Investigation of the Release of Growth Factors from Apheresis Platelet Concentrate (APC) Loaded Three Layered Composite Nanofiber Surface

Objective To establish a new method for quantitating leukocyte fragments (LFs) in apheresis platelet concentrates (AP-PCs) by using real-time quantitative polymerase chain reaction (RQ-PCR) and flow cytometry(FCM) and discuss the factors influencing LFs concentrations such as storage time, filtration and PLT concentration. Methods 67 qualified donors were selected. Each of them donated one therapeutic dose of AP-PCs. AP-PCs samples were collected as soon as possible and divided into si xfractions. One was analyzed by hematology analyzer. For the Others, DNA was extracted under differen tconditions (filtrated or unfiltrated, before or after centrifugation) at 4 hours, 24 hours, 48 hours, 72 hours, 96 hours after blood draw, respectively. Then the amounts of albumin gene of the AP-PCs and the cell-free DNA in supematant were quantitatively determined using RQ-PCR and the results were calculated into leukocytes equivalent(WBCs/μl). Intact leucocytes were counted by FCM. The concentrations of LFs were calculated by subtracting cell-frce DNA and intact leucocytes from the total DNA amount. Then the differences of LFs concentrations among groups with different storage time were compared and the differences of LFs concentrations between unfihrated and filtrated groups were also compared. After grouping all the AP-PCs according to their PLT concentrations, LFs contents of AP-PCs before filtration among groups were compared. Meanwhile, bivariate correlation analysis between PLT concentrations and LFs contents was carried out. Results LFs contents of all the AP-PCs samples were quantitated successfully. The concentrations of LFs in AP-PCs before filtration in 4 hours,24 hours,48 hours,72 hours , 96 houres after blood draw were(31.4±17. 6), (47.5±25.3), (100.7±53.5), (89.5 ±47.2) and (16.1±7.8) WBCs/μl ; After filtration the results were (16. 9±8. 7), (24. 3 ± 12. 2), (83. 1±42. 6), (78.2 ±40. 2) and (13.6 ± 6. 6) WBCs/μl respectively. There were statistically significant differences among groups of different storage time (Fwithin subjects = 472. 756,P 0.05). Conclusions RQ-PCR and FCM can be used to quantitate LFs in AP-PCs. The concentration of LFs in AP-PCs is influenced by storage time and filtration, but it is not affected by PLT concentration. Key words: Plaleletpheresis; Blood platelets; Leukocyte count; Polymerase chain reaction; Flow cytometry

Objective:In the last decade, substantial evidence has accumulated about the use of cryopreserved platelet concentrates, especially in trauma. However, little reference has been made in these studies to the morphological and functional changes of platelets. Recently platelets have been shown to be activated by cryopreservation processes and to undergo procoagulant membrane changes resulting in the generation of platelet-derived microparticles (PMPs), platelet degranulation, and release of platelet-derived growth factors (PDGFs). We assessed the viabilities and the PMP and PDGF levels of cryopreserved platelets, and their relation with thrombin generation.Materials and Methods:Apheresis platelet concentrates (APCs) from 20 donors were stored for 1 day and cryopreserved with 6% dimethyl sulfoxide. Cryopreserved APCs were kept at -80 °C for 1 day. Thawed APCs (100 mL) were diluted with 20 mL of autologous plasma and specimens were analyzed for viabilities and PMPs by flow cytometry, for thrombin generation by calibrated automated thrombogram, and for PDGFs by enzyme-linked immunosorbent assay testing.Results:The mean PMP and PDGF levels in freeze-thawed APCs were significantly higher (2763±399.4/µL vs. 319.9±80.5/µL, p<0.001 and 550.9±73.6 pg/mL vs. 96.5±49 pg/mL, p<0.001, respectively), but the viability rates were significantly lower (68.2±13.7% vs. 94±7.5%, p<0.001) than those of fresh APCs. The mean endogenous thrombin potential (ETP) of freeze-thawed APCs was significantly higher than that of the fresh APCs (3406.1±430.4 nM.min vs. 2757.6±485.7 nM.min, p<0.001). Moreover, there was a significant positive poor correlation between ETP levels and PMP levels (r=0.192, p=0.014).Conclusion:Our results showed that, after cryopreservation, while levels of PMPs were increasing, significantly higher and earlier thrombin formation was occurring in the samples analyzed despite the significant decrease in viability. Considering the damage caused by the freezing process and the scarcity of evidence for their in vivo superiority, frozen platelets should be considered for use in austere environments, reserving fresh platelets for prophylactic use in blood banks.

