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Главная > № 4 (2019) > Самодай

LYOPHILIZED ALLOGENIC GROWTH FACTORS IN TRAUMATOLOGY AND ORTHOPEDICS AS A PROMISING DIRECTION OF REGENERATIVE MEDICINE

Samoday V.G., Starikov A.O., Kalashnikov P.I.

Voronezh State Medical University named after N.N. Burdenko, Voronezh, Russia

 During the recent years, the problem of high energy trauma is looming large over the whole world. It is associated with rapid urbanization, technical progress, increasing rate of technogenic disasters, and increasing proportion of high energy trauma among all injuries.
For polytrauma defined as concomitant injury, skeletal injuries consist 93 % of cases [1], with slow fracture union in half of them (50 %), and pseudoarthrosis [2-4]. Despite of great use of resources for prevention of injuries, one does not observe any trends of decreasing amount of patients with slow fracture union and formation of pseudoarthrosis [5]. Such situation is associated with the important issue on ways of prevention of complications, which are inevitable after treatment of associated injuries.
Treatment of patients with false joints, and their return to normal life style take one year and more, resulting in high economic losses [6]. As result, new technologies for normalization of bone tissue regeneration are required.
Currently, active researches are directed to endogenous factors, which influence on the reparative process. The factors are mutually related in unique natural relationship. The offer to use platelet-rich plasma (PRP) made by Marshall R. Urist, and the growth factors discovered by Rita Levi-Montalchini and Sten Coen in 1986 allowed making a new step in development of injuries to both soft injuries and bone tissue.
It is known that platelets play the important role in recovery of injured tissues of the body. Platelets released the growth factors from alpha-granules during tissue adhesion or destruction [7, 8]. The growth factors stimulate histiogenesis, chemotaxis and cell differentiation [9].
Usually, autoblood is used for PRP therapy. However, the use of this treatment method is limited for patients with somatic pathology and severe condition. Disorder of regenerative capability often happens in such patients.
One of the main problems of autoPRP is impossibility of long term storage and keeping it as reserve. The storage time of platelets is not more than 3 days. The increase in this period initiates the release of pro-inflammatory cytokines and other undesirable substances [10, 11].
The main problems of use of autoPRP consist in limited storage period, limited time of preparation from collection of the whole blood to introduction for the patient, and costs for plasma production. According to our opinion, the search for ways for solving this problem is initiated from research of action of PRP from allogenic blood.
The study of problems of slow fracture union and pseudoarthrosis, and techniques for normalization of reparative osteogenesis is being conducted at the department of traumatology and orthopedics at Voronezh State Medical University named after N.N. Burdenko since 2005 [12-18].
There are a lot of discussions of transplantology problems at the present time. There is an unsolved problem of development of immune response to introduction of allogenic PRP during spontaneous transplantation tolerance. A platelet is a cellular structure. Therefore, introduction of alloPRP will correspond to all laws and principles of transplantology and immunology.
Immune homeostasis is achieved by means of continuous interaction of systemic analysis and previous experience of the body. It has been found that alloantigens are not recognized by the system of inborn immunity [19].
Activation of direct immunity appears immediately after transplantation. Donor passenger leukocytes migrate from the graft to lymphocytic organs, and mature, acquiring the properties of donor-specific antigen-presenting cell (APC). It has been shown that most immune processes are related to presence of immunocompetent cells –leukocytes. Platelet transfusion cannot initiate release of HLA anti-bodies by itself since platelets do not contain antigens of class 2, which are required for T-helper activation of B-cells and production of anti-bodies [20].
There are some advances in leukodepletion process which are used for preparation of platelet concentrates. Filters in apheresis devices prevent the appearance of leukocytes in the concentrate. It makes the use of alloPRP more attractive from position of the immune response [21].
During realization of the indirect way, processing of donor proteins happens in recipient’s APC. Synthesis of donor proteins is controlled by minor genes of histocompatibility. As known, activation of the indirect pathway presents the main role in development of chronic rejection. The second pathway of recipient’s sensibilization requires for antigenic presentation through cells, which express HLA of class 2, which is absent in platelets [10, 20].
The available international literature contains some data on use of alloPRP for tissue regeneration [23-26]. The efficiency of combination of allogenic platelet-rich plasma and collagen for treatment of femoral bone defects in rats has been investigated [27].
The use of alloPRP significantly simplifies the scheme of preparation and administration of PRP since donor blood is used. But the problem of storage is always important.
One of the perspective techniques for preservation of functional capability of growth factors is sublimate drying. Liophilisation is the modern technique for drying of substances, whereby a substance is frozen, and the solvent is sublimated in vacuum conditions. Owing to absence of high temperatures, the protein does not denature and does not loss its structural and functional integrity. Lyophilized tissues and samples recover their primary properties when moistened [28]. At the department of traumatology and orthopedics, Voronezh State Medical University named after N.N. Burdenko, “Technology of lyophilization of platelet-rich plasma with preservation of vitality of TGF, PDGF and VEGF” has been developed in 2012 [18]. It has been shown that protein structure of growth factors is preserved during lyophilization. In lyophilized plasma, the level of growth factors is almost the same as in autoPRP [4]. We suppose that allogenic lyophilized platelet-derived growth factors (alloPDGF) can stimulate osteogenesis like autoPRP, and they are preserved for a long time. In our experiment, lyophilisate was kept at room temperature (~26˚ С) within 16 days. Lyophilisate can be used in any form: gel, powder or solution. It makes the procedure both efficient and simple.
A way for production of alloPDGF was offered abroad [29]. However, the practical use of this technology is impossible due to absence of information on studies and evidences of safety of the agent.
Objective – to develop a technique for normalizing osteogenesis in fractures of long tubular bones using a complex of allogenic lyophilized growth factors in an experiment with laboratory rats. 

