Investigation of the effect of anticoagulants on platelet recovery, enrichment factor, PDGF, TGF-β1 and optimal dose in the preparation of platelet-rich plasma

doi:10.21203/rs.3.rs-3138006/v1

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Investigation of the effect of anticoagulants on platelet recovery, enrichment factor, PDGF, TGF-β1 and optimal dose in the preparation of platelet-rich plasma

https://doi.org/10.21203/rs.3.rs-3138006/v1

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Objective

To explore the effect of anticoagulant on platelet recovery and enrichment coefficient of platelet-rich plasma and the optimal dose.

Methods

Nine New Zealand rabbits were divided into 9 groups with 1 rabbit in each group. Platelet recovery rate, enrichment coefficient, platelet-derived factor (PDGF), transforming growth factor (TGF-β1) contents were observed.

Results

The enrichment coefficient and platelet recovery of conventional blood + 120% sodium citrate group were the highest, the high dose blood + 120% sodium citrate group was the lowest, the conventional blood + 120% sodium citrate group was the lowest, and the low dose blood + 80% sodium citrate group was the most. Before plasma activation, the expressions of PDGF and TGF-β1 were significantly increased in the other groups compared with the control group, and were higher in the high-concentration blood group. Compared with before activation, TGF-β1 was significantly decreased in the low concentration blood group after activation; TGF-β1 was significantly decreased in the high concentration blood + 80% sodium citrate group; PDGF showed a downward trend in the high concentration blood group; the expression of PDGF was significantly decreased in each group after activation; TGF-β1 was significantly increased in the conventional blood + 120% sodium citrate group. TGF-β1 was significantly decreased in patients treated with low concentration of 80%, 100%, 120% sodium citrate and high concentration of blood + 80% sodium citrate.

Conclusion

Anticoagulant 120% sodium citrate in conventional blood could separate more platelets, and the enrichment coefficient and platelet recovery rate were the highest, and the biological function was good.

anticoagulant

platelet rich

platelet recovery

enrichment factor

PDGF

TGF-β1

Platelet-rich plasma (PRP) is a kind of blood product that is centrifugatively separated and concentrated from whole blood [1]. It was first isolated and prepared in 1977 and applied in cardiac surgery to prevent impaired platelet function and postoperative bleeding during cardiopulmonary bypass. After combining human plasma PRP with CaCl2 and thrombin, Can make platelet activation; PRP was introduced in oral surgery in 1997. PRP was applied to large segment defect of mandible in 1998 to promote osteogenesis. When activated, platelets in PRP can release a variety of growth factors, including platelet-derived factor (PDGF), vascular growth factor (VEGF), transforming growth factor (TGF-β), etc. [2]. These factors have synergistic effects on cell proliferation and extracellular matrix synthesis. In addition, synthetic PRP cells, mainly leukocytes, neutrophils, monocytes and lymphocytes, have an important role in the inflammatory response and the development of infections and immune responses in the body[3]. It has also been shown in the past[4] that PRP contains a variety of antimicrobial proteins that exert antimicrobial effects by inhibiting the growth of bacteria and fungi. PRP has been reported to be used in a wide range of disciplines[5–6] including tissue repair in a number of fields including dentistry, orthopaedics, orthopaedics, ENT, neurosurgery etc. PRP has been used for the treatment of acne marks, repair of refractory wounds, survival of fat cells after autologous fat transplantation, healing of wounds after laser treatment, smoothing of skin [7]PRP has been shown to improve endometrial tolerance, improve ovarian reserve function, promote structural and functional recovery of reproductive organs after chemotherapy and ischemia, and has a positive effect on infertility [8].

