Eduardo Anitua

High-throughput proteomic characterization of plasma rich in growth factors (PRGF-Endoret)-derived fibrin clot interactome.

Plasma rich in growth factors (PRGF®‐Endoret®) is an autologous technology that contains a set of proteins specifically addressed to wound healing and tissue regeneration. The scaffold formed by using this technology is a clot mainly composed of fibrin protein, forming a three‐dimensional (3D) macroscopic network. This biomaterial is easily obtained by biotechnological means from blood and can be used in a range of situations to help wound healing and tissue regeneration. Although the main constituent of this clot is the fibrin scaffold, little is known about other proteins interacting in this clot that may act as adjuvants in the healing process. The aim of this study was to characterize the proteins enclosed by PRGF–Endoret scaffold, using a double‐proteomic approach that combines 1D‐SDS–PAGE approach followed by LC–MS/MS, and 2‐DE followed by MALDI–TOF/TOF. The results presented here provide a description of the catalogue of key proteins in close contact with the fibrin scaffold. The obtained lists of proteins were grouped into families and networks according to gene ontology. Taken together, an enrichment of both proteins and protein families specifically involved in tissue regeneration and wound healing has been found. Copyright

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

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.

Ultrasound-guided plasma rich in growth factors injections and scaffolds hasten motor nerve functional recovery in an ovine model of nerve crush injury.

In the present study we evaluated the motor recovery process of peripheral nerve injury (PNI), based on electrophysiological and histomorphometric criteria, after treatment with plasma rich in growth factors (PRGF) injections and scaffolds in an ovine model. Three groups of sheep underwent a nerve crush lesion: the first group (n = 3) was left to recover spontaneously (SR); the second group was administered saline injections (SI; n = 5) and a third group (n = 6) received PRGF injections and scaffolds immediately after the crush injury. At post‐intervention week 8, 70% of sheep in the PRGF group were CMAP‐positive, with no electrophysiological response in the rest of the groups. Histomorphometric analysis 12 weeks after the surgical intervention revealed that the average axonal density of the SR (1184 ± 864 axons/µm2) and SI (3109 ± 2450 axons/µm2) groups was significantly inferior to the control (8427 ± 2433 axons/µm2) and also inferior to the PRGF group (5276 ± 4148 axons/µm2), showing no significant differences between the control and PRGF groups. The axonal size of the SR and SI groups was significantly smaller compared with the control group (18 ± 4 µm2), whereas the axonal size of the PRGF group (6 ± 5 µm2) did not show statistical differences from the control. Morphometry of the target muscles indicated that the PRGF group had the lowest percentage volume reduction 12 weeks after the crush injury. The PRGF group had larger muscle fibre areas than the SI and SR groups, although the differences did not reach statistical significance. Overall, these data suggest that the PRGF injections and scaffolds hastened functional axon recovery and dampened atrophy of the target muscles in an ovine model. Copyright

Implementation of a more physiological plasma rich in growth factor (PRGF) protocol: Anticoagulant removal and reduction in activator concentration

Abstract Plasma rich in growth factors (PRGF) is a biological therapy that uses patient’s own growth factors for promoting tissue regeneration. Given the current European regulatory framework in which anticoagulant solution in blood extraction tubes could be considered as a medicinal product, a new PRGF protocol has been developed. The actual protocol (PRGF-A) and the new one (PRGF-B) have been performed and compared under Good Laboratory Practices. PRGF-A protocol uses extraction tubes with 0.9 mL of trisodium citrate as anticoagulant and 50 μL of calcium chloride/mL PRGF to activate it. The PRGF-B reduces the amount of sodium citrate and calcium chloride to 0.4 mL and to 20 μL, respectively. Basic hematological parameters, platelet function, the scaffold obtaining process, growth factors content, and the biological effect were compared between both PRGF obtaining protocols. Results: PRGF-B protocol led to a statistically significant higher enrichment and recovery of platelets regarding to the PRGF-A. Hypotonic stress response by platelets was significantly better in the new protocol. A statistically significant decrease in the basal platelet activation status of PRGF-B compared to PRGF-A was also observed. The duration of the lag phase in the platelet aggregation assay was statistically lower for the PRGF-B protocol. Both the clotting and the clot retraction time were significantly reduced in the B protocol. A higher growth factor concentration was detected in the plasma obtained using the PRGF-B protocol. The new PRGF obtaining protocol, with a reduction in the amount of anticoagulant and activator, has even improved the actual one.

Closing regulatory gaps: new ground rules for platelet-rich plasma

The Spanish Agency of Medicines and Medical Devices (AEMPS) has drawn up a comprehensive report and resolution that regulates for the first time the use of platelet-rich plasma (PRP) as a human-use medicinal product. This regulatory framework offers emerging challenges to adapt the use of PRP to the new requirements of safety and efficacy. The heterogeneity of the different products can hinder their regulation, which today differs substantially in the different worldwide regulatory frameworks.

Human-Based Biological and Biomimetic Autologous Therapies for Musculoskeletal Tissue Regeneration

Repairing and regenerating damaged musculoskeletal tissues is one of the greatest challenges in regenerative medicine. Blood contains the essential ingredients to biologically engineer drug delivery devices that provide spatiotemporal control over the availability of a wide range of autologous agents, including small molecules, cytokines, and growth factors. This opinion article summarizes our current knowledge of blood-derived biological drug delivery therapies. The potential mechanisms that control protein release are discussed, along with the biological rationale and effects of their use in different relevant musculoskeletal tissues, including articular cartilage, bone, tendon, muscle, and nerve tissue injuries. Finally, we finally describe the current challenges facing the field and recent advances that should drive novel solutions for the musculoskeletal system.

