Perftoran, experimental fracture, microcirculation, laser doppler

flowmeter, osteogenesis
G. M. Dubrovin, A. V. Ivanov, O. V. Yakovleva, Chen Chi Hsiang,
D. A. Ivanov
The influences of Perftoran on the local circulation and
osteoreparation for the experimental high-energetic diaphysis
fractures depend on the method of introductions
Kursk State Medical University, Kursk
There are increased portions of high-energetic injury in general
structual trauma in the every year [3]. The frequency of development is
slowly in the consolidation with prosthetic joints in ones according to the
different authors , there are attaint to the amount in 33.2% , ones in given
data from the consequences of high-energetic fractures in long inferior
extrmities[1,2,16].
With given evidents of the important stages in speed and quality of
consolidation in fracture, is depending in the situation upon the
hemodynamics of injurious extrmities[2,3,612,13]. In the one so called
priority tendency in the development of modern traumatology is the
elaboration of the well-based pathogenetic methods which is the most
significant, to acquired by means of restore the adquate tissues perfusion
through the stimulation in osteoreparation[6,10]. With these views of
points were to represent the application of Perftoran as the majority of
interests in study regeneration in traumatology.
Perftoran- the blood substitute with air-transportive function and in
which there are possessing hemodynamics, rheologics,membrane-
satblizings,cardio-proectorings,diuretics and the properties of sorption[5].
The application of Perftoran which be able to afford as, to enhance the
content of oxygen transpotation, to standardize the utilization of tissues
oxygen and to create the pre-requisite for relive the hypoxia[8,9,15]. The
Perfluorocarbon emulsion has possesses the resources of formed so called
perfluorophages. These Perfluorophges are to preserve the functionl
activity and to take a part in the process of regeneration till to healing the
wound, by means of this, it given the process of regeneration to be
accelerated. With the revealing phenomenon a steady increases of the
partial pressure of the oxygen in the wounded tissues at the zone of
Perftorans which were presented, there are appeared to be assured of the
tissues oxygenation, and to stimulating the reparation and the
regeneration, and to preventing the pathological fibro- and
collagenesis[4]. There are basic mechanisms in the reduction of the
hypoxic tissues, to implement in the oxygen expenses, by the gradients of
partial pressure makes the solution of Perftoran to transitory in tissues, be
expressed to reduce the frequency of infectious complications and to
increase quantity of viable cells in the area of traumatic destruction of
bones[11,14].
In the present time, the aims of using Perftoran are to keep the
tissues’ viabilities in the zones of fire-arm shotting fractures[11], plus
with treatment in opened fractures by using Ilizarov method[15] and so
on.. However abrupt infrigement of regional blood current is not allow to
eliminate the local hypoxia in the place of fracture – it is that we wish the
gas exchange not be processing and, neither intravenous introduction nor
for Perftoran perfusion in the zones of fractures.
The aim of our investigation – to study the influences of the
dynamics in osteoreparation under the experimental high-energetic
fracutres on the long tubular bones for different methods of Perftroan
introductions.
Materials and methods
The experimental investigation is carried out on the 100 male rats
(Wistar), and the weight of each is about 220-260 grams. The operative
intervention and morbid manipulation for animals all were conducted to
narcotic in accordance with «The regulations of humane treatment with
laboratory animals». In the during operation and after opertation periods
rats were all applied with antibiotics therapy.
First stage in the experiment were modelization high-energetic
fractures at the upper 3rd femoral bones of right posterior extremity: it is
after the transverse osteotomy was conducted with the removal of
periosteum and endosteum, at 0.5 cm. from the line of fractures, on the
distal and proximal fragments, plus with mashed off soft tissues in the
zone of fractures with the help of clamp. Further was conducted with the
intramedullary osteosynthesis slim drill, then the wound be sutured
tightly.
The animals after modelization in fractures were allocated into four
groups: one control group and three trial groups, in each group there is 25
rats (Tab.1).
Table 1 Scheme of experiment
 Groups Scheme of experiment: quantity of animals, periods of introduction in
preparation and conclusion of animals from trials

Modelization of fractures
Introduction of conclusion of animals from trials
preparation
7th 10th 13th 15th 21st 30th 45th 60th
days days days days days days days days
1st Controls
25 25 25 5 5 5 5 5
2nd Introduction of Perftoran in intraosseous
25 25 25 5 5 5 5 5
3rd Introduction of Perftoran in intraperitoneum
25 25 25 5 5 5 5 5
4th Introduction of Perftoran in interfragments zone
25 25 25 5 5 5 5 5