PRP is simple to prepare, low cost, non-toxic, less prone to allergic reactions and it contains high concentrations of growth factors[9]. The levels of growth factors on which PRP depends for its action, the concentration of platelets and the structure of the PRP gel may be influenced by more than one factor[10]. In the preparation of PRP, sodium citrate is a commonly used anticoagulant, but the dose of anticoagulant of choice is unclear. Therefore, this study focused on the effects of different doses of anticoagulants on platelet recovery, enrichment factor, PDGF and TGF-β1 in the preparation of platelet-rich plasma, and to find the optimal dose of anticoagulants so as to prepare the most suitable platelet-rich plasma and provide a theoretical basis for the clinical treatment of tissue repair and regeneration and wound healing.

2.1 Reagents, apparatus and animals

2.1.1 Main reagents and instruments

Sodium citrate (Shaanxi Panlong Yihai Pharmaceutical Co., Ltd.); Rabbit transforming growth factor β1 (TGF-β1) kit ( MM-0239O1, Jiangsu Enzyme Free Industry Co., Ltd.); Rabbit platelet-derived growth factor (PDGF) kit ( MM-0248O1, Jiangsu Enzyme Free Industry Co., Ltd.); Fully automated blood cell analyzer (Kangtai Medical Systems Co., Ltd.).

2.1.2 Experimental animals

Nine New Zealand rabbits (2.0 kg, purchased from Hunan Taiping Biotechnology Co., Ltd, license number: SCXK(Xiang)2020-0005) were housed at 20–26°C, 40–70% humidity, 12h/12h light/dark conditions. The animals were allowed to eat and drink freely, and the experimental process of animal feeding, handling and execution methods were in accordance with moral and ethical standards, and had passed the review of our animal ethics committee.

2.2 Methodology

2.2.1 Blood dilution and sodium bicarbonate treatment

After 1 week of acclimatization, nine 30mL syringes were prepared to draw 2.4mL of 10% sodium citrate solution and wet the tubes. After the rabbits were anesthetized, blood was drawn from the rabbits' abdominal aorta to 30mL to obtain the regular blood + 80% sodium citrate group. The blood was placed in 50mL centrifuge tubes and 10% sodium citrate was added to 100% and 120% according to the grouping to obtain the regular group blood sample. Low dilution blood sample preparation: Add saline to the regular group blood sample to dilute the blood concentration to 85%. High dilution blood sample preparation: add saline to the regular blood sample to dilute the blood concentration to 60%. In the routine blood + 100% sodium citrate group, 0.5ml was taken for routine blood testing of normal blood platelet levels.They were divided into conventional blood + 80% sodium citrate group, conventional blood + 100% sodium citrate group, conventional blood + 120% sodium citrate group, low-dose blood + 80% sodium citrate group, low-dose blood + 100% sodium citrate group, low-dose blood + 120% sodium citrate group, high-dose blood + 80% sodium citrate group, high-dose blood + 100% citrate group Sodium sulfate group, high dose blood + 120% sodium citrate group.

2.2.2 Testing platelet levels

All blood samples collected were stored in an EDTA-K2 anticoagulant tube. The blood samples of all patients were tested by machine using automatic blood cell analyzer, the reagent and instrument used in the study were matched, the blood specimens were smeated 2 sheets according to the standard operating specifications, and the specimens were stained with Reishi - Jimsa dye solution. All specimens were examined by Olympus CX-23 microscope by professionals. The main test is the platelet count in the blood, and the test results are recorded in detail.

2.2.3 Preparation of platelet-rich plasma, activation of platelet-rich plasma

Platelet-rich plasma was prepared by the Aghaloo method. 10ml of rabbit ear margin venous blood was collected and placed in a 40g/L sodium citrate anticoagulation tube, shaken well and transferred to a sterile EP tube, centrifuged for the first time at 215 x g10min, the lower layer of red blood cells was discarded, the plasma above the white film layer after centrifugation was placed in another centrifuge tube and centrifuged for the second time at 863 x g10min, the upper middle layer supernatant was discarded and the supernatant was collected as platelet-poor plasma, the The supernatant was transferred to another centrifuge tube and the remaining 0.8mL was blown to make platelet-rich plasma.

