OPPORTUNITIES AND PROSPECTS FOR THE USE OF PLASMA ENRICHED IN PLATFOLES IN THE TREATMENT OF FRACTURES AND BONE DEFECTS Burykin K.I., Parshikov M.V., Yarygin N.V., Svetlov D.V., Govorov M.V., Chemyanov I.G., Prosvirin A.A.
Department of traumatology, orthopedics and disaster medicine, A.I. Yevdokimov Moscow State Medical and Dental University, Moscow, Russia
Treatment of bone fractures, primarily multi-fragmentary ones, causes significant difficulties for achievement of precise reposition of fragments and for recovery of functioning of an injured segment and extremity at whole. The situation is complicated by presence of defects with bone tissue deficiency in the fracture site, resulting in significant limitations for achievement of satisfactory results. To solve these problems, including replacement of this deficiency and optimization of the reparative process, scientists of various specialties have been searching for more advanced osteosynthesis techniques, new composite materials, special coverings for implants etc. To answer this question about achievement appropriate bone tissue regeneration in the fracture site in maximally short time intervals, we have analyzed publications for the last five years (from 2015 to 2020).
Objective − studying the possible prospects for the use of enriched plasma platelets in the treatment of bone fractures and bone deficiency replacement tissue with use of available systems and implants for optimization of bone tissue recovery.
One of the directions of scientific activity for research of the bone regeneration process in various conditions was creation of systems and implants, which optimize union and remodeling, particularly, possibility for coverage of osteosynthesis implants with special materials.
So, according to results of the experimental study by Akhtyamov I.F. et al. (2016), it was found that the use of constructs with titanium nitride and hafnium in intramedullary fixation of the tibia was accompanied by formation of cortical plate with higher density as compared to the comparison group; some changes, which are common for vessels dilatation in the injury site in the postsurgical period, were found, and they can be reviewed as a positive moment in formation of primary callus [1].
The researchers from Rochester University (NY, USA) developed the periosteal coverage with use of polycaprolactone, collagen and nano-hydroxyapatite composite nanofibers (TEP). Osteotomy with formation of a longitudinal 4 mm defect was performed in the femoral bone of mice. To correct the defect, the allograft, which was covered with biomimetric periosteum sheets, was fixed with intramedullary fixation. The result of treatment was evident recovery of periosteal layer, remodeling of the allograft in relation to the donor bone, evident improvement in osteointegration in the site of injured periosteum. It gives some hopes for prospectivity of further use of this approach in clinical practice [2].
Also multiple comparison studies and the search for optimal replacer of bone tissue are being conducted. However, the results are sometimes poor. For example, at Bern University Clinic (Switzerland), for estimation of influence of three types of the bone substitute made of deproteinized bovine bone mineral, beta tricalcium phosphate and mixture of alfa-TCP and hydroxyapatite, the researchers performed a study with implementation of these materials into artificial bone defects of tibial bone defects in dogs. As result, it was found that absorption of biomaterial was minimal at all stages of the study, and the influence of all types of materials was insignificant and did not promote formation of a new bone [3].
The special attention should be given to calcium orthophosphate (tricalcium phosphate) − non-organic substance, which is widely spread in nature, and is a part of various minerals, including hydroxyapatite, which is the main mineral component of bones. Various synthetic derivatives of calcium phosphate are the most common materials for bone substitute which are widely used and are studied by researchers in the whole world. So, at Iran University of Medical Science, a study (in vivo) of influence of tricalcium phosphate (β-TCP) and collage-based composite materials on bone formation and recovery of defects was conducted. 3 cranial defects (8 mm diameter) were created in New Zealand white rabbits. The defects were distributed into 2 groups: treatment with combination of collagen and β-TCP; only collagen; the comparison group without treatment. The results were estimated after 4 and 8 weeks. The macroscopic estimation after 8 weeks showed no complete healing in all 3 defects. However, the defect with collagen/β-TCP was scarcely different from surrounding bone tissue and was motionless. Defects, which were prepared with only collagen, had less bone mass than collagen/β-TCP. Histological estimation of collagen/β-TCP group showed formation of the immature bone by the 4th week. The defect was covered with the new bone by the 8th week. In the only collagen group, only slight formation of the bone could be observed by the 8th week, and most defects were hollow. In the group without treatment, all square of the defect was covered with fibrous tissue by the 8th week, and the signs of formation of the new bone could be found only on the edges. The study showed that a combination of collagen/β-TCP had a potential for use as the bone substitute in clinical practice. However, the possibilities of this material will be evident only after future studies and analysis of long term results [4].
