Early changes in S100B maternal blood levels can predict fetal intrauterine growth restriction.

OBJECTIVES: Intrauterine growth restriction (IUGR) represents one of the main causes of perinatal mortality and morbidity. Nowadays, IUGR early diagnosis is mandatory in order to limit the occurrence of multiorgan failure, especially the brain. Therefore, we investigated whether longitudinal S100B assessment in maternal blood could be a trustable predictor of IUGR. METHODS: We conducted a prospective study in 480 pregnancies (IUGR: n=40; small for gestational age, SGA: n=40; controls: n=400) in whom S100B was measured at three predetermined monitoring time-points (T1: 8-18 GA; T2: 19-23 GA; T3: 24-28 GA). RESULTS: Lower S100B in IUGR fetuses than SGA and controls (p<0.05, for all) at T1-T3. Receiver operating characteristic curve showed that S100B at T1 was the best predictor of IUGR (sensitivity: 100 %; specificity: 81.4 %) than T2, T3. CONCLUSIONS: The early lower S100B concentration in pregnant women lately complicated by IUGR support the notion that non-invasive early IUGR diagnosis and monitoring is becoming feasible. Results open the way to further studies aimed at diagnosing and monitoring fetal/maternal diseases at earliest time.

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.

S100B Maternal Blood Levels in Gestational Diabetes Mellitus Are Birthweight, Gender and Delivery Mode Dependent.

Gestational Diabetes Mellitus (GDM) is one of the main causes of perinatal mortality/morbidity. Today, a parameter offering useful information on fetal central nervous system (CNS) development/damage is eagerly awaited. We investigated the role of brain-protein S100B in the maternal blood of GDM pregnancies by means of a prospective case-control study in 646 pregnancies (GDM: n = 106; controls: n = 530). Maternal blood samples for S100B measurement were collected at four monitoring time-points from 24 weeks of gestation to term. Data was corrected for gender and delivery mode and correlated with gestational age and weight at birth. Results showed higher (p < 0.05) S100B from 24 to 32 weeks and at term in GDM fetuses than controls. Higher (p < 0.05) S100B was observed in GDM male new-borns than in females from 24 to 32 weeks and at term, in GDM cases delivering vaginally than by caesarean section. Finally, S100B positively correlated with gestational age and weight at birth (R = 0.27; R = 0.37, respectively; p < 0.01). The present findings show the usefulness of S100B in CNS to monitor high-risk pregnancies during perinatal standard-of-care procedures. The results suggest that further investigations into its potential role as an early marker of CNS growth/damage in GDM population are needed.

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.

PLA-grafting of collagen chains leading to a biomaterial with mechanical performances useful in tendon regeneration.

With the aim to obtain a scaffold with improved mechanical properties with respect to collagen for tendon augmentation and regeneration, a novel collagen-based material was prepared via heterogeneous phase derivatization of type I collagen sponges using polylactic acid. Compared to the untreated collagen, the functionalized sponge (Coll-PLA) was characterized by higher tensile properties and lower swelling capability; the degradation rate of Coll-PLA, in the presence of collagenase, was lower than that of the untreated collagen sponge. These results are related to an increased hydrophobic character of the collagen matrix due to the presence of PLA chains. In vitro tests, performed with human primary fibroblasts, showed that cell adhesion and proliferation rate on Coll-PLA were comparable to those obtained with the non-functionalized collagen. These findings suggest that the new biomaterial could be suitable as scaffold in tendon augmentation and regeneration.

A Single-Center Pilot Prospective Study of Topical Application of Platelet-Derived Eye Drops for Patients with Ocular Chronic Graft-versus-Host Disease.

Ocular involvement of chronic graft-versus-host disease (cGVHD) is a complication that occurs in up to 60% of patients after allogeneic hematopoietic stem cell transplantation. Conventional therapeutic options include medical and surgical procedures that are administered depending on the severity of the condition, but most of them have provided unsatisfactory results and, to date, there is no consensus about treatment. We considered that topical application of a platelet lysate, administered as eye drops, might be considered an alternative worthwhile of investigation to treat ocular surface disorders in patients suffering from cGVHD. Therefore, we conducted a single-center prospective pilot study to assess the efficacy and safety of using eye drops made from reconstituted lysed platelet concentrate. Twenty-six patients with ocular cGVHD were eligible for the study; all but 2 completed their scheduled 1-year treatment and complied with the hematologic and ophthalmic regimen. At their first assessment interviews, after 30 days of treatment, 91% of patients reported an improvement in their symptoms and for 32%, substantive objective differences were measured. Remission of corneal damage was seen for 86% of our cohort, and improved National Institutes of Health scores for 73%, of whom 8% achieved the best score of 0 (ie, non-dry eye). Similar results were seen at later time points. Comparing outcomes for our patient cohort to those determined retrospectively for patients in our institutional database revealed a 5-year overall survival (OS) of 65%. This OS is comparable to patients with limited cGVHD (75%) and is superior to that of patients with nonocular extensive cGVHD or without cGVHD (30% and 59%, respectively) (P = .013). Our results suggest that platelet-derived eye drops are a safe, practical, and well-tolerated therapeutic option that offers substantial benefits for most patients affected by ocular cGVHD at onset. The favorable OS of our patient cohort suggests that this topical therapy, rather than systemic immunosuppression, may be the treatment of choice.