MATERIALS AND METHODS

The experiment was conducted at the basis of Research Institute of Voronezh State Medical University named after N.N. Burdenko at the department of traumatology and orthopedics in 2018-2019. The study included two stages.
The experimental protocol and the protocol of animal management and experiment completion corresponded to the bioethical principles and the rules for laboratory practice presented in “The Manual for Management and Use of Laboratory Animals” (1996), and in the Order of Health Ministry of Russia No. 266, June 19, 2003. All manipulations were made with adherence to the rules of human treatment of animals (Report of the AVMA Panel on Euthanasia JAVMA, 2001), in compliance with demands by European Convention for the Protection of Vertebrate Animals Used for Experimental and other Scientific Purposes (Directive 86/609 EEC). The copies of all materials are kept by the authors. The study protocol was approved by the ethical committee of Voronezh State Medical University named after N.N. Burdenko (the protocol No. 3, November 15, 2018).
The first stage of the experiment required for calculation of platelet level in plasma of laboratory animals. The mean level of platelets in laboratory rats in the first stage of the experiment was 5-8.5 × 10⁸ per ml. During preparation of alloPDGF, the level of platelets increases approximately 3 times. It is known that each platelet contains 1,200 molecules of PDGF with mass of 26-30 kDa [30]. After calculation of mass of PDGF per 1 ml of whole blood, we receive 15.6 × 10¹² – 30.6 × 10¹² kDa. It is known that stimulation of direct effect in regeneration is sufficient for level of PDGF of 5-20 ng/ml [31]. Conversion of kDa into ng is required for confirmation of efficiency of our technique. It is known that 1 kDa is 1.66043 ± 0.0031 × 10^–12 ng. [32]. As result, 1 ml of whole blood contains PDGF level of 25.9-50.8 ng/ml. Therefore, 0.5 ml can be used to obtain 12.95-22.415 ng of PDGF. Therefore, we can conclude that 0.5 ml of whole blood used for preparation of PDGF is sufficient for influence on reparative osteogenesis.
Five convectional non-lineal stock male rates (age of 10 months) were used for the first stage of the study (blood collection from animals and preparation of alloPRP lyophilisate). Manipulations were conducted under inhalation narcosis with isoflurane solution with additional delivery of oxygen for prevention of asphixy. After achievement of surgical stage of narcosis, the region of the probable puncture was shaven, and the skin was disinfected with alcoholic solution of chlorhexidine. Cardiac impulse was estimated in palpatory manner. The blood was taken by means of cardiac puncture with use of vacuum containers with 3.8 % sodium citrate [33]. Totally, 30 ml of whole blood was used in the experiment. The blood was divided into 5 sterile test tubes with 6 ml for each group.
Then, PRP was obtained from whole blood with use of Messora technique [34]. The numerated test tubes were simultaneously centrifuged with moment of 160 g within 20 minutes (Fig. 1). The separated plasma was placed into empty sterile test tubes. Recurrent centrifugation of test tubes was carried out with moment of 400 g within 15 minutes (Fig. 2). The bottom fraction was taken, with concentration of 15-29 ×10⁸ of platelets per ml. The final platelet concentrate was rapidly frozen in the refrigerator at the temperature of -40 °C. Then it was exposed to sublimation drying in the lyophilic chamber LS-1000 within at least 15 minutes at the temperature of 2-30 °C (Fig. 3). The final lyophilisate (5 test tubes) was sterilized in the ozone chamber Orion with at least 140 minutes of exposure. It was placed into a sterile hermetic container and was kept in dry place.

