Fanglian Yao

Hydrophilic PCU scaffolds prepared by grafting PEGMA and immobilizing gelatin to enhance cell adhesion and proliferation

Gelatin contains many functional motifs which can modulate cell specific adhesion, so we modified polycarbonate urethane (PCU) scaffold surface by immobilization of gelatin. PCU-g-gelatin scaffolds were prepared by direct immobilizing gelatins onto the surface of aminated PCU scaffolds. To increase the immobilization amount of gelatin, poly(ethylene glycol) methacrylate (PEGMA) was grafted onto PCU scaffolds by surface initiated atom transfer radical polymerization. Then, following amination and immobilization, PCU-g-PEGMA-g-gelatin scaffolds were obtained. Both modified scaffolds were characterized by chemical and biological methods. After immobilization of gelatin, the microfiber surface became rough, but the original morphology of scaffolds was maintained successfully. PCU-g-PEGMA-g-gelatin scaffolds were more hydrophilic than PCU-g-gelatin scaffolds. Because hydrophilic PEGMA and gelatin were grafted and immobilized onto the surface, the PCU-g-PEGMA-g-gelatin scaffolds showed low platelet adhesion, perfect anti-hemolytic activity and excellent cell growth and proliferation capacity. It could be envisioned that PCU-g-PEGMA-g-gelatin scaffolds might have potential applications in tissue engineering artificial scaffolds.

Proliferation and migration of human vascular endothelial cells mediated by ZNF580 gene complexed with mPEG-b-P(MMD-co-GA)-g-PEI microparticles

Herein, we developed a novel biodegradable gene carrier for rapid endothelialization of endothelial cells (ECs) in vitro. Three triblock amphiphilic copolymers, methoxy-poly(ethylene glycol)-block-poly(3(S)-methyl-2,5-morpholinedione-co-glycolide)-graft-polyethyleneimine (mPEG-b-P(MMD-co-GA)-g-PEI) with different 3(S)-methyl-2,5-morpholinedione and glycolide contents were synthesized. Microparticles (MPs) were obtained via self-assembly of these copolymers. The hydrophobic core composed of P(MMD-co-GA) segments provide crosslinking points for numbers of PEG and short PEI chains to form a highly hydrophilic and positively charged corona/shell of MPs. Using these MPs, potential genes (ZNF580) for rapid endothelialization were efficiently transported into EA.hy926 cells. Because of the hydrophilic PEG chains and low molecular weight PEI in the triblock copolymers, the cytotoxicity of these MPs and their complexes with pEGFP-ZNF580 was decreased significantly. The transfection efficacy of MPs/pEGFP-ZNF580 complexes was as high as Lipofectamine™ 2000 reagent to EA.hy926 cells in vitro. The proliferation and migration of EA.hy926 cells were improved greatly by the expression of pEGFP-ZNF580 after 60 hours. Our results indicated that the mPEG-b-P(MMD-co-GA)-g-PEI based MPs could be a suitable non-viral gene carrier for ZNF580 gene to enhance rapid endothelialization.

Formation and characterization of natural polysaccharide hollow nanocapsules via template layer-by-layer self-assembly.

With natural polysaccharides carrageenan (Car) and chitosan (Cs) as the polyanion and polycation, respectively, multilayer hollow nanocapsules have been fabricated via sequential layer-by-layer (LbL) electrostatic self-assembly from the sacrificed templates nanospheres (SiO(2)-NH(2)). The LbL assembly process with the polysaccharides on SiO(2)-NH(2) core was followed by ζ-potential and size analysis. The fabrication of (Car/Cs)(x) nanocapsules and the removing of the SiO(2)-NH(2) core templates were confirmed by TGA and EDS analysis. The morphology of SiO(2)(Car/Cs)(x) nanospheres and (Car/Cs)(x) nanocapsules were observed by TEM analysis. The size analysis of (Car/Cs)(x) nanocapsules indicated that the cyst wall thickness and cavity volume of the nanocapsules are pH and ionic strength dual responsive. Due to the biocompatibility of the natural polysaccharides carrageenan and chitosan and the responsiveness of nanocapsules to pH and ionic strength, the (Car/Cs)(x) multilayer nanocapsules are expected to be used as nanoreactors or nanocontainers to control the synthesis, encapsulation, and releasing behaviors of bioactive molecules.