Preservation of Biological Activity of Plasma and Platelet-Derived Eye Drops After Their Different Time and Temperature Conditions of Storage.

Purpose: To analyze whether plasma rich in growth factors (PRGF) eye drops preserve their biological characteristics and activity after storage for 3 and 6 months at −20°C, at 4°C, and at room temperature for 72 hours, compared with fresh samples (t0). Methods: Blood from 6 healthy donors was harvested and centrifuged to obtain PRGF free of leukocytes. Resulting PRGF eye drops were stored for 3 and 6 months at −20°C. At each time, 2 aliquots were maintained at room temperature or at 4°C for 72 hours. Platelet-derived growth factor-AB, transforming growth factor-&bgr;1, vascular endothelial growth factor, epidermal growth factor, insulin-like growth factor-1, angiopoietin-1, and thrombospondin-1 were quantified at each time and temperature of storage. Also, the effect of PRGF eye drops on proliferation of primary human keratocytes was evaluated. Results: All the analyzed growth factor levels remained constant at each time and storage condition. No differences were observed in the proliferative activity of keratocytes after treatment with PRGF eye drops at any studied time or temperature. Finally, there was no microbial contamination in any of the PRGF eye drops. Conclusions: The preservation of the PRGF eye drops at −20°C for up to 3 and 6 months does not mean reduction of the main growth factors and proteins implicated in ocular surface wound healing. Eye drop characteristics and in vitro biological activity were not affected by their usage and conservation for 72 hours at 4°C or at room temperature.

Allogeneic Platelet-Rich Plasma: At the Dawn of an Off-the-Shelf Therapy?

A new therapeutic tool is emerging based on a biomimetic strategy: platelet-rich plasma (PRP) technology. Allogeneic off-the-shelf PRP represents a safe and cost-effective treatment for osteoarthritis and related diseases. This breakthrough opens the door not only to improved treatments and opportunities but also to new challenges with multiple uncertainties.

Platelet Rich Plasma (PRP) Biotechnology: Concepts and Therapeutic Applications in Orthopedics and Sports Medicine

Regenerative medicine is the augmentation or substitution of diseased or injured cells or tissues by one of two means: (1) an improvement in the ability of endogenous cells to reform damaged tissue or (2) the use of exogenous cells or tissues to replace damaged cells or tissues. Advances in regenerative medicine essentially depend on improving our understanding of cell biology and molecular signaling. Cell signaling is complex and incompletely understood due to the multiple interactions and cross-talk among system components. The human body has some 100 trillion cells, which in the healthy state coordinate their actions through an exchange of chemical signals to maintain body homeostasis. Every cell phenotype secretes signaling proteins that influence their own behavior (autocrine) or the behavior of other neighboring cells (paracrine) through interactions with specific transmembrane receptors located in the cellular membrane. Currently, a great deal of research is directed towards improving our understanding of intercellular communication and the intracellular transduction of these signals; in the field of regenerative medicine, this knowledge will help to disentangle the mysteries of tissue repair and to achieve proper tissue repair and regeneration. Moreover, to reach this goal we must integrate all the information and understanding derived from basic research into novel therapies that yield quicker and more efficient tissue regeneration.

Intradiscal and intra articular facet infiltrations with plasma rich in growth factors reduce pain in patients with chronic low back pain

Context: Low back pain (LBP) is a complex and disabling condition, and its treatment becomes a challenge. Aims: The aim of our study was to assess the clinical outcome of plasma rich in growth factors (PRGF Endoret) infiltrations (one intradiscal, one intra articular facet, and one transforaminal epidural injection) under fluoroscopic guidance control in patients with chronic LBP. PRGF Endoret which has been shown to be an efficient treatment to reduce joint pain. Settings and Design: The study was designed as an observational retrospective pilot study. Eighty six patients with a history of chronic LBP and degenerative disease of the lumbar spine who met inclusion and exclusion criteria were recruited between December 2010 and January 2012. Subjects and Methods: One intradiscal, one intra articular facet, and one transforaminal epidural injection of PRGF Endoret under fluoroscopic guidance control were carried out in 86 patients with chronic LBP in the operating theater setting. Statistical Analysis Used: Descriptive statistics were performed using absolute and relative frequency distributions for qualitative variables and mean values and standard deviations for quantitative variables. The nonparametric Friedman statistical test was used to determine the possible differences between baseline and different follow up time points on pain reduction after treatment. Results: Pain assessment was determined using a visual analog scale (VAS) at the first visit before (baseline) and after the procedure at 1, 3, and 6 months. The pain reduction after the PRGF Endoret injections showed a statistically significant drop from 8.4 ± 1.1 before the treatment to 4 ± 2.6, 1.7 ± 2.3, and 0.8 ± 1.7 at 1, 3, and 6 months after the treatment, respectively, with respect to all the time evaluations (P < 0.0001) except for the pain reduction between the 3rd and 6th month whose signification was lower (P < 0.05). The analysis of the VAS over time showed that at the end point of the study (6 months), 91% of patients showed an excellent score, 8.1% showed a moderate improvement, and 1.2% were in the inefficient score. Conclusions: Fluoroscopy guided infiltrations of intervertebral discs and facet joints with PRGF in patients with chronic LBP resulted in significant pain reduction assessed by VAS.