Figure 1. Results of test tube centrifugation with moment of 160gb during 20 minutes



Figure 2. Result of recurrent centrifugation with moment of 400 gb during 15 minutes



Figure 3. Lyophilizated allogenic growth factors of allogenic blood

The second stage of the experiment included 10 subgroups of laboratory animals: non-lineal convectional rats, males, age of 5-6 months, weight of 450-550 g, 12 rats in each subgroup (the table 1).

Table 1. Characteristics of studied groups of laboratory animals

Characteristics of groups	Number of subgroup	Terms of estimation of osteogenesis after osteoclasis	Amount of animals
Experimental group	`1.1	5th day	12
	`1.2	14th day	12
	`1.3	21st day	12
	`1.4	32nd day	12
	`1.5	44th day	12
Control group	`2.1	5th day	12
	`2.2	14th day	12
	`2.3	21st day	12
	`2.4	32nd day	12
	`2.5	44th day	12

The first group was experimental (60 rats), with estimation of osteogenesis on the 5th day (12 rats) (in the future, the authors plan to use this subgroup for histochemical analysis since the signs of bone formation are not evident, and immunogenic processes are within the full range), on the 14th day (12 rats), on 21st day (12 rats), on 32nd day (12 rats) and on the 44th day (12 rats).
The second group (control, 60 rats) included 5 subgroups with 12 subjects in each one (in correspondence to equivalent time intervals in experimental subgroups).
 Before the experiment, the animals were under observation within two weeks. The rats were kept in small cages with limited space for moving. The animals received food according to the existing standards, identically in both groups. All manipulations were made under narcosis with isoflurane. Manual osteoclasia of the right femur was performed for formation of a closed fracture in the middle lower one-third. AlloPDGF from each tube was added to 6 ml of 0.9 % NaCl. This solution was introduced into the fracture site (0.25 ml for the first and second days after osteoclasia). The control group received 0.9 % NaCl (0.25 ml) in the same manner than in the experimental one. Before completion of the experiment, X-ray images in single plane were made with use of stationary veterinary radiologic system HF-525plus EcoRay, with mode of 30 mA 0.07 kW and exposure of 1 sec. During X-ray imaging, all animals were under narcosis. The images were taken in supine position. The examined extremity was stretched along the body (Fig. 4). The experiment was completed by means of use of lethal exposure of isoflurane. The extremity was amputated in the hip joint. The bone fragment with callus was fixed in 10 % of neutral formalin. Then it was placed into decalcifying medium. The standard procedure of routing was performed, and the material was placed into paraffin. Paraffin media (thickness of 5-7 µm) were stained with hematoxilin eosine and according to Masson. Microscopy was carried out with the optical microscope with equipment for microphotography. The final information was analyzed with ImageG software. The same software was also used for morphometry.

Figure 4. X-ray imaging of animals in supine position; an examined limb was stretched along the body

For qualitative and quantitative estimation of reparative osteogenesis, we calculated the squares of connective tissue component of hyaline cartilage and compact bone tissue for estimation of time course in absolute and relative values. Also the total level of chondrocytes and osteocytes was calculated in callus tissues. For information interpretation of the results, the morphologic statistical analysis with Statistica 8.0 and SSPS 13, with use of parametrical tests, was conducted. The results were presented as the mean (M) and standard error of the mean (m). The significance of differences was estimated with Student’s test. The statistically significant value was p < 0.05.

RESULTS AND DISCUSSION

The similar signs were observed in both groups on the 5th day. The fracture lines were clear.1 The fragment edges were sharp. The signs of callus formation were not visualized. Therefore, we could state the absence of radiologic signs of fracture union in both groups. There were not any differences in the experimental group and the controls after X-ray imaging examination on the 14th day (Fig. 5). However we could observe the beginning of the reparative process, residual signs of fracture site, and smooth edges of bone fragments. The contours of cortical layer were interrupted. It meant periosteum hypertrophy, and initiation of periosteal callus.

