Recombinant human gelatin substitute with photoreactive properties for cell culture and tissue engineering.

The human recombinant collagen I α1 chain monomer (rh-gelatin) was modified by the incorporation of an azidophenyl group to prepare photoreactive human gelatin (Az-rh-gelatin), with approximately 90% of the lysine residues conjugated with azidobenzoic acid. Slight changes in conformation (circular dichroism spectra) and thermal properties (gelation and melting points) were noticed after modification. Ultraviolet (UV) irradiation could immobilize the Az-rh-gelatin on polymer surfaces, such as polystyrene and polytetrafluoroethylene. Az-rh-gelatin was stably retained on the polymer surfaces, while unmodified gelatin was mostly lost by brief washing. Human mesenchymal cells grew more efficiently on the immobilized surface than on the coated surface. The immobilized Az-rh-gelatin on the polymer surfaces was able to capture engineered growth factors with collagen affinity, and the bound growth factors stimulated the growth of cells dose-dependently. It was also possible to immobilize Az-rh-gelatin in micropatterns (stripe, grid, and so on) using photomasks, and the cells grew according to the patterns. These results suggest that the photoreactive human gelatin, in combination with collagen-binding growth factors, will be clinically useful for surface modification of synthetic materials for cell culture systems and tissue engineering.

Photo-immobilization of proteins on carbons.

The photofunctionalization of three different carbons with two proteins was studied at room temperature. Water solutions of bovine serum albumin, BSA, and α-amylase, AA, were photolyzed at 21 °C in the presence of graphite microparticles (6.20 µm), MPG, graphene oxide, MPGO, and graphene oxide modified with SO2, mMPGO. The insertion of BSA on carbon matrixes occurred with a deoxygenation reaction, most likely due to a dehydration step of a water molecule. XPS, TOC and TGA, showed that the BSA photo-insertion on MPG was highly efficient with 34.9% of the weight of MPG after photolysis, with an initial concentration of 1 g∙L-1 of BSA. A high yield of AA photoinsertion on the carbons was also obtained. The calculated weight of AA inserted on MPG and MPGO after photolysis was 22.30% and 18.08%, respectively, with respect to the initial weight of carbon, when the initial concentration of AA was 60 mg∙L-1. AA immobilized on MPG was active while the enzyme on MPGO showed a smaller activity, within the experimental error. Although a certain extent of denaturalization of both proteins was observed during photolysis, the molecular weight and composition changed very little during the photolysis, which would produce mainly conformational changes and isomerization reactions.

Anti-biofouling and functionalizable bioinspired chitosan-based hydrogel coating via surface photo-immobilization.

Zwitterionic polymer is a new generation of anti-fouling materials with its good resistance to protein and bacterial adhesion. Constructing the anti-fouling surfaces with zwitterionic polymer has been regarded as an effective approach for improving the biocompatibility and biofunctionality of clinic devices. Herein, we reported a facile approach to construct a biodegradable anti-biofouling and functionalizable hydrogel coating via photo-immobilization using commercial polyethylene terephthalate (PET) films as the substrate, based on zwitterionic glycidyl methacrylate-phosphorylcholine-chitosan (PCCs-GMA). The surface structure and physicochemical properties of zwitterionic PCCs-GMA hydrogel coating were investigated by X-ray photoelectron spectroscopy (XPS), atomic force microscope (AFM) and static water contact angle measurement, and its functionalizable sites were detected by fluorescence labeling. Compared with the pristine PET and cationic chitosan - GMA and hydroxypropyltrimethyl ammonium chloride chitosan (HTCC) - GMA hydrogel coatings, zwitterionic PCCs-GMA hydrogel coating exhibited excellent biocompatibility, and significantly reduced protein adsorption for three model proteins of fibrinogen, immunoglobulin and lysozyme, repelled platelet adhesion, as well as showed a high resistance to bacterial attachment of Escherichia coli and Staphylococcus aureus and superior anti-fouling properties to MRC-5 cells. The results indicated that photo-immobilized zwitterionic PCCs-GMA hydrogel coating has perspective as a dual functional platform with integrated antifouling and further biofunctional properties for various biomedical applications.

Patterned Polyvinyl Alcohol Hydrogel Dressings with Stem Cells Seeded for Wound Healing.

