Porcine Platelet-rich Plasma Research Articles

The shear rate promotes pinocytosis of extracellular dextran in platelets.

Several conventional studies focused on platelet pinocytosis for possible utilization as drug delivery systems. Although platelet pinocytosis is important in such utilization, the impact of the shear rate on pinocytosis is unclear. Our objective was to investigate the relationship between shear rate and platelet pinocytosis in vitro. In addition, this study addressed the change in platelet aggregation reactivity with adenosine diphosphate (ADP) stimulation after pinocytosis. Porcine platelet-rich plasma was mixed with fluorescein isothiocyanate (FITC)-conjugated dextran and incubated for 15 min under shear conditions of 0, 500, and 1500 s-1. After incubation, confocal microscopic scanning and three-dimensional rendering were performed to confirm the internalization of FITC-dextran into platelets. The amount of FITC-dextran accumulated via platelet pinocytosis was compared using flow cytometry at each shear rate. In addition, light transmission aggregometry by ADP stimulation was applied to platelets after pinocytosis. The amount of intracellular FITC-dextran increased with higher shear rates. Platelets with increased amounts of intracellular FITC-dextran did not show changes in the aggregation reactivity to ADP. A higher shear rate promotes platelet pinocytosis, but enhanced pinocytosis does not affect aggregation sensitivity, which is stimulated by ADP.

Effects of specific pathogen-free porcine platelet-rich plasma activated by the novel activator ectoine on cell proliferation and biological function

During the past two decades, cell culture has played an important role in many biochemical and biomedical fields. However, finding an alternative to animal serum is an important area in cell culture methods and related biotechnology industries. In a previous study, we demonstrated that the platelet-rich plasma (PRP) from specific pathogen-free pigs was a rich source of cytokines and growth factors and might serve as a replacement for animal serum. The selection of an activator is a crucial step in PRP activation and affects the availability of bioactive molecules. The present study is the first to demonstrate the ability of ectoine to activate PRP and to investigate the effects of ectoine-activated PRP on cell proliferation and biological function. The amounts of bioactive molecules, including TGF-β, PDGF-AB, EGF, VEGF, KGF and IGF, secreted from PRP activated by ectoine were measured. In addition, two types of primary cells and three types of cell lines were cultured with various concentrations of ectoine-activated PRP under animal serum-free conditions. Then, the effects of ectoine-activated PRP on cell growth kinetics, extracellular matrix secretion and melanin synthesis were also determined. Finally, our results demonstrated that ectoine is a novel and effective PRP activator and that ectoine-activated PRP can serve as an excellent replacement for animal serum in cell culture and related applications.

In situ polymerizable hydrogel incorporated with specific pathogen-free porcine platelet-rich plasma for the reconstruction of the corneal endothelium

Corneal inflammation and injury result in scarring and the loss of transparency of the cornea, accounting for a million cases of blindness worldwide each year. Hence, tissue engineering for the reconstruction of the corneal endothelium is a potential therapy. In this study, hydrogels composed of various ratios of hyaluronic acid (H) and Pluronic F-127 (F) were synthesized. These hydrogels can transit between liquid below 15 °C and hydrogel at 37 °C and were injectable for treating corneal wounds via in situ polymerization. The hydrogel preparations were assessed by Fourier transform infrared spectroscopy and scanning electron microscopy, and their physical–chemical properties, including transparency, viscoelastic properties, biodegradation and swelling properties, were also evaluated. The results demonstrated that the two ratios of hydrogels, H:F=1:15 (H1F15) and 1:20 (H1F20), exhibited thermal responsive rheological properties, biodegradation and swelling properties. Specially, the H1F15 hydrogel incubated with 10,000U of a lysozyme solution still retained over 70% of its remaining weight for 7days. Moreover, this study further compared the characteristics of bovine corneal endothelial cells (BCECs) cultured on the hydrogels with and without porcine platelet rich plasma (P-PRP) using cytotoxicity, migration, apoptosis/necrosis and cell cycle assays. The results showed that the H1F15 hydrogel with P-PRP displayed higher cell viability and cell migration. In addition, the incorporation of P-PRP positively affected the hydrogel to reduce apoptosis and to enhance the cell cycle of BCECs. Taken together, these results confirmed that the H1F15 hydrogel lacks cytotoxicity and is biodegradable. BCECs can survive, grow and retain normal characteristics in the H1F15 hydrogel with P-PRP. These results provide an opportunity for corneal endothelium reconstruction using a tissue engineering approach with the H1F15 hydrogel with P-PRP.