Figure 5. X-ray imaging of injured femur on the day 14: (to the left) control group, (to the right) experimental group

On the day 21, X-ray imaging (Fig. 6) showed an increase in tissue regeneration in fracture site in the experimental group, despite of significant displacement of fragments. The fracture line was not visible in this group. Periosteal response was evident. However, the control group still showed the fracture line, with sparsity of bone tissue in the fracture site, which indicated osteopenia.

Figure 6. X-ray imaging of fracture on the day 21: (to the left) control group, (to the right) experimental group

On the day 32, X-ray images (Fig. 7) showed evident callus in animals of the experimental group. In the control group, the X-ray signs showed high similarity with the experimental group on the day 21. It meant `0-12 days of delay in regeneration process.

Figure 7. X-ray imaging on the day 32 of the experiment: (to the left) control group, (to the right) experimental group

X-ray images showed the complete fracture union in the experimental group on the day 44 (Fig. 8). The fracture site was unclear. There were some clear signs of paraosseous callus. The control group showed some signs of ongoing formation of bone callus. The X-ray signs corresponded to fracture union in the experimental group on the day 32.

Figure 8. X-ray imaging on the day 44 of the experiment: (to the left) control group, (to the right) experimental group

The quantitative analysis of tissue composition of bone callus was carried out by means of calculation of absolute and relative values of square of connective, cartilaginous and bone tissues, and total number of chondrocytes and osteocytes The animals of the control group showed predominance of dense fibrous tissue, and absence of bone tissue on the day 14 (the tables 2, 3, Fig. 9).

Table 2. Ratio of tissue components and their square, µm

	14th day			21st day			32nd day			44th day
	Square of connective tissue (µm)	Square of hyaline cartilage (µm)	Square of bone tissue (µm)	Square of connective tissue (µm)	Square of hyaline cartilage (µm)	Square of bone tissue (µm)	Square of connective tissue (µm)	Square of hyaline cartilage (µm)	Square of bone tissue (µm)	Square of connective tissue (µm)	Square of hyaline cartilage (µm)	Square of bone tissue (µm)
Control	427.81	11.03	0	341.5	195.2917	110.9583	259.43	191.97	137.62	237.875	184	169.5
	97 %	3 %		53 %	30 %	17 %	44 %	33 %	23 %	40 %	31 %	29 %
Experiment	286	41.7	13.64	204.875	237.7083	196.4167	197.98	211.35	201.2	187.7083	183.5833	209.1667
	84 %	12 %	4 %	32 %	37 %	31 %	32 %	35 %	33 %	32 %	32 %	36 %

 
Table 3. Number of cells per field of vision (CU) 

	5th day		14th day		21st day		32nd day		44th day
	Amount of chondrocytes	Amount of osteocytes	Amount of chondrocytes	Amount of osteocytes	Amount of chondrocytes	Amount of osteocytes	Amount of chondrocytes	Amount of osteocytes	Amount of chondrocytes	Amount of osteocytes
Control	0	0	8.34	0	220.7917	66.66667	974.56	101.39	1243.708	129.0833
Experiment	0	0	67.9	9.34	1863.125	188.3333	1234.83	279.22	385.0833	519.375

Figure 9. The rat’s hip fracture site in 14 days after osteoclasia. The control subgroup No.2.2, hematoxylin and eosin staining (x 100). Extensive fields of rough connective tissue.

The features of tissuecomposition of bone callus differed from the control group on the day 14: absolute and relative number of dense fibrous tissue was lower, and the square of cartilaginous and bone tissue increased (the table 2). The total number of chondrocytes and osteocytes increased reliably in comparison with the control group (the table 3, Fig. 10).

Figure 10. The rat’s hip fracture site in 14 days after osteoclasia. The experimental subgroup No.1.2, hematoxylin and eosin staining (x 100). A wide strip of connective tissue, and signs of cartilage formation.

The microsamples from the fracture union site showed some fragments of compact bone tissue in the control group on the day 21. Normal red bone marrow was visible in the center. Near one of the edges, the bone callus was located, which mostly consisted of dense fibrous tissue with single full-blooded vessels of capillary type on the surface (Fig. 11).

