Отправить письмо автору 
Написать комментарий 
RATIONALE FOR THE USE OF BONE AUTOREGENERATE FOR STIMULATION OF REPAIR OSTEOGENESIS PROCESSES (AN EXPERIMENTAL STUDY)
Mukhanov M.L., Blazhenko A.N., Afaunov A.A., Bogdanov S.B., Sotnichenko A.S., Rusinova T.B., Aliev R.R.
Kuban State Medical University, Krasnodar, Russia
Currently,
due to an increase in the level of trauma [1] and an increasing frequency of
fractures accompanied by a defect in bone tissue [2, 3, 4], there is a need not
only to shorten the time of consolidation of fractures [3], but also an
osteoinductive material that allows replacement bone defects of large volumes.
The arsenal of an orthopedic traumatologist includes a number of methods
for treating fractures accompanied by a defect in bone tissue, signs of delayed
consolidation or emerging pseudarthrosis. These methods can be used both
independently and in combination, for example, distraction-compression
osteosynthesis [5, 6], as well as a number of different methods of bone
grafting, such as autologous bone grafts, allografts and bone graft substitutes
[7, 8].
Another area of optimizing the processes of reparative osteogenesis is
bone and tissue engineering, a promising area of personalized medicine.
Achievements of tissue engineering are used in many specialties, including traumatology
and orthopedics, where synthetic scaffolds with cells and growth factors
applied to them are used to replace bone tissue defects. However, in this
direction, many issues of efficiency, safety and cost have not been resolved,
which does not allow at this stage to widely introduce these methods into
practical medicine [9, 10].
The methods of local stimulation of reparative osteogenesis using growth
factors are the most widely used [2, 3, 11]. These include the use of
platelet-rich plasma (PRP) [2], the introduction of a suspension of bone marrow
aspirate concentrate (BMAC) into the fracture zone [3, 11], the use of
synthetic growth factors such as bone morphogenetic proteins (BMPs) [3],
fibroblast growth factor (FGF) [3], etc.
Most modern strategies for accelerating bone
regeneration give relatively satisfactory results, which is confirmed by
conflicting publications about their clinical and economic efficiency [13]. In
addition, at present, there are no heterologous or synthetic bone substitutes that
would have higher or even the same biological or mechanical properties as
compared to bone. Therefore, there is a need to develop new methods of local
stimulation of reparative osteogenesis using growth factors as an adjunct to
standard methods of fracture treatment [2, 3, 14].
Objective - in vitro, to determine the ratio of the main growth
factors in the zone of active reparative osteogenesis and to conduct a
comparative analysis with known methods of local stimulation of reparative
osteogenesis.
MATERIALS AND METHODS
This
study was carried out on laboratory animals: rams of the
"Romanovskaya" breed - two heads over the age of 1 year, weighing
31.2 and 28.6 kg.
All
manipulations with animals were carried out in accordance with the rules
adopted by the European Convention for the Protection of Vertebrate Animals
Used for Experimental and other Scientific Purposes (ETS 123, Strasbourg,
1986), was carried out examination of the study in the independent ethical
committee of Kuban State Medical University (protocol No. 80 of September 27, 2019).
In
accordance with the study design, biological media taken from laboratory
animals and used for local stimulation of reparative osteogenesis were
subjected to comparative analysis, namely, bone marrow aspirate suspension
concentrate (BMAC), platelet-rich plasma (PRP), native plasma and
autoregenerate.
The
autoregenerate was obtained according to the original method as follows: after
the access was performed using a chisel, an osteotomy was performed in the
iliac crest and a bone wound was formed (Fig. 1) up to 50.0 mm long, up to 10.0
mm wide and up to 30.0 mm (15,000 mm3 = 15.0 ml), in which an
autoregenerate was formed from the hematoma within 5-7 days, which is an
"organizing" clot (Fig. 2), a part of which was taken for enzyme
immunoassay and morphological analysis in order to determine the number of
growth factors and cellular composition in the regenerate sample.
Figure 1. Osteotomy of the
iliac wing
Figure 2. Autoregenerate
obtained from the iliac wing
To
obtain red bone marrow, a puncture of the wing of the ilium was performed with
a collection of red bone marrow in a volume of 15-20 ml, as well as blood
sampling for the preparation of platelet-rich plasma and native blood plasma,
venipuncture was performed with a venous blood sampling in a volume of 15-20
ml.
Plasma
platelet enrichment was performed using Hettich Eba 20 tabletop centrifuge
using the Plasmolifting technology using test tubes containing a separation
gel.
