Aims: To compare levels and activity of the growth factors between fresh and lyophilized platelet-rich plasma (PRP). Methods: Analysis of platelet concentration using fibroblast and human umbilical vein endothelial cell cultures were compared between fresh and lyophilized PRP obtained from peripheral blood. Results: After lyophilization, 54% of platelets were intact whereas the fresh showed no aggregation with agonists (levels under 20%). The concentration of growth factors (VEGF, EGF, TGF-β and PDGF) in both products were similar. Fresh and lyophilized PRPs induced proliferation in the fibroblasts at 24 h (0.303 vs 0.300, respectively). Conclusion: Lyophilized PRP appears to be an alternative to fresh PRP and the results evidenced the role of growth factors as a key element in the activity of this product.

IntroductionPost-thrombotic syndrome (PTS) is a limiting long-term complication present in 20-50% of patients with deep venous thrombosis (DVT) of the lower limbs. A panel of biomarkers with potential relevance to enhance knowledge on the pathophysiology of PTS was investigated. MethodsThis case-control study included 93 patients with DVT in the lower limbs, 31 with severe PTS (cases) and 62 with mild/no PTS (controls), over 24 months after an acute episode. Thirty-one healthy individuals (HI) with no history of DVT were included as a reference to the population. FVIII activity, D-dimer, inflammatory cytokines, endothelial dysfunction markers, matrix metalloproteinases, and their inhibitors, tissue remodeling and growth factor levels were evaluated. The classification of PTS was, by the Villalta scale. ResultsPatients with severe PTS showed elevated levels of CRP, sICAM-1, sE-selectin, and decreased MMP-9 and MCP-1 levels when compared to patients with mild/no PTS. Moreover, DVT patients presented higher levels of FVIII and D-dimer when compared to HI. ConclusionsDVT patients present an inflammatory status, endothelial dysfunction and altered proteolysis MMPs activity, even a long time after the acute thrombotic episode, which is more significant in severe PTS. These results suggest a possible role of these mediators in the maintenance and worsening of PTS severity.

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Platelet-rich Plasma (PRP) has been widely used in different fields of medicine as autologous therapeutic product. The main component that appears to be associated with the therapeutic effect is the presence of growth factors (GF). However, many protocols available induce high methodology variability. In addition, is still unclear what is the best platelet activator and the necessity for the clinical practice. The traditional PRP used (Fresh PRP) can vary to each preparation and it has also been difficult to use in a time manager, especially for emergency care. Freeze-drying processes come out as a PRP standardization possibility, offering a low-risk proliferative microorganisms. This study aims to compare in vitrothe fresh PRP with the lyophilized PRP, in terms of platelet concentration capacity, and the GF potential release. For fresh and lyophilized PRP production, plasma from twenty-two male healthy individuals were obtained, with mean age of 28.6 ± 5.6 years. The blood was collected with ACD tubes (BD Vacutainer), than centrifuged twice: first with a spin at 300 g for 5 minutes and second with 700 g for 17 minutes. At the end of the double spin, the top layer plasma was characterized as platelet poor plasma and the lower layer was considered the PRP. The pellet were homogenized slowly, and adjusted to 1,2x106 platelets/µL before being frozen at -80ºC. For lyophilized PRP a stabilizing buffer were add and samples were frozen for 1 hour at -80ºC. After that, the PRP was lyophilized by Christ Alpha Plus for 20 hours. To compare fresh and lyophilized PRP, the platelets were evaluated for number of concentration, functionality, and the capacity of GF release, such as VEGF, PDGF, EGF and TGFβ. Non-parametric statistics were used in all analysis (Graph Pad 5.0). The PRP was able to recover high concentrations of platelets. The mean of platelet concentration was 1622 x 103 cells/µl, which represents 5.3 folds higher from the basal number (303 x 103 cells/µl). The recovery of platelets after freeze-drying was 54% compared to the initial concentration (1200 x 103 cells/µl). Platelet function was evaluated pre and post fresh PRP preparation and after freeze-drying, with two agonists ADP and epinephrine, to check the capacity of then to induce platelet aggregation. Results were evaluated trough amplitude of aggregation curve. Interestingly, high amplitude was observed only for samples from pre fresh PRP preparation (ADP median 86% from 71% to 100 % and epinephrine median 86 % from 74% to 103%). Despite the high concentration obtained from PRP (5 folds higher the basal number), no amplitude curve for platelet aggregation was observed for samples post fresh PRP preparation (ADP median 19% from 0% to 85% and epinephrine median 2% from 1% to 37%), even for lyophilized PRP (ADP median 1% from 0% to 2% and epinephrine median 1.5 % from 0 % to 3%), figure 1. The GF levels were similar for both products, with any grow factor loss after freeze-drying. The mean and standard deviation for level of GF were: PDGF 49365 pg/ml ± 17410 for fresh PRP and 60207 pg/ml ± 18472 for lyophilized PRP; VEGF 1250 pg/ml ±1171 for fresh PRP and 954,3 pg/ml ± 644,6 for lyophilized PRP; TGFβ 140373 pg/ml ± 91454 for fresh PRP and 111991 pg/ml ± 19827for lyophilized PRP; EGF 771,6 pg/ml ± 320,4 for fresh PRP and 739,1 pg/ml ± 324,1 for lyophilized PRP, figure 2. The results showed that fresh or lyophilized PRP were unable to show normal aggregation function, suggesting that these samples had been already activated by several conditions of preparation, such as the manual manipulation, temperature, pressure that the platelet is subjected inside the needle, among others. This result confirms that platelet activation with thrombin, or calcium chloride before PRP application is not crucial. Anyhow, the GF that is considered an important component for PRP regarding the therapeutic effect, were preserved. In addition, the lyophilized PRP appears as a possible replacement of fresh PRP, adding minor technical variability with a single process production, enabling a large-scale, with shelf life increased. Figure 1 Represents aggregation percentage generated after two antagonists addition. The results demonstrate that post-PRP has no platelet activation. Figure 2. Represents growth factor measurement in Fresh PRP and lyophilized PRP. The only difference between both preparations was PDGF (p=0, 0464). Figure 1. Represents aggregation percentage generated after two antagonists addition. The results demonstrate that post-PRP has no platelet activation. Figure 2. Represents growth factor measurement in Fresh PRP and lyophilized PRP. The only difference between both preparations was PDGF (p=0, 0464). Disclosures No relevant conflicts of interest to declare.

