The Impacts of the Sterilization Method and the Electrospinning Conditions of Nanofibrous Biodegradable Layers on Their Degradation and Hemocompatibility Behavior.

The use of electrospun polymeric biodegradable materials for medical applications is becoming increasingly widespread. One of the most important parameters regarding the functionality of nanofiber scaffolds during implantation and the subsequent regeneration of damaged tissues concerns their stability and degradation behavior, both of which are influenced by a wide range of factors (the properties of the polymer and the polymer solution, the technological processing approach, the sterilization method, etc.). This study monitored the degradation of nanofibrous materials fabricated from degradable polyesters as a result of the sterilization method applied (ethylene oxide and gamma irradiation) and the solvent system used to prepare the spun polymer solution. Aliphatic polyesters PCL and PLCL were chosen for this study and selected with respect to the applicability and handling in the surgical setting of these nanofibrous materials for vascular bandaging. The results revealed that the choice of solvent system exerts a significant impact on degradation during sterilization, especially at higher gamma irradiation values. The subsequent enzyme-catalyzed degradation of the materials following sterilization indicated that the choice of the sterilization method influenced the degradation behavior of the materials. Whereas wave-like degradation was evident concerning ethylene oxide sterilization, no such behavior was observed following gamma-irradiation sterilization. With concern for some of the tested materials, the results also indicated the potential for influencing the development of degradation within the bulk versus degradation from the surface of the material. Both the sterilization method and the choice of the spinning solvent system were found to impact degradation, which was observed to be most accelerated in the case of PLCL (L-lactide-co-caprolactone copolymer) electrospun from organic acids and subsequently sterilized using gamma irradiation. Since we planned to use these materials in cardiovascular applications, it was decided that their hemocompatibility would also be tested. The results of these tests revealed that changes in the structures of the materials initiated by sterilization may exert thrombogenic and anticoagulant impacts. Moreover, the microscopic analysis suggested that the solvent system used in the preparation of the materials potentially affects the behavior of erythrocytes; however, no indication of the occurrence of hemolysis was detected.

A novel bifunctional multilayered nanofibrous membrane combining polycaprolactone and poly (vinyl alcohol) enriched with platelet lysate for skin wound healing.

Skin wound healing is a complex physiological process that involves various cell types, growth factors, cytokines, and other bioactive compounds. In this study, a novel dual-function multilayered nanofibrous membrane is developed for chronic wound application. The membrane is composed of five alternating layers of polycaprolactone (PCL) and poly (vinyl alcohol) (PVA) nanofibers (PCL-PVA) with a dual function: the PCL nanofibrous layers allow cell adhesion and growth, and the PVA layers enriched with incorporated platelet lysate (PCL-PVA + PL) serve as a drug delivery system for continuous release of bioactive compounds from PL into an aqueous environment. The material is produced using a needleless multi-jet electrospinning approach which can lead to homogeneous large-scale production. The bioactive PCL-PVA + PL membranes are cytocompatible and hemocompatible. A spatially compartmented co-culture of three cell types involved in wound healing - keratinocytes, fibroblasts and endothelial cells - is used for cytocompatibility studies. PCL-PVA + PL membranes enhance the proliferation of all cell types and increase the migration of both fibroblasts and endothelial cells. The membranes are also hemocompatible without any deleterious effect for thrombogenicity, hemolysis and coagulation. Thus, the beneficial effect of the PCL-PVA + PL membrane is demonstrated in vitro, making it a promising scaffold for the treatment of chronic wounds.

Adipose-Derived Stem Cells in Reinforced Collagen Gel: A Comparison between Two Approaches to Differentiation towards Smooth Muscle Cells.

Scaffolds made of degradable polymers, such as collagen, polyesters or polysaccharides, are promising matrices for fabrication of bioartificial vascular grafts or patches. In this study, collagen isolated from porcine skin was processed into a gel, reinforced with collagen particles and with incorporated adipose tissue-derived stem cells (ASCs). The cell-material constructs were then incubated in a DMEM medium with 2% of FS (DMEM_part), with added polyvinylalcohol nanofibers (PVA_part sample), and for ASCs differentiation towards smooth muscle cells (SMCs), the medium was supplemented either with human platelet lysate released from PVA nanofibers (PVA_PL_part) or with TGF-ß1 + BMP-4 (TGF + BMP_part). The constructs were further endothelialised with human umbilical vein endothelial cells (ECs). The immunofluorescence staining of alpha-actin and calponin, and von Willebrand factor, was performed. The proteins involved in cell differentiation, the extracellular matrix (ECM) proteins, and ECM remodelling proteins were evaluated by mass spectrometry on day 12 of culture. Mechanical properties of the gels with ASCs were measured via an unconfined compression test on day 5. Gels evinced limited planar shrinkage, but it was higher in endothelialised TGF + BMP_part gel. Both PVA_PL_part samples and TGF + BMP_part samples supported ASC growth and differentiation towards SMCs, but only PVA_PL_part supported homogeneous endothelialisation. Young modulus of elasticity increased in all samples compared to day 0, and PVA_PL_part gel evinced a slightly higher ratio of elastic energy. The results suggest that PVA_PL_part collagen construct has the highest potential to remodel into a functional vascular wall.

