physics of
Hungary and by the Hungarian Optical Works.

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