Non-Hematopoietic Essential Functions of Bone Marrow Cells: A Review of Scientific and Clinical Literature and Rationale for Treating Bone Defects.

Hematopoiesis as the only essential function of bone marrow cells has been challenged for several decades through basic science (in vitro and in vivo) and clinical data. Such work has shed light on two other essential functions of bone marrow cells: osteopoiesis and angio-genesis/vasculogenesis. Clinical utility of autologous concentrated bone marrow aspirate (CBMA) has demonstrated both safety and efficacy in treating bone defects. Moreover, CBMA has been shown to be comparable to the gold standard of iliac crest bone graft (ICBG), or autograft, with regard to being osteogenic and osteoinductive. ICBG is not considered an advanced therapy medicinal product (ATMP), but CBMA may become regulated as an ATMP. The European Medicines Agency Committee for Advanced Therapies (EMA:CAT) has issued a reflection paper (20 June 2014) in which reversal of the 2013 ruling that CBMA is a non-ATMP has been proposed. We review bone marrow cell involvement in osteopoiesis and angiogenesis/vasculogenesis to examine EMA:CAT 2013 decision to use CBMA for treatment of osteonecrosis (e.g, of the femoral head) should be considered a non-ATMP. This paper is intended to provide discussion on the 20 June 2014 reflection paper by reviewing two non-hematopoietic essential functions of bone marrow cells. Additionally, we provide clinical and scientific rationale for treating osteonecrosis with CBMA.

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.

Platelet lysate modulates MMP-2 and MMP-9 expression, matrix deposition and cell-to-matrix adhesion in keratinocytes and fibroblasts.

Cell-matrix interactions are an essential element of wound healing, while platelet derivatives are used in clinical settings for the treatment of chronic wounds. We used a platelet lysate (PL), which had been previously shown to accelerate in vitro the wounding of HaCaT keratinocytes and fibroblasts (J Cell Mol Med, 13, 2009, 2030; Br J Dermatol, 159, 2008, 537), to study the modulation of MMP-2 and MMP-9 collagenase expression, collagen type I and III production and syndecan-4 expression and rearrangement in these cells. Zymography and Western blot analyses showed that exposure to 20% (v/v) PL for 24 h induced an apparently ERK1/2- and p38-dependent, NF-kappaB-independent, translational upregulation of MMP-9 in HaCaT, while HaCaT MMP-2 and fibroblast collagenases were almost unaffected. The use of in-cell ELISA showed that PL induced an increase in the collagen III production of fibroblasts. In-cell ELISA and immunofluorescence microscopy revealed an increase in the expression of syndecan-4 and its rearrangement to form focal adhesions in both cell types after PL exposure. Taken together, data indicate that PL promotes keratinocyte epithelialization and regulates fibroblast matrix deposition, thus providing a molecular basis for the ability of this platelet derivative to heal severe and problematic wounds without leading to heavy scarring and keloid formation.

One-Step Cartilage Repair with Bone Marrow Aspirate Concentrated Cells and Collagen Matrix in Full-Thickness Knee Cartilage Lesions: Results at 2-Year Follow-up.

OBJECTIVE: The purpose of our study was to determine the effectiveness of cartilage repair utilizing 1-step surgery with bone marrow aspirate concentrate (BMAC) and a collagen I/III matrix (Chondro-Gide, Geistlich, Wolhusen, Switzerland). MATERIALS AND METHODS: We prospectively followed up for 2 years 15 patients (mean age, 48 years) who were operated for grade IV cartilage lesions of the knee. Six of the patients had multiple chondral lesions; the average size of the lesions was 9.2 cm(2). All patients underwent a mini-arthrotomy and concomitant transplantation with BMAC covered with the collagen matrix. Coexisting pathologies were treated before or during the same surgery. X-rays and MRI were collected preoperatively and at 1 and 2 years' follow-up. Visual analog scale (VAS), International Knee Documentation Committee (IKDC), Knee injury and Osteoarthritis Outcome Score (KOOS), Lysholm, Marx, SF-36 (physical/mental), and Tegner scores were collected preoperatively and at 6, 12, and 24 months' follow-up. Four patients gave their consent for second-look arthroscopy and 3 of them for a concomitant biopsy. RESULTS: Patients showed significant improvement in all scores at final follow-up (P < 0.005). Patients presenting single lesions and patients with small lesions showed higher improvement. MRI showed coverage of the lesion with hyaline-like tissue in all patients in accordance with clinical results. Hyaline-like histological findings were also reported for all the specimens analyzed. No adverse reactions or postoperative complications were noted. CONCLUSION: This study showed that 1-step surgery with BMAC and collagen I/III matrix could be a viable technique in the treatment of grade IV knee chondral lesions.