Industrial-scale processing of activated platelet-rich plasma from specific pathogen-free pigs and its effect on promoting human hair follicle dermal papilla cell cultivation

Abstract Due to the controversial nature of fetal bovine serum (FBS) production and safety concerns, there is a major interest in finding a potential FBS replacement, such as human platelet-rich plasma (PRP). However, traditional PRP production is largely limited by yield and is unsuitable for large-scale production. In this study, our first aim is to optimize the porcine PRP production protocol, and render it capable of manufacturing a large amount of porcine PRP that is suitable as an FBS replacement. Additionally, our secondary aim is to determine the effects of porcine PRP on human follicle dermal papilla cells (HFDPCs). In our process, the platelets are separated and concentrated via small- (with a capacity of 2.4 L/h) or large-scale preparations (with a capacity of 200–500 L/h). The porcine PRP is activated using a combination of collagen, thrombin, and calcium chloride, resulting in three distinct PRP formulations. Growth factor levels and stabilities of PRP under different treatments were measured using ELISA, MTT and real-time PCR assays. Our results showed that porcine PRP has higher growth factor levels than FBS, and is stable during freeze-drying and surfactant treatments, whereas HFDPCs treated with freshly prepared or freeze-dried PRP showed no significant difference in proliferation compared to FBS. In conclusion, our study is the first to confirm the commercial viability of porcine PRP as an FBS replacement and hair growth promoter.

Inhibition of complement component C5 prevents clotting in an ex vivo model of xenogeneic activation of coagulation.

Xenogeneic thrombotic microangiopathy (TMA) and acute vascular rejection (AVR) prevent long-term survival of porcine xenografts after transplantation into non-human primates. Preformed xenoreactive natural antibodies (XNA) cause endothelial damage and activate the complement system. Mechanisms of xenogeneic coagulation and platelet activation are only poorly characterized. A microfluidic flow chamber was used to study platelet activation and thrombus formation of human platelet-rich plasma (PRP) upon perfusion over wild-type (WT) or α-1,3- galactosyltransferase knockout (GTKO) and human CD46 (hCD46) transgenic porcine aortic endothelial cells (PAEC). Activation of plasma coagulation (thrombin-anti-thrombin complex; TAT) and complement (C3a, C5a) was studied in human platelet-free plasma (PFP) after co-incubation with PAEC. The activation of PAEC (E-Selectin, tissue factor, ICAM-1, ICAM-2, VCAM-1) was studied after incubation with human serum. Eculizumab (200 μg/ml) was used to inhibit terminal complement activation in all experiments. WT-PAEC perfused with human PRP showed thrombus formation at different shear rates (3 dyn/cm(2) : 23 ± 10%; 10 dyn/cm(2) : 17 ± 10% of flow chamber viewing field). GTKO/hCD46 PAEC exhibited reduced, but not fully prevented thrombus formation (3 dyn/cm(2) : 12 ± 12%). Porcine PRP caused little or no thrombus formation (3.0 ± 4% and 0.5 ± 0.9%, respectively). Flow cytometry of human platelets after perfusion over WT-PAEC revealed an increase in platelet CD62P expression (29.5 ± 3%), compared to non-perfused PRP (7 ± 2%) or PRP running through empty flow chambers (12.7 ± 0.3%). Incubation of human PFP with PAEC resulted in an increase of TAT that correlated with C5a activation. Specific inhibition of complement by eculizumab prevented thrombus formation (WT-PAEC: 1.6 ± 2% at 3 dyn/cm(2) and 0.24 ± 0.33% at 10 dyn/cm(2) , GTKO/hCD46 PAEC: 0.2 ± 0.3% at 3 dyn/cm(2) ) as well as activation of coagulation and platelets. Induction of endothelial E-Selectin and VCAM-1 in WT-PAEC upon incubation with human serum was significantly reduced by eculizumab. Eculizumab did not reduce thrombin generation capacity of human PRP or normal platelet aggregation. Thrombus formation in this ex vivo model of xenogeneic TMA was closely linked with complement activation. Specific inhibition of complement C5 by eculizumab prevented endothelial cell activation, but also coagulation and platelet activation without compromising thrombin generation capacity of human blood or normal platelet function.