The Effect of the Controlled Release of Platelet Lysate from PVA Nanomats on Keratinocytes, Endothelial Cells and Fibroblasts.

Platelet lysate (PL) provides a natural source of growth factors and other bioactive molecules, and the local controlled release of these bioactive PL components is capable of improving the healing of chronic wounds. Therefore, we prepared composite nanofibrous meshes via the needleless electrospinning technique using poly(vinyl alcohol) (PVA) with a high molecular weight and with a high degree of hydrolysis with the incorporated PL (10% w/w). The morphology, wettability and protein release from the nanofibers was then assessed from the resulting composite PVA-PL nanomats. The bioactivity of the PVA-PL nanomats was proved in vitro using HaCaT keratinocytes, human saphenous endothelial cells (HSVECs) and 3T3 fibroblasts. The PVA-PL supported cell adhesion, proliferation, and viability. The improved phenotypic maturation of the HaCaT cells due to the PVA-PL was manifested via the formation of intermediate filaments positive for cytokeratin 10. The PVA-PL enhanced both the synthesis of the von Willebrand factor via HSVECs and HSVECs chemotaxis through membranes with 8 µm-sized pores. These results indicated the favorable effects of the PVA-PL nanomats on the three cell types involved in the wound healing process, and established PVA-PL nanomats as a promising candidate for further evaluation with respect to in vivo experiments.

The Effect of a Polyester Nanofibrous Membrane with a Fibrin-Platelet Lysate Coating on Keratinocytes and Endothelial Cells in a Co-Culture System.

Chronic wounds affect millions of patients worldwide, and it is estimated that this number will increase steadily in the future due to population ageing. The research of new therapeutic approaches to wound healing includes the development of nanofibrous meshes and the use of platelet lysate (PL) to stimulate skin regeneration. This study considers a combination of a degradable electrospun nanofibrous blend of poly(L-lactide-co-ε-caprolactone) and poly(ε-caprolactone) (PLCL/PCL) membranes (NF) and fibrin loaded with various concentrations of PL aimed at the development of bioactive skin wound healing dressings. The cytocompatibility of the NF membranes, as well as the effect of PL, was evaluated in both monocultures and co-cultures of human keratinocytes and human endothelial cells. We determined that the keratinocytes were able to adhere on all the membranes, and their increased proliferation and differentiation was observed on the membranes that contained fibrin with at least 50% of PL (Fbg + PL) after 14 days. With respect to the co-culture experiments, the membranes with fibrin with 20% of PL were observed to enhance the metabolic activity of endothelial cells and their migration, and the proliferation and differentiation of keratinocytes. The results suggest that the newly developed NF combined with fibrin and PL, described in the study, provides a promising dressing for chronic wound healing purposes.

The assessment of electrospun scaffolds fabricated from polycaprolactone with the addition of L-arginine.

Polycaprolactone (PCL) was electrospun with the addition of arginine (Arg), an α-amino acid that accelerates the healing process. The efficient needleless electrospinning technique was used for the fabrication of the nanofibrous layers. The materials produced consisted mainly of fibers with diameters of between 200 and 400 nm. Moreover, both microfibers and beads were present within the layers. Higher bead sizes were observed with the increased addition of arginine. The arginine content within the layers as well as the weight of the resultant electrospun materials were enhanced with the increased addition of arginine to the electrospinning solution (1, 5 and 10 wt%). The PCL + 1% Arg nanofibrous layer contained 5.67 ± 0.04% of arginine, the PCL + 5% Arg layer 22.66 ± 0.24% of arginine and the PCL + 10% Arg layer 37.33 ± 0.39% of arginine according to the results of the elemental analysis. A high burst release within 5 h of soaking was recorded for the PCL + 5% and PCL + 10% nanofibrous layers. However, the release rate of arginine from the PCL + 1% Arg was significantly slower, reaching a maximum level after 72 h of soaking. The resulting materials were hydrophobic. Hemocompatibility testing under static conditions revealed no effect on hemolysis following the addition of arginine and the prolongation of the prothrombin time with the increased addition of arginine, thus exerting an influence on the extrinsic and common pathway of coagulation activation. The results of the dynamic hemocompatibility assessment revealed that the numbers of blood cells and platelets were not affected significantly by the various electrospun samples during incubation. The TAT, ß-thromboglobulin and SC5-b9 concentrations were characterized by a moderate increase in the PCL group compared to those of the control group. The presence of arginine resulted in a decrease in the investigated hemocompatibility markers. The PMN elastase levels were comparable with respect to all the groups.

Comparison of titer results obtained using immediate spin one-dilution techniques to a reference method.

