Gelatin-siloxane nanoparticles to deliver nitric oxide for vascular cell regulation: synthesis, cytocompatibility, and cellular responses.

Nitric oxide (NO) is an important mediator in cardiovascular system to regulate vascular tone and maintain tissue homeostasis. Its role in vascular cell regulation makes it promising to address the post-surgery restenosis problem. However, the application of NO is constrained by its high reactivity. Here, we developed a novel NO-releasing gelatin-siloxane nanoparticle (GS-NO NP) to deliver NO effectively for vascular cell regulation. Results showed that gelatin-siloxane nanoparticles (GS NPs) could be synthesized via sol-gel chemistry with a diameter of ∼200 nm. It could be modified into GS-NO NPs via S-nitrosothiol (RSNO) modification. The synthesized GS-NO NPs could release a total of ∼0.12 µmol/mg NO sustainably for 7 days following a first-order exponential profile. They showed not only excellent cytocompatibility, but also rapid intracellularization within 2 h. GS-NO NPs showed inhibition of human aortic smooth muscle cell (AoSMC) proliferation and promotion of human umbilical vein endothelial cell (HUVEC) proliferation in a dose-dependent manner, which is an important approach to prevent restenosis. With GS-NO NP dose at 100 µg/mL, the proliferation of AoSMCs could be slowed down whereas the growth of HUVECs was significantly promoted. We concluded that GS-NO NPs could have potential to be used as a promising nano-system to deliver NO for vascular cell regulation.

Enhancing mesenchymal stem cell response using uniaxially stretched poly(ε-caprolactone) film micropatterns for vascular tissue engineering application.

Regeneration of tunica media with anisotropic architecture still remains a challenging issue for vascular tissue engineering (TE). Herein, we present the development of flexible poly(ε-caprolactone) (PCL) film micropatterns to regulate mesenchymal stem cells (MSCs) function for tunica media construction. Results showed that uniaxial thermal stretching of PCL films resulted in topographical micropatterns comprising of ridges/grooves, and improved mechanical properties, including yield stress, Young's modulus, and fracture stress without sacrificing film elasticity. Culturing on such PCL film micropatterns, MSCs self-aligned along the ridges with a more elongated morphology as compared to that of the un-stretched film group. Moreover, MSCs obtained a contractile SMCs-like phenotype, with ordered organization of cellular stress filaments and upregulated expression of the contractile makers, including SM-α-actin, calponin, and SM-MHC. The PCL film micropatterns could be rolled into a small-diameter 3D tubular scaffold with circumferential anisotropy of ridges/grooves, and in the incorporation of MSCs, which facilitated a hybrid sandwich-like vascular wall construction with ordered cell architecture similar to that of the tunica media. These results provide insights of how geometric cues are able to regulate stem cells with desired functions and have significant implications for the designing of a functionalized vascular TE scaffold with appropriate topographical geometries for guiding tunica media regeneration with microscale control of cell alignment and genetic expression.

Biomimetic three-dimensional anisotropic geometries by uniaxial stretching of poly(ε-caprolactone) films: degradation and mesenchymal stem cell responses.

Geometric cues have been used for a variety of cell regulation and tissue regenerative applications. While the function of geometric cues is being recognized, their stability and degradation behaviors are not well known. Here, we studied the influence of degradation on uniaxial-stretch-induced poly(ε-caprolactone) (UX-PCL) ridge/groove arrays and further cellular responses. Results from accelerated hydrolysis in vitro showed that UX-PCL ridge/groove arrays followed a surface-controlled erosion, with an overall geometry remained even at ∼45% film weight loss. Compared to unstretched PCL flat surfaces and/or ridge/groove arrays, UX-PCL ridge/groove arrays achieved an enhanced morphological stability against degradation. Over the degradation period, UX-PCL ridge/groove arrays exhibited an "S-shape" behavior of film weight loss, and retained more stable surface hydrophilicity and higher film mechanical properties than those of unstretched PCL surfaces. Human mesenchymal stem cells (MSCs) aligned better toward UX-PCL ridge/groove arrays when the geometries were remained intact, and became sensitive with gradually declined nucleus alignment and elongation to the geometric degradation of ridges. We speculate that uniaxial stretching confers UX-PCL ridge/groove arrays with enhanced stability against degradation in erosive environment. This study provides insights of how degradation influences geometric cues and further cell responses, and has implications for the design of biomaterials with stability-enhanced geometric cues for long-term tissue regeneration.

Biomimetic three-dimensional anisotropic geometries by uniaxial stretch of poly(ε-caprolactone) films for mesenchymal stem cell proliferation, alignment, and myogenic differentiation.

Anisotropic geometries are critical for eliciting cell alignment to dictate tissue microarchitectures and biological functions. Current fabrication techniques are complex and utilize toxic solvents, hampering their applications for translational research. Here, we present a novel simple, solvent-free, and reproducible method via uniaxial stretching for incorporating anisotropic topographies on bioresorbable films with ambitions to realize stem cell alignment control. Uniaxial stretching of poly(ε-caprolactone) (PCL) films resulted in a three-dimensional micro-ridge/groove topography (inter-ridge-distance: ~6 µm; ridge-length: ~90 µm; ridge-depth: 200-900 nm) with uniform distribution and controllable orientation by the direction of stretch on the whole film surface. When stretch temperature (Ts) and draw ratio (DR) were increased, the inter-ridge-distance was reduced and ridge-length increased. Through modification of hydrolysis, increased surface hydrophilicity was achieved, while maintaining the morphology of PCL ridge/grooves. Upon seeding human mesenchymal stem cells (hMSCs) on uniaxial-stretched PCL (UX-PCL) films, aligned hMSC organization was obtained. Compared to unstretched films, hMSCs on UX-PCL had larger increase in cellular alignment (>85%) and elongation, without indication of cytotoxicity or reduction in cellular proliferation. This aligned hMSC organization was homogenous and stably maintained with controlled orientation along the ridges on the whole UX-PCL surface for over 2 weeks. Moreover, the hMSCs on UX-PCL had a higher level of myogenic genes' expression than that on the unstretched films. We conclude that uniaxial stretching has potential in patterning film topography with anisotropic structures. The UX-PCL in conjunction with hMSCs could be used as "basic units" to create tissue constructs with microscale control of cellular alignment and elongation for tissue engineering applications.

Novel gelatin-siloxane nanoparticles decorated by Tat peptide as vectors for gene therapy.

In principle, the technique of gene delivery involves taking complete or parts of genes that can code specific messages and delivering them to selected cells in the body. Such a transfer of plasmid DNA into mammalian cells has posed major challenges for gene therapy. A series of gelatin-siloxane nanoparticles (GS NPs) with controlled size and surface charge were synthesized through a two-step sol-gel process. In order to increase the efficiency of cellular uptake, HIV-derived Tat peptide was further grafted to GS NPs. In vitro co-location and endocytosis inhibition experiments suggested that the as-synthesized TG NPs may enter HeLa cells via a combined pathway of lipid-raft- and receptor-dependent endocytosis, and only cause little cell damage. Moreover, this study shows the encapsulation of a plasmid DNA in TG NPs to be obtained as a non-viral gene vector. This kind of encapsulation provides complete protection to the plasmid DNA from the external DNase and serum environment, and generates the hope that the resulting formulation can be developed into a potential vector for effective gene delivery. In order to check this potential, the reporter gene pSVß-gal was encapsulated, and in vitro transfection efficiency of this system was found to be nearly 130% compared to the commercially available transfection reagent Lipofectamine™.