Plasma Microparticles Have Different Effects on Thrombin Generation in Platelet-Rich and Platelet-Poor Plasma.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1760-1760
Author(s):  
Gleb E. Ivanov ◽  
N. Macartney ◽  
E. Stephens ◽  
N. Bowen ◽  
S. Lees ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Platelet Rich Plasma ◽  
Maximal Velocity ◽  
Physiological Level ◽  
Wide Range ◽  
Coagulation Tests ◽  
Platelet Concentration ◽  
Vitro Effect ◽  
Free Plasma

Abstract Circulating peripheral blood microparticles (MPs) of various cell origin have been described and measured in physiological and a wide range of pathological conditions. MPs are likely to play a role in coagulation either by exposure of procoagulant phospholipids or expression of tissue factor (TF), but the degree of this contribution to global haemostasis is not yet clear. We studied thrombin generation (TG) parameters (lag, peak thrombin, initial velocity (Vini) and maximal velocity (Vmax) in platelet-free (PFP) and platelet-rich plasma (PRP) of normal volunteers (n=9) in presence of corn trypsin inhibitor, using calibrated automated thrombography (CAT). MP-rich plasma was prepared by ultracentrifugation of PFP and reconstitution of pelleted MPs in a reduced volume of autologous MP-free plasma. TG was also measured in MP-depleted supernatant and platelet-free plasma (PFP) filtered through 0.1 μm filter. In MP-rich plasma, triggered with 5pM TF, with no addition of exogenous phospholipids, we found significantly increased peak TG, compared with PFP and supernatant (70.8 +/− 6.3 vs 51.4 +/− 5.0 vs 28.4 +/− 2.2 nM/L thrombin, p=0.024 and p<0.0001 respectively). MP-rich fraction also produced raised Vini (10.3 +/− 0.9 vs 5.0 +/− 0.6 thrombin nM/L/min, p=0.019) and Vmax (18.3 +/− 2.4 vs 6.8 +/− 1.0 thrombin nM/L/min, p=0.004) compared with MP-depleted supernatant. Ultracentrifugation resulted in reduction of peak TG almost by half, compared with native PFP. The augmenting effect of MP-rich plasma on thrombin peak and velocity was shown to be abolished by filtration. In our experiments removal of MPs by filtration of PFP did not affect routine clinical coagulation tests, but resulted in a significant reduction of peak TG (from 51.4 +/− 5.0 to 23.9 +/− 1.4 thrombin nM, p=0.0002), Vini (from 10.2 +/− 0.4 to 5.6 +/− 0.6, p=0.02) and Vmax (from 15.2 +/− 1.8 to 5.9 +/− 0.2, p=0.02) as compared to PFP. In order to assess the contribution of MPs to TG in presence of platelets, MP-rich plasma was added to various dilutions of PRP, using low concentration of TF (0.5pM) as a trigger. Interestingly, addition of MP-rich fraction only marginally augmented PRP with a platelet concentration of 150x109/L, but the enhancement of peak and velocity of TG became more pronounced when platelet concentration was reduced to 1.5x109/L. In a separate set of experiments, we studied TG in PRP in which MP concentration was reduced by dilution with filtered MP-free plasma as compared to PRP diluted with MP-containing PFP. Reduction in PRP MP content did not lead to a significant decrease in TG even at a low platelet concentration (1.5x109/L), when MP concentration was reduced to about 100 times below the physiological level. Our results indicate that MPs contained in PFP of normal donors significantly affect thrombin generation peak and velocity when compared to PFP in which MPs were eliminated by either ultracentifugation or filtration. The in vitro effect of an increased number of MPs on TG is less noticeable in presence of near-physiological platelet count, but contribution of MPs to TG at low platelet concentrations may potentially protect from bleeding in thrombocytopenic states and explain differences in bleeding phenotype. CAT measurement of TG in MP-rich vs MP-poor plasma could serve as a useful tool in assessing these differences.