Immunological
and histomorphological studies were carried out in the central research
laboratory of Kuban State Medical University.
An
enzyme-linked immunosorbent assay was performed for the concentration of the
following cytokines: platelet derived growth factor AB (PDGFAB), transforming
growth factor beta 1 (TGFb1), bone morphogenetic protein 6 (BMP6), bone
morphogenetic protein 7 (BMP7), insulin like growth factor 1 (IGF1), fibroblast
growth factor 1 (FGF1). ELISA was used with the appropriate test-systems
(Cloud-Clone Corp, USA): SEA436Ov ELISA Kit For Platelet Derived Growth Factor
AB, SEA124Ov ELISA Kit For Transforming Growth Factor Beta 1, SEA646Ov ELISA
Kit for Bone Morphogenetic Protein 6, SEA799Ov ELISA Kit for Bone Morphogenetic
Protein 7, ELISA Kit for Insulin Like Growth Factor 1, SEA032Ov ELISA Kit for
Fibroblast Growth Factor Acidic according to the manufacturer's protocol on a
microplate reader Filter Max F5 (USA).
For
each biological sample, 4 measurements were made. The data are presented as the
median, the first and third quartile (Me [Q1; Q3]).
The
histomorphological assessment of the tissues was carried out according to the
generally accepted algorithm. The biological material was fixed for 3–5 days in
10 % solution of neutral buffered formalin (Histolab, Sweden) and washed in
running water for 60 min. The materials were transported according to the
standard technique by the automatic method on Leica TP1020 histoprocessor
(Germany). Paraffin blocks with samples of the studied materials were prepared
on Leica EG1150H modular setup (Germany). Leica RM2235 rotary microtome
(Germany) was used for cutting the preparations. The obtained material sections
with a thickness of 5 μm were stained with hematoxylin and eosin according to
the standard technique. Microscopy of the preparations was carried out using a
Olympus CX41 microscope (Japan).
Statistical
processing of the research results was performed using the StatSoft 2009
software (USA). Since the sample was small and the distribution did not differ
from normal, the results are presented as the median, first and third quartiles
(Me [Q1; Q3]). The significance of differences was assessed using the
Mann-Whitney U-test.
RESULTS
For the first time, experimental data were obtained on the quantitative content of cytokines: PDGFAB, TGFb, FGF1, IGF, BMP6 and BMP7 in bone autoregenerate, which reflect the physiological ratio of factors involved in local stimulation of reparative osteogenesis, which can be used for therapeutic and diagnostic purposes. In addition, the concentration of the corresponding parameters in blood plasma, in platelet-rich plasma and in suspension of bone marrow aspirate (BMAC) was determined. The results obtained are presented graphically in the table.
Table. The content of cytokines regulating osteosynthesis and repair in various biological samples of sheep (Me [Q1; Q3])
|
Biological samples
Cytokines |
Bone regenerate |
Bone marrow aspiration
concentrate (BMAC) |
Platelet-rich plasma (PRP) |
Blood plasma |
|
Insulin-like growth factor (IGF1), ng/ml |
17.2 |
40.9* |
6.7# |
0^ |
|
Fibroblast growth factor (FGF1), pg/ml |
8.96 |
6.11* |
0# |
3.07^ |
|
Transforming growth factor b (TGFb), pg/ml |
16.66 |
34.74* |
0# |
16.16 |
|
Platelet growth factor AB (PDGFAB), ng/ml |
2.67 |
2.52 |
7.22# |
3.05 |
|
Bone morphogenetic protein 6 (BMP6), pg/ml |
57.30 |
96.50* |
23.15# |
26.02^ |
|
Bone morphogenetic protein 7 (BMP7), pg/ml |
1736.50 |
1086.00* |
300.00# |
366.50^ |
Note: * – the significance of the differences between autoregenerate and BMAC – p < 0.05; # – significance of differences between autoregenerate and PRP – p < 0.05; ^ – the significance of the differences between autoregenerate and blood plasma – p < 0.05.
