Treatment of severe pressure ulcers with protein-enriched filtered platelet-rich plasma (PEFPRP): a possible management.

Background: Biological dressings with non-transfusion blood components are among the treatments available for pressure ulcers (PUs). Biological dressings contain active concentrated pro-regenerative molecules that can modify and switch off local inflammatory pathways. This re-establishes the physiological homing, which results in healing. In our study, we used a biological component obtained by ultrafiltration of plasma-platelet concentrate: protein-enriched filtered platelet-rich plasma (PEFPRP) with a higher platelet and higher plasma protein concentration. We tested whether treatment with PEFPRP could improve healing in advanced-stage pressure ulcers with a large surface area. All the patients in this study had a surgical indication but were not able to undergo surgery for various reasons. Materials and methods: Ten patients with severe neurological disability and advanced-stage sacral pressure ulcers were treated with allogenic PEFPRP. The mean lesion surface area at T0 was 13.4 cm2 ( ± 9.8 SD). PEFPRP was derived from allogenic plasma-platelet apheresis that had been pre-ultrafiltered with a ProSmart™ filter (Medica, Italy) to obtain a concentration after filtration of the plasma protein (12-16 g/dL) and platelet (1-1.2 x 106 microL). Results and Conclusion: All cases showed a reduction in the surface area of the pressure ulcer and in the Pressure Ulcer Scale for Healing (PUSH) score. The mean reduction values at week 6 were as follows: -52% for surface area and -21% for PUSH. Rapid wound healing is fundamental to avoid infections and improve patients' quality of life. This blood component builds new tissue by creating a new extracellular matrix. This, in turn, promotes rapid restoration of the three-dimensional structure of the tissue necessary for healing deeper wounds. PEFPRP shrinks the PU and improves its morphological features (reducing undermining and boosting granulation tissue). PEFPRP also promotes tissue restoration, obtaining an optimal scar. It is a safe and feasible treatment, and these preliminary results support the use of PEFPRP in the treatment of pressure ulcers. PEFPRP dressings could be integrated in the standard treatment of advanced-stage PU.

Perinatal asphyxia partly affects presepsin urine levels in non-infected term infants.

OBJECTIVES: Standard of care sepsis biomarkers such as C-reactive protein (CRP) and procalcitonin (PCT) can be affected by several perinatal factors, among which perinatal asphyxia (PA) has a significant role. In this light, new early sepsis biomarkers such as presepsin (P-SEP) are needed to enact therapeutic strategies at a stage when clinical and laboratory patterns are still silent or unavailable. We aimed at investigating the potential effects of PA on longitudinal P-SEP urine levels. METHODS: We conducted an observational case-control study in 76 term infants, 38 with PA and 38 controls. Standard clinical, laboratory, radiological monitoring procedures and P-SEP urine measurement were performed at four time-points (first void, 24, 48, 96 h) after birth. RESULTS: Higher (p<0.05) CRP and PCT blood levels at T1-T3 were observed in PA than control infants whilst no differences (p>0.05, for all) at T0 were observed between groups. P-SEP urine levels were higher (p<0.05) in PA at first void and at 24 h while no differences (p>0.05) at 48 and 96 h were observed. No significant correlations were found (p>0.05) between P-SEP and urea (R=0.11) and creatinine (R=0.02) blood levels, respectively. CONCLUSIONS: The present results, showed that PA effects on P-SEP were limited up to the first 24 h following birth in absence of any kidney function bias. Data open the way to further investigations aimed at validating P-SEP assessment in non-invasive biological fluids as a reliable tool for early EOS and LOS detection in high-risk infants.

Endothelial response boosted by platelet lysate: the involvement of calcium toolkit.

Wound repair is a dynamic process during which crucial signaling pathways are regulated by growth factors and cytokines released by several kinds of cells directly involved in the healing process. However, the limited applications and heterogeneous clinical results of single growth factors in wound healing encouraged the use of a mixture of bioactive molecules such as platelet derivatives for best results in wound repair. An interesting platelet derivative, obtained from blood samples, is platelet lysate (PL), which has shown potential clinical application. PL is obtained from freezing and thawing of platelet-enriched blood samples. Intracellular calcium (Ca2+) signals play a central role in the control of endothelial cell survival, proliferation, motility, and differentiation. We investigated the role of Ca2+ signaling in the PL-driven endothelial healing process. In our experiments, the functional significance of Ca2+ signaling machinery was highlighted performing the scratch wound assay in presence of different inhibitors or specific RNAi. We also pointed out that the PL-induced generation of intracellular ROS (reactive oxygen species) via NOX4 (NADPH oxidase 4) is necessary for the activation of TRPM2 and the resulting Ca2+ entry from the extracellular space. This is the first report of the mechanism of wound repair in an endothelial cell model boosted by the PL-induced regulation of [Ca2+]i.

Platelet-rich plasma induces mixed osteogenic/osteoclastogenic phenotype in osteosarcoma SaOS-2 cells: role of TGF-beta.

