Research article
The inclusion of leukocytes into platelet rich plasma reduces scaffold stability and hinders extracellular matrix remodelling.

https://doi.org/10.1016/j.aanat.2021.151853Get rights and content

Highlights

  • 3D biomaterials should mimic the native ECM with a controllable degradation rate.

  • Fibrin is the natural wound healing matrix.

  • PRP is a source of both autologous growth factors and 3D fibrin scaffold.

  • There is no consensus regarding the obtaining conditions and composition of PRPs.

  • Leukocytes’ inclusion into PRP reduced scaffold stability and cell remodelling.

Abstract

Background

Scaffolds should have controllable degradation rate and allow cells to produce their own extracellular matrix. Platelet rich plasma (PRP) is a source of autologous growth factors and proteins embedded in a 3D fibrin scaffold. There is no consensus regarding the obtaining conditions and composition of PRPs. The aim of this study was to evaluate how the inclusion of leukocytes (L-PRP) in plasma rich in growth factors (PRGF) may alter the process of fibrinolysis. The effect of different combinations of cellular phenotypes with PRGF and L-PRP clots on both the fibrinolysis and matrix deposition process was also determined.

Methods

PRGF and L-PRP clots were incubated for 14 days and D-dimer and type I collagen were determined in their conditioned media to evaluate clots’ stability. For remodelling assays, gingival fibroblasts, alveolar osteoblasts and human umbilical vein endothelial cells (HUVEC) were seeded onto the two types of clots for 14 days. D-dimer, type I collagen, and laminin α4 were measured by ELISA kits in their conditioned media. Morphological and histological analysis were also performed. Cell proliferation was additionally determined

Results

PRGF clots preserved their stability as shown by the low levels of both D-dimer and collagen type I compared to those obtained for L-PRP clots. The inclusion of both gingival fibroblasts and alveolar osteoblasts stimulated a higher fibrinolysis in the PRGF clots. In contrast to this, the degradation rates of both PRGF and L-PRP clots remained unchanged after culturing with the endothelial cells. In all cases, type I collagen and laminin α4 levels were in line with the degree of clots’ degradation. In all phenotypes, cell proliferation was significantly higher in PRGF than in L-PRP clots.

Conclusion

The inclusion of leukocytes in PRGF scaffolds reduced their stability, decreased cell number and slowed down cell remodelling.

Introduction

The promising field of tissue engineering and regenerative medicine aims at restoring damaged tissues by combining cells, scaffolds and signalling molecules. Scaffolds act as template for tissue regeneration therefore becoming a key piece of the puzzle (Chaires-Rosas et al., 2019, Dolcimascolo et al., 2019). In fact, they are often combined with cells (but not always necessary) or with growth factors. 3D biomaterials for scaffolds developing are expected to mimic the native structure and composition of extracellular matrix (ECM) (Keane and Badylak, 2014, Okur et al., 2020). Despite each anatomic site has distinctive characteristics, overall, the ECM is a dynamic and complex structure that provides physical support and spatial organization. The cells-surrounding matrix provides a microenvironment that acts controlling cell behaviour, including cell survival, adhesion, proliferation, migration, differentiation, and angiogenesis (Christman, 2019, Dickinson and Gerecht, 2016). Bone remodelling is an extremely coordinated continuous process concerning multiple cell types, including osteoblasts, osteoclasts, fibroblasts, endothelial cells and immune cells (Li et al., 2020). Fibroblasts represent a dynamic and versatile cell population that are responsible for synthesizing and depositing extracellular matrix components thus modulating the neighbour cells’ behaviour. These qualities confer them a great potential for tissue engineering application (Costa-Almeida et al., 2018). Supporting functional angiogenesis is critical for the success of tissue engineered constructs. Different cell types including endothelial cells are involved in ensuring the supply of nutrients, oxygen diffusion and waste products removal, to finally guarantee fully vascularization (Lee et al., 2021).