It is difficult to contradict that the appropriate implant must be highly biocompatible, without immunological response, and with features of osteoinduction and osteoconduction. Moreover, availability of antimicrobial properties is the evident advantage on the way to good results. An example of successful use of antimicrobial features, including zinc, is a study of Indian school of biological sciences. The study was oriented to combination of nano-hydroxyapatite and ZnO, with use of this combination as the bone graft. The researchers had a task to create the optimal structure, balanced biocompatibility and osteoregeneration. Biocompatibility of the developed model in vitro was estimated by means of cultivation of MG-6 cells and a study of influence on vitality of these cells, proliferation, protein absorption, alkaline phosphatase activity and biomineralization. Various biochemical test systems identified two-fold increase in vitality and proliferation of cells. Presence of ZnO showed intense antimicrobial efficiency in studies with gram-negative (E. coli) and gram-positive (S. aureus) bacteria. The authors found the optimal concentration of ZnO (10 % max.), with balanced values of mechanical strength, antimicrobial effect and biocompatibility. The use of such biosubstrate can be perspective for bone tissue regeneration [5].
Bone morphogenetic proteins (BMP) take the individual place in bone tissue regeneration. They are directly involved in regulation of fracture union. These proteins play the main role in formation and development of bone and cartilage tissue, osteoblast differentiation and tissue development [6]. Various studies are being conducted for researching and using of BMP and their influence on regeneration. For example, in 2016, the researchers from Taiwan National University developed the biodegradable thermo-sensitive hydrogel system as the carrier for delivery of BMP in treatment of rabbits with femoral bone defects. Femoral bone osteotomy was conducted with formation of 10-mm defect. The femur was stabilized with the stainless steel plate, and the defect was filled with 2.3 mm implants consisting of the autobone and 1 ml hydrogel system with BMP and without it. Various levels of BMP (from 5 µg per ml to 20 µg per ml) were used for determination of the most appropriate concentration to achieve union. The results were estimated with X-ray imaging, histological staining, microcomputer tomography (mcCT) and biomechanical testing after 4, 8 and 12 weeks from surgery. As result, the successful healing of bone defects and better quality of bone tissue were observed in the groups with BMP. Moreover, the higher levels accelerated union. The only autograft group showed the poorest results in regeneration time, and the comparison groups and the groups with hydrogel system did not show any signs of union. The studied hydrogel system was sufficiently safe for wide use in clinical practice, and, according to the authors' opinion can be the perspective alternative strategy for correction of bone tissue deficiency [7].
Another similar hybrid system for delivery of BMP into the defect site was developed in Parker Institute of Bioengineering and Bioscience (USA) in 2018. It is based on nano-fibers with alfa-alginate. The construct is stabilized with the fixing plate. The graft was saturated with BMP in basic condition as the comparison group. The recovery was controlled with X-ray imaging and histological examination after 2, 4, 8 and 12 weeks from surgery. The following results were achieved: the hybrid system of delivery resulted in higher bone formation and improvement in biomechanical properties of torque failure as compared to the autograft after 12 weeks. However, the density of the new bone was lower in the group of hybrid system as compared to the control group at all stages of the study. However, the rigidity ratio in the group with hybrid system exceeded the values in the control group [8].
The benefits of BMP as growth factors of bone and cartilage formation are well known and have been proved. Currently, BMP are produced with gene engineering that requires for special equipment, specialized laboratories, trained staff and legal laws for use and activity in this field. All of these requires for economic investments and creates some additional difficulties for wide use and economic benefits. Moreover, it is known that mutations in BMP and in their inhibitors determine some human diseases, including oncologic ones. It requires for careful approach to use of proteins of this group [9].
Adequate blood circulation in the fracture site is the key for successful union and fast recovery of an injured segment. Therefore, it resulted in occurrence of the urgent issue in terms of formation of vascular network by means of stimulation of osteogenesis in the fracture or defect site. There are some new data, which shows that silicate-based biomaterials can cause neovascularization by means of stimulation of endothelial vascular growth factor and, as result, can promote bone regeneration. So, at Korean Institute of Tissue Regeneration Engineering (ITREN), hydrogel fiber with silicate coating for its placement onto biopolymer for improvement of its biological properties has been developed. The release of silicate-ion is highly efficient for stimulation of expression of RNA of angiogenic markers (VEGF, KDR, eNOS, bFGF and HIF 1-α) in endothelial cells. In vivo studies with rats which received this biopolymer into subcutaneous fat showed more intense formation of blood vessels around biopolymer with silicate coating as compared to absence of silicate. After implantation into the artificial skull defect, the group of silicate use demonstrated significant increase in bone formation according to volume and density, as well as presence of the concurrent sign of proangiogenesis in comparison with the control group. This study can testify the possibility for recovery of an injured bone by means of local stimulation of angiogenesis, and presents the perspective direction for bone tissue regeneration [10].
