Jun Lin

Light-induced cell detachment for cell sheet technology.

The phenomenon of light-induced cell detachment is reported. Mouse calvaria-derived, pre-osteoblastic (MC3T3-E1) cells were cultured on a TiO(2) nanodot-coated quartz substrate. After 20 min of UV365 illumination, over 90% of the cells would detach from the surface. Moreover, intact cell sheets could be obtained in the same way. It was found that the as-obtained cells showed good viability, and could be used for further culture processes and other applications. Also, biocompatibility and safety characterizations indicated that the use of TiO(2) nanodots and UV365 illumination was safe for such cell detachment. It is suggested that adsorbed extracellular matrix proteins play key roles in developing cell sheets and ensuring biocompatibility. The present light-induced cell detachment method demonstrates a promising way for rapid cell/cell sheet harvesting.

Preparation and antibiotic drug release of mineralized collagen coatings on titanium

In this study, a mineralized collagen coating was electrolytically deposited onto titanium. The results showed that the mineralized collagen coatings with dense or porous morphology could be obtained. The mineral phase was mainly hydroxyapatite. In vitro evaluation showed the mineralized collagen coatings were stable in Kokubo’s simulated body fluid, and displayed a good cytocompatibility in the cell multiplication test. The mineralized collagen coatings loaded with vancomycin hydrochloride showed an inhibitory effect on the growth of S.aureus. The present mineralized collagen coating demonstrates good suitability for surface modification of orthopedic metal implants.

Synthesized silicon-substituted hydroxyapatite coating on titanium substrate by electrochemical deposition

Silicon-substituted hydroxyapaptite (Si-HA) coatings were prepared on titanium substrates by electrolytic deposition technique in electrolytes containing Ca2+, PO43− and SiO32− ions with various SiO32−/(PO43−xa0+xa0SiO32−) molar ratios(ηsi). The deposition was all conducted at a constant voltage of 3.0xa0V, with titanium substrate as cathode and platinum as anode, for 1xa0h at 85°C. The coatings thus prepared were characterized with inductively coupled plasma (ICP), X-ray diffraction (XRD), fourier transform infrared spectroscopy (FTIR), field-emission-type scanning electron microscope (FSEM). The results show that the silicon amount in the coatings increases linearly to about 0.48xa0wt% at first with increasing ηsi between 0 and 0.03, then increases slowly to about 0.55xa0wt% between 0.03 and 0.10 and finally maintains almost at a level around 0.55xa0wt% between 0.10 and 0.30. The tree-like Si-HA crystals are observed in the coatings prepared in the electrolyte of ηsixa0=xa00.20. And the presence of silicon in electrolytes decreases the thickness of the coatings, with effect being more significant as ηsi increased. Additionally, the substitution of Si causes some OH− loss and changes the lattice parameters of hydroxyapatite (HA).

Incorporation of chitosan nanospheres into thin mineralized collagen coatings for improving the antibacterial effect.

It is desired that the coatings on metallic implants have both excellent biological responses and good loading-release capacities of biological factors or drugs. So far, the challenge still remains, because the morphology and composition of the bioactive coatings are usually not favorable for accommodating drug molecules. In this study, we adopted an approach of incorporating chitosan nanospheres into a thin mineralized collagen coating; this approach is based on the good loading-release behavior of the nanospheres and the good cytocompatibility of the thin coating. The incorporation of chitosan nanospheres into the mineralized collagen coatings was realized by electrolytic co-deposition. The morphologies and microstructures of the resulting coatings were characterized by SEM, and the phase and chemical compositions of the coatings were measured by XRD and FTIR. The loading-release capacity for vancomycin hydrochloride (VH) was determined by ultraviolet spectrophotometry. MTS assay was used to evaluate cytocompatibility, and in vitro bacterial adhesion was tested for assessing the antibacterial effects of the VH-loaded coatings. The chitosan nanospheres adhered tightly to collagen fibrils. The incorporated coatings facilitated the sustained release of VH, and had a clear antibacterial effect. The incorporation of chitosan nanospheres into mineralized collagen coatings demonstrates an effective way to improve the drug loading-release capacity for the thin coatings. This formulation had a highly effective biological response.

Surface hydroxyl groups direct cellular response on amorphous and anatase TiO2 nanodots

In this study, we investigated the differences between amorphous and anatase TiO2 at the biomolecular level which could explain differences in the osteoblast response on these surfaces. The number of surface hydroxyl groups in the TiOHT form on amorphous and anatase TiO2 was found to be the most important factor, resulting in adsorption of bovine serum albumin as a monolayer on amorphous TiO2 nanodots but as a multilayer on anatase TiO2 nanodots. The reason for this is that the presence of more TiOHT groups on amorphous TiO2 nanodots attracts more -NH3+ groups on BSA molecules, causing the conformation of surface-bound BSA molecules to differ from those adsorbed on