The platelet rich plasma and whole blood were activated before use with an activator and the supernatant was extracted from 1000 ∪ porcine thrombin lyophilised powder and 5 mL of 10% calcium chloride solution. After addition of the activator, the tubes containing the samples were incubated in a 37°C water bath for 30 min. The tubes were then removed and centrifuged in a centrifuge at 3000 x g RCF for 20 min.

2.2.4 Detection of platelet recovery, enrichment factor

Calculation of platelet recovery, enrichment factor in several groups of platelet rich plasma. Calculated based on whole blood volume, volume of PRP, platelet concentration in whole blood, and platelet concentration. Platelet recovery (%) = [volume of PRP (mL) × platelet concentration in PRP ( 109 /L)] / [volume of whole blood ( mL) × platelet concentration in whole blood ( 109 /L)], platelet enrichment factor (times) = platelet concentration in PRP (109 /L) / platelet concentration in whole blood (109 /L).

2.2.5 ELISA for PDGF, TGF-β1 in platelet-rich plasma

The content of platelet-derived growth factor PDGF and transforming growth factor TGF-β1 in normal plasma, platelet-rich plasma before and after activation of the three groups were measured by ELISA kits. The platelet-rich plasma was centrifuged at 1000×g for 15 min at 2–8℃, and the supernatant was extracted and the absorbance (OD) at 450nm was measured in strict accordance with the instructions of the kit.

2.3 Statistical analysis

SPSS 26.0 was used to analyse the data. The measurement data were expressed as (mean ± standard deviation). Comparisons between two groups were made using the independent samples t-test, comparisons between multiple groups were made using one-way ANOVA, F-test, and two-by-two comparisons were made using the S-N-K method. The level of significant difference was tested at P < 0.05.

3.1 Platelet count and platelet recovery and enrichment factor for routine blood tests

The results of blood routine showed that the platelet number, platelet recovery and enrichment coefficient of platelet-rich plasma prepared by different blood concentrations and different doses of sodium citrate anticoagulant were different, and the platelet number of platelet-rich plasma prepared by rabbit plasma of conventional blood + 100% sodium citrate group was 189*10^9/L. Conventional blood was treated with 120% sodium citrate anticoagulant, the highest enrichment coefficient and platelet recovery rate, and the lowest platelet count was 169*10^9/L. The maximum platelet count in low-dose blood + 80% sodium citrate group was 370*10^9/L, while the enrichment coefficient and platelet recovery in high-dose blood + 120% sodium citrate group were the lowest.

3.2 ELISA for PDGF and TGF-β1 expression before and after activation in each group

Before plasma activation, the expression of PDGF and TGF-β1 was significantly increased in all other groups compared to the control group, and was higher in the high concentration blood group. TGF-β1 was significantly lower in the low concentration blood group compared to the pre-activation group, and in the high concentration blood group TGF-β1 was significantly lower upon 80% sodium citrate treatment. There was a tendency for PDGF to decrease in the high concentration blood group compared to pre-activation. PDGF expression was significantly decreased in all groups after activation compared to pre-activation. TGF-β1 was significantly increased in conventional blood at 120% sodium citrate treatment, and significantly decreased in low concentration at 80%, 100%, and 120% sodium citrate treatment, and in high concentration blood group at 80% sodium citrate treatment(Fig. 1).