Surface functionalization of carbon nanofibers by sol–gel coating of zinc oxide

Platelet (PLT) growth factors released by thrombin activation of autologous PLT concentrates (PCs) are used in clinics as PLT gels or releasates for tissue repair and wound healing applications. If allogeneic products are to be used for clinical or cell culture applications, a method of viral inactivation of the PC source of growth factors is desirable. PLT-derived growth factor-AB (PDGF-AB), transforming growth factor-beta1 (TGF-beta1), epidermal growth factor (EGF), and insulinlike growth factor-1 (IGF-1) in apheresis PC subjected to solvent/detergent (S/D) treatment with or without prior activation by CaCl(2) and/or bovine thrombin were measured. Mean (+/- standard deviation) PDGF-AB, TGF-beta1, EGF, and IGF-1 content was 13.8 +/- 14.3, 16.6 +/- 14.3, less than 0.0007, and 83.4 +/- 33.4 ng per mL, respectively, in the starting PC. They increased to 184.4 +/- 80.2, 192.2 +/- 37.4, 2.2 +/- 1.6, and 88.4 +/- 33.5 after 1 percent tri-n-butyl phosphate (TnBP)-1 percent Triton X-45 treatment, respectively. Mean content was 84.6 +/- 35.5, 63.8 +/- 14.1, 0.9 +/- 0.6, and 117.2 +/- 34.9 ng per mL, respectively, in CaCl(2)-activated PC and remained stable after subsequent S/D treatment (88.3 +/- 45.9, 68.6 +/- 27.2, 1.40 +/- 1.0, and 112.4 +/- 39.7 ng/mL, respectively). Two percent TnBP treatment yielded similar release as with TnBP-Triton X-45. Addition of bovine thrombin did not increase the release of growth factors. S/D treatment efficiently releases PDGF-AB, TGF-beta1, and EGF from nonactivated apheresis PCs and may be of interest to prepare virally inactivated allogeneic growth factors for clinical and cell culture applications.

Background: Gaseous ozone therapy is a new modality that has shown increasingly promising results in the healing of diabetic foot ulcers and an increased release of growth factors from platelets but presents health risks. Since aqueous ozone is safer and may also increase the release of growth factors, it was used in this multifaceted five-part study to determine its safety and effect on the release of platelets in diabetic participants. The data is expected to provide a proof of concept for a future study to quantify the effect of the external application of aqueous ozone on diabetic foot ulcers. Methods: Experiment 1: real time platelet aggregation and adenosine triphosphate (ATP) release from the platelets of four non-diabetic participants was analysed via the Chrono-log lumi-aggregometer after immediate exposure and a 2hr incubation with phosphate buffered saline (PBS), ozonized PBS (O3PBS) or adenosine diphosphate (ADP). Experiment 2: Six non-diabetic and six diabetic’s blood plasma collected in citrate tubes was treated with either PBS, O3PBS, or ADP and incubated for either 30 min or 2 hrs. Analysis of the growth factors was performed via platelet derived growth factor (PDGF-BB) ELISA assay kit. Experiment 3: Protocol is identical to experiment 2 however the level of transforming growth factor β (TGF-β) was analysed via an ELISA assay kit. Experiment 4: Four non-diabetic and four diabetic’s blood plasma was treated with either PBS, O3PBS or ADP and incubated for either 2hrs or 4hrs. Analysis of oxidative stress was determined via a protein carbonyl assay. Experiment 5: two diabetic volunteers with a diabetic foot ulcer consented to an aqueous ozone wash three times per week for 5 weeks or until the ulcer had healed. Results: The level of ATP release was higher in the O3PBS condition compared to the PBS condition in both immediate and 2 hr incubation groups (p < 0.001). No significant difference was seen in level of aggregation between O3PBS and ADP and PBS groups (p = 0.1, p = 0.5). In experiment 2 the level of PDGF was higher in the ADP group compared to O3PBS and PBS group after 2hrs incubation (p < 0.001). No other significant differences were seen in any other conditions. No significant differences in level of TGF-β were seen in experiment 3. The level of protein carbonyl in experiment 4 was significantly higher for both O3PBS and ADP in the diabetic after 2hrs of incubation (p < 0.05) compared to PBS. Aqueous ozone significantly decreased ulcer size of both diabetic participants after 5 weeks of treatment (p < 0.008) in experiment 5. Discussion: These five experiments give evidence towards a healing effect of aqueous ozone without causing notable oxidative stress or aggregation in platelets of diabetics. Although O3PBS could cause release of dense-alpha granules, demonstrated by ATP release, this healing effect may not be due to growth factor interaction as previously thought. For the purpose of this study however, we have demonstrated some evidence that aqueous ozone may be a beneficial treatment for diabetic foot ulcers but via a different pathway then previously predicted.

