Elsevier

Journal of Controlled Release

Volume 202, 28 March 2015, Pages 31-39
Journal of Controlled Release

Infiltration of plasma rich in growth factors enhances in vivo angiogenesis and improves reperfusion and tissue remodeling after severe hind limb ischemia

https://doi.org/10.1016/j.jconrel.2015.01.029Get rights and content

Abstract

PRGF is a platelet concentrate within a plasma suspension that forms an in situ-generated fibrin-matrix delivery system, releasing multiple growth factors and other bioactive molecules that play key roles in tissue regeneration. This study was aimed at exploring the angiogenic and myogenic effects of PRGF on in vitro endothelial cells (HUVEC) and skeletal myoblasts (hSkMb) as well as on in vivo mouse subcutaneously implanted matrigel and on limb muscles after a severe ischemia. Human PRGF was prepared and characterized. Both proliferative and anti-apoptotic responses to PRGF were assessed in vitro in HUVEC and hSkMb. In vivo murine matrigel plug assay was conducted to determine the angiogenic capacity of PRGF, whereas in vivo ischemic hind limb model was carried out to demonstrate PRGF-driven vascular and myogenic regeneration. Primary HUVEC and hSkMb incubated with PRGF showed a dose dependent proliferative and anti-apoptotic effect and the PRGF matrigel plugs triggered an early and significant sustained angiogenesis compared with the control group. Moreover, mice treated with PRGF intramuscular infiltrations displayed a substantial reperfusion enhancement at day 28 associated with a fibrotic tissue reduction. These findings suggest that PRGF-induced angiogenesis is functionally effective at expanding the perfusion capacity of the new vasculature and attenuating the endogenous tissue fibrosis after a severe-induced skeletal muscle ischemia.

Introduction

A remarkable clinical motivation for building microvessels arises from the need to reestablish or improve blood flow and tissue function in compromised or damaged tissues. The latter occurs in many pathological conditions including chronic wound healing, arterial obstructive syndromes and coronary artery and peripheral arterial diseases among others [1], [2], [3]. As an approach to the challenge of optimizing tissue protection and regeneration through promotion of angiogenesis and minimization of the formation of fibrotic scar, several different strategies have been developed by using a sustained delivery of growth factors (reviewed in [4]). The application of endogenous intramuscular infiltrations of plasma rich in growth factors (PRGF) [5] is an innovative biological approach grounded in the regenerative potential of platelets, fibronectin, plasma biomolecules, and fibrin scaffold [6], [7], [8]. There is an increasing body of evidence that points towards growth factors as instrumental in angiogenesis and muscle regeneration (reviewed in [9]). Once PRGF is activated, it polymerizes in situ into a three-dimensional fibrin scaffold that releases progressively a pool of proteins, growth factors, cytokines and even exosomes, acting as a local growth factor delivery system [7], whose release kinetics has been recently described [10]. This transient fibrin scaffold presents important features: (i) heparan sulfate binding domains, that may sequester and then release heparin-binding growth factors including PDGF, FGF, HGF, and VEGF and (ii) cell adhesion motifs that may contribute to cell survival [11], [12], [13], [14].

PRGF muscle infiltrations are aimed at recruiting, activating and mobilizing mainly myogenic progenitor cells and resident macrophages which contribute to muscle reparation processes by cell signaling soluble factors, in addition to the already activated endothelial cells, macrophages, and platelets in the injured area [15].

The aim of the current study was to explore and assess the angiogenic and myogenic effect of PRGF on in vitro experiments with endothelial cells (ECs) and skeletal myoblasts as well as on in vivo mouse subcutaneously implanted matrigel and on limb muscles after a severe ischemia.

Section snippets

PRGF preparation and characterization

For the preparation of PRGF, 27–36 mL of peripheral venous blood was withdrawn from 3 healthy donors into 9 mL tubes containing sodium citrate (3.8%). None of the donors had taken medication in the last week that could alter the hematological parameters. This study was conducted following the ethical principles for medical research contained in the Declaration of Helsinki amended in 2008. PRGF was obtained using the Endoret protocol (BTI-Biotechnology Institute, Vitoria, Spain). Briefly, blood

PRGF characterization

The hematological results were consistent with the PRGF standardized protocol. A description of relevant parameters (platelets and leukocytes) in peripheral blood and PRGF, as well as the platelet enrichment efficacy is indicated in Supplementary Table 1.

PRGF prevents apoptosis and induces muscle and endothelial cell proliferation in vitro

Primary human umbilical vein ECs (HUVECs) and skeletal myoblasts (hSkMbs) were incubated with different doses of PRGF and proliferated in a dose-dependent manner in response to PRGF (Fig. 2A). Endothelial cells cultured with 20% and 40% of PRGF

Discussion

To date, the biological benefits [19], [20] and the therapeutic applications of PRGF have yielded promising clinical and surgical outcomes in musculoskeletal system pathologies [21] as well as in other medical fields [22]. In addition to many bioactive mediators present in the platelet α-granules (TGF-β, PDGF, VEGF, FGF, EGF, IGF-1, HGF, BMPs, BDNF) and dense granules (histamine, serotonin, calcium and ATP/ADP), there are other plasma constituents, namely, IGF-1, HGF, prothrombin, fibrinogen,

Conclusions

These findings suggest that PRGF-induced angiogenesis is functionally effective not only at providing early vessel growth and maintaining the long-term stability of these new vessels but also at expanding the perfusion capacity of the new microvasculature by generating a suitable cell microenvironment for myogenesis and angiogenesis.

The following is the supplementary data related to this article.

Conflict of interest

E Anitua is the scientific director and R Prado, S Padilla and JJ Aguirre are scientists at BTI-Biotechnology Institute, the company that has developed the PRGF-Endoret technology.

Acknowledgments

This work was supported by grants from the MICCIN PI13/02144 and CP09/00333.

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