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Comparative study of different anticoagulants and coagulants in the evaluation of clinical application of platelet-rich plasma (PRP) standardization

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Abstract

To investigate the impact of different anticoagulants and coagulants with autologous platelet-rich plasma (PRP) in order to evaluate the clinical application of PRP standardization. Bone marrow stem cells (BMSCs) were seeded into autologous PRP gel scaffolds with different anticoagulants (EDTA, heparin sodium HS, and sodium citrate SC) as well as control group (the whole blood group). Quality of PRP was evaluated and flow cytometric assay was used to detect the activity of the platelet (CD62p, PAC-1). BMSCs were also seeded into PRP with different coagulants (Thrombin, Collagen-I, ADP) as well as PRP un-activated (negative group) and L-DMEM complete culture without PRP (control group). The effects of different coagulants with PRP on proliferation, osteogenic differentiation of BMSCs were analyzed by methyl thiazolyl tetrazolium assay (MTT), ALP staining, Von Kossa staining, Confocal microscopic observation, RT-PCR and Western Blot at the morphological, cellular and molecular levels. Different anticoagulants (EDTA, HS, and SC) could affect the quality of PRP. EDTA group revealed the best quality and activity (CD62p, PAC-1). With different coagulants (Thrombin, Collagen-I and ADP) in the proliferation of BMSCs, the MTT assay showed that the proliferation of BMSCs was increased in all groups with time. On the sixth day of culture, the cell number of each PRP group was significantly higher than that in the control group (P < 0.05), while the most rapidly increasing was found in Collagen-I group. The cumulative release of growth factor (TGF-β1, PDGF) at each time point in the PRP gel of the four groups was higher than that in the control group (P < 0.05). Collagen-I was considered as the best PRP coagulant. When thrombin was used as a platelet coagulant, the release of growth factor in PRP was rapid and direct, while the release of growth factor in Collagen-I-activated PRP was sustained and slow, and the total release of ADP-activated PRP growth factors was the lowest. The study demonstrated the similar outcome in osteogenic differentiation. In terms of gene expression and western bolt, the PCR results showed that the expression levels of OCN gene and RUNX2 protein in each PRP group were higher than that in the control group (P < 0.05). Different anticoagulants caused different degrees of lysis and spontaneous activation of platelets, which lead to different quality of PRP. Compared with HS and SC, EDTA could maintain the structural integrity of platelets, reduce their spontaneous activation, and increase the release of PRP growth factors for a longer period of time, thus ensuring the biomass of PRP. In addition, different coagulants also showed different results in the proliferation as well as osteogenic differentiation of BMSCs. Compared with Thrombin and ADP, Collagen-I may be a better choice.

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Abbreviations

PRP:

Platelet-rich plasma

BMSCs:

Bone marrow stem cells

MTT:

Methyl thiazolyl tetrazolium assay

TGF:

Transforming growth factor

ELISA:

Immunosorbent assay

PDGF-AB:

Platelet-derived growth factor

RIPA:

Radio immuno precipitation assay

SDS:

Sodium dodecyl sulfate

ECL:

Enhanced chemiluminescence

SC:

Sodium citrate

HS:

Heparin sodium

CTAD:

Citrate-theophylline-adenine-dipyridamole

ACD:

Citrate dextrose

TRAP:

Thrombin receptor agonist peptide-6

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Funding

This study was funded by the specialized Research Fund for Military Medicine Innovation (Grant No. 13CXZ030).

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    Authors and Affiliations

    1. Department of Orthopedics, Center of Sport Medicine, Chinese PLA General Hospital, Fuxing-Road, Haidian, Bei**g, 100853, China

      Ning Zhang, Ketao Wang & Zhongli Li

    2. The Second Department of Orrhopaedics, Bethune International Peace Hospital of PLA, Shijiazhuang, 050082, China

      Tao Luo

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    1. Ning Zhang
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    2. Ketao Wang
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    3. Zhongli Li
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    4. Tao Luo
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    Contributions

    ZL: Guarantor of integrity of the entire study; study concepts; study design; definition of intellectual content; manuscript review. NZ and KW: Literature research; clinical studies; experimental studies. NZ: Data acquisition; manuscript preparation; manuscript editing. NZ, KW and TL: Data analysis; statistical analysis.

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    Correspondence to Zhongli Li.

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    Ning Zhang, Ketao Wang have contributed equally to this work.

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    Zhang, N., Wang, K., Li, Z. et al. Comparative study of different anticoagulants and coagulants in the evaluation of clinical application of platelet-rich plasma (PRP) standardization. Cell Tissue Bank 20, 61–75 (2019). https://doi.org/10.1007/s10561-019-09753-y

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