The preparation of as-spun silver–polyacrylonitrile composite nanofibers (Ag/PAN Com) and the in situ synthesis of silver nanoparticles anchored on the surface of PAN nanofibers were presented. The former were directly electrospun from the solution of PAN and silver nitrate (AgNO3). The latter (AgNPs/PAN) were prepared by immersing as-spun PAN nanofibers in AgNO3 aqueous solutions with different concentrations under the radiation of UV light, as a facilitator for the reduction of Ag ions into AgNPs. A comparison between these materials, which are based on silver and polyacrylonitrile but via two different synthetic methods, as antibacterial composite nanofiber membranes against Escherichia coli (E. coli) and Bacillus subtilis (B. subtilis), was made. The success of synthesizing was confirmed by scanning electron microscopy and transmission electron microscopy (TEM). Chemical groups on the surfaces of nanofibers were detected by Fourier transform infrared spectroscopy. The crystallinity of PAN nanofibers, the crystalline alterations of Ag/PAN Com due to the penetration of silver ions into the polymer matrix of nanofibers, and the structural models of newly formed silver nanoparticles were ascertained by x-ray diffraction (XRD). The gradual transformation of Ag ions into AgNPs, which occurred near the surfaces of Ag/PAN Com nanofibers without any catalyst agents, was observed by TEM. The occurrence was also clarified by analyzing FTIR and XRD spectra. The inhibition zones of Ag/PAN Com membranes at the first cycle of bactericidal test appeared the most expansive against both strains of bacteria even with lowering the release amount of silver. However, the AgNPs/PAN exhibited a more sustainable antibacterial ability after the second and the third incubation cycles.

The Effect of Thrombocytopenia on Dermal Wound Healing

Hyaluronan Metabolism in Human Keratinocytes and Atopic Dermatitis Skin Is Driven by a Balance of Hyaluronan Synthases 1 and 3

Improved myocardial performance in infarcted rat heart by co-injection of basic fibroblast growth factor with temperature-responsive Chitosan hydrogel

Photoluminescence sensing of Pb2+ using cellulose acetate nanofiber decorated with Au nanoparticles