A thermoresponsive poly(N-vinylcaprolactam-co-sulfobetaine methacrylate) zwitterionic hydrogel exhibiting switchable anti-biofouling and cytocompatibility

Non-specific protein adsorption adversely affects the application of thermoresponsive polymers in the biomedical field. To overcome this disadvantage, thermoresponsive N-vinylcaprolactam (VCL) and anti-biofouling zwitterionic sulfobetaine methacrylate (SBMA) monomers with various VCL/SBMA ratios were used for the synthesis of poly(VCL-co-SBMA) (P(VCL-co-SBMA)) copolymers via free radical solution polymerization. The P(VCL-co-SBMA) copolymers exhibited both a lower critical solution temperature (LCST) and an upper critical solution temperature (UCST) in aqueous solutions. Furthermore, both the UCST and LCST of the copolymer shift to higher temperatures with the increase of PSBMA segments, and they shift to lower temperatures with the increase of salt concentrations in the solution. Based on these results, P(VCL-co-SBMA) hydrogels were prepared using N,N′-methylenebisacrylamide (MBAA) as the crosslinker. Compared with the PVCL hydrogel, the P(VCL-co-SBMA) hydrogels exhibit better mechanical properties. Notably, the P(VCL-co-SBMA) hydrogel retained the temperature sensitivity of PVCL, and it could be modulated by varying the PVCL/PSBMA segment ratios. In addition, all the hydrogels exhibit good cytocompatibility. More importantly, the protein adsorption and cell adhesion of the hydrogel can be controlled by temperature. The non-specific protein adsorption was effectively suppressed at physiological temperatures. The switchable anti-biofouling nature of P(VCL-co-SBMA) hydrogel together with their temperature sensitivity can be potentially used in drug, cell or enzyme delivery.

Modulation of mesenchymal stem cells behaviors by chitosan/gelatin/pectin network films

In this article, the chitosan/gelatin/pectin (CGP) network films were prepared to build appropriate physicochemical and mechanical microenvironment for attachment, proliferation, and differentiation of mesenchymal stem cells (MSCs). Results suggested that the hydrophilicity and mechanical character of CGP composites films could be modulated via adjusting the pectin content in the composites. The investigations of attachment and proliferation behaviors of mesenchymal stem cells (MSCs) on the CGP films were carried out. The morphology of cells was observed with hematoxylin/eosin staining (HE) and scanning electron microscope (SEM). The osteogenic differentiation of MSCs was investigated via ALP and polymerase chain reaction (PCR). Results suggested that the CGP films have excellent biocompatibility. MSCs seeded on CGP (0.1) film show higher proliferation capacity compared with other samples. Moreover, osteogenic differentiation of MSCs also depends on the properties of the substrate. The MSCs seeded on CGP (0.5) expressed the highest ALP activity, osteogenic gene expression and mineral formation capacity. These results suggest that the composition of the CGP network films could effectively modulate their physicochemical and mechanical properties and further regulate the cell behaviors of MSCs.

Physically crosslinked poly(vinyl alcohol)–carrageenan composite hydrogels: pore structure stability and cell adhesive ability

Poly(vinyl alcohol) (PVA) hydrogels have gained comprehensive attention in the biomedical area. However, their resistance to cell adhesion is a drawback for applications such as tissue engineering. Besides, the controllability of the porous structure of PVA-based hydrogels during lyophilization needs to be further improved. Herein, we prepared PVA–carrageenan (CAR) composite hydrogels as tissue engineering scaffolds using the freeze–thaw technique. The hydrogels were found to possess deformation resistance, preserving their shape during the lyophilization process without shrinkage. Besides, ATDC5 cells showed good adherence and proliferation activity on the composite hydrogels. In addition, the PVA–CAR composite hydrogels possess good hemocompatibility and did not cause any adverse effects in the inflammatory response from RAW 264.7 macrophage cells. Overall, the results obtained indicate that the PVA–CAR composite hydrogels show potential applications in the field of tissue engineering based on their good structural stability, excellent biocompatibility and mild fabrication process.

Physical Cross-Linking Starch-Based Zwitterionic Hydrogel Exhibiting Excellent Biocompatibility, Protein Resistance, and Biodegradability

In this work, a novel starch-based zwitterionic copolymer, starch-graft-poly(sulfobetaine methacrylate) (ST-g-PSBMA), was synthesized via Atom Transfer Radical Polymerization. Starch, which formed the main chain, can be degraded completely in vivo, and the pendent segments of PSBMA endowed the copolymer with excellent protein resistance properties. This ST-g-PSBMA copolymer could self-assemble into a physical hydrogel in normal saline, and studies of the formation mechanism indicated that the generation of the physical hydrogel was driven by electrostatic interactions between PSBMA segments. The obtained hydrogels were subjected to detailed analysis by scanning electron microscopy, swelling ratio, protein resistance, and rheology tests. Toxicity and hemolysis analysis demonstrated that the ST-g-PSBMA hydrogels possess excellent biocompatibility and hemocompatibility. Moreover, the cytokine secretion assays (IL-6, TNF-α, and NO) confirmed that ST-g-PSBMA hydrogels had low potential to trigger the activation of macrophages and were suitable for in vivo biomedical applications. On the basis of these in vitro results, the ST-g-PSBMA hydrogels were implanted in SD rats. The tissue responses to hydrogel implantation and the hydrogel degradation in vivo were determined by histological analysis (Hematoxylin and eosin, Van Gieson, and Massons Trichrome stains). The results presented in this study demonstrate that the physical cross-linking, starch-based zwitterionic hydrogels possess excellent protein resistance, low macrophage-activation properties, and good biocompatibility, and they are a promising candidate for an in vivo biomedical application platform.