The
data obtained show that such cytokines as fibroblast growth factor 1 - FGF1 (p
= 0.026; p = 0.001; p = 0.009 in relation to BMAC, PRP, native plasma,
respectively) and bone morphogenetic protein 7 - BMP7 (p = 0.043; p = 0.009; p
= 0.009 in relation to BMAC, PRP, native plasma, respectively) compared with
all studied biological samples, which determines the key role of these factors
in the formation of connective tissue during reparative osteogenesis. The rest
of the indicators in the autoregenerate differ significantly in different
directions in relation to other studied biological samples, which demonstrates
the specificity of the functions of all studied cytokines in maintaining
homeostasis at the tissue and systemic levels. It should be noted that platelet-rich
plasma contains a large amount of platelet growth factor AB - PDGFAB (p = 0.07;
p = 0.012; p = 0.043, respectively, for BMAC, PRP, native plasma), which is at
least 2 times higher in comparison with other biological media. In the
suspension of bone marrow aspirate, the concentration of the following
cytokines prevails: TGFb (p = 0.048; p = 0.001; p = 0.048 in relation to BMAC,
PRP, native plasma, respectively) and BMP6 (p = 0.012; p = 0.041; p = 0.033 in
relation to BMAC, PRP, native plasma, respectively).
To
visualize and compare the profiles of cytokines in the studied biological
samples, the value of the concentrations of cytokines in the bone
autoregenerate was taken as 100 percent, and a diagram was constructed (Fig.
3).
Figure 3. Comparative analysis of the cytokine profile in bone
autoregenerate, red bone marrow (BMAC), platelet-rich plasma (PRP) and blood
plasma (in percent, relative to values for bone autoregenerate)
Note: *, #, ^ – the significance of the differences – p <0.05 in all biological samples in relation to the autoregenerate indicators.
An
analysis of the diagram showed that the profiles of cytokines in plasma and
plasma enriched with platelets are quite close in comparison with the data for
bone marrow, except for PDGFAB, the increase in the concentration of which is
apparently explained by the high content of activated platelets. The data
obtained indicate the small role of platelet growth factor and transforming
growth factor in bone tissue repair and the inexpediency of using
platelet-enriched plasma in the treatment of bone tissue defects. The closest
to autoregenerate in terms of the content of growth factors is suspension of
red bone marrow, while the relatively high content of IGF1 and TGFb may be due
to the processes of enhanced formation of bone matrix due to stimulation of
collagen synthesis.
Thus,
in the autoregenerate, which in essence represents an organizing hematoma in
the fracture area, in the course of this experiment, we revealed the optimal
ratio of the main cytokines necessary to optimize the processes of reparative
osteogenesis: FGF1 - 8.96 pg/ml, BMP7 - 1736.5 pg/ml, IGF1 - 17.2 ng/ml, TGFb
-16.66 pg/ml, BMP6 - 57.3 pg/ml, PDGF - 2.67 ng/ml. The
data
are
presented
in
the
diagram
(Fig.
3).
During
the histological analysis of the autoregenerate, it was found that the samples
were imbibed by fibrin, mononuclear cells were detected in the infiltrate, and
a large number of proliferating fibroblasts and newly formed thin-walled
capillaries were found along the entire plane of the cut with the formation of
granulation tissue, which may be a consequence of the high content of
fibroblast growth factor tested samples (Fig. 4a, 4b).
Figure 4. Histological examination. A fragment of the regenerate obtained on 7th day:
a) an increase ×10;
b) an increase ×20
The
data of morphological analysis indicate a high reparative activity in the zone
of autoregenerate production and that its transplantation will contribute to
the stimulation of osteogenesis. Based on the enzyme immunoassay and
morphohistological analysis of the autoregenerate, it can be concluded that it
is an effective and promising means of local stimulation of reparative
osteogenesis. Therefore, it is necessary to further study it on experimental
models (animals) and assess the possibilities of practical application of the
results obtained.
Thus,
as a result of the study, the difference between the autoregenerate obtained by
the original method and the suspension of bone marrow aspirate (BMAC) and a
significant difference from platelet-rich blood plasma (PRP), which are the
most widespread methods of local stimulation of reparative osteogenesis, was
established.
CONCLUSION
Currently,
many techniques are used in traumatology and orthopedics that allow local
stimulation of reparative bone tissue regeneration, of which A-PRP therapy is
the most widespread, affordable and safe. A number of common biological effects
are associated with platelets due to known growth factors (transforming growth
factor β - TGF-β, platelet growth factor - PDGF, IGF-II, vascular endothelial
growth factor - VEGF, epidermal growth factor - EGF, endothelial cell growth
factor - ESGF, insulin-like growth factor - IGF-I, fibroblast growth factor
-FGF), located in a-granules; platelets include ions K+, Ca++,
ATP, ADP, cytokines (serotonin, histamine, dopamine, prostaglandins),
coagulation factors, chemokines, acid hydrolases, elastases, lysozyme,
cathepsin D and E, proteases, and antibacterial and fungicidal proteins.
owever,
according to the data presented in our study (Fig. 3), platelet-rich plasma has
a stimulating effect on reparative osteogenesis, but does not have pronounced
osteoinductive properties, and the effect can be explained by the natural
ability of platelets to influence healing processes by stimulating the
regenerative potential of bone tissue for account of nonspecific growth
factors.