BACKGROUND: Many transfusion services determine the titer of potentially incompatible plasma-containing products by performing a one-dilution titer at their selected titer threshold. This study compared the results of immediate spin (IS) one-dilution titers determined by three methods with a reference standard method. METHODS: Plasma-containing products from group A and O donors were titered using the participant's routine IS one-dilution titer method. No time or temperature incubations were performed, and antihuman globulin reagent was not used. The samples were then tested using a reference method, which was a saline tube test with a 1-hour room temperature incubation; antihuman globulin was not used in the reference method. The results of the one-dilution titer were then compared to that obtained in the reference method. RESULTS: Nine centers participated in this study. There were 698 antibodies from 374 units tested by the manual IS tube one-dilution titer method; sensitivity was 0.88 (95% confidence interval [CI], 0.83-0.92), and specificity was 1.00 (95% CI, 0.98-1.00). There were 412 antibodies from 206 units tested by the manual and automated IS buffered gel card one-dilution titer method; sensitivity was 0.95 (95% CI, 0.91-0.98), and specificity was 0.87 (95% CI, 0.81-0.91). There were 98 antibodies from 49 units tested by an automated microplate IS one-dilution titer method; sensitivity was 0.76 (95% CI, 0.71-0.93), and specificity was 0.96 (95% CI, 0.92-0.99). All three methods had an accuracy rate of 90% or greater. CONCLUSION: The manual and automated one-dilution titer methods are suitable for screening plasma-containing units, although more evaluation of the automated microplate method might be required.

Comprehensive assessment of electrospun scaffolds hemocompatibility.

Biodegradable polyesters, namely polycaprolactone (PCL) and copolymer of polylactide and polycaprolactone (PLCL) were electrospun into various fibrous structures and their hemocompatibility was evaluated in vitro. Firstly, hemolytic effect was evaluated upon incubation with diluted whole blood. The results showed that the degree of hemolysis depended on chemical composition and fibrous morphology. Electrospun polycaprolactone induced slight degree of hemolysis depending on its molecular weight and fibrous morphology; copolymer PLCL did not cause detectable hemolysis. The influence of coagulation pathways was examined by measurement of coagulation times. It was showed that intrinsic coagulation pathway assessed by activated partial thromboplastin time (APTT) was moderately accelerated after incubation with PCL and prolonged after incubation with copolymer PLCL. Extrinsic activation of coagulation tested by prothrombin time (PT) was slightly accelerated after incubation with all tested electrospun samples. Thrombogenicity assessment of fibrous samples revealed high thrombogenic properties of fibrous materials that was comparable to high degree of collagen thrombogenicity. The level of platelet activation was dependent on chemical composition and surface morphology of tested materials.

APOE polymorphism as a potential determinant of functional fitness in the elderly regardless of nutritional status.

OBJECTIVES: Life expectancy is determined by a combination of genetic predisposition (~25%) and environmental influences (~75%). Nevertheless a stronger genetic influence is anticipated in long-living individuals. Apolipoprotein E (APOE) gene belongs among the most studied candidate genes of longevity. We evaluated the relation of APOE polymorphism and fitness status in the elderly. MATERIAL AND METHODS: We examined a total number of 128 subjects, over 80 years of age. Using a battery of functional tests their fitness status was assessed and the subjects were stratified into 5 functional categories according to Spirduso´s classification. Biochemistry analysis was performed by enzymatic method using automated analyzers. APOE gene polymorphism was analysed performed using PCR-RFLP. RESULTS: APOE4 allele carriers had significantly worse fitness status compared to non-carriers (p=0.025). Multiple logistic regression analysis showed the APOE4 carriers had higher risk (p=0.05) of functional unfitness compared to APOE2/E3 individuals. CONCLUSIONS: APOE gene polymorphism seems be an important genetic contributor to frailty development in the elderly. While APOE2 carriers tend to remain functionally fit till higher age, the functional status of APOE4 carriers deteriorates more rapidly.

Markers of platelet activation and apoptosis in platelet concentrates collected by apheresis.

BACKGROUND: A product with well-preserved haemostatic function of platelets is the ultimate goal of platelet concentrate production. However, platelet activation and apoptosis are induced by both collection and storage of platelet concentrates. AIM OF STUDY: Platelet concentrates obtained either by two blood separators with different technology of apheresis (Haemonetics MCS+, Haemonetics Corp. Braintree, USA and Trima Accel, Gambro BCT Inc., Lakewood, USA, respectively) or derived from buffy-coat were compared using evaluation of pH, LDH, lactate, glucose, annexin V, and sP-selectin levels immediately after collecting and at the end of expiration to estimate the differences in the activation and apoptosis of platelets in these products. RESULTS: The lowest degree of platelet activation was found in products obtained by Haemonetics MCS+ apparatus at the time of collection. Platelet concentrates obtained by apheresis revealed higher rise of LDH, annexin V and sP-selectin compared to buffy-coat derived platelets. Products from Haemonetics MCS+ showed higher rise of annexin V in comparison with products from Trima separator. Increase of LDH and sP-selectin in both apheresis products was comparable. CONCLUSIONS: On the basis of changes of sP-selectin and annexin V levels it could be concluded that initial platelet activation, which is induced by apheresis, is very likely without any further impact on quality of platelets during storage. Development of platelet storage lesions is influenced especially by storage conditions and platelet concentration in products.