Various disperse systems for better filling of a bone defect are being studied. So, Srnec R., Skoric М., the researchers from Czech University of Veterinary and Pharmaceutical Sciences, used the synthetic hydropeptide-gel enriched with poly-caprolactone nano-fibers for bone defect filling. The in vivo experiment included Wistar rats. Single penetrating defects (diameter of 5 mm) were created in the femoral diaphysis in both extremities. The first defect was filled with hydropeptide gel. The defect in another extremity was not treated. The results were estimated after 2, 4 and 6 weeks after surgical intervention. The difference between control and experimental bone defects was observed only at the stage of healing, two weeks after implantation, when a trend to higher formation of new bone trabecules in the defect prepared with composite hydropeptide gel was observed. At later stage of healing in experimental and control groups (age of defect − 4-6 weeks), the healing process and remodeling of the graft did not have any evident morphological differences. This fact can be an advantage, especially at the early stage of bone tissue regeneration [11].
Currently, the materials are being searched which can be the best in recovery of bone tissue defects. The ideal implant should have a lot of important properties: osteoinduction, osteoconductivity, immunogenecity, biocompatibility, safety etc. However, due to complexity of developed technologies, absent experience in their use and economic component, most works stay at the level of laboratory experimental studies and are not appropriate for wide use.
Therefore, another priority direction is optimization of reparative processes in the fracture site with use of platelet-riched plasma (PRP), and research of its possibilities in various combinations with bone grafts and composite materials.
Currently, it is known that platelets include lots of active substances, which are involved in tissue recovery by means of stimulation of such processes as chemotaxis, cell proliferation and differentiation, angiogenesis, immunomodulation, antimicrobial activity and remodeling. The most interesting substances for researching (growth factors) are platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), transforming growth factor (TGF-β1), insulin-like growth factor (IGF-1), vascular and endothelial growth factors (VEGF, VGF), cytokines (IL-1, IL-6, TNF-α) and others. There are some findings that PDGF, FGF, TGF-β1 and IGF-l can stimulate proliferation, migration and osteogenic differentiation of mesenchimal stem cells, stimulate growth of osteoblasts and synthesis of intercellular matrix. Also TGF-β1 can suppress the activity of osteoclasts, with prevention of resorption of the new bone [12, 13]. It is difficult to ignore the helpful features of growth factors which promote regeneration. Over the whole world, physicians of various specialties have been using PRP for treatment of osteoarthritis, lesions of tendons and ligaments, in costemology, stomatology etc. There are much less findings relating to use of PRP for treatment of fractures and recovery of bone defects. Currently, the number of studies has increased which review the properties of PRP in combination with various composite materials or grafts for treatment of bone defects.
So, at Bogomolts National Medical University, bioglass was implanted into the defect site of the tibial bone in the first group of animals (rabbits). The second group of rabbits received implantation of bioglass in combination with PRP. The follow-up intervals were 4 and 12 weeks. For morphological studies, defected leg fragments were separated and histological slices (7-9 µm) were made. Results of histological studies of samples were the findings on increasing of formation of bone tissue in the defect by 9 % with use of PRP as compared to the control group. An increase in osteoconductive effect was observed, as well as anti-inflammatory action in the implantation site was observed [14].
An experiment was conducted at Sklifosovsky Institute of Emergency Medicine. It was directed to research of influence of PRP in combination with collagen on the process of bone regeneration in the distal femur in rats. Allogenic collagen was received from rat tails. It was transformed into gel. PRP was prepared with Messora technique. The concentration of platelets in PRP was 1,100-1,300 × 103/µl. A defect (2 mm diameter) was formed in the femoral condyle. In the first group (comparison), the wound was sutured without filling with plastic material. In the second group, bone defects were filled with rat collagen (150-200 µl). In the third group, bone defects were filled with mixture of rat collagen and allogenic PRP with 1:1 ratio. The time course of bone recovery in the defect site was examined with histological samples stained with hematoxilin and eosin according to Van-Gieson. After analysis of the results, the authors concluded that use of PRP in combination with collagen could release growth factors from platelet granules, resulting in 2-fold decrease in time of reparation of a bone defect in rats. Also the use of PRP decreased the intensity of inflammatory response in the bone defect site [15].