Figure 11. The rat’s hip fracture site in 21 days after osteoclasia. The control subgroup No.2.3, hematoxylin and eosin staining (x 100). Wide periosteum, and small islets of new cartilaginous tissu

Also the quantitative analysis of tissue composition of bone callus was made by means of calculation of absolute and relative values of square of connective, cartilaginous and relative number of chondrocytes and osteocytes. Dense fibrous tissue prevailed in tissue composition of bone callus. The second place was taken by cartilaginous tissue. Bone tissue was visualized as small fragments of bone rods (the table 2, 3).
The microsamples from the animals of the experimental group showed some fragments of compact bone tissue on the day 21 of the follow-up. Dense fibrous tissue included some fragments of hyaline cartilage covered by wide perichondrium with high amount of chondroblasts. Approximately a half studied samples showed some regions of developing bone rods (Fig. 12).

Figure 12. The rat’s hip fracture site in 21 days after osteoclasia. The experimental subgroup No.1.3, hematoxylin and eosin staining (x 100). Big islets of hyaline cartilage and signs of bone formation

Also absolute and relative values of tissue composition of callus differed from the control group: absolute and relative amount of dense fibrous tissue fibers was lower, but the ratio between cartilaginous and bone tissue was almost the same. The total amount of chondrocytes and osteocytes was reliably higher than the similar values in the temporary control group (the table 2, 3).
Therefore, on the day of 21 of the follow-up, the animals of both control and experimental groups showed some signs of bone callus. However, the group of animals without alloPDGF had callus mainly of dense fibrous tissue and single foci of osteogenesis. The experimental group showed higher intensity of osteogenesis signs, mainly in the site of new cartilaginous tissue (Fig. 9, 10).
The quantitative analysis of absolute and relative values of callus composition showed dominance of rough fibrous connective tissue in the control group on the day 32. The second place was taken by cartilaginous tissue. Bone tissue was presented by small fragments and small spongy bone rods (the tables 2, 3, Fig. 13).

Figure 13. The rat’s hip fracture site in 32 days after osteoclasia. The control subgroup No.2.4, hematoxylin and eosin staining (x 100). Islets of cartilaginous tissue with “crevices” in the center; quite high amount of connective tissue around

The comparison of absolute and relative values of tissue composition of callus showed the evident decrease in volume of dense fibrous tissue in the experimental group. The values of cartilaginous and bone tissues were higher. Also the amount of chondrocytes and osteocytes increased (the table 2, 3, Fig. 14).

Figure 14. The rat’s hip fracture site in 32 days after osteoclasia. The experimental subgroup No.1.4, hematoxylin and eosin staining (x 100). Cartilaginous tissue in view of “a strip”. The bone forms both from cartilage and connective tissue

The microsamples of the control group showed some big regions of cartilaginous tissue with quite wide periosteum on the day 44. The space between bone rods was mainly filled with loose fibrous connective tissue (Fig. 15).

Figure 15. The rat’s hip fracture site in 44 days after osteoclasia. The control group No. 2.5, hematoxylin and eosin staining (x 100). “A strip of cartilage” with signs of bone formation on one side

The microsamples from the animals of the experimental group showed hyaline matrix on the day 44. It was weakly visible, optically dense, with high amount of chondrocytes. The signs of formation of new chondrocytes from chondroblasts were evident in one end, and transition of chondrocytes to osteocytes on the other side (Fig. 16).

Figure 16. The rat’s hip fracture site in 44 days after osteoclasia. The experimental subgroup No.1.5, hematoxylin and eosin staining (x 100). Active bone formation in site of a thin strip of cartilage