BackgroundThe objective of this investigation was to develop a new class of antibacterial material in the form of nanofibers coated with silver nanoparticles (AgNPs) using a modified coaxial electrospinning approach. Through manipulation of the distribution on the surface of nanofibers, the antibacterial effect of Ag can be improved substantially.MethodsUsing polyacrylonitrile (PAN) as the filament-forming polymer matrix, an electrospinnable PAN solution was prepared as the core fluid. A silver nitrate (AgNO3) solution was exploited as sheath fluid to carry out the modified coaxial electrospinning process under varied sheath-to-core flow rate ratios.ResultsScanning electron microscopy and transmission electron microscopy demonstrated that the sheath AgNO3 solution can take a role in reducing the nanofibers’ diameters significantly, a sheath-to-core flow rate ratio of 0.1 and 0.2 resulting in PAN nanofibers with diameters of 380 ± 110 nm and 230 ± 70 nm respectively. AgNPs are well distributed on the surface of PAN nanofibers. The antibacterial experiments demonstrated that these nanofibers show strong antimicrobial activities against Bacillus subtilis Wb800, and Escherichia coli dh5α.ConclusionCoaxial electrospinning with AgNO3 solution as sheath fluid not only facilitates the electrospinning process, providing nanofibers with reduced diameters, but also allows functionalization of the nanofibers through coating with functional ingredients, effectively ensuring that the active antibacterial component is on the surface of the material, which leads to enhanced activity. We report an example of the systematic design, preparation, and application of a novel type of antibacterial material coated with AgNPs via a modified coaxial electrospinning methodology.

The leukocyte- and platelet-rich fibrin block (L-PRF block) is a composite graft that combines a xenograft that is acting as a scaffold with L-PRF membranes that serve as a bioactive nodule with osteoinductive capacity. This study evaluated the properties of the L-PRF block and its components in terms of release of growth factors, cellular content, and structure. The concentration of transforming growth factor-β1 (TGF-β1), vascular endothelial growth factor (VEGF), platelet-derived growth factor-AB (PDGF-AB) and bone morphogenetic protein-1 (BMP-1) released by a L-PRF membrane (mb) and a L-PRF block were examined with ELISA for five time intervals (0 to 4 hours, 4 hours to 1 day, 1 to 3 days, 3 to 7 days, 7 to 14 days). Those levels in L-PRF exudate and liquid fibrinogen were also evaluated. The cellular content of the liquid fibrinogen, L-PRF membrane and exudate was calculated. The L-PRF block was also analyzed by means of a microCT scan and scanning electron microscopy (SEM). TGF-β1 was the most released growth factor after 14 days, followed by PDGF-AB, VEGF, and BMP-1. All L-PRF blocks constantly released the four growth factors up to 14 days. L-PRF membrane and liquid fibrinogen presented high concentration of leukocytes and platelets. The microCT and SEM images revealed the bone substitute particles surrounded by platelets and leukocytes, embedded in a dens fibrin network. The L-PRF block consists of deproteinized bovine bone mineral particles surrounded by platelets and leukocytes, embedded in a fibrin network that releases growth factors up to 14 days.

Membrane Type-1 Matrix Metalloproteinase Potentiates Basic Fibroblast Growth Factor-Induced Corneal Neovascularization

On-surface synthesis of metal nanostructures on solid and hydrated polymer nanofibers coated with polydopamine

We evaluated the effects of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) on the healing of large traumatic tympanic membrane perforations (TMPs). Prospective clinical study. Tertiary university hospital. A randomized, prospective analysis was performed between June 2013 and August 2014 on the treatment of traumatic TMPs larger than 25% of the TM. Closure rate, closure time, hearing gain, and rate of otorrhea were compared between EGF and bFGF groups, as well as to an observation-only group. Final analysis was performed on 86 patients at 3 months. The closure rates of perforation in the EGF, bFGF, and observation groups were 86.2%, 89.3%, and 72.4%, respectively. The closure rates in the EGF and bFGF groups were 14% to 17% higher than in the observation group, although the difference was not statistically significant for the total closure rate among the three groups (P = 0.200). The average closure time was significantly longer (P < 0.01) in the observation group than in the EGF and bFGF groups. However, the closure times in the EGF and bFGF groups were not significantly different (P = 0.92). In addition, differences in purulent otorrhea rates among the groups were not statistically significant (P = 0.82). Both EGF and bFGF can accelerate the closure of human large traumatic TMPs. The healing outcomes among the two growth factors were not significantly different.