According
to our study, the greatest potential for stimulating reparative osteogenesis was
shown by red bone marrow and autoregenerate obtained according to the original
method. Based on the data of the enzyme-linked immunosorbent assay, the
qualitative and quantitative indicators of growth factors were determined,
which were necessary to optimize the processes of reparative osteogenesis.
Moreover, the indicators regarding the qualitative results obtained by us
coincide with the results published by other researchers [15] regarding the
importance of cytokines such as TGF for bone tissue regeneration, which are a
large group of proteins, including TGF-P1 and BMPs [16, 17 ].
Thus,
the use of autoregenerate in order to optimize the processes of reparative
osteogenesis can be attributed to one of the safest methods of local
stimulation of osteogenesis processes based on the following advantages:
absolute biocompatibility; minimal risk of infection; the content of cytokines
in an optimal ratio for local stimulation of reparative osteogenesis.
The
possibilities of using this technology in clinical medicine, namely in
traumatology and orthopedics, require further research aimed at creating
protocols for performing the proposed procedure.
CONCLUSION
1.
Based on the results of a comparative enzyme-linked immunosorbent assay of
autoregenerate, we were able to determine the optimal concentration of the main
growth factors that stimulate reparative osteogenesis: FGF1 - 8.96 pg/ml, BMP7
- 1736.5 pg/ml, IGF1 - 17.2 ng/ml, TGFb - 16.66 pg/ml, BMP6 - 57.3 pg/ml, PDGF
- 2.67 ng/ml.
2.
Based on the results of enzyme immunoassay and morphohistological analysis of
autoregenerate, it can be concluded that it is an effective and promising means
of local stimulation of reparative osteogenesis.
Funding information and conflicts of interest
The
study was carried out with the financial support of the Russian Foundation for
Basic Research within the framework of scientific project No. 19-415-233004/19
(20) "r_mol_a" dated April 22, 2019.
The
authors declare no obvious and potential conflicts of interest related to the publication
of this article.
REFERENCES:
1. Alekseenko SN, Redko AN,
Karipidi RK, Zakharchenko YuI. Primary disability of the adult population of
the Krasnodar territory duetoroad accidents // Bulletin of the All-Russian Society of Specialists in Medical and
Social Expertise, Rehabilitation and Rehabilitation Industry. 2017; (4): 44-48. Russian (Алексеенко С.Н., Редько А.Н.,
Карипиди Р.К., Захарченко Ю.И. Первичная инвалидность взрослого населения
краснодарского края вследствие дорожно-транспортных происшествий //Вестник
Всероссийского общества специалистов по медико-социальной экспертизе,
реабилитации и реабилитационной индустрии. 2017. № 4. С. 44-48)
2. Blazhenko
AN, Rodin IA, Ponkina ON, Mukhanov ML, Samoylova AS, Verevkin AA, et al. The effect of A-PRP therapy on the reparative regeneration of bone
tissue in fresh fractures of limb bones. Innovative
Medicine of the Kuban. 2019; № 15(3): 32-38. Russian (Блаженко А.Н., Родин И.А., Понкина О.Н., Муханов М.Л., Самойлова А.С., Веревкин А.А. и др. Влияние A-PRP-терапии на
репаративную регенерацию костной ткани при свежих переломах костей конечностей
//Инновационная медицина Кубани. 2019. 3(15). C. 32-38)
3. Sadykov RI, Akhtyamov IF,
Local factors of reparative osteogenesis stimulation (literature review). Department of Traumatology and Orthopedics.
2020; (3): 23-30. Russian (Садыков Р.И., Ахтямов И.Ф., Локальные факторы стимуляции репаративного остеогенеза (обзор литературы) //Кафедра травматологии и ортопедии. 2020. № 3.