The group of researchers from Kioto Medical University (Japan, 2018) conducted a study of influence of PRP combined with β-TCP sponge on bone formation in the defect of vertebral body in rats. A defect of critical size was formed in the lumbar vertebral body in each rat. The animals were divided into the groups. The group 1 received β-TCP and PRP, the group 2 − gelatin sponge with phosphate-saline buffer. The group 3 did not receive treatment. The results were estimated after 4, 8 and 12 weeks from surgery. In the PRP group, a significant growth of bone tissue was observed as compared to the group without PRP. Histological study showed additional increase in bone tissue in β-TCP and PRP as compared to groups without PRP. Histological study showed additional growth of bone tissue in β-TCP and PRP as compared to sponges without PRP. One can suppose that PRP could show its osteogenic properties in combination with excellent osteal integrity of tricalcium phosphate. Moreover, biomechanical tests showed high rigidity of vertebral bodies in the PRP group [16].
Continuing the discussion of influence of PRP on bone tissue regeneration, two studies from University of Natural Sciences in Poland can be presented as examples. First of all, the experiment with use of autological PRP for treatment of artificial femoral bone defects in rabbits was carried out. In both femoral bones of each rabbit, some monocortical defects (4 mm) were formed. Defects in left lower extremities were intact (the comparison group). Defects to the right were filled with PRP which was prepared by means of standard centrifugation and thrombin activation. X-ray imaging of the studied segments was conducted each 7th day from surgery. The experiment was completed after 8 weeks. The results were estimated according to radiological, biochemical, histological and biomechanical characteristics. Analyzing the received data, the authors noted some signs of increasing volume of bone tissue in the defect site in the experimental group, but biomechanical tests testified low density of formed bone callus. The amount of low density tissue fraction significantly exceeded the amount of high density tissue.
The same authors estimated another similar experiment of influence of autological PRP on remodeling of hydroxyapatite tricalcium phosphate in experimental defects of femoral bones in rabbits. The animals were divided into two groups. A monocortical diaphyseal defect (4 mm diameter) was formed in each femoral bone in both groups. In the group 1, the defects were intact on one side. On other side, they were filled with tricalcium phosphate and hydroxyapatite implant (80 %). In the group 2, one side was intact, whereas on other side, defects were filled with tricalcium phosphate (80 %) and hydroxyapatite (20 %) in combination with 0.7 ml of autological PRP. The experiment lasted for 8 weeks. After that, the results were estimated according to radiological, histological and biomechanical properties. Radiological and histological estimation did not show any positive effect of PRP in combination with bone substitute as compared to filling of defects with only material for bone replacement. However, PRP in combination with hydroxyapatite tricalcium phosphate made the positive influence on the new bone, increasing its density [17, 18].
The analysis of results of these studies leads to a fair question: why the use of the same experimental substrates (PRP and tricalcium phosphate) give good results in one case and converse ones in other case? Certainly, the studies are similar only at first glimpse. It is necessary to consider the difference in the experimental models, in the type and concentrations of tricalcium phosphate, and, of course, in the way of PRP preparation, in general conditions of experiment realization and in other points.
At the department of traumatology and orthopedics of Turkish Medical University, a study of influence of PRP on fracture healing was conducted. The experimental model included rats which were divided into 3 identical groups. By means of osteotomy, a monocortical defect was created in femoral bones of rats in the groups 1 and 2. The animals of the first group were left without treatment. The group 2 received PRP. The group 3 did not receive osteotomy and presented the comparison group for biomechanical tests. PRP was prepared by means of collection of venous blood and after addition of natrium citrate as anticoagulant, with single-shot centrifugation within 8 minutes at 1,800 rpm. After collection of enriched fraction, platelets were exposed to activation with chloride natrium solution and were introduced into the defect. 4 weeks later, histological estimation of results was conducted, 9 weeks later − biomechanical estimation, with comparison of the studied groups with control one without osteotomy. As result, on the basis of histological signs, more full-featured recovery of bone defects was observed in the group with PRP as compared to the group without treatment. Also, by the 9th week, biomechanical characteristics of the new bone were better as compared to the group without injection of plasma [19].