The quantitative analysis and estimation of relative values showed the higher square of connective tissue in the control group, whereas the square of cartilaginous tissue was similar. However, the square of bone tissue was reliably higher in the experimental group (the table 2, 3).
Therefore, we can conclude that the morphological signs and quantitative estimation of tissue composition of callus in animals of the control group on the day 44 of the experiment were similar with the values in the experimental group on the day 21 of the experiment (the tables 2, 3).
The quantitative analysis and estimation of relative values of square of main tissue components of callus showed the approximately equal ratio of connective, cartilaginous and bone tissues in the experimental group on the day 44. Moreover, the square of bone tissue was higher than in the temporary control group. The total amount of chondrocytes and osteocytes showed evident two-directed time course as compared to the total amount of these cells in the control group, meaning the terminal phase of the regenerative process in bone tissue (the table 2, 3).
Therefore, the signs of bone tissue formation were found on the 44th day of the experiment. These signs were absent in callus as compared to the animals of the control group that was testified by high amount of osteogenesis foci in the cartilage and in connective tissue. Also the group of animals receiving alloPDGF demonstrated the high amount of vessels in thickness of callus. Probably, these vessels increased the trophism and promoted faster osteogenesis.
The received results prove the reliability of changes in the square of each stromal tissue in relation to the previous period (p < 0.05).
So, for connective tissue (control), the changes in the square of connective tissue are statistically reliable and dynamic for the time intervals of 5-14 days and 14-21 days, as well as for 21-32 days. The changes in the square of connective tissue on the day 44 in relation to the values on the day 32 exist, but they are not statistically significant (p > 0.05). For the square of connective tissue (experiment), some statistically significant differences are observed only for time interval of 14-21 days. For other time intervals, the changes in the square of connective tissue component were insignificant and unreliable.
The changes in the square of hyaline cartilaginous tissue were reliable in the control group on the days 14-21. For other time intervals, the changes in the square were unidirectional, with a trend to decrease without reliable changes in relation to previous period of follow-up. In the experimental group, the square of hyaline cartilage showed some statistically significant changes on the days 14-21 and 32-44, whereas the changes were insignificant on the days 5-14 and 21-32 (p > 0.05).
The changes in the square of bone tissue were statistically reliable for all time intervals in the control group. The experimental group demonstrated some evident changes in the square of bone tissue on the day 21 as compared to the day 14. Subsequently, the changes in the square of bone component were insignificant and statistically unreliable at the background of the general trend of the increase as compared to previous time intervals. The evident and statistically significant changes in the square of bone tissue are supported by all time intervals as compared to the day 14 of the experiment (p < 0.05).
The square of primary cartilaginous callus formed by chondrocytes and osteocytes was higher in the experimental group as compared to the control group on the day 14 of the experiment, with the subsequent decrease in the time course of the follow-up to minimal values on the day 21, with lower values than in the control group (Fig. 17).

Figure 17. The amount of chondrocytes and osteocytes in time course of the experiment. Note: * - reliability of changes in relation to the control group; p < 0.05.

Therefore, the rate of formation of cartilaginous callus with mature chondrocytes show the highest intensity in the first time intervals of the follow-up in the experimental group as compared to the control one (p < 0.05).
The total square of hyaline cartilage in the union site was increasing after 21 days. Then it was at the maximal level, with gradual decrease by the end of the experiment, with only residual foci. The values in the control group were similar, but with approximately 10 days of delay (p < 0.05) (Fig. 18).

Figure 18. The square of connective and bone tissue in site of fracture union during the experiment.



Note: * - reliability of changes in relation to the control group; p < 0.05.

The process of bone formation on the site of primary callus began on the day 21 in the experimental group, with small square in view of islets in the deep of hyaline cartilage, whereas this process was almost noteless in the control group (p < 0.05).
From the day 21, the experimental group was demonstrating the statistically reliable decrease in the level of chondrocytes. The time course of changes in number of chondrocytes was the same in the control group, but with slight delay. 

CONCLUSION

1. Allogenic lyophilized platelet-rich plasma can stimulate the reparative osteogenesis. It is shown by the analysis of radiological signs in the control and experimental groups. The radiological and morphological signs of osteogenesis show faster regeneration in the experimental group (a lead of 10-12 days). It indicates the stimulating influence of allPDGF on regenerative capability of bone tissue.
2. One of the main advantages of alloPRP is long term storage and keeping as a reserve. It allows using the agent when required in any medical facility and even in outpatient conditions.
3. Receive of alloPDGF is a quite simple technological process, which does not require for big costs for equipment and storage.
4. AlloPDGF can stimulate osteogenesis like the traditional autoPRP, and can be used for patients with somatic pathology and severe condition since only donor blooded is used for preparation of the agent.
5. Lyophilisate can be used in view of powder, gel or injection solution. The injection form is most convenient and low-invasive way of delivery of the agent to the injury site or developing pseudoarthrosis. It makes the procedure efficient and simple.
6. The conducted experiment with use of allogenic lyophilisate of growth factors did not fund any side-effects in clinical, radiological and morphological aspects. On can state the fact of absence of response from immune system after introduction of alloPDGF.
7. The statistical analysis of the result of examinations showed the efficiency of the complex of autogenous lyophilized platelet factors. 

Information on financing and conflict of interests

The study was conducted without sponsorship.
The authors declare the absence of any clear or potential conflicts of interest relating to publication of this article. 

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