Polyvinyl alcohol (PVA) hydrogel and stem cell therapy have been widely used in wound healing. However, the lack of bioactivity for PVA and security of stem therapy limited their application. In this study, an adipose-derived stem cells (ADSCs)-seeded PVA dressing (ADSCs/PVA) was prepared for wound healing. One side of the PVA dressing was modified with photo-reactive gelatin (Az-Gel) via ultraviolet (UV) irradiation (Az-Gel@PVA), and thus ADSCs could adhere, proliferate on the PVA dressings and keep the other side of the dressings without adhering to the wound. The structure and mechanics of Az-Gel@PVA were determined by scanning electron microscopy (SEM) and material testing instruments. Then, the adhesion and proliferation of ADSCs were observed via cell counts and live-dead staining. Finally, in vitro and in vivo experiments were utilized to confirm the effect of ADSCs/PVA dressing for wound healing. The results showed that Az-Gel was immobilized on the PVA and showed little effect on the mechanical properties of PVA hydrogels. The surface-modified PVA could facilitate ADSCs adhesion and proliferation. Protein released tests indicated that the bioactive factors secreted from ADSCs could penetrated to the wound. Finally, in vitro and in vivo experiments both suggested the ADSCs/PVA could promote the wound healing via secreting bioactive factors from ADSCs. It was speculated that the ADSCs/PVA dressing could not only promote the wound healing, but also provide a new way for the safe application of stem cells, which would be of great potential for skin tissue engineering.

Preparation of azidophenyl-low molecular chitosan derivative micro particles for enhance drug delivery.

Systems for delivering damaged tissue by immobilization of a bioactive substance or a protein drug for rapid recovery of a patient are being studied. To immobilize drugs using natural polymer, photo-immobilization method has been designed. Immobilization through photo-reaction is a new technology that stabilizes drugs or growth factors for sustained release. Introduction of photo-reactive functional groups into biocompatible natural polymers produces materials applicable to the medical field. Since chitosan is a natural polymer with stability and biocompatibility, this study attempts to use chitosan as a mediator of drug delivery. In addition, If the form of the immobilized biomaterial is made into a micro-sized particle, it can be utilized as an injectable material in addition to the stability of the photo-immobilization. In photo-immobilization in particle form, the probability of exposure to the enzyme in the body is lower than if it is injected into the body in the conventional free state. In addition, since it can be freely injected into a desired target site, it can be used for various medical applications. Therefore, it is expected that various effects of growth factors and drugs can be utilized and additional effects can be obtained by photo-immobilization together with various effects.

Visible and UV-curable chitosan derivatives for immobilization of biomolecules.

Chitosan, which has many biocompatible properties, is used widely in medical field like wound healing, drug delivery and so on. Chitosan could be used as a biomaterial to immobilize protein-drug. There are many methods to immobilize protein-drug, but they have some drawbacks such as low efficiency and denaturation of protein. Therefore, photo-immobilization method is suggested to immobilize protein-drug. Photo-immobilization method is simple-reaction and also needs no additional crosslinking reagent. There has been some effort to modify chitosan to have an ability of photo-immobilization. Generally, visible and UV light reactive chitosan derivatives were prepared. Various types of photo-curable chitosan derivatives showed possibility for application to medical field. For example, they showed ability for protein-immobilization and some of them showed wound-healing effect, anti-adhesive effect, or property to interact directly with titanium surface. In this study, we introduce many types of photo-curable chitosan derivative and their possibility of medical application.

Preparation and characterization of latex films photo-immobilized with IFN-α.

We developed a biomaterial by photo-immobilizing interferon-α (IFN-α) on the surface of latex condom films for the prevention and treatment of cervicitis, cervical cancers and diseases caused by cervical virus. The IFN-α modification by photoactive N-(4-azidobenzoyloxy) succinimide was characterized on a nano-scale by spectroscopy analysis and micro morphology. The anti-bacterial, anti-cancer, and anti-viral effects of the modified bioactive latex films were evaluated by antibacterial susceptibility testing, Gram staining, flow cytometry, immunofluorescence, and Western blotting. Our results showed that the photo-immobilized IFN-α latex films effectively inhibited the growth of both Neisseria gonorrhoeae and human cervical cancer HeLa cells. Moreover, the expression of anti-viral proteins, including P56, MxA, and 2', 5'-OAS, in the human cervical epithelial cell line NC104 was significantly increased by photo-immobilized IFN-α latex films. Taken together, these results suggest that photo-immobilized IFN-α latex films may have therapeutic effects against cervicitis, cervical cancers, and cervical virus.