In Vitro Effect of Danaparoid Sodium (Orgaran®) on Thrombin Generation after Minimal Tissue Factor Pathway Activation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4151-4151
Author(s):  
Ismail Elalamy ◽  
Anna D. Petropoulou ◽  
Mohamed Hatmi ◽  
Meyer M. Samama ◽  
Grigoris T. Gerotziafas
Keyword(s):  
Molecular Weight ◽  
Tissue Factor ◽  
Thrombin Generation ◽  
Lag Time ◽  
Dependent Manner ◽  
Pathway Activation ◽  
Coagulation Tests ◽  
Vitro Effect ◽  
Routine Coagulation

Abstract Introduction: Orgaran® (Org 10172) is a low molecular weight heparinoid which consists of natural sulphated glycosaminoglycans (heparan, dermatan, chondroitin sulphate). It has a mean molecular weight of approximately 6 kDa (4–10 kDa), an excellent bioavailability following subcutaneous administration and an anti-Xa/anti-IIa activity ratio superior to 22. It is the anticoagulant of choice in patients developping Heparin-Induced Thrombocytopenia (HIT), whereas its’ use is also proposed for surgical thromboprophylaxis. Orgaran® has no effect on routine coagulation tests (aPTT, PT, TT). Thrombin generation test(TG) is a global clotting assay proven to be sensitive to the anticoagulant effect of LMWHs and specific FXa inhibitors (i.e. fondaparinux and BAY-597939). In this in vitro study, we determined the tissue factor (TF)-induced TG inhibition potency of Orgaran® using the Thrombogram-Thrombinoscope® assay. Materials and Methods: TG was assessed after TF pathway activation in Platelet Rich Plasma (PRP) (1.5x105 platelets/μl) using diluted thromboplastin (Dade Innovin®, 1:1000 final dilution). The clotting process is provoked by a physiologically relevant TF concentration. Orgaran® was added to control plasma from 8 healthy volunteers at five different final concentrations (0.2, 0.4, 0.6, 0.8 and 1IU anti-Xa/ml). TG was initiated by adding the triggering solution containing CaCl2 and the fluorogenic substrate. The analyzed TG parameters are the lag time, the maximal concentration of thrombin (Cmax), the time to reach Cmax (Tmax), the TG velocity and the endogenous thrombin potential (ETP). Results: Orgaran® prolonged significantly the lag time and the Tmax at a concentration over 0.40 IU anti-Xa/ml (p<0.05). At the lowest studied concentration (0.20 IU anti-Xa/ml), lag time and Tmax were only prolonged by 12%, whereas their maximal prolongation (around 50%) was observed at 1IU anti-Xa/ml. Furthermore, Orgaran® inhibited ETP, Cmax and TG velocity in an almost linear dose dependent manner. A significant inhibition of ETP, Cmax and TG velocity was obtained at concentrations superior to 0.20 IU anti-Xa/ml. (p<0.05). At the highest studied concentration (1IU anti-Xa/ml) Orgaran® suppressed all TG parameters by about 80% (Table 1). Conclusion: Orgaran® exhibited a significant inhibitory activity of in vitro TG. At concentrations achieved in clinical practice (prophylactic or therapeutic dose), Orgaran® modified in vitro TG profile while it has no effect on routine coagulation tests. Thus, TG assay is a sensitive method for monitoring Orgaran® and this test requires a clinical prospective evaluation. Table 1. Determination of IC20 and IC50 anti-Xa inhibitory concentrations of Orgaran® on TG parameters Lag Time Tmax ETP Cmax Velocity IC: Inhibitory Concentration * or Concentration increasing 20% and 50% the lag time and the Tmax respectively IC 20 (IU/ml) 0.30 0.30 0.18 0.18 0.15 IC 50 (IU/ml) 0.83 >1 0.30 0.50 0.35 1IU anti-Xa/ml 53% 47% 68% 76% 84%

Effects of argatroban, danaparoid, and fondaparinux on trombin generation in heparin-induced thrombocytopenia