In the 1st control group the preparation for animals were not be
introduced.
In the 2nd group at 7,10, 13 days after operation in intramedullary
with introduced of Perftoran (Open Joint stock compny, SPF
“PERFTORN”, Pushchino city. Moscow) at the rate of 1 ml. per 100 g.
weigt of animal (in average 2.4 ml. Perftroan, dosage selected by means
of experiment). In the 3rd group animals in these same periods introduced
intraperitoneum 3 ml. in Perftoran.
In 4th group upon the indication days, Perftoran is in injection at
quantity of 2 ml. introduced in interfragments zone.
Using the amount of 5 rats from each every groups on the
experiment at 15th,21st,30th, 45th, and 60th days of correspond with
after operation (it is in introduction to carry out the approximate
measured doage of Chloral hydrate intoduced into intraperitonium).
There are animals for all groups were in the fact of hatching for trials to
carry out, the during life time’s investigation of microcirculation.
The method of control standard microcirculation in the zone of
injury were chose in Laser Doppler Flowmeter (LDF), which can
definites the stages of blood current compensation and was so called one
of the basic methods for this aspect on the present days, it is able to
affords the analize of microcirculation in the realistic scale times[7,17].
The dynamics of indicators on the all groups were to be estimated by the
help of the apparatus in model of Biopac MP-100 with the needle like
sensor in TSD-144 type. It is to computes the intergral exponent of
microcirculation in the marked by BPU (Blood Perfusion Unit) and
which can characterizes the perfusion of tissues. The investigation of
blood current in the capillaries of the tissues in the zones of injury can
divide into 3 ground levels: it is the interfragments callus (the tissue
reside between fragments), the periosteum of distal fragment at the
distance of 0.5 cm. from the line of fracture, the muscular tissue in the
area of fracture. The recording and processing of data to implement by
the software program AcqKnowledge-38. The digital data to processes
into statistics with the help of analytical package of MS Excel.
For the morphological investigation is to excises the fragments on
the extrmities, that is including the zones of experiment influences on
with the surrounding tissues. After the decalcification of the tissues, with
further processing in histological preparation and utilizing the micotome
to slicing the tissues into 10-12 micro meter dying the preparations with
the methods of Van Gieson stain, Mallory and Hemotoxylin-Eosin. The
morphometry with the conduct help of software program Image J to
converses into the statistical data.
The consolidation of fracture was evaluated by roentgenological
method (x-ray ) to estimate for data using.
Results
There are all of the animals which were participants in the
experiment, the wound were healed with initial tension ( without pus in
cleaned wound ). The results of the study in the microcirculation of in
different periods of observations were reflected on the diagrams.
Picture 1. The Chang of microcirculation in the interfragments.

Y-axis: BPU (Blood perfusion unit), X-axis: Days

The denotes of colors:
Deep blue: 1st group (------) (control group)
Light blue: 2nd group (____) (Perftoran intraosseous)
Light green: 3rd group (____) (Perftoran interperitoneum)
Red: 4th group (___) (Perftoran interfractures zones)
BPU

микроциркуляция в межфрагментарной мозоли

250
227,77
211,98
200 180,96 182,38
177,64

150
115,34 118,11
94,22
100
73,54 92,2 89,04
67,36
76,32

66,66 72,12
50 64,89
59,36

0
15 21 30 45 60 сутки
1 группа (контрольная) 2 группа (перфторан в/к)
3 группа (перфторан в/брюшинно) 4 группа (перфторан межотл. зона)
 Picture 2. The Chang of microcirculation in the periosteum.
Y-axis: BPU (Blood perfusion unit), X-axis: Days
The denotes of colors:
Deep blue: 1st group (------) (control group)
Light blue: 2nd group (____) (Perftoran intraosseous)
Light green: 3rd group (____) (Perftoran interperitoneum)
Red: 4th group (___) (Perftoran interfractures zones)
BPU

микроциркуляция в надкостнице
350

300 304

250
202
200 203
167
150 129
107 124
107 113 106 91
100
94 92
60 77 83
50
46
0
15 21 30 45 60 сутки
1 группа (контрольная) 2 группа (перфторан в/к)
3 группа (перфторан в/брюшинно) 4 группа (перфторан межотл. зона)
 Picture 3. The Chang of microcirculation in the muscles.
Y-axis: BPU (Blood perfusion unit), X-axis: Days
The denotes of colors:
Deep blue: 1st group (------) (control group)
Light blue: 2nd group (____) (Perftoran intraosseous)
Light green: 3rd group (____) (Perftoran interperitoneum)
Red: 4th group (___) (Perftoran interfractures zones)
BPU