Carboxymethyl chitosan-poly(amidoamine) dendrimer core–shell nanoparticles for intracellular lysozyme delivery

Intracellular delivery of native, active proteins is challenging due to the fragility of most proteins. Herein, a novel polymer/protein polyion complex (PIC) nanoparticle with core-shell structure was prepared. Carboxymethyl chitosan-grafted-terminal carboxyl group-poly(amidoamine) (CM-chitosan-PAMAM) dendrimers were synthesized by amidation and saponification reactions. (1)H NMR was used to characterize CM-chitosan-PAMAM dendrimers. The TEM images and results of lysozyme loading efficiency indicated that CM-chitosan-PAMAM dendrimers could self-assemble into core-shell nanoparticles, and lysozyme was efficiently encapsulated inside the core of CM-chitosan-PAMAM dendrimer nanoparticles. Activity of lysozyme was completely inhibited by CM-chitosan-PAMAM Dendrimers at physiological pH, whereas it was released into the medium and exhibited a significant enzymatic activity in an acidic intracellular environment. Moreover, the CM-chitosan-PAMAM dendrimer nanoparticles did not exhibit significant cytotoxicity in the range of concentrations below 3.16 mg/ml. The results indicated that these CM-chitosan-PAMAM dendrimers have excellent properties as highly potent and non-toxic intracellular protein carriers, which would create opportunities for novel applications in protein delivery.

Iota-carrageenan/chitosan/gelatin scaffold for the osteogenic differentiation of adipose-derived MSCs in vitro.

In this study, we have developed ι-carrageenan/chitosan/gelatin (CCG) scaffold containing multiple functional groups (-NH2 , -OH, -COOH, and -SO3 H) to resemble the native extracellular matrix (ECM), using the ion-shielding technology and ultrasonic dispersion method. Fourier transform infrared spectroscopy (FTIR) of the CCG scaffolds suggests that the formation of CCG network involves electrostatic interactions between ι-carrageenan (ι-CA) and chitosan/gelatin, and the covalent cross-linking among amino groups of chitosan and/or gelatin. Scanning electron microscopic (SEM) observation reveals that the porous structure of scaffolds can be modulated by the ratio of ι-CA to chitosan/gelatin. The swelling ratio of the hydrogels increases as the ι-CA contents increase. Using differential scanning calorimetry, we found that the double helix structure of ι-CA is only stabilized at low contents of ι-CA in the CCG scaffolds (e.g., 5 wt %). The scaffolds containing 5% ι-CA showed the best protein adsorption capacity (4.46 ± 0.63 μg protein/mg scaffold) and elastic modulus (5.37 ± 1.03 MPa). In addition, the CCG scaffolds exhibit excellent support for adipose-derived mesenchymal stem cells (ADMSCs) attachment and proliferation, and they can improve the osteogenic differentiation and neovascularization capacities of ADMSCs. Overall, we conclude that the CCG may represent an ideal scaffold material for bone tissue engineering.

Development of Electrically Conductive Double-Network Hydrogels via One-Step Facile Strategy for Cardiac Tissue Engineering.

Cardiac tissue engineering is an effective method to treat the myocardial infarction. However, the lack of electrical conductivity of biomaterials limits their applications. In this work, a homogeneous electronically conductive double network (HEDN) hydrogel via one-step facile strategy is developed, consisting of a rigid/hydrophobic/conductive network of chemical crosslinked poly(thiophene-3-acetic acid) (PTAA) and a flexible/hydrophilic/biocompatible network of photo-crosslinking methacrylated aminated gelatin (MAAG). Results suggest that the swelling, mechanical, and conductive properties of HEDN hydrogel can be modulated via adjusting the ratio of PTAA network to MAAG network. HEDN hydrogel has Youngs moduli ranging from 22.7 to 493.1 kPa, and its conductivity (≈10(-4) S cm(-1)) falls in the range of reported conductivities for native myocardium tissue. To assess their biological activity, the brown adipose-derived stem cells (BADSCs) are seeded on the surface of HEDN hydrogel with or without electrical stimulation. Our data show that the HEDN hydrogel can support the survival and proliferation of BADSCs, and that it can improve the cardiac differentiation efficiency of BADSCs and upregulate the expression of connexin 43. Moreover, electrical stimulation can further improve this effect. Overall, it is concluded that the HEDN hydrogel may represent an ideal scaffold for cardiac tissue engineering.