At Kuban Agrarian University in cooperation with Kuban State Medical University, two studies were carried out which investigated the properties of PRP in relation to bone tissue regeneration. First of all, the experiment was held, which estimated the role of PRP-therapy in stimulation of osteoregeneration. Under general anesthesia, an opened transverse fracture of the middle one-third of the left tibial diaphysis was formed. After that, reposition and fixation of a fracture was performed with 4 K-wires. PRP was prepared with the centrifuge (3,500 prm) and test-tubes with separation gel. PRP was introduced into the fracture site for animals of the group 1 on the day 5. In the animals of the group 2, the fracture union was without influence of any agents. Rabbits were devitalized on the day 30. The results were compared on the days 5, 7, 10, 15, 20, 25 and 30 after introduction of PRP. The results of the study were estimated with histological and radiological signs. In the group of animals treated with PRP, the time of healing of the bone defect reduced by 8-10 %.
An analysis of results of treatment of 16 women with fractures of distal epiphyseal cartilage of the radial bone was carried out. After A-PRP therapy, it was found that the time trends of correction of clinical and radiological signs of a fracture was more intense in patients of the study group (n = 6) as compared to the control group (n = 10). According to the results of the study, the time intervals of fracture union decreased by 3-4 days: 9.5 ± 1.1 % from average time of fracture union in the control group. The evident changes in bone tissue in view of radiological signs of union were found by the day 20. At the background of stimulation of reparative osteogenesis with PRP, soft callus became X-ray-contrast by the day 20 [20, 21].
At Heinrich Heine University Clinic (Germany), a study of properties of PRP in combination with autograft for recovery of radial bone diaphyseal defects in rabbits was conducted. Two groups were studied. The group 1 received the autograft and PRP, another one − only the autograft. Histological and radiological estimation confirmed higher efficiency of PRP as compared to the control group [22].
At the Central Institute of Orthopedics (Beijing, China), a study of combined action of calcium sulphate (CS) and PRP on recovery of long bones was conducted. The experimental model was presented by a defect (12 mm) in the radial bone of a rabbit. Defects were initiated by osteotomy. Rabbits were divided into 4 groups. The group 1 received treatment with combination of CS/PRP, the group 2 − CS, the group 3 − PRP. The group 4 was intact (the comparison group). PRP was prepared from autological blood with use of two-stage process of centrifugation. 3 ml of peripheral blood was collected from each rabbit. Ethylenediamine tetraacetic acid was used as anticoagulant. Blood was centrifugated at 1,130 rpm to separate red blood cells from platelets and plasma. The layer consisting of platelets and plasma was collected and centrifugated at 1,1130 rpm for platelet buttoning. The final concentration exceeded the physiological levels by 8-10 times. Radiological and histological results were estimated after 10 weeks from implantation. As result, recovery of a defect was identified only in the group of CS/PRP. The group with isolated use of CS or PRP did not show fracture union despite of slight formation of callus [23].
Calcium sulphate, which does not have properties of osteoinduction, but having good osteointegrative ability in combination with PRP with osteogenic properties, may present the successful model for bone deficiency compensation. But why the single use of PRP does not give expected results in this experiment, whereas we can see positive results in other mentioned studies? Considering the fact that the studies were conducted by various authors in absolutely different conditions, one should pay attention to differences in protocols of receiving and use of PRP. It creates obstacles for adequate comparative assessment of studies to globally estimate the role of PRP.
The researchers from Alexandroupolis University Hospital (Greece) conducted an experiment of influence of autological PRP in combination with xenogenic demineralized bone matrix (DBM) on recovery of a defect of critical size in the ulnar bone (2-2.5 times bigger than the diameter of the ulnar bone) in New Zealand white rabbits. The unilateral defect of critical size was created surgically in each animal. The contralateral extremity was intact. The tested animals were distributed into 3 groups. Only PRP was used in the group 1 (A). The group 2 (B) received PRP in combination with DBM. The group 3 (C) was without treatment. PRP was prepared according to the protocol of commercial producer (the data on rate and time of centrifugation are not disclosed). All experimental animals were exposed to radiological estimation of frontal extremities immediately after surgery and in the postsurgical period on the weeks 4 and 12. After completion of the experiment, the histological and biomechanical analysis was conducted. The group B had higher radiological and histological values as compared to the groups A and C. The defects in the group B were highly filled with the new bone, whereas bone formation was minimal or absent in the groups A and C. The macroscopic examination showed bone union only in the group B. Despite of this, biomechanical estimation of prepared and intact contralateral extremities in the group B showed a decrease in bone density in the experimental extremity. The final results set one thinking about quality of the new bone and justifiability of this technique [24].