Platelet-derived factors involved in tissue repair-from signal to function.

Topical treatment with platelet derivatives has increasingly been described as being capable of accelerating wound healing and to aid in tissue repair. In vitro data indicate that platelets and their contents have chemotactic, migration-inducing, and mitogenic activities, and a major role of these factors in tissue repair has thus been advocated. However, how platelet-derived factors orchestrate tissue repair at the cellular level remains quite obscure even to those individuals who prescribe platelet derivatives as topical wound healing therapy. The primary objective of this review was to provide the practitioner, inexpert in biochemistry, an overview about signal transduction within cells in response to platelet-derived factors. Concepts from the literature were selected to illustrate how a relatively few units of information can be put together in specific order to allow for complex biologic functions to be elicited. To illustrate how functional complexity emerges from a narrow set of messengers, an analogy between signal transduction and language, or contrapunctual music, has been drawn.

Role of ERK1/2 in platelet lysate-driven endothelial cell repair.

Mechanisms of endothelial repair induced by a platelet lysate (PL) were studied on human (HuVEC, HMVEC-c) and non-human (PAOEC, bEnd5) endothelial cells. A first set of analyses on these cells showed that 20% (v/v) PL promotes scratch wound healing, with a maximum effect on HuVEC. Further analyses made on HuVEC showed that the ERK inhibitor PD98059 maximally inhibited the PL-induced endothelial repair, followed in order of importance by the calcium chelator BAPTA-AM, the PI3K inhibitor wortmannin and the p38 inhibitor SB203580. The PL exerted a chemotactic effect on HuVEC, which was abolished by all the above inhibitors, and induced a PD98059-sensitive increase of cell proliferation rate. Confocal calcium imaging of fluo-3-loaded HuVEC showed that PL was able to induce cytosolic free Ca(2+) oscillations, visible also in Ca(2+)-free medium, suggesting an involvement of Ins3P-dependent Ca(2+) release. Western blot analysis on scratch wounded HuVEC showed that PL induced no activation of p38, a transient activation of AKT, and a sustained activation of ERK1/2. The complex of data indicates that, although different signalling pathways are involved in PL-promoted endothelial repair, the process is chiefly under the control of ERK1/2.

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.

Platelet lysate promotes in vitro wound scratch closure of human dermal fibroblasts: different roles of cell calcium, P38, ERK and PI3K/AKT.

There is a growing interest for the clinical use of platelet derivates in wound dressing. Platelet beneficial effect is attributed to the release of growth factors and other bioactive substances, though mechanisms are mostly unknown. We studied wound-healing processes of human primary fibroblasts, by exposing cells to a platelet lysate (PL) obtained from blood samples. Crystal violet and tetrazolium salt (MTS) assays showed dose-response increase of cell proliferation and metabolism. In scratch wound and transwell assays, a dose of 20% PL induced a significant increase of wound closure rate at 6 and 24 hrs, and had a strong chemotactic effect. BAPTA-AM, SB203580 and PD98059 caused 100% inhibition of PL effects, whereas wortmannin reduced to about one third the effect of PL on wound healing and abolished the chemotactic response. Confocal imaging showed the induction by PL of serial Ca2(+) oscillations in fibroblasts. Data indicate that cell Ca2(+) plays a fundamental role in wound healing even without PL, p38 and ERK1/2 are essential for PL effects but are also activated by wounding per se, PI3K is essential for PL effects and its downstream effector Akt is activated only in the presence of PL. In conclusion, PL stimulates fibroblast wound healing through the activation of cell proliferation and motility with different patterns of involvement of different signalling pathways.

Tissue regeneration and in loco administration of platelet derivatives: clinical outcome, heterogeneous products, and heterogeneity of the effector mechanisms.

BACKGROUND: In loco administration of platelet (PLT) derivatives is a relatively new auxiliary treatment for tissue regeneration to be hastened. Enthusiastic reports are faced by more critical ones. The more obvious rationale for the in vivo administration of PLT derivatives resides in their growth factor content. STUDY DESIGN AND METHODS: The relevant literature was systematically reviewed. Close scrutiny of the technical details was carried out to find out the procedural differences accounting for conflicting results. RESULTS: An impressively vast heterogeneity of conduct was found in both in vitro and in vivo studies. Major outcome-affecting variables were recognized such as those associated with PLT preparation; growth factor measurement; proliferation test; dose, timing, and administration of the PLT derivatives; study design; and primary endpoints. CONCLUSIONS: So many variables were found making standardization or confrontation of the in vitro and the in vivo studies barely conceivable or manageable. The mechanisms of action are very complex. The attribution of tissue regeneration capacity of PLT derivatives solely to the PLT-derived growth factors is simplistic. The results obtained through in vitro experiments are indicative for general mechanisms. Their simplistic hold to the complex in vivo environment may be misleading.