микроциркуляция в мышце
1600
1515
1400

1200 1240
1157
1009 1085 1060
1000
735
800 876
607
781 641 666 554
600
586
400 527
418 323
200

0
15 21 30 45 60 сутки
1 группа (контрольная) 2 группа (перфторан в/к)
3 группа (перфторан в/брюшинно) 4 группа (перфторан межотл. зона)
 In the tissues of interfractures zones revealed the evidence of the
microcirculation (BPU) at 15 days of the experiment by the statistics in
authenticly higher from the series of with intramedullary (227.98±83.3)
introduction of Perftoran and the directly introduction of Perftoran into
the interfractures zones (211.98±44.9) in compared with the others groups
(pic.1).At the 21st days after modelization into fractures, it is records from
the statistics in authenticly at 2-2.5 times higher in the 2nd group be
compared with the others groups, it is the evidence of the circulation from
the formation of callous. Moreover, from above of the statistics that this
exceeding keeps on till 45 days time. The BPU of the 4 th group (the
introduction of Perftoran into the interfractures) beginning at the 21 days
till the ends of experiment which was lowering in the value,it is don’t
have authentic distinction with first and 3rd groups. In the point of
interfragment callous the first and third both groups on the authenticly of
the statitics does not had distinct evidence in with the micrcirculation of
all the extent of experiment.
The study in the microcirculation at the periosteum gave the
evidence of the maximun BPU (203±84.6) value from the interfractures
coexist with the introduction of Perftoran at 15 days (pic. 2). However,
from the 21 days and till the ends of experiment proved the
microcirculation on the 2nd group (intraosseous introduction of Perftoran)
had excceded BPU than other groups, moreover, at 21 days this
exceeding had have statistical authentic signaficance (304±54.8). In the
control and 3rd groups had proved that the microcirculation in the
statistics does not have differences and be with the least meanings.
In the muscular tissues, by the data which were gained from the
LDF, the better blood circulation by the observed on the intraperitoneum
with method of introduction of Perftoran at 30 days and 45 days (pic.3).In
the other groups revealed that BPU were lower and essential distinctions
between each others were not revealed.
For all the investigating groups till at the 60 days in the
experiment, proved there are the microcirculation on the intrafractures
tissues, the periosteum and the muscles all practically made the
equalization in statistics in BPU and not had the differences among
themsleves too.
The analysis of roentgenogram (X-ray) bring out from the trial of
animals proved as follows: in the control group have had one of the cases
in consolidation of fracture that begun at the 45 days and the other case is
at the 60 days. In the other cases, it is developed the artificial joint ( pic.
4).
Picture 4. The roentgenogram (x-ray) and microphotography of fracture.

Control gruop (45 days): artificial joint; The presence of cartilage tissues and

cavity of different volumes with biased to merging in the intrafracture zone

(Hemotoxylin-Eosion, zoom in ×100).

In the 2nd group there are the consolidation were observed on, it is
from the one of the animals at 30 days, from the fours at the 45 days and
from the fours by the introduction of trials at the 60 days ( pic. 5). Only
there is one case in the result of migration of drill was to turn out in the
artificial joint. The similar results gained from the 4th group: there are 2
cases in consolidations at the 45 days and other 2 cases at the 60 days.
Picture 5. The roentgenogram (x-ray) and microphotography of fracture.

The grup of investigation №2 (45 days): the consolidation of fracture;

The formed callus from the fibrous tissues; (dyeing, Hemotoxylin-Eosion,

zoom in ×100).