2013 ◽  
Vol 109 (03) ◽  
pp. 504-509 ◽  
Author(s):  
Marion Combe ◽  
Michèle Piot ◽  
Céline Chapelle ◽  
Majid Akrour ◽  
Bernard Tardy ◽  
...  
Keyword(s):  
Clinical Studies ◽  
Thrombin Generation ◽  
Platelet Rich Plasma ◽  
Study Objective ◽  
Heparin Induced Thrombocytopenia ◽  
Thrombin Generation Assay ◽  
Vitro Effect ◽  
Vitro Data ◽  
In Vitro Data

SummaryThere is no in vitro data on the comparison of the effects of danaparoid, argatroban and fondaparinux on thrombin generation in patients with heparin-induced thrombocytopenia. It was the study objective to compare the in vitro anticoagulant potential of argatroban, danaparoid and fondaparinux using a thrombin generation assay TGA on a mixture of control platelet-rich plasma (PRP) and HIT patient platelet-poor plasma (PPP). The plasma of seven patients with a clear HIT diagnosed at our institution was selected. Mixtures of donor PRP and patient PPP were incubated with unfractionated heparin 0.2 U.mL-1, argatroban at 600 ng.mL-1, argatroban at 400 ng.mL-1, danaparoid at 0.65 IU.mL-1 and fondaparinux at 1 [uni03BC]g.mL-1. Thrombin generation was assessed by calibrated thrombinography. The percentage of inhibition of the endogenous thrombin potential observed with argatroban at 600 ng.mL-1 was statistically significantly higher compared with those observed with fondaparinux (median: 53.6% vs. 3.9%; p = 0.031) but not compared with argatroban at 400 ng.mL-1 and danaparoid. The percentage of inhibition of the thrombin peak observed with argatroban at 600 ng.mL-1 was statistically significantly higher compared with those observed with danaparoid (median: 71.2 vs. 56.8; p = 0.031) and fondaparinux (mean: 71.2 vs. 30; p = 0.031) but not with argatroban at 400 ng.mL-1. In conclusion, the in vitro effect of argatroban and danaparoid on thrombin generation seems to corroborate the results of clinical studies of these drugs in the treatment of HIT in term of efficiency. Fondaparinux showed a very small effect on thrombin generation evaluated by calibrated thrombinography.

Anti-Inflammatory Effects of Novel Standardized Platelet Rich Plasma Releasates on Knee Osteoarthritic Chondrocytes and Cartilage in vitro

2019 ◽  
Vol 20 (11) ◽  
pp. 920-933 ◽  
Author(s):  
Lucía Gato-Calvo ◽  
Tamara Hermida-Gómez ◽  
Cristina R. Romero ◽  
Elena F. Burguera ◽  
Francisco J. Blanco
Keyword(s):  
Gene Expression ◽  
Ex Vivo ◽  
Platelet Rich Plasma ◽  
Cartilage Explants ◽  
Ex Vivo Model ◽  
Chondrocyte Viability ◽  
Platelet Concentration ◽  
The Absolute ◽  
Anti Inflammatory

