In vitro study of the role of thrombin in platelet rich plasma (PRP) preparation: utility for gel formation and impact in growth factors release

Huber Sc ,Da Silva Lq ,Lana Jf ,Rodrigues Bl ,Cunha Júnior Jl ,Paffaro Au ,Silmara Aparecida De Lima Montalvão ,Da Silva Fa ,Jm Annichino-Bizzacchi

doi:10.46582/jsrm.1201002

Huber Sc , Da Silva Lq + Show 7 more

Open Access

https://doi.org/10.46582/jsrm.1201002

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Abstract

The use of PRP has been studied for different fields, with promising results in regenerative medicine. Until now, there is no study in the literature evaluating thrombin levels in serum, used as autologous thrombin preparation. Therefore, in the present study we evaluated the role played by different thrombin concentrations in PRP and the impact in the release of growth factors. Also, different activators for PRP gel formation were evaluated. Thrombin levels were measured in different autologous preparations: serum, L-PRP (PRP rich in leukocytes) and T-PRP (thrombin produced through PRP added calcium gluconate). L-PRP was prepared according to the literature, with platelets and leukocytes being quantified. The effect of autologous thrombin associated or not with calcium in PRP gel was determined by measuring the time of gel formation. The relationship between thrombin concentration and release of growth factors was determined by growth factors (PDGF-AA, VEGF and EGF) multiplex analysis. A similar concentration of thrombin was observed in serum, L-PRP and T-PRP (8.13 nM, 8.63 nM and 7.56 nM, respectively) with a high variation between individuals (CV%: 35.07, 43 and 58.42, respectively). T-PRP and serum with calcium chloride showed similar results in time to promote gel formation. The increase of thrombin concentrations (2.66, 8 and 24 nM) did not promote an increase in growth factor release. The technique of using serum as a thrombin source proved to be the most efficient and reproducible for promoting PRP gel formation, with some advantages when compared to other activation methods, as this technique is easier and quicker with no need of consuming part of PRP. Noteworthy, PRP activation using different thrombin concentrations did not promote a higher release of growth factors, appearing not to be necessary when PRP is used as a suspension.

Highlights

The use of Platelet rich plasma (PRP) has been studied for different fields, with promising results in regenerative medicine
In order to assess the amount of thrombin in the different types of preparations, all analyses were performed in the same individual sample for serum, L-PRP and T-PRP
The levels of growth factors released were close to 8500 pg/ml of PDGF-AA and 500 pg/ml of EGF. When these levels were compared with our results we demonstrated that EGF levels were very similar when using the same thrombin concentration, the PDGF-AA levels were much higher in our study (43820 ± 16927) using the protocol 1

Summary

Introduction

The use of PRP has been studied for different fields, with promising results in regenerative medicine. Platelets contain over 1100 proteins including growth factors, messengers of the immune system, enzymes, enzyme inhibitors and other bioactive compounds These factors can improve tissue repair by diverse mechanisms including regulation of inflammation, angiogenesis, synthesis and remodeling of new tissues[2, 3]. For these reasons, PRP has been used in different fields: odontology[4], plastic surgery[5], orthopedics[6], wound healing[7] and aesthetics[8] with promising results. In vitro studies evidenced that the different methodologies used in the preparation of PRP can affect biological aspects and clinical effects, which depend on several variables, platelet and growth factor concentration, presence or absence of leukocytes and the type of activation[2]

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