The biological activity of PRP is determined by the activity of a series of cellular molecules in serial tandem, which are paracrine/autocrine proteins at multiple levels involved in the process of wound repair as well as in tissue regeneration[11]. The mechanism of action is as follows: upon activation of PRP, platelets release a large number of activating factors that contribute to neovascularisation. The activating factors complete the process of tissue remodelling by influencing the proliferation and migration of relevant cells, which in turn modulates the inflammatory response and promotes the synthesis of the extracellular matrix[12]. PRP can construct a microenvironment conducive to tissue remodelling by releasing a large number of signalling molecules. In addition, the growth factors and cytokines contained in PRP play an important role in injury repair and tissue remodelling[13]. These factors modulate cell surface receptors, regulate cell proliferation and differentiation, and participate in cell degradation and tissue remodelling. The large number of factors secreted by PRP also promotes the production of neovascularisation, which provides new cells to the injured tissue and removes residual tissue, facilitating blood circulation and nutrient replenishment to the damaged tissue and repairing the wound.[14] Different doses of anticoagulants have different effects on the quality of platelet-rich plasma and the production of PDGF and TGF-β1. It is therefore particularly important to find the optimal dose of anticoagulant.

When platelet counts are high[15], the platelets are more easily separated by blood separators and the number of cycles in the device is reduced, resulting in lower residual white and red blood cell counts and higher platelet levels in the product. It is now well documented that platelet concentrations are not proportional to their own biological effects and that too high or too low a concentration of platelets can be detrimental to wound healing[16]. Previous studies have shown[17] that platelet aggregation ranges from about 1.4 to 8.1 times, while in 75% PRP, platelet enrichment is more than 4 times; in 73% PRP, platelet recovery is greater than 60% Marx, platelet enrichment factors should be more than 3 times to ensure the effectiveness of the treatment. Previous studies have shown[18] that the most suitable concentration of platelets for endothelial cell proliferation in vascular endothelial cells cultured in vitro is 1.5 million mL per mL, in addition to promoting wound repair requires platelets at 4–5 times the baseline concentration, and that the aggregation capacity of MSCs increases substantially with higher platelet levels over time. It has been reported that[19], that platelet counts above 2 million per ml inhibit the biological behaviour of tendon cells and that the optimal platelet level is 1 to 1.5 million ml per ml. Optimal platelet concentrations can therefore play an optimal role in the repair of disease or trauma. The results of routine blood tests in this study show that the platelet counts, platelet recovery and enrichment factors of PRP prepared from different concentrations of blood with different doses of anticoagulant vary. Different concentrations of different doses of the preparation of sodium citrate anticoagulant blood platelet rich plasma number of platelets, platelet recovery, enrichment coefficient of different, routine blood + 100% sodium citrate preparation of rabbit plasma platelet rich plasma platelet number is 189 * 10^ 9 / L. Conventional blood was treated with 120% sodium citrate anticoagulant, the highest enrichment coefficient and platelet recovery rate, and the lowest platelet count was 169*10^9/L. The maximum platelet count in low-dose blood + 80% sodium citrate group was 370*10^9/L, while the enrichment coefficient and platelet recovery in high-dose blood + 120% sodium citrate group were the lowest. Conventional blood treated with 120% anticoagulant showed an enrichment factor above 3, platelet recovery greater than 60% and platelet counts closest to the optimal concentration. This indicates that PRP prepared from conventional blood treated with 120% anticoagulant has the best bioactivity and is more conducive to wound repair. This is consistent with the above findings.