Background: Platelet Rich Plasma (PRP) has recently emerged as a potential treatment for osteoarthritis (OA), but composition heterogeneity hampers comparison among studies, with the result that definite conclusions on its efficacy have not been reached. Objective: 1) To develop a novel methodology to prepare a series of standardized PRP releasates (PRP-Rs) with known absolute platelet concentrations, and 2) To evaluate the influence of this standardization parameter on the anti-inflammatory properties of these PRP-Rs in an in vitro and an ex vivo model of OA. Methods: A series of PRPs was prepared using the absolute platelet concentration as the standardization parameter. Doses of platelets ranged from 0% (platelet poor plasma, PPP) to 1.5·105 platelets/µl. PRPs were then activated with CaCl2 to obtain releasates (PRP-R). Chondrocytes were stimulated with 10% of each PRP-R in serum-free culture medium for 72 h to assess proliferation and viability. Cells were co-stimulated with interleukin (IL)-1β (5 ng/ml) and 10% of each PRP-R for 48 h to determine the effects on gene expression, secretion and intra-cellular content of common markers associated with inflammation, catabolism and oxidative stress in OA. OA cartilage explants were co-stimulated with IL-1β (5 ng/ml) and 10% of either PRP-R with 0.75·105 platelets/µl or PRP-R with 1.5·105 platelets/µl for 21 days to assess matrix inflammatory degradation. Results: Chondrocyte viability was not affected, and proliferation was dose-dependently increased. The gene expression of all pro-inflammatory mediators was significantly and dose-independently reduced, except for that of IL-1β and IL-8. Immunoblotting corroborated this effect for inducible NO synthase (NOS2). Secreted matrix metalloproteinase-13 (MMP-13) was reduced to almost basal levels by the PRP-R from PPP. Increasing platelet dosage led to progressive loss to this anti-catabolic ability. Safranin O and toluidine blue stains supported the beneficial effect of low platelet dosage on cartilage matrix preservation. Conclusion: We have developed a methodology to prepare PRP releasates using the absolute platelet concentration as the standardization parameter. Using this approach, the composition of the resulting PRP derived product is independent of the donor initial basal platelet count, thereby allowing the evaluation of its effects objectively and reproducibly. In our OA models, PRP-Rs showed antiinflammatory, anti-oxidant and anti-catabolic properties. Platelet enrichment could favor chondrocyte proliferation but is not necessary for the above effects and could even be counter-productive.

scholarly journals The in Vitro Effect on Thrombin Generation of Adding rFVIII or rFIX to Hemophilia a or b Plasma in the Absence or Presence of Concizumab

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 22-23
Author(s):  
Marianne Kjalke ◽  
Søren Andersen
Keyword(s):  
Drug Interaction ◽  
Thrombin Generation ◽  
Hemophilia A ◽  
Plasma Samples ◽  
Publicly Traded ◽  
Plasma Pool ◽  
Generation Capacity ◽  
Drug Drug Interaction ◽  
Vitro Effect