Growth Factor Release from Lyophilized Porcine Platelet-Rich Plasma: Quantitative Analysis and Implications for Clinical Applications.

Freeze-dried platelet-rich plasma (FD PRP) is of potential value for clinical applications. However, growth factors released from FD PRP have not been well studied. Our study investigates growth factor release from FD PRP preparations, compared with other PRP samples, to further facilitate such clinical use. We used four experimental groups: (1) Fresh porcine PRP (PRP), (2) PRP activated by calcium chloride (CaCl2) (Ca PRP), (3) PRP activated by CaCl2, followed by freeze drying (Ca-FD PRP), and (4) PRP freeze-dried first, then activated by CaCl2 (FD-Ca PRP). All FD PRP samples were kept for up to 4 weeks at room temperature (22 °C) and reconstituted prior to analysis. Transforming growth factor-β1 (TGF-β1), platelet-derived growth factor AB (PDGF-AB), and vascular endothelial growth factor (VEGF) were quantitated by ELISA at 15 min and 1 h incubation times. The concentrations of all growth factors in Ca PRP, measured at 1 h, were significantly higher than those in PRP (p < 0.05). PDGF-AB concentrations in FD-Ca PRP were not significantly different than in Ca PRP (p > 0.05). Levels of VEGF in Ca-FD PRP were not significantly different than in Ca PRP (p > 0.05). However, TGF-β1 concentrations in Ca-FD PRP, measured at 15 min, were higher than those in Ca PRP (p < 0.05). PRP was activated efficiently by calcium chloride. Freeze-dried PRP remained rich in growth factors after storage for 4 weeks at room temperature, indicating its ease of use and wider possibilities for clinical applications. This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266.

Optimization of the processing of porcine platelet-rich plasma and its application on human mesenchymal stem cell cultivation

Because of ethical and scientific controversy, the utilization of fetal bovine serum (FBS) for cell culture medium must be minimized. This study develops porcine platelet-rich plasma (P-PRP) as a FBS substitute for human mesenchymal stem cell (hMSC) cultivation. Concentrating porcine blood by serial centrifugation to obtain P-PRP leads to activation by different agonist combinations to stimulate the secretion of growth factors. The concentration of growth factor in P-PRP is significantly increased by activation (p<0.05). The concentration of PDGF, KGF and TGF-β in activated P-PRP is significantly higher than that in FBS. Design-expert was used to decide whether Co−T+Ca−, Co+T−Ca−, and Co+T+Ca− are optimal agonist formulations. MSC cultivation shows that the attachment rate, proliferation rate and viability of P-PRP supplemented media are significantly higher than those for FBS-supplemented and commercial media (p<0.05). The results demonstrate that P-PRP is an optimal FBS substitute that supports in vitro h-MSCs expansion for subsequent biomedical applications.

Flow cytometry analysis of porcine platelets: Optimized methods for best results

Animal models are essential tools for the in vivo evaluation of pharmacological modulation of platelet function and the mechanisms underlying thrombosis. In particular, pigs are being increasingly used in cardiovascular and platelet research. One standard method for the investigation of platelet function under experimental conditions is flow cytometry. However, this approach is limited by a shortage of feasible antibodies and a lack of incubation protocols with regard to porcine platelets. This study aimed to establish a method for the investigation of porcine platelets in flow cytometry. Platelets from pigs and human donors were stained with various commercially available specific antibodies against platelet receptors CD41a, CD42bα, CD62P, activated CD41/CD61, and platelet-bound fibrinogen. Staining procedures were performed in undiluted or diluted whole blood (WB) or platelet-rich plasma (PRP). Samples were treated with PBS buffer as control or with adenosine diphosphate (ADP) to induce platelet activation. Flow cytometry was performed using standard methodology. Furthermore, platelet counts were determined and ADP-induced platelet aggregations of both species were examined to confirm that the agonist ADP reliably activates human as well as porcine platelets. Five of the investigated antibodies bound to human, but not to porcine platelets only. However, two chicken-derived antibodies directed against CD62P (09-143) and fibrinogen (09-038) as well as a monoclonal mouse anti-CD62P (KO2.5) and a polyclonal rabbit anti-fibrinogen antibody (F0111) allowed reliable detection of porcine platelet activation. Moreover, binding intensity of the 09-143 antibody was increased when incubated in porcine PRP compared to WB, whereas antibody binding of both anti-fibrinogen antibodies to porcine platelets was only observed when incubated in a WB-buffer solution. KO2.5 antibody binding was detectable employing PRP as well as the WB-buffer solution. The feasibility of our new incubation protocols was confirmed by successful investigation of platelet activation in a porcine in vivo cardiopulmonary bypass model. In conclusion, we describe a reliable method to detect the activation of porcine platelets and therefore provide a useful tool for platelet flow cytometry in porcine models. Notably, the applied incubation protocol and medium, in which platelets are suspended, have major effects on antibody-binding properties.

