Nitric Oxide-Releasing Porous Coating with Antibacterial Activity and Blood Compatibility.

Nitric oxide (NO)-releasing coating is promising to enhance the biocompatibility of medical devices. In this study, polyurethane (PU) and S-nitrosated keratin (KSNO) were dissolved with dimethyl sulfoxide (DMSO) and tetrahydrofuran (THF) to prepare a coating solution. This solution is facile to form a porous coating on various substrates based on solvent-evaporation-induced phase separation (SEIPS). The coating could continuously release NO up to 200 h in the presence of ascorbic acid (Asc). In addition, the coating could accelerate endothelialization by promoting the viability of human umbilical vein endothelial cells (HUVECs) while inhibiting the proliferation of human umbilical artery smooth muscle cells (HUASMCs). Furthermore, the coating had good antibacterial activity and blood compatibility. Taken together, this universal coating provides wider potential applications in the field of cardiovascular implants.

Catalytic Generation of Nitric Oxide from Poly(ε-caprolactone)/Phosphobetainized Keratin Mats for a Vascular Tissue Engineering Scaffold.

Tissue-engineered vascular graft (TEVG) is a promising alternative to meet the clinical demand of organ shortages. Herein, human hair keratin was extracted by the reduction method, followed by modification with zwitterionic 2-methacryloyloxyethyl phosphorylcholine (MPC) through thiol-Michael addition to improve blood clotting nature. Then, phosphobetainized keratin (PK) was coelectrospun with poly(ε-caprolactone) (PCL) to afford PCL/PK mats with a ratio of 7:3. The surface morphology, chemical structure, and wettability of these mats were characterized. The biocomposite mats selectively enhanced adhesion, migration, and growth of endothelial cells (ECs) while suppressed proliferation of smooth muscle cells (SMCs) in the presence of glutathione (GSH) and GSNO due to the catalytic generation of NO. In addition, these mats exhibited good blood anticoagulant activity by reducing platelet adhesion, prolonging blood clotting time, and inhibiting hemolysis. Taken together, these NO-generating PCL/PK mats have potential applications as a scaffold for vascular tissue engineering with rapid endothelialization and reduced SMC proliferation.

Nitric oxide releasing poly(vinyl alcohol)/S-nitrosated keratin film as a potential vascular graft.

Nitric oxide (NO) releasing vascular graft is promising due to its merits of thromboembolism reduction and endothelialization promotion. In this study, keratin-based NO donor of S-nitrosated keratin (KSNO) was blended with poly(vinyl alcohol) (PVA) and further crosslinked with sodium trimetaphosphate (STMP) to afford PVA/KSNO biocomposite films. These films could release NO sustainably for up to 10 days, resulting in the promotion of HUVECs growth and the inhibition of HUASMCs growth. In addition, these films displayed good blood compatibility and antibacterial activity. Taken together, these films have potential applications in vascular grafts.

Facile fabrication of copper-incorporating poly(ε-caprolactone)/keratin mats for tissue-engineered vascular grafts with the potential of catalytic nitric oxide generation.

Tissue-engineered vascular grafts (TEVGs) provide a new alternative for vascular construction. Nitric oxide (NO) is capable of promoting vascular tissue regeneration and reducing restenosis caused by vascular implantation. Therefore, in situ production of NO by catalytic decomposition of the endogenous donor is a promising strategy to fabricate a TEVG. In this study, poly(ε-caprolactone) (PCL) was first electrospun with keratin (Ker) to afford PCL/Ker mats and then incorporated with Cu(II) ions through multiple interactions. This strategy is very simple, green, and facile. Particularly, the incorporated Cu(II) ions were partially reduced to Cu(I) ions due to the reducibility of keratin. The chelated copper ions were expected to catalyze the generation of NO from endogenous S-nitrosothiol (RSNO). As a result, PCL/Ker-Cu mats selectively accelerated the adhesion, migration, and growth of human umbilical vein endothelial cells (HUVECs), while inhibiting the proliferation of human umbilical artery smooth muscle cells (HUASMCs). Furthermore, these mats exhibited excellent blood compatibility and significant antibacterial activity. Vascular implantation in vivo indicated that the tubular mats could inhibit thrombus formation and retain patency for 3 months after implantation in the rabbit carotid artery. More importantly, vascular remodeling was observed during follow-up, including a complete endothelium and smooth muscle layer. Taken together, the PCL/Ker-Cu mats have great potential application in vascular tissue regeneration.

Antioxidant and multi-sensitive PNIPAAm/keratin double network gels for self-stripping wound dressing application.

Hydrogel is a potential wound dressing material due to its ability to maintain a humid environment, the strong absorptive capacity of exuded tissue fluid, and gas exchange function. Herein, poly(N-isopropyl acrylamide)/keratin double network (PNIPAAm/keratin DN) gels were fabricated through covalent and ionic double cross-linking strategy. The effects of PNIPAAm/keratin ratios on the morphology and swelling rate of gels were characterized. The DN gels could swell up from 2600% to 4600% in proportion to the keratin content, demonstrating their great ability to absorb tissue fluid. The gels possessed thermo-sensitiveness, imparting self-stripping property. Moreover, the antibacterial chlorhexidine acetate (CHX) was loaded into gels with a post-fabrication drug-loading strategy. The release behavior showed that CHX-loaded DN gels exhibited multiple responsive characteristics (temperature, pH, and ROS). Furthermore, the drug-loaded gels showed greater antibacterial activity than free CHX due to the sustained drug release effect. Meanwhile, the antioxidant efficiency of PNIPAAm/keratin DN gels was ca. 33.1%, while the PNIPAAm gel was just ca. 18.2%, indicating the strong oxidation resistance of DN gels. In the Sprague Dawley (SD) rat skin defect model, the hydrogel had better tissue regeneration ability than the commercial film. Taken together, the multifunctional PNIPAAm/keratin DN gels are potential candidates for clinical wound treatment.