The repairing effect of platelet rich plasma on human tissues is dependent on its high content of growth factors. Platelet alpha particles contain a variety of growth factors such as PDGF, transforming growth factor, insulin-like growth factor, vascular endothelial growth factor, epidermal growth factor and fibroblast growth factor. Many of the growth factors have synergistic effects and have functions such as promoting neovascularisation and extracellular matrix synthesis, thereby facilitating the formation of the extracellular matrix[20]. In platelet-rich plasma, PDGF and TGF are the main factors influencing cell regeneration and tissue remodelling. PDGF is a kind of cationic polypeptide with acid resistance, high temperature resistance and easy to be destroyed by trypsin. It is a dimer composed of two polyamino acid chains, and its molecular weight is between 28 and 35 KD[21]. Each segment of PDGF contains two disulfide bonds, which are pdgf-AA, -BB, -AB, -CC and -DD, respectively. Different cell surface receptors bind to different types of PDGF. The biological activity of PDGF is due to the formation of dimers between the four polyaminergic molecules. PDGF-AA and AB mainly exist in platelets and are activated through α particle release. PDGF is a mitogen that regulates osteoblasts and mesenchymal cells; it has functions such as the secretion of collagenase, collagen synthesis, and promotes the migration of macrophages and neutrophils, the proliferation of fibroblasts and, in the presence of chemokines and TGF-β, causes wound inflammatory infiltration[22]. PDGF released from platelet-rich plasma promotes cell proliferation by promoting the synthesis of inositol diphosphate and diacylglycerol, increasing the amount of calcium ions in cells, and promoting G0/G1 into S phase[23]. TGF-β is a superfamily member associated with cell growth and differentiation with a molecular weight of 25 KD. In addition, TGF-β also has 5 homologous isomers. TGF-β is a multifunctional cytokine that regulates cell growth, proliferation, cell adhesion and apoptosis. TGF-β enhances collagen contraction and fibrosis with the participation of PDGF and IGF. The effect of TGF-β on bone repair mainly depends on the selection of vectors and the participation of bone cells. TGF-β can promote the proliferation, migration and stroma secretion of human dental pulp cells in vitro. TGF-β1 plays a pivotal role in chondrogenesis, growth plate development, joint formation, cartilage maintenance, and intervertebral disc formation and development. It promotes endothelial cell proliferation, regulates the division of endothelial cells and fibroblasts, regulates collagen synthesis and collagenase secretion, stimulates endothelial cell chemotaxis and angiogenesis, and inhibits the proliferation of macrophages and lymphocytes. It inhibits degeneration of intervertebral discs, promotes cell synthesis, promotes cell proliferation, inhibits apoptosis, suppresses inflammatory response and reduces the aggregation of inflammatory cells[24]. In addition, platelet-rich plasma plays a role in bone repair by attracting osteoblasts to the implant site through the release of TGF-β. It can promote the growth of osteoblasts and inhibit the generation of osteoclasts to a certain extent, thus promoting the differentiation of fibroblasts and osteoblast precursor cells, increasing the number of osteoblasts, and promoting the generation of collagen and non-collagen proteins to generate new capillaries at the transplant site. In this study, the concentrations of PDGF and TGF-β1 were measured by ELISA after the preparation of platelet-rich plasma. The results showed that the expression of PDGF and TGF-β1 in plasma was significantly increased in all other groups before plasma activation compared to the control group, and the expression was higher in the high blood concentration group. Compared to the pre-activation period, TGF-β1 was significantly lower in the low concentration blood group after activation and significantly lower in the high concentration blood group upon 80% sodium citrate treatment. There was a trend of decrease in PDGF in the high concentration blood group compared to pre-activation. The expression of PDGF decreased significantly in all groups after activation compared to pre-activation. TGF-β1 was significantly increased in conventional blood at 120% sodium citrate treatment, and decreased significantly in low concentrations at 80%, 100%, and 120% sodium citrate treatment, and in the high concentration blood group at 80% sodium citrate treatment. PDGF and TGF-β1 have the broadest role among the many biologic factors released by PRP and are the two most beneficial in promoting wound healing. Previous studies have also shown[25] that TGF-β1 released by PRP is involved in the induction of seed cell osteogenesis and chondrogenesis. It has also been shown[26] that PRP participates in tissue repair by promoting the expression of PDGF and exerts a role in reducing bleeding, anti-infection, accelerating soft tissue healing and bone tissue regeneration. A certain concentration of TGF-β1 can promote the proliferation of periodontal membrane cells, and PDGF can directly promote the absorption of osteoclasts to bone through its receptor, thus stimulating the proliferation of periodontal membrane fibroblasts and improving the ability of cell division[27–28]. 120% sodium berbate anticoagulant of conventional blood can isolate a higher number of platelets during PRP preparation, with the highest enrichment factor and platelet recovery and good biological function. 120% is the best dose of sodium berbate The optimal dose of anticoagulant.