Introduction: Lack of factor VIII/IX (FVIII/FIX) in hemophilia A/B (HA/HB), respectively, results in reduced thrombin generation, leading to recurrent/spontaneous bleeds. Concizumab is an anti-tissue factor pathway inhibitor (TFPI) monoclonal antibody, currently under clinical investigation for subcutaneous prophylaxis of HA/HB patients with/without inhibitors. Breakthrough bleeds occurring in HA/HB patients while on concizumab prophylaxis may be treated with FVIII/FIX. We aimed to compare the in vitro effect of recombinant FVIII (rFVIII) and FIX (rFIX) in HA and HB plasma, respectively, in the absence or presence of concizumab. Methods: rFVIII/rFIX was added to HA/HB pooled plasma at 0.25, 0.5 or 1 IU/mL (corresponding to post-administration plasma concentrations of 12.5, 25 and 50 IU/kg rFVIII and 12.5−25, 25−50 and 50−100 IU/kg rFIX) in the absence or presence of concizumab (1,500, 4,500 or 15,000 ng/mL). In a separate experiment, 33 plasma samples from eight HA patients, who were on concizumab prophylaxis as part of the phase 2 explorer5 trial (NCT03196297), were spiked with 0.5, 1 and 1.5 IU/mL rFVIII. Pre-dose samples (before concizumab prophylaxis) from seven of these patients were also included. Thrombin generation was measured after initiation with 1 pM tissue factor (PPP-Low, Thrombinoscope). Statistical analysis of the effects conferred by each (combination of) drug(s) was performed by ANOVA analyses. Results: A significant (p&lt;0.001) and concentration-dependent increase in thrombin peak was observed when HA plasma pool samples were spiked with rFVIII, both in the absence and presence of concizumab. Likewise, concizumab increased the thrombin peak both in the absence and in presence of rFVIII. Increasing concizumab from 1,500 to 4,500 and 15,000 ng/mL only slightly increased the thrombin peak further, demonstrating that a close-to-maximal effect on thrombin peak was achieved at 1,500 ng/mL concizumab. The effects of concizumab and rFVIII were mainly additive with an up to 20% additional effect caused by drug-drug interaction. The addition of rFVIII to explorer5 patient plasma samples resulted in a significant and concentration-dependent increase in thrombin peak. The effects observed for rFVIII and concizumab were exclusively additive. The thrombin peak obtained with 1.0 IU/mL rFVIII before concizumab administration was lower than with 0.5 IU/mL rFVIII in the presence of concizumab. This suggests that a 2-fold reduced rFVIII dose may be sufficient to achieve the same plasma thrombin generation capacity as with the standard rFVIII dose in the absence of concizumab. The addition of rFIX to a HB plasma pool increased the thrombin peak significantly (p&lt;0.001) and in a concentration-dependent manner both in the absence and presence of concizumab (1,500 ng/mL). Likewise, concizumab increased the thrombin peak at all rFIX concentrations (p&lt;0.001). Increasing concizumab from 1,500 to 4,500 and 15,000 ng/mL had no or limited further effect. The effects of concizumab and rFIX were mainly additive with an up to 10% effect conferred by negative drug-drug interaction for 1 IU/mL rFIX combined with concizumab &gt;1,500 ng/mL and 0.5 IU/mL rFIX combined with 15,000 ng/mL concizumab, i.e., a 10% smaller effect of rFIX was observed in the presence of concizumab than in its absence. The thrombin peak obtained upon adding 1.0 IU/mL rFIX to plasma without concizumab was similar to the thrombin peak in the presence of concizumab and 0.5 IU/mL rFIX. This suggests that in the presence of concizumab, a 2-fold reduced dose of rFIX would be sufficient to obtain the same plasma thrombin generation capacity as with 1.0 IU/mL rFIX in the absence of concizumab. Conclusion: rFVIII/rFIX increased the thrombin peak in HA and HB plasma, respectively, both in the absence and presence of concizumab. The combined effects of rFVIII/rFIX with concizumab were mainly additive with an up to 20% additional effect caused by drug-drug interaction with rFVIII and a 10% reduction with rFIX. No signs of exaggerated thrombin generation were observed by combining concizumab with rFVIII/rFIX. Therefore, the data support rFVIII/rFIX use for bleed treatment in patients on concizumab prophylaxis. As rFVIII/rFIX and concizumab have additive effects in terms of thrombin generation capacity, data suggest that clinical effectiveness could be achieved with rFVIII/rFIX doses in the lower range recommended for such products. Disclosures Kjalke: Novo Nordisk A/S: Current Employment, Current equity holder in publicly-traded company. Andersen:Novo Nordisk A/S: Current Employment, Current equity holder in publicly-traded company.

Comparison of the effect of fondaparinux and enoxaparin on thrombin generation during in-vitro clotting of whole blood and platelet-rich plasma

2004 ◽  
Vol 15 (2) ◽  
pp. 149-156 ◽  
Author(s):  
Grigoris T Gerotziafas ◽  
François Depasse ◽  
Tahar Chakroun ◽  
Patrick Van Dreden ◽  
Meyer M Samama ◽  
...  
Keyword(s):  
Whole Blood ◽  
Thrombin Generation ◽  
Platelet Rich Plasma

In vitro effect of autologous platelet‐rich plasma on H 2 O 2 ‐induced oxidative stress in human spermatozoa

Andrology ◽  
2019 ◽  
Vol 8 (1) ◽  
pp. 191-200 ◽  
Author(s):  
R. Bader ◽  
J. N. Ibrahim ◽  
M. Moussa ◽  
A. Mourad ◽  
J. Azoury ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Platelet Rich Plasma ◽  
Human Spermatozoa ◽  
Autologous Platelet Rich Plasma ◽  
Vitro Effect ◽  
Autologous Platelet

scholarly journals Effect of Combined Leukocyte-Poor Platelet-Rich Plasma and Hyaluronic Acid on Bone Marrow–Derived Mesenchymal Stem Cell and Chondrocyte Metabolism