Besides resembling the native ECM, scaffolds should have controllable degradation rate to match to that of tissue to repair and to allow cells to produce their own extracellular matrix (Li et al., 2015, Muhleder et al., 2018). In this context, the naturally occurring materials such as fibrin have certain advantages for their suitability for tissue engineering. Fibrin is the natural wound healing matrix and it has been widely used for several tissue engineering applications. The fibrin network provides a physical support and a set of biological cues that modulate cell behaviour (Brown and Barker, 2014). It also contains numerous cell and ECM binding sites and is susceptible to proteolytic degradation that enables cell colonization and tissue remodelling (Brown and Barker, 2014, Christman, 2019). Fibrinolysis is extremely important in tissue engineering. Plasmin breaks down the fibrin through the fibrinolytic system. The process is initiated by two enzymes, the urokinase plasminogen activator (uPA) and the tissue plasminogen activator (tPA) that transform plasminogen into plasmin (Chaires-Rosas et al., 2019, Muhleder et al., 2018, Wyganowska-Swiatkowska et al., 2014). The resultant plasmin is responsible for the degradation of fibrin and many other substrates, thus contributing to the degradation and turnover of the ECM (Chang et al., 2003). Soluble fibrin degradation products (FDPs) include fragments E, fragments D and D-dimer. These by-products are nontoxic and have potent biological activity (Ahmann et al., 2010, Brown and Barker, 2014).

Platelet rich plasma (PRP) is a source of autologous growth factors and proteins embedded in a 3D fibrin scaffold. There is no consensus regarding the obtaining conditions and composition of these PRPs which leads to different products with different biological potential (Anitua et al., 2013a, Anitua et al., 2017, Torres et al., 2008). The inclusion or not of leukocytes is one of the currently most controversial issues in this field. It has been demonstrated that leukocytes contain and produce cytokines that are primarily catabolic or inflammatory, including metalloproteinases, reactive oxygen species and proinflammatory cytokines that may influence the biological outcomes of PRP technology by perpetuating a proinflammatory environment (Anitua et al., 2015c, Kobayashi et al., 2016, McCarrel et al., 2012, Sundman et al., 2011, Xu et al., 2017a). The structure and composition of the fibrin scaffold can also be affected by the cellular composition of PRP protocols. Leukocyte inclusion reduces fibres density throughout the fibrin mesh (Anitua et al., 2015d) and affects the viscoelastic properties (Anitua et al., 2015c).

Therefore, the aim of this study was to evaluate how the inclusion of leukocytes (L-PRP) in plasma rich in growth factors (PRGF), a PRP free of white blood cells with a moderate platelet concentration, may alter the process of fibrinolysis. In addition to this, the effect of different cellular phenotypes (fibroblasts, osteoblasts and endothelial cells) on both the fibrinolysis and matrix deposition process in PRGF and L-PRP clots was also determined.

Section snippets

PRP preparations and haematology characterization

The study was performed following the principles established in the Declaration of Helsinki of 1964 amended in 2013.

Blood from healthy donors was collected into 9-mL tubes with 3.8% (wt/v) sodium citrate, after written informed consent was provided. Four donors were used in the fibrin stability and remodel assays. However, in the proliferation assay, only one donor’s blood was employed. Blood was centrifugated at 580 g for 8 min at room temperature (Endoret System, BTI Biotechnology Institute,

PRP preparations and haematology characterization

The haematological analysis of PRP preparations showed that no significant differences were found in platelets count between PRGF and L-PRP. Concerning the other two measured parameters, almost no leukocytes nor erythrocytes were detected in PRGF. However, leukocyte concentration in L-PRP was 3.9 ± 0.9-fold higher than in blood, being significantly greater than that detected in the PRGF. In addition, the presence of red blood cells was also detected in the L-PRP, responsible for its reddish

Discussion

The implanted matrices for tissue engineering purpose must supply a template to guide the restoration of the original tissue architecture. Thus, the ideal scaffold material should provide a certain level of biomechanical strength as well as a controlled degradation rate, mimicking the biological functionality of native extracellular matrix (ECM) (Williams, 2019, Zhao et al., 2018). In this sense, fibrin matrices from PRPs could be a promising approach due to their transcendental properties as

Conclusion

In summary, despite the limitation of the sample size, the results shown in this experimental work indicate that PRGF scaffolds would be consider as a promising and safe option for tissue engineering, since they would provide a cellular support whose degradation rate was in line with the extracellular matrix deposition. Additionally, leukocyte content into the L-PRP could contribute to the scaffold instability and the reduced capacity to induce cell proliferation, thus hindering the

Ethics approval and consent to participate

The study was performed following the principles established in the Declaration of Helsinki of 1964 amended in 2013, and after written informed consent was provided.

Conflict of interest

The authors declare the following competing financial interests: E.A. is the Scientific Director of and M.Z., M.T., R.T., and MHA are scientists at BTI Biotechnology Institute, a dental implant company that investigates in the fields of oral implantology and PRGF-Endoret technology.

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    These authors contributed equally to this work.

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