Platelet-rich plasma (PRP) is widely used to promote tissue repair and accelerate osteogenesis, but there is no agreement about its mechanism of action. We characterized the modulatory effect of PRP on the in vitro osteoblast model SaOS-2, by using cell motility/chemoattraction and osteogenesis/mineralization assays, and a series of osteogenic/ osteoclastogenic genomic markers. Scratch wound assay showed that PRP stimulates cell motility, while transwell assay revealed a strong chemoattraction. Alkaline phosphatase (ALP) and alizarin red-S assays showed that PRP induces slight, but significant, stimulations of ALP activity and mineralization. The TGF-ß inhibitor SB431542 reversed these effects, showing a main role for TGF-ß1 released by PRP. Analyses of gene expression by qRT-PCR, showed the upregulation of osteocalcin, osteopontin, osteoprotegerin, receptor activator of NFκB (RANK), and runt-related transcription factor 2 (RUNX2) genes, with a total reversion by SB431542 for osteoprotegerin and RANK, and a partial reversion for ostecalcin, osteopontin, and RUNX2. The use of PCR array technique revealed the upregulation of the cathepsin K gene. These data show that PRP induces the development of mixed osteogenic/osteoclastogenic traits in the SaOS-2 model. Such a behavior may favour in vivo bone resorption and reconstitution at post-surgery or post-traumatic sites.

"Reasonable compromise" to define the quality standards of platelet concentrate for non-transfusion use (CPunT).

Platelets concentrate for non-transfusion use (CPunT) is a blood component specific for regenerative medicine. This blood component has found regenerative applications in many clinical fields (orthopedic, plastic surgery, maxillofacial surgery) since platelets contain growth factors, cytokines and bioactive molecules. Plasticity and ease of preparation of this blood component has often led the user to prepare it without using standardized procedures and references to quality product standards, but to evaluate the effectiveness of treatments and to standardize clinical protocols, is essential. The complexity of establish functional and non-functional parameters to define CPunT properties is linked to three fundamental steps: variability and bioavailability of biomolecules content in platelets, variability in product preparation. Then it is very difficult to understand which are the real parameters to evaluate, but it seems a "reasonable compromise" to establish content of platelets×ml (1×10(9)ml) as reference realistic parameter for CPunT qualification.

Platelet concentrates for topical use: bedside device and blood transfusion technology. Quality and versatility.

More or less after a decade of experimental and pioneering manual procedures to prepare platelet-rich plasma (PRP) for topical use, several portable and bedside devices were made available to prepare the PRP at the point-of-care. This technical opportunity increased the number of patients who got access to the treatment with autologous PRP and PRP-gel. Since topical treatment of tissue with PRP and PRP-gel was restricted to autologous preparation, blood transfusion centers that professionally prepare donor-derived platelet concentrates were not able to cover the overwhelming request for autologous PRP supply. Principally for logistic and organization reasons blood transfusion centers usually fail the challenge of prompt delivery of PRP to the physician over large territory. Nevertheless the blood bank production of platelet concentrates is associated with high standardization and quality controls not achievable from bedside and portable devices. Furthermore it easy to demonstrate that high-volume blood bank-produced platelet concentrates are less expensive than low-volume PRP produced by portable and bedside devices. Taking also in consideration the ever-increasing safety of the blood components, the relationship between bedside device-produced and blood-bank-produced PRP might be reconsidered. Here we discuss this topic concluding that the variety of sources of PRP production is an opportunity for versatility and that, ultimately, versatility is an opportunity for the patient's care.

Long-term effect of platelet lysate on primary fibroblasts highlighted with a proteomic approach.

The use of platelets and platelet derivatives has acquired clinical relevance as a means of accelerating wound healing. Platelet beneficial effect is attributed to the release of growth factors and other bioactive substances able to regulate cellular activities. The purpose of this study was to evaluate the biological effects of platelet lysate (PL) on human primary skin fibroblasts. We studied cell viability, MAPK signalling and proteomic profile of fibroblasts exposed to a platelet lysate (PL) obtained from blood sample. Crystal violet and neutral red uptake assays showed the dose-response effects of PL on cell viability and metabolism at 3 and 6 days of exposure. Western blot demonstrated a more sustained activation of p38 than of ERK1/2. A proteomic approach was applied to identify soluble cellular components in primary fibroblasts that are differentially expressed in response to PL exposure. Protein identification was performed by mass spectrometry. The data demonstrate that human fibroblasts respond to PL exposure by modifying a number of proteins, related principally to stress response, metabolism and the cytoskeleton.

Scratch wound closure of C2C12 mouse myoblasts is enhanced by human platelet lysate.

The effect of a platelet lysate (PL) on muscle wound healing, based on in vitro scratch wound of C2C12 mouse myoblasts, has been investigated. Cell viability assays show that PL induced an increase in cell proliferation at concentrations of 1-20%, but was slightly cytotoxic at 100%. PL promoted wound closure after scratch wounding of cell monolayers. The p38 inhibitor SB203580 and the PI3K inhibitor, wortmannin, decreased the PL effect, whereas the ERK inhibitor, PD98059, did not. Transwell migration of cells was also increased by PL, and although SB203580 abrogated this effect, wortmannin reduced it, whereas PD98059 was ineffective. Western blot analyses of scratch wounded cells showed activation of AKT and p38, while in the presence of PL there was a faster and sustained activation of AKT and p38 (up to 6h), and a transient activation of ERK1/2. Taken together, the data show that PL promotes C2C12 wound healing by enhancing cell proliferation and motility.