Cartilage ◽  
2019 ◽  
pp. 194760351985873 ◽  
Author(s):  
Alexander M. Satin ◽  
Jolanta B. Norelli ◽  
Nicholas A. Sgaglione ◽  
Daniel A. Grande
Keyword(s):  
Bone Marrow ◽  
Hyaluronic Acid ◽  
Cellular Proliferation ◽  
Culture Media ◽  
Platelet Rich Plasma ◽  
Interleukin 1 ◽  
Cellular Growth ◽  
Sprague Dawley ◽  
Vitro Effect

ObjectiveGiven the potential applications of combined biologics, the authors sought to evaluate the in vitro effect of combined platelet-rich plasma (PRP) and hyaluronic acid (HA) on cellular metabolism.DesignBone marrow–derived mesenchymal stem cells (BMSCs) and chondrocytes were obtained from the femurs of Sprague-Dawley rats. An inflammatory model was created by adding 10 ng/mL interleukin-1-beta to culture media. Non-crosslinked high-molecular-weight HA, activated-PRP (aPRP), and unactivated-PRP (uPRP) were tested. Cellular proliferation and gene expression were measured at 1 week. Genes of interest included aggrecan, matrix metalloproteinase (MMP)-9, and MMP-13.ResultsCombined uPRP-HA was associated with a significant increase in chondrocyte and BMSC proliferation at numerous preparations. There was a trend of increased chondrocyte aggrecan expression with combined PRP-HA. The greatest and only significant decrease in BMSC MMP-9 expression was observed with combined PRP-HA. While a significant reduction of BMSC MMP-13 expression was seen with PRP and HA-alone, a greater reduction was observed with PRP-HA. MMP-9 chondrocyte expression was significantly reduced in cells treated with PRP-HA. PRP-alone and HA-alone at identical concentrations did not result in a significant reduction. The greatest reduction of MMP-13 chondrocyte expression was observed in chondrocytes plus combined PRP-HA.ConclusionsWe demonstrated a statistically significant increase in BMSC and chondrocyte proliferation and decreased expression of catabolic enzymes with combined PRP-HA. These results demonstrate the additive in vitro effect of combined PRP-HA to stimulate cellular growth, restore components of the articular extracellular matrix, and reduce inflammation.

scholarly journals The Effect of Procoagulative Phospholipids on the Synthetic Substrate Determined Thrombin Generation in Vitro

1977 ◽  
Author(s):  
F. Schulte ◽  
O. Klug ◽  
Ursula Roth
Keyword(s):  
Thrombin Generation ◽  
Chromogenic Substrate ◽  
Chromogenic Substrates ◽  
Platelet Factor ◽  
Synthetic Substrate ◽  
Thrombin Activity ◽  
Vitro Effect ◽  
Hydrolysis Of

The effect of procoagulative phospholipids (Procops) with platelet factor-3 like activity on the generation of thrombin in plasma can be determined in vitro with the aid of synthetic chromogenic substrates. Standard citrated plasma of two manufacturers and from different batches incubated with amounts of 2-10 meg Procops as 1:100 - 1:500 diluted Tachostyptan (micellar Procops in form of a pharmaceutical speciality) was activated. The generated amount of thrombin activity catalyses the hydrolysis of chromogenic substrate to a tripeptide and to p-nitroaniline which was measured kinetically with a spectrophotometer at 405 nm. At optimum concentrations of Procops, activities adequate to 80 (SD ± 7, VK 8. 8%) - 115 i. u. (SD ± 2. 5, VK 2.2%) thrombin per ml plasma were measured depending on the conditions of incubation and activation. Having made the basis of appropriate procedures for incubation and activation the in vitro effect of Procops on the thrombin generation in plasma is reproducibly measurable with the aid of chromogenic substrate.

scholarly journals Centrifugation Conditions in the L-PRP Preparation Affect Soluble Factors Release and Mesenchymal Stem Cell Proliferation in Fibrin Nanofibers