At the Jilin university clinic (China), an experiment with osteointegration of decellularized bone matrix in combination with PRP-therapy for a radial bone defect of critical size in rabbits was conducted. A 15 mm defect of the radial bone was initiated, with removal of periosteum on both sides of a defect to prevent periosteal calcification. DBM was prepared from femoral bones of pigs with subsequent ultrasonic preparation, protein denaturation, cell lysis and lyophilization. PRP was prepared by means of staged centrifugation of blood of rabbits. After activation with natrium citrate, primary centrifugation was performed within 16 minutes at 2,000 rpm to remove red blood cell mass, then, within 12 minutes at 1,500 rpm to achieve platelet bottoming. Rabbits were distributed into three experimental groups. The first group was left without treatment. The second group received only DBM, the third group − combination of DBM and PRP. The implants were placed into a defect and sutured to muscular fascia without additional fixation. The results were estimated after 4, 8 and 12 weeks from implantation. Comparing the radiological data by the 12th week after implantation, the authors could see that the group without treatment and in the group with only DBM did not show any evident signs of bone connection, and unsuccessful osteointegration was evident. Reconstruction of the graft was observed by the week 8 in the group with combined use of PRP-therapy. Histological estimation of samples of the group without treatment showed only signs of osteochondrosis in the fracture site by 12th week. In the group of DBM, the surface of the graft was covered with big mass of fibrous connective tissue without clear visualization of cortical layer. In the group with combined use of PRP, the surface of the graft was covered with the cortical bone, which was similar to natural structure of cortical layer of the radial bone. The authors considered these observations more closed to natural regeneration of the bone that can testify positive effects of PRP and successful completion of the experiment [25].
Such comparison of influence of PRP and DBM on critical defects of the bone of a rat was conducted at Hacettepe Medical University (Turkey). The created defects of the radial bone were filled with combination of PRP and DBM, PRP and DBM in pure form, and alone. Also the group without treatment was studied. The experiment was completed by the week 10 from implantation. Then, after removal of the inferior layer, the second centrifugation was conducted at 4,000 rpm within 7 minutes. The inferior layer with high amount of platelets was collected. The superior layer with lower amount of platelets was removed. Radiological estimation of the results showed that ossification was more efficient in the group of PRP followed by DBM. It was confirmed by histological studies [26].
An analysis of results of these similar studies of combination of PRP and DMB raised the question: why the result of one study is the dominating efficiency of PRP in combination with DBM, and the result of other study is higher efficiency of single use of PRP as compared to combined use with DMB? Possibly, the answer is the multi-faceted approach to realization of a study and, as it was mentioned before, conditions and procedure of testing. Probably, high variety in results from other authors in researching of the bone substitute is caused by absence of the uniform standard in use of bone fillers, and, possibly, various protocols for PRP preparation can influence on efficiency and amount of released growth factors and their subsequent role in regeneration process.
The world society is not limited by studies of PRP properties in animal tests. Due to the fact that derivation and preparation of PRP does not require for high tech provision, currently, some physicians try to treat various bone fractures in humans with use of PRP. So, in India, a study was carried out to estimate the role of PRP in regeneration of bone tissue. It included 72 patients with diaphyseal fractures of the femoral bone. Patients were distributed into the groups. One group received intramedullary fixation of the femoral bone in combination with introduction of PRP into the fracture site. Another group received only intramedullary fixation. For preparation of PRP, 70 ml of venous blood was collected. Centrifugation was performed at 2,000 rpm. After separation of the inferior layer with red blood cells, recurrent centrifugation was carried out at 2,800 rpm. PRP was activated with autological thrombin and was introduced into the fracture site with use of a syringe. The results of the study were estimated only with radiological signs, assessing the ratio between natural cortical layer and cortical layer in the callus zone. The signs of radiological union were not observed in any patients before 4th month. By the 6th month, all patients had radiological signs of union. Over the whole period, the authors did not note statistically significant differences between the compared groups. They concluded that PRP did not influence on femur fracture union in combination with closed intramedullary fixation and can provide only the effect of artificial hematoma at the initial stage of union. Discussing the results of the study, they supposed that efficiency of PRP can be too weak to cause bone formation in a defect with low recovery potential. The positive result in most animal studies is associated with presence of defects in spongy bone with good perfusion. Moreover, the combined use with autografts can make additional osteogenic effect [27].
Another study was conducted in India. It included patients with slow fracture union (leg, hip, forearm, shoulder) of long bones (94 patients). Patients received PRP. 15-20 ml of PRP (>2,000,000 platelets/µl) were introduced into the fracture site. Radiological assessment was performed each month. As result, by the 4th month, 82 patients (among 94) had evident signs of fracture union. Other 12 patients did not have any signs of callus formation and were recognized as unsuccessful [28]. It is difficult to adequately estimate this study owing to its high heterogeneity, but it is possibly to suppose that fracture union in patients included into the study can be the result of action of platelet growth factors used in treatment.