С. 23-30)
4. Shastov AL. The problem of
replacement of post-traumatic defects of long bones in the domestic
traumatological and orthopedic practice (literature review). Genius of Orthopedics. 2018;
24(2): 252-257. Russian (Шастов А.Л. Проблема замещения посттравматических
дефектов длинных костей в отечественной травматолого–ортопедической практике
(обзор литературы) //Гений ортопедии. 2018. Т. 24, № 2. С. 252-257)
5. Aronson J.
Limb-lengthening, skeletal reconstruction, and bone transport with the Ilizarov
method. J Bone Joint Surg Am. 1997;
79(8): 1243-1258
6. Green SA,
Jackson JM, Wall DM, Marinow H, Ishkanian J. Management of segmental defects by
the Ilizarov intercalary bone transport method. Clin Orthop Relat Re. 1992; 28: 136-142
7. Giannoudis PV,
Dinopoulos H, Tsiridis E. Bone substitutes: an update. Injury. 2005; 36 (Suppl 3): 20-27
8. Giannoudis PV,
Einhorn TA. Bone morphogenetic proteins in musculoskeletal medicine. Injury. 2009; 40 (Suppl 3): 1-3
9. Salgado AJ, Coutinho OP, Reis RL. Bone tissue engineering: state of the
art and future trends. Macromol Biosci.
2004; 4(8): 743-765. 10.1002/mabi.200400026
10. Rose FR, Oreffo
RO. Bone tissue engineering: hope vs hype. Biochem
Biophys Res Commun. 2002; (2): 1-7. doi: 10.1006/bbrc.2002.6519
11. Korzh NA,
Vorontsov PM, Vishnyakova IV, Samoilova EM. Innovative methods for optimizing
bone regeneration: mesenchymal stem cells (message 2) (literature review) Orthopedics, Traumatology and Prosthetics.
2018;
(1): 105-116. Russian (Корж Н.А.,
Воронцов П.М., Вишнякова И.В., Самойлова Е.М. Инновационные методы оптимизации
регенерации кости: мезенхимальные стволовые клетки (сообщение 2) (обзор
литературы) //Ортопедия, травматология и протезирование. 2018. № 1. С.
105-116)
12. Akhtyamov IF,
Zhitlova EA, Tsyplakov DE, Boychuk SV, Shakirova FV, Korobeynikova DA. X-ray
morphological parallels of the osteoregenerative process when using a drug
based on lanthanide ethidronates.
Polytrauma.
2017; (4): 16-22. Russian (Ахтямов И.Ф., Житлова Е.А., Цыплаков Д.Э., Бойчук С.В., Шакирова
Ф.В., Коробейникова Д.А. Рентгеноморфологические параллели
остеорегенеративного процесса при использовании препарата на основе этидронатов
лантаноидов //Политравма. 2017. № 4. С. 16-22)
13. Piuzzi
NS, Dominici M, Long M, Pascual-Garrido C, Rodeo S, Huard J, et al. Proceedings
of the signature series symposium «cellular therapies for orthopaedics and
musculoskeletal disease proven and unproven therapies-promise, facts and
fantasy», international society for cellular therapies, Montreal, Canada, May
2, 2018. Cytotherapy. 2018; 20(11): 1381-1400.
doi: 10.1016/j.jcyt.2018.09.001
14. Talashova IA,
Osipova NA, Kononovich NA. Comparative quantitative assessment of the
reparative process during implantation of biocompositional materials in bone
defects. Genius of Orthopedics. 2012; (2): 68.
Russian (Талашова И.А.,
Осипова Н.А., Кононович Н.А. Сравнительная количественная оценка репаративного
процесса при имплантации биокомпозиционных материалов в костные дефекты //Гений
ортопедии. 2012. № 2. С. 68)
15. Pavlova LA, Pavlova TV,
Nesterov AV. Modern understanding of osteoinductive mechanisms of bone tissue
regeneration. Review of the state of the problem. Actual Problems of Medicine. 2010; 10(81): 5-11. Russian (Павлова Л. А., Павлова Т. В.,
Нестеров А. В. Современное представление об остеоиндуктивных механизмах
регенерации костной ткани. Обзор состояния проблемы //Актуальные проблемы
медицины. 2010. № 10(81). С. 5-11)
16. Andrades
JA, Han B, Nimni ME, Ertl DC, Simpkins RJ, Arrabal MP, et al. A modified
rhTGF-beta1 and rhBMP-2 are effective in initiating a chondro-osseous
differentiation pathway in bone marrow cells cultured in vitro. Connect Tissue Res. 2003; 44(3-4): 188-97.
doi: 10.1080/03008200390229912
17. Lieberman
JR, Daluiski A, Einhorn TA. The role of growth factors in the repair of bone.
Biology and clinical applications. J Bone
Joint Surg Am. 2002; 84(6): 1032-1044. doi:
10.2106/00004623-200206000-00022
Статистика просмотров
Ссылки
- На текущий момент ссылки отсутствуют.