In summary, the effect of different dosages of anticoagulants on platelet-rich plasma preparation varied. During PRP preparation, conventional blood 120% sodium berbate anticoagulant isolated a greater number of platelets with the highest enrichment coefficient and platelet recovery, and good biological function.

The limitations of this study may include the small sample size, which may affect the statistical power and generalizability of the results. Additionally, the study was conducted on New Zealand rabbits, and the findings may not be directly applicable to human subjects. Moreover, the study only examined the effects of one anticoagulant (sodium citrate) and did not compare it with other anticoagulants commonly used in clinical practice. Furthermore, the study only evaluated the effect of anticoagulant on platelet recovery and enrichment coefficient of platelet-rich plasma and did not measure any clinical outcomes, such as wound healing or tissue regeneration. Finally, the study did not explore the optimal dose of anticoagulant for different clinical applications, which may limit its clinical relevance.

Ethical Approval

Written informed consent was obtained from all the participants prior to the enrollment (or for the publication) of this study (or case report).

Competing interests

The authors declared that they have no conflicts of interest regarding this work.

Authors' contributions

杨军，统筹，实验设计、指标制定、技术路线设定、人员调配等；陈超，负责试验全过程，数据分析及整理，文章撰写；雷均，具体试验，数据收集及整理；易鑫明，具体试验，文献查阅，资料收集，数据整理。

Jun Yang, coordination, experiment design, index formulation, technical route setting, personnel deployment, etc.; Chao Chen, responsible for the whole test process, data analysis and collation, article writing; Jun Lei, specific test, data collection and collation; Xinming Yi , specific test, literature review, data collection and data collation.

Funding

This study was supported by the Xinyang Vocational and Technical College Key scientific research projects in 2021(2021-XZYZD-008).

Availability of data and materials

The experimental data used to support the findings of this study are available from the corresponding author upon request.

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Table1 Platelet counts and platelet recovery and enrichment factors for routine blood tests in each group

Sample number	Grouping	Platelet Count (10^9/L)	Enrichment factor	Platelet recovery rate
1	Regular blood +80% sodium citrate group	233	6.976744186	27.90697674
2	Regular blood +100% sodium citrate group	189	14.76356589	59.05426357
3	Regular blood +120% sodium citrate group	169	15.05813953	60.23255814
4	Low dose blood +80% sodium citrate group	370	12.15503876	48.62015504
5	Low dose blood +100% sodium citrate group	274	14.41472868	57.65891473
6	Low dose blood +120% sodium citrate group	243	10.69767442	42.79069767
7	High dose blood +80% sodium citrate group	298	3.07751938	12.31007752
8	High dose blood +100% sodium citrate group	319	3.387596899	13.5503876
9	High dose blood +120% sodium citrate group	230	2.542635659	10.17054264

No competing interests reported.

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Investigation of the effect of anticoagulants on platelet recovery, enrichment factor, PDGF, TGF-β1 and optimal dose in the preparation of platelet-rich plasma

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Abstract

Objective

Methods

Results

Conclusion

Figures

1. Introduction

2. Materials and methods

2.1 Reagents, apparatus and animals

2.1.1 Main reagents and instruments

2.1.2 Experimental animals

2.2 Methodology

2.2.1 Blood dilution and sodium bicarbonate treatment

2.2.2 Testing platelet levels

2.2.3 Preparation of platelet-rich plasma, activation of platelet-rich plasma

2.2.4 Detection of platelet recovery, enrichment factor

2.2.5 ELISA for PDGF, TGF-β1 in platelet-rich plasma

2.3 Statistical analysis

3. Results

3.1 Platelet count and platelet recovery and enrichment factor for routine blood tests

3.2 ELISA for PDGF and TGF-β1 expression before and after activation in each group

4. Discussion

Declarations

References

Tables

Additional Declarations

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