The specialists from Egypt tried to use PRP in treatment of femoral neck fractures. The study included 60 patients which were distributed into 2 groups: the group 1 received classic osteosynthesis of femoral neck with 3 cannulated screws. The group 2 received the same osteosynthesis and PRP therapy. As result, 53 patients achieved reliable fracture union: 25 patients in the group without PRP and 28 in the group with PRP. 3 patients had avascular necrosis of the femoral head (2 patients in the group 1, 1 patient in the group 2). 6 patients required for revision surgery. Although the statistical result showed that the mean arithmetic and radiological time of fracture union were lower after use of PRP as compared to the group without PRP, the results of the study show that the risk of non-union and development of avascular necrosis after femoral neck osteosynthesis did not change significantly. The use of PRP did not show expected positive effect [29].
A study was performed at Iran University of Medical Sciences. It was directed to improvement in functional results after fractures of the radial bone in common site. The experiment included 30 patients. A half of them received PRP after closed reposition under radiological control percutaneously into the region of the radiocarpal joint. The results were estimated after 3 and 6 months according to pain score and range of movements in the joint. The result showed pain decreasing and increasing range of movements in the group with PRP [30].
Despite of the positive result of PRP use in treatment of fractures in patients in the above mentioned studies, it is impossible to adequately estimate the role of PRP. We have few reliable data to consider the properties of platelets as the measure to optimize bone regeneration. Probably, we are not ready to use PRP for treatment of fractures in our patients.
For efficient using of PRP technique, it is necessary to have full understanding of properties of each growth factor influencing on regeneration ability. Also one must understand how to prepare PRP to maximally use properties of platelets, which substance is better to use for activation, and in which period of union growth factors can show their potential to the full degree? To answer these and other questions, we reviewed some in vitro studies of PRP features.
The stage of activation before introduction of PRP is included into a lot of protocols, usually by means of addition of thrombin and/or calcium chloride (CaCl2), but some physicians prefer to introduce PRP with its resting form, relying on spontaneous activation of platelets after influence on natural collagen in connective tissue [31].
At the department of orthopedic surgery of Korean Medical University (Korea Republic), the kinetics of release of cytokines (growth factors) in PRP was estimated in dependence on various protocols of activation. To prepare PRP, the blood was collected from 14 clinically healthy patients. According to the first protocol (A), it was exposed to one-stage centrifugation at 900 g during 5 minutes, and to the protocol with double centrifugation − at 900 g during 5 minutes, and then during 15 minutes (B). Both PRP were activated with one of three protocols of activation: without activation, activation with only calcium (Ca) and calcium with low dose of thrombin (50 IU per 1 ml of PRP). The kinetics of cytokine release was estimated with levels of PDGF, TGF, VEGF, FGF, IL-1 and MMP-9 with immune enzyme analysis. As result, it was found that level of cytokines released from PRP changed over time and depended on various activation protocols. So, activation with Ca in the protocol A increased the levels of VEGF, FGF and IL-1 as compared to the protocol B. For activation with Ca/thrombin and the protocol A and B, one could observe high levels of PDGF and VEGF, and the levels of TGF and FGF were low. Moreover, for activation with Ca/thrombin, the general release of cytokines was at the therapeutic level up to 7 days. Therefore, one can suppose that the difference in preparation of PRP and activation method makes direct influence on quality of the basic product and, therefore, on efficiency of regeneration properties [32].