Background: Increasing prevalence of diabetes and obesity has registered a simultaneous increase in chronic wounds and defects in tissue repair. Recently PRP was described to be used in the treatment of chronic ulcers. PRP contains cytokines, chemokines and growth factors (GF) derived from platelets which induce molecular and cell mechanisms of natural wound healing. The possible use of GF derived from platelets, and the potential to prolong the shelf life of platelet concentrates, freeze-drying becomes one particularly suitable method to improve wound healing treatment. The use of lyophilized PRP appears to be interesting for greater convenience: only one blood collection, after hydration its soon ready for use, and also due to the highest concentration of bioactive molecules. This study aims to compare intra-lesional freeze-dried and fresh PRP for treatment of acute wound in a pre-clinic model. Methods: Fresh and freeze-dried PRP was prepared from two human platelet concentrate bags using double spin method. PRP characterization included platelet number and quantification of PDGF-AA, EGF, VEGF and TGF-β1 by Luminex (Millipore). Animal wound model was performed after shaving the dorsum of the rat, and a full-thickness excisional wound (1 cm2) was made to the level of the panniculus carnosus muscle. Thirty animals were divided according to the type o treatment in 3 groups: Fresh PRP (n=10), Freeze-dried PRP (FD-PRP) (n=10) and control saline (n=10). The animals received one perilesional application of 500 µL in the day of wound induction. The monitoring of wound closure was made through macroscopical analysis of wound size in days 3, 7 and 10 after wounding. Animals were sacrificed in the tenth day and the skin was removed for histology with hematoxilin-eosin and Masson´s Trichrome, and immunohistochemistry with α-actin smooth muscle for myofibroblasts and blood vessels, and quantified through image J. Results: The platelet number of PRP was 5714 x 103 cells / µL, presenting a high platelet recovery. The FD-PRP presented higher level of all GF when compared to fresh PRP from 1.64 to 3.72 folds. The application of PRP or FD-PRP did not induce significantly changes in wound healing kinetics compared to control during all evaluated days. The mean and standard deviation (SD) of area in D3 was 112.9 ± 16.6%, 107.9 ± 21.3 and 100.8 ± 30.2 for control, PRP and FD-PRP, respectively. In D7 it was 54.3 ± 28.9, 63.9 ± 22.8 and 61.6 ± 20.6 for control, PRP and FD-PRP, respectively. In D10 it was 9.9 ± 6.1, 13.9 ± 11.2, 7.7 ± 6.0 for control, PRP and FD-PRP, respectively. In immunohistochemistry of deep epidermis FD-PRP presented a significantly higher concentration of myofibroblasts in comparison with fresh PRP (16.61 ± 9.04 vs. 13.99 ± 14.07, p=0.0095). A significantly higher number of blood vessels was observed in the group treated with FD-PRP (in percentage of area) in comparison to the controls, both in superficial and deep regions of epidermis (0.43 ± 0.5 vs. 0.21 ± 0.22, P=0.01; 0.38 ± 0.44 vs. 0.24 ± 0.25, P=0.03, respectively). Discussion and conclusion: The most interesting result of this study was the increased number of blood vessels. VEGF plays a central role in promoting angiogenesis during wound repair. Previous studies demonstrated accelerated wound closure with collagen-binding VEGF topical treatment. However, the use of one growth factor has limited success and heterogeneity of clinical results, probably due to the need of multiple bioactive molecules that are necessary in the cascade of complexes events of healing. The use of multiples factors necessary to healing can orchestrated the physiological events resulting in chemotaxis, proliferation and differentiation of cells and the angiogenic process. The use of FD-PRP in this study of acute model of wounds showed positive results, especially in angiogenesis. The practicality and the improve in the shelf life of PRP have a great value in clinical practice and becomes very attractive, showing low cost, the possibility to increase the concentration of GF, various samples ready-to-use and a single blood collection. The investigation of FD-PRP in other clinical situations in which fresh PRP have been used is an interesting approach to be to evaluated to determine its effectiveness. Figure Quantification of blood vessels through imunohistochemistry using smooth α-actin and treated by Image J software; A) Superficial epidermis; B) Deep epidermis. Figure. Quantification of blood vessels through imunohistochemistry using smooth α-actin and treated by Image J software; A) Superficial epidermis; B) Deep epidermis. Disclosures No relevant conflicts of interest to declare.