In Vitro Characterization of Hypoxia Preconditioned Serum (HPS)—Fibrin Hydrogels: Basis for an Injectable Biomimetic Tissue Regeneration Therapy

Abstract

Blood-derived growth factor preparations have long been employed to improve perfusion and aid tissue repair. Among these, platelet-rich plasma (PRP)-based therapies have seen the widest application, albeit with mixed clinical results to date. Hypoxia-preconditioned blood products present an alternative to PRP, by comprising the complete wound healing factor-cascade, i.e., hypoxia-induced peripheral blood cell signaling, in addition to platelet-derived factors. This study set out to characterize the preparation of hypoxia preconditioned serum (HPS), and assess the utility of HPS–fibrin hydrogels as vehicles for controlled factor delivery. Our findings demonstrate the positive influence of hypoxic incubation on HPS angiogenic potential, and the individual variability of HPS angiogenic factor concentration. HPS–fibrin hydrogels can rapidly retain HPS factor proteins and gradually release them over time, while both functions appear to depend on the fibrin matrix mass. This offers a means of controlling factor retention/release, through adjustment of HPS fibrinogen concentration, thus allowing modulation of cellular angiogenic responses in a growth factor dose-dependent manner. This study provides the first evidence that HPS–fibrin hydrogels could constitute a new generation of autologous/bioactive injectable compositions that provide biochemical and biomaterial signals analogous to those mediating physiological wound healing. This therefore establishes a rational foundation for their application towards biomimetic tissue regeneration.

Comparative Evaluation of the Angiogenic Potential of Hypoxia Preconditioned Blood-Derived Secretomes and Platelet-Rich Plasma: An In Vitro Analysis

“…Over the past decade, there has been a growing interest in the therapeutic application of autologous blood-derived products for the treatment of various skin pathologies and chronic wounds [1][2][3][4][5]. Under physiological conditions, a wound can rapidly regenerate through a series of well-defined wound healing stages, namely haemostasis, inflammation, proliferation and angiogenesis, and eventually tissue remodelling [6,7].…”

“…Thus, the main purpose of the application of blood-derived secretomes in wounded or ischaemic tissues is the targeted stimulation and support of the cellular responses that naturally drive angiogenesis and tissue repair, via protein growth factor signalling. The utilization of autologous growth factors provides a means of offering a personalised treatment to each individual patient, while autologous blood-derived products present unique advantages compared to allogeneic/xenogeneic therapies, by minimizing the risk of infection and adverse immunological reactions [2,5,16,[19][20][21].…”

“…Utilization of hypoxia as a tool to stimulate angiogenesis on-demand harnesses the innate biological mechanism that naturally promotes new vessel formation in the body, in both physiological (e.g., embryogenesis) and pathological states (e.g., ischemia, wound healing, tumour formation) [16][17][18]42,43]. Hypoxia preconditioned blood-derived secretomes can be generated through the process of "extracorporeal wound simulation", which entails peripheral blood incubation under physiological temperature (37 • C) and hypoxia (1-10% O 2 ) ( Figure 1B) [2,5,19,44]. As previously demonstrated, pericellular hypoxia can be achieved in situ within the blood incubation chamber, by adjusting the blood volume per unit area (BVUA), i.e., the PBC seeding density, and hence cellular O 2 consumption, thus overcoming the need for an oxygen-controlling incubator [2,13,19].…”

“…Hypoxia preconditioned blood-derived secretomes can be generated through the process of "extracorporeal wound simulation", which entails peripheral blood incubation under physiological temperature (37 • C) and hypoxia (1-10% O 2 ) ( Figure 1B) [2,5,19,44]. As previously demonstrated, pericellular hypoxia can be achieved in situ within the blood incubation chamber, by adjusting the blood volume per unit area (BVUA), i.e., the PBC seeding density, and hence cellular O 2 consumption, thus overcoming the need for an oxygen-controlling incubator [2,13,19]. It is already established that PBCs respond to stress (e.g., hypoxia, ischemia, inflammation, ultrasound) by upregulating a range of pro-angiogenic growth factors such as VEGF [2,5,[45][46][47][48], bFGF [46][47][48], IL-8 [2,47,48] and MMP-9 [2,47].…”

“…As previously demonstrated, pericellular hypoxia can be achieved in situ within the blood incubation chamber, by adjusting the blood volume per unit area (BVUA), i.e., the PBC seeding density, and hence cellular O 2 consumption, thus overcoming the need for an oxygen-controlling incubator [2,13,19]. It is already established that PBCs respond to stress (e.g., hypoxia, ischemia, inflammation, ultrasound) by upregulating a range of pro-angiogenic growth factors such as VEGF [2,5,[45][46][47][48], bFGF [46][47][48], IL-8 [2,47,48] and MMP-9 [2,47]. For the purpose of a personalised therapeutic approach, PBCs represent an ideal autologous cell type, since their simple harvest and ample availability makes them easy to employ [2,5,19].…”