Hydrophilic, semipermeable membranes fabricated with poly(ethylene oxide)–polysulfone block copolymer

Semipermeable membranes may be fabricated from mixtures of poly(ethylene oxide)/polysulfone block copolymer (PEO- b-PSF) and polysulfone. Membranes fabricated with PEO- b-PSF possess a hydrophilic surface. PEO- b-PSF segregates to the membrane surface during phase inversion fabrication of the membrane rendering the surface hydrophilic. Changes in surface hydrophilicity were demonstrated by a dramatic reduction in the dynamic contact angle in water. With regard to the similar microporous hollow fiber membranes, a PEO- b-PSF membrane had a dynamic water contact angle of 33°±2 compared to a 111°±3 for a polysulfone membrane. Studies on porcine platelet-rich plasma in vitro demonstrated that the hydrophilic PEO- b-PSF membrane was resistant to platelet adhesion compared to a polysulfone membrane. An order of magnitude fewer adherent platelets were observed on a PEO- b-PSF membrane compared to a polysulfone membrane. The hydrophilicity of PEO- b-PSF makes it a unique material for the fabrication of membranes for medical devices.

The potent platelet inhibitory effects of S-nitrosated albumin coating of artificial surfaces

OBJECTIVESWe studied the antithrombotic effect of coating glass, collagen and metal stent surfaces with bovine serum albumin (BSA) covalently modified to carry S-NO functional groups denoted (pS-NO-BSA).METHODSVideo-enhanced light microscopy was used to visualize canine blood platelet adhesion and aggregation in a parallel plate glass chamber. Platelet adhesion was observed for 60 min on glass, glass coated with BSA, glass coated with pS-NO-BSA, collagen I (CO) surface, CO coated with BSA and CO coated with pS-NO-BSA. We also coated Palmaz-Shatz (P-S) stents with pS-NO-BSA. Coated and uncoated stents were then immersed in porcine platelet-rich plasma for two min and the platelet cyclic GMP level was measured. In six anesthetized pigs, coated and uncoated stents were placed in the carotid arteries and [111In]-labeled platelets were circulated for 2 h. The stented arteries were then removed and placed in a gamma well counter.RESULTSThere was significantly less platelet attachment, adhesion and aggregation on the pS-NO-BSA coated surfaces compared with the BSA coated and uncoated surfaces. The pS-NO-BSA coating increased the platelet cGMP levels to 5.9 ± 0.7 pmoles/108platelets compared with 2.7 ± 0.9 pmoles/108platelets for control (p < 0.01). The average gamma ray count from [111In]-labeled platelets that attached to the coated stents was 90,000 ± 42,000/min and 435,000 ± 290,000/min for the uncoated stents (p < 0.01).CONCLUSIONSThe pS-NO-BSA coating of thrombogenic surfaces reduces platelet adhesion and aggregation, possibly by increasing the platelet cGMP. This inhibitory effect appears to be a consequence of the direct antiplatelet actions of NO combined with the antiadhesive properties of albumin.

Soluble saccharides block the inhibition of agonist-induced human platelet aggregation observed after in vitro incubation of human platelet-rich plasma with porcine aortic endothelial cells.