Nitric oxide-releasing poly(ε-caprolactone)/S-nitrosylated keratin biocomposite scaffolds for potential small-diameter vascular grafts.

Rapid endothelialization and regulation of smooth muscle cell proliferation are crucial for small-diameter vascular grafts to address poor compliance, thromboembolism, and intimal hyperplasia, and achieve revascularization. As a gaseous signaling molecule, nitric oxide (NO) regulates cardiovascular homeostasis, inhibits blood clotting and intimal hyperplasia, and promotes the growth of endothelial cells. Due to the instability and burst release of small molecular NO donors, a novel biomacromolecular donor has generated increasing interest. In the study, a low toxic NO donor of S-nitrosated keratin (KSNO) was first synthesized and then coelectrospun with poly(ε-caprolactone) to afford NO-releasing small-diameter vascular graft. PCL/KSNO graft was capable to generate NO under the catalysis of ascorbic acid (Asc), so the graft selectively elevated adhesion and growth of human umbilical vein endothelial cells (HUVECs), while inhibited the proliferation of human aortic smooth muscle cells (HASMCs) in the presence of Asc. In addition, the graft displayed significant antibacterial properties and good blood compatibility. Animal experiments showed that the biocomposite graft could inhibit thrombus formation and preserve normal blood flow via single rabbit carotid artery replacement for 1 month. More importantly, a complete endothelium was observed on the lumen surface. Taken together, PCL/KSNO small-diameter vascular graft has potential applications in vascular tissue engineering with rapid endothelialization and vascular remolding.

PCL/sulfonated keratin mats for vascular tissue engineering scaffold with potential of catalytic nitric oxide generation.

Heparin-like polymers have a good anticoagulant effect due to some groups similar to heparin. Herein, sulfonated keratin(SK) was prepared by chain transfer radical polymerization of 3-sulfopropyl methacrylate(SPMA) to improve blood coagulation nature of keratin. Poly(ε-caprolactone)(PCL)/SK nanofibrous mats with the ratio of 7 and 3 were then fabricated by electrospinning. In vitro cytotoxicity and blood compatibility tests were performed to assess the biocompatibility. Viability of NIH 3T3 cells on PCL/SK mats was higher than that on the pristine PCL mats, indicating their good cytocompatibility. These sulfonated keratin-containing mats enhanced endothelial cell growth, while inhibited smooth muscle cell proliferation and reduced platelet adhesion in the presence of GSH and GSNO, as a result of NO generation. Furthermore, the biocomposite mats prolonged the activated partial thromboplastin time(APTT) effectively without hemolysis. Taken together, PCL/SK mats are potential for applications in vascular tissue engineering.

Fabrication of PCL/keratin composite scaffolds for vascular tissue engineering with catalytic generation of nitric oxide potential.

Tissue-engineered vascular grafts (TEVGs) have been proposed as a promising approach to fulfill the need for small-diameter blood vessel substitutes. However, common failure caused by thrombosis and neointimal proliferation after implantation has restricted their use in the clinic. Herein, a NO-generating scaffold for vascular tissue engineering was developed by coelectrospinning poly(ε-caprolactone) (PCL) with keratin. The morphology and surface chemical composition were characterized via SEM, ATR-FTIR spectroscopy and XPS. The biocomposite scaffold selectively enhanced the adhesion and growth of endothelial cells (ECs) while suppressing the proliferation of smooth muscle cells (SMCs) in the presence of GSH and GSNO due to the catalytic generation of NO. In addition, these mats displayed excellent blood compatibility by prolonging the blood-clotting time. In summary, these NO-generating PCL/keratin scaffolds have potential applications in vascular tissue engineering with rapid endothelialization and reduced SMC proliferation.

Isolation of Nanjing local strains of HHV-7 and their biological and immunological characteristics.

To investigate the biological and immunological characteristics of the Nanjing local strains of HHV-7, four strains of herpesvirus were isolated from saliva specimens of one healthy individual and three children suffering from a kidney disease in Nanjing. The viruses were identified by transmission electron microscopy (EM), indirect immunofluorescence assay (IFA) with a specific monoclonal antibody; nested polymerase chain reaction, restriction mapping and DNA sequencing. The virus-infected cells showed the typical cytophathic effect (CPE) under microscopy and could be detected by IFA with the human herpesvirus-7 (HHV-7) specific antibody. Under EM, herpesvirus-like and virions capsids could be found in their cytoplasm or nucleoplasm. HHV-7 DNA fragments amplified from infected cells by nested PCR were confirmed by restriction mapping and DNA sequencing. Similarly to DC strain, an known HHV-7 strain used in the present study as the positive control, the virus could be inactivated by ultraviolet irradiation for 10 min, heated at 45 centigrade degree for 30 min, pH<5 or>9 at 4 centigrade degree for 2 h and ether or chloroform for 10 h. The virus induced the production of TNF-alpha, IL-10 and IL-12 p 70 while inhibited IFN-alpha secretion, increased the percentage of CD2(+) cells while decreased that of CD4(+) or CD 45 RA(+) cells. The results indicate that the viruses isolated in Nanjing are HHV-7, which has similar biological characteristic to the known HHV-7 strain, DC. Infection with HHV-7 in vitro could affect immune function of lymphocytes by disturbing cytokine production and CD antigen expression.