Another study of properties of PRP in dependence on way of activation was conducted in Italian Institute of Orthopedics. Activation of PRP prepared with the uniform protocol was realized by means of addition of 10 % CaCl, 10 % autological thrombin, 10 % mixture of CaCl2 + thrombin and 10 % collagen of type 1. After 30 minutes, 1, 2 and 24 hours from activation, immune enzyme analysis was conducted for testing the release of VEGF, TGF-1, PDGF-AB, IL-1 and TNF-α. It was found that PRP activated with collagen of type 1 caused total decrease in release of growth factors as compared to other activators. At that, all activators lead to immediate release of high amount of PDGF and TGF-β 1, and their values were stable in the beginning. Conversely, VEGF showed a trend to increasing level from 30 minutes to 24 hours. It confirms that activation with PRP makes high influence on release time and amount of growth factors and directly influences on treatment outcome [33]. At Choson University (Korea Republic), the influence of PRF-enriched fibrin growth factors on proliferation and differentiation of MG-63 osteoblasts was estimated. PRF is the modified form of PRP which can be got after 1 cycle of centrifugation without addition of anticoagulants or heterogeneous thrombin. It is physiological and, like PRP, can release growth factors, which are identified in platelets, including PDGF, TGF, insulin-like growth factor (IGF) etc. To prepare PRF, the blood was connected. Without anticoagulant, centrifugation was performed at 2,000 rpm during 10 minutes. A fibrin clot appeared in the middle part of the test-tube, whereas the superior part included cell-free plasma, and the inferior part − red blood cells. Then the influence of PRF growth factors on proliferation and differentiation of MG-63 cells was studied in vitro. Activity of alkaline phosphatase increased in MG-63 cells prepared with 10 % PRF. Also calcification and mineralization of MG-63 cells were identified. PRF activated the genes of biomarkers such as collagen of type 1, BMP-2 and osteocalcin which are associated with formation of a bone in MG-63. By the example of osteoblasts MG-63, this study shows that PRF makes positive influence on bone tissue regeneration by means of properties of growth factors [34].
Another study dedicated to release of growth factors from PRP was conducted at Bern Medical University (Switzerland). This study included comparison of release of growth factors from PRP, PRF and A-PRF (modified PRF) over time and research of 7 growth factors: PDGF-AA; PDGF-AB; PDGF-BB; transforming growth factor beta 1 (TGFB1); vascular endothelial growth factor (VEGF); epidermal growth factor (EGF) and insulin-like growth factor after 15 minutes, 60 minutes, 8 hours, 1 day, 3 days and 10 days after incubation. PRP was made by means of centrifugation of 10 ml of the donor blood during 7 minutes at 1,000 rpm per minute and subsequent centrifugation after plasma detachment within 10 minutes at 3,000 rpm. PRF was also prepared by means of centrifugation of 10 ml of donor blood without anticoagulant at 2,700 rpm during 12 minutes, whereas A-PRF was centrifugated at 1,500 rpm per minute within 14 minutes. Incubation was conducted by means of transfer of clots of PRP, PRF and A-PRF into 6-well (in vitro) plastic cups with addition of 5 ml of nutrition medium for cultivation. The amount of released growth factors was estimated with EIA. Analyzing the results, the authors could observe the advantage of PRP in release of higher amounts of growth factors in earlier time intervals, whereas PRF demonstrates constant and persistent release of growth factors during 10 days. It can suppose the appropriateness of combined use of PRP and PRF [35].
Despite of available fundamental data on positive properties of growth factors for tissue regeneration, some authors believe that clinical findings are insufficient for wide use of PRP. Theoretically, PRP may have some advantages relating to high release of cytokines and growth factors appearing as result of supraphysiological levels of platelets, which promote recovery, regeneration and remodeling of various tissues, including bone tissue. But in practice, not all clinical studies lead to positive results [36].
Making the systematic reviews of literature relating to PRP for bone tissue regeneration, some authors believe that the available literature is characterized by serious limitations from the perspective of low quality of and high heterogeneity, limiting the possibility for optimization of treatment of fractures and bone defects with PRP. Also there is an assumption that conditions of realization of studies, namely, types of animals, PRP preparation protocols, bone defect types, presence of secondary graft or implant etc., could influence on efficiency of growth factors and change the final results [37, 38].
Future high quality big clinical studies will be critical for formation of notion about properties of PRP and its role in the process of bone tissue regeneration. Heterogeneity of PRP agents at the present time and previously does not allow making wide recommendations concerning its usefulness. This heterogeneity also complicated interpretation of available literature [39].
CONCLUSION
The analysis of the data showed a lot of interesting, perspective and evidence-based studies from authors in the whole world in favor of use of growth factors for optimization of bone tissue regeneration. However, the clinical efficiency of PRP and blood-derived growth factors is still to be proved. Currently, there is not any uniform protocol for PRP preparation. At the same time, there a lot of protocols for preparation and variants of use. It hinders the comparison of results of studies. We still do not have the uniform notion about individual influence of each interesting growth factor on a specific stage of bone tissue regeneration. There are not any reliable data on time of positive effect, its strength and rate of occurrence, long term results of PRP-therapy and its safety. There is an opened question about use of platelet-enriched plasma for treatment of acute injury as the method for replacement of bone tissue deficiency in the fracture site, as the secondary element for reorganization of the graft of various origin in the defect site. It requires for systematization, general protocols of using and further investigation.
Information on financing and conflict of interests
The study was conducted without sponsorship. The authors declare the absence of any clear and potential conflicts of interests relating to publication of this article.