Platelet aggregation is a prominent feature in the hyperacute process of vascularized allografts and xenografts. In a study of extracorporeal connection of pig kidneys to the blood circulation of human volunteers, we observed in one case considerable destruction of human platelets in the pig kidney without signs of hyperacute rejection or microthrombi formation. In the present study, we have investigated the agonist-induced aggregation of human platelets in mixtures with porcine aortic endothelial cells (PAEC). In vitro incubation of human platelet-rich plasma (PRP) with PAEC inhibited platelet aggregation induced by ADP, collagen and arachidonic acid in a time-dependent manner and partially inhibited adrenalin-induced aggregation. Aggregation of the human platelets could not be induced by high concentrations of ADP (20 microM) to overcome the inhibition capacity of the PAEC. The PAEC inhibiting effect could be transferred by the supernatants of PAEC/PRP and PAEC/PPP incubation mixtures. Preincubation of the PAEC with aspirin, but not with NG-methyl-L-Arg, reduced the aggregation inhibitory effect. Control experiments mixing human umbilical vein endothelial cells (HUVEC) and human PRP or mixing porcine PRP and PAEC did not elicit any inhibition of ADP-induced platelet aggregation. The aggregation inhibition effect could partially be blocked by preincubation of PRP with soluble Gal alpha 1-3Gal, Gal alpha 1-3 beta 1-4GlcNAc, lactose, galactose, and glucose, but not by lactosamine, galactosamine, or glucosamine. The Gal alpha 1-3Gal disaccharide was most effective in blocking aggregation inhibition, and to a similar extent as its ability to block the human anti-pig lymphocytotoxicity reaction. In conclusion, the data indicate that PAEC, upon stimulation by human anti-pig xenoantibodies in a nondynamic system, inhibits agonist-induced human platelet aggregation, and that this effect is probably at least partially caused by prostacyclin released from the PAEC.

Soluble saccharides block the inhibition of agonist-induced human platelet aggregation observed after in vitro incubation of human platelet-rich plasma with porcine aortic endothelial cells

Abstract Platelet aggregation is a prominent feature in the hyperacute process of vascularized allografts and xenografts. In a study of extracorporeal connection of pig kidneys to the blood circulation of human volunteers, we observed in one case considerable destruction of human platelets in the pig kidney without signs of hyperacute rejection or microthrombi formation. In the present study, we have investigated the agonist-induced aggregation of human platelets in mixtures with porcine aortic endothelial cells (PAEC). In vitro incubation of human platelet-rich plasma (PRP) with PAEC inhibited platelet aggregation induced by ADP, collagen and arachidonic acid in a time-dependent manner and partially inhibited adrenalin-induced aggregation. Aggregation of the human platelets could not be induced by high concentrations of ADP (20 µM) to overcome the inhibition capacity of the PAEC. The PAEC inhibiting effect could be transferred by the supernatants of PAEC/PRP and PAEC/PPP incubation mixtures. Preincubation of the PAEC with aspirin, but not with NG-methyl-L-Arg, reduced the aggregation inhibitory effect. Control experiments mixing human umbilical vein endothelial cells (HUVEC) and human PRP or mixing porcine PRP and PAEC did not elicit any inhibition of ADP-induced platelet aggregation. The aggregation inhibition effect could partially be blocked by preincubation of PRP with soluble Galαl–3Gal, Galα1–3β1–4GlcNAc, lactose, galactose, and glucose, but not by lactosamine, galactosamine, or glucosamine. The Galα1-3Gal disaccharide was most effective in blocking aggregation inhibition, and to a similar extent as its ability to block the human anti-pig lymphocytotoxicity reaction. In conclusion, the data indicate that PAEC, upon stimulation by human anti-pig xenoantibodies in a nondynamic system, inhibits agonist-induced human platelet aggregation, and that this effect is probably at least partially caused by prostáiyclin released from the PAEC.

The Calcium Modulator Nifedipine Exerts Its Antiaggregatory Property via a Nitric Oxide Mediated Process

The in vitro effect of nifedipine, a calcium channel blocker of the dihydropyridine (DHP) type, on platelet aggregation was reinvestigated considering especially the capability of platelets to form endogenous nitric oxide (NO). We studied the dose-dependent antiaggregatory property of nifedipine in porcine platelet rich plasma. Aggregation was stimulated by collagen (7.5 micrograms/ml). Nifedipine inhibited collagen-induced platelet aggregation with an IC50 of 380 nmol/l. The antiaggregatory effect of nifedipine could be significantly diminished by N-nitro-L-arginine (NNA) in a concentration dependent manner, whereas oxy haemoglobin (4 microM), a NO scavenger, totally abolished the effect of nifedipine. L-Arginine, the precursor of NO, dose-dependently inhibited the collagen-induced platelet aggregation but did not potentiate the effects of nifedipine. Therefore, we propose that in platelet rich plasma the nifedipine induced inhibition of platelet aggregation is mediated by NO, a potent endogenous inhibitor of aggregation. We could confirm this hypothesis by measuring NO directly with a specific electrode.