Molecules ◽  
2019 ◽  
Vol 24 (15) ◽  
pp. 2729 ◽  
Author(s):  
Melo ◽  
Luzo ◽  
Lana ◽  
Santana
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor ◽  
Platelet Rich Plasma ◽  
Clinical Practices ◽  
Soluble Factors ◽  
Tnf Α ◽  
Platelet Concentration ◽  
Tgf Β1

Leukocyte and platelet-rich plasma (L-PRP) is an autologous product that when activated forms fibrin nanofibers, which are useful in regenerative medicine. As an important part of the preparation of L-PRP, the centrifugation parameters may affect the release of soluble factors that modulate the behavior of the cells in the nanofibers. In this study, we evaluated the influences of four different centrifugation conditions on the concentration of platelets and leukocytes in L-PRP and on the anabolic/catabolic balance of the nanofiber microenvironment. Human adipose-derived mesenchymal stem cells (h-AdMSCs) were seeded in the nanofibers, and their viability and growth were evaluated. L-PRPs prepared at 100× g and 100 + 400× g released higher levels of transforming growth factor (TGF)-β1 and platelet-derived growth factor (PDGF)-BB due to the increased platelet concentration, while inflammatory cytokines interleukin (IL)-8 and tumor necrosis factor (TNF)-α were more significantly released from L-PRPs prepared via two centrifugation steps (100 + 400× g and 800 + 400× g) due to the increased concentration of leukocytes. Our results showed that with the exception of nanofibers formed from L-PRP prepared at 800 + 400× g, all other microenvironments were favorable for h-AdMSC proliferation. Here, we present a reproducible protocol for the standardization of L-PRP and fibrin nanofibers useful in clinical practices with known platelet/leukocyte ratios and in vitro evaluations that may predict in vivo results.

scholarly journals Evaluation of allogeneic freeze-dried platelet lysate in cartilage exposed to interleukin 1-β in vitro

2019 ◽  
Vol 15 (1) ◽  
Author(s):  
Livia Camargo Garbin ◽  
C. Wayne McIlwraith ◽  
David D. Frisbie
Keyword(s):  
Musculoskeletal Diseases ◽  
Platelet Rich Plasma ◽  
Autologous Blood ◽  
Interleukin 1 ◽  
Platelet Lysate ◽  
Freeze Dried ◽  
Platelet Concentration ◽  
High Concentrations ◽  
Disease Modifying Treatment

Abstract Background Platelet-rich plasma (PRP) as well as other platelet-derived products have been used as a potential disease-modifying treatment for musculoskeletal diseases, such as osteoarthritis (OA). The restorative properties of such products rely mainly on the high concentrations of growth factors, demonstrating encouraging results experimentally and clinically. Yet, the autologous blood-derived nature of the PRP product lead to limitations that precludes it’s widespread use. The main limitations for PRP use are; product variability, the need for minimum laboratory settings in most cases, and the need for storage at low temperatures to preserve its properties. Based on these limitations, the objective of this study was to investigate an allogeneic off-the-shelf platelet lysate (PL) in cartilage exposed to interleukin 1β (IL-1β). For this purpose, blood and cartilage were harvested from eight skeletally mature and healthy horses. Blood was processed into PL aliquots and divided into three groups (Frozen, Freeze-dried and Filtered freeze-dried), used in autologous and allogeneic conditions and in three different concentrations (1.5, 3 and 6-fold). Different PL preparations were then applied in cartilage culture with interleukin-1 beta and cultured for 10 days. Cartilage and media samples were collected and analyzed for total GAG and 35SO4-labeled GAG content. Results No significant differences between the controls and PL groups in cartilage and media were demonstrated. The effects of PL on cartilage matrix were concentration dependent and intermediate concentrations (3-fold) in PL showed increased 35SO4-labelled GAG in cartilage. Conclusion In conclusion, the allogeneic freeze-dried PL presented equivalent effects compared to frozen autologous PL. Intermediate platelet concentration on average demonstrated improved results, demonstrating less GAG loss compared to other concentrations.

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