From the acquisitions of data by the roentgenographic investigation

there are conform to with the morphological investigations of the
preparations ( pic. 4, 5). There is revealing that the intramedullary
introduction of Perftoran have the essential to hasten the organization of
bony calluses. In such a way, more narrowly at the 30 days after
modelization of fracture, the thickness of calluses was to formed at the
quantity of 0.17±0.08 mm, it is the statistics in which the authenticly to
distinguish from the control groups and the series of experiment with the
intramuscular introduction of Perftoran. At the 45 days in the most
preparations of the interfractures spaces were occupied by coarse fibrous
connective tissues with the islets of cartilage tissues, which is observed to
in the formation of the trabecular bones with morphological indications
of ossification.
Discussion
There are many authors to mentioned about, that owing to the
broad spectrum of the activity of Perftoran, which is sufficiently with
effective for the intravascular infusion and also the same with the local
introduction (applications)[11,14,15]. But these methods of intrduction of
preparations does not solve the problems of local hypoxia, which is
occurred especially in the bone tissues due to the high-energetic fracture.
The analysis of dopplergram revealed the important increasement
evidences in microcirculation of BPU at the interfractures tissues with the
intraosseous introduction of Perftoran as practically along on the whole
experitment. In the begun with the 21 days to the 45 days of after the
initial experiment there were had 2-2.5 times in the increasement of the
level of the microcirculation in the develop of intramedullary callus in
the compare with the other groups. Ther is elevated evidences in
microcirculation connected with the increasement of the amount of
functionl capillaries in the intrefragment callus, and in this period going
on the most intensive callus formation, it is as same as the conformation
by the hitologics and roentgenologics investigations. This is may to
explains why there are exist Perftoran as follows, it is that intraosseous
introduction at the distal metaphysis Perftoran can partially found directly
inside the intraosseous vessels, it is partially to found Perftoran
expression at the interfracture space, that it is allowed to lower down the
hypoxia in the place of fracture and it is in some cases directly inside the
bone tissues.
There are autheticly at most of the lower evidences in BPU at the
direct introduction of Perftroan in the interfracture zone, but for all the
lower ones in BPU there have exceeds more lower evidences as the
introduction of Perftoran in the intraperitoneum (it is as similar as to the
intravenous introduction) and as for the control groups both. This to
indicated that the injection of the introduction of preparation in the
injured tissues with infringed on the blood current will not allow in the
full value to realize its property, however it is more by preference than the
intravenous passage to introduction of Perftoran. The intravenous
introduction of Perftoran not be able to afford to have an influence at the
local perfusion in the tissues of the zone of high-energetic injury: there
are evidences in BPU in authenticly not to distinguish from the control
groups.
Another changes in the microcirculation for the measurement at the
non- damage periosteum and muscles in the zone of fracture. The highest
meanings of BPU for the interfractures of introduction of Perftoran at
wounds in times of 15 days speak about has possbility to improves the
microculation in the non-damage soft tissues with on the direct contact of
Perftoran. The intravenous introduction of Perftoran is named as the
optimal for the improvement of microcirculation in the structual non-
damage muscles in the zone of fracture.The more lower evidences of
circulation in the non-damage periosteum and muscles of the intraosseous
of the introduction of Perftoran to testify about, that for the given method
of introduction of preparation not act on the «the relatively well» tissues.
The investigation proved that the intraosseous method of the introduction
of Perftoran to be able to afford to raise its concentration at aiming the
ischemic tissues at the zone of fracture.
The stated changes of microcirculation in all the groups straight to
conform with the dynamics of osteoreparation. The revelation about that
in the intraosseous utilization of Perftoran, which maximum increased the
amount of active functional elements of the microcirculative channels,
and these are provide for the conditions for active of osteorepapration.
Only the intraosseous intorduction of Perftoran bolding in the most cases
to get union of fracture. About for all the remains groups were to
prevailed in the development of the artificial joint.
Thus, the carrying out of the investigation were confirmed that the
capacity of Perftoran to assure the early timing in more accelerate than in
the control groups of the level of the microcirculation in the zone of
fracture, that to assist of the strengthening of the osteogenesis. The
intraosseous introduction of Perftoran for the treatment of the high-
energetic bone wounds were possessed advantages than the intravenous
and local applications, it is supply the optimal oxygenation in injured
tissues in the zone of fracture, it stimulates reparative processes in the
bone tissues and to impedes the development in the amount of artifical
joints.
Conclusions
1. The formation of bone calluses was depending on the
conditions of microcirculation. The high-energetic fracture it causes the
imprigement of the local blood circulation, hypoxia, and as the result of
observed cause the indirect osteogenesis. In the control series of
expeiment were prevailed in the retarded union, the non-connecting
fracture and the artificial joints.
2. The intravenous and local applications of Perftoran not
to afford the important to influence on the local hypoxia and
microcirculation in the zone of fracture because of the sharp
impringement of the local blood current in the injurious tissues.
3. The registration of increase in BPU in the group with
intramedullary method of the introduction of Perftoran try to prove, that
namely this method is apparently with optimum for the high-energetic
fractures. The intraosseous introduction of Perftoran optimal local
microcirculation on the damaged tissues in the zone of fracture, it
stimulates reparative processes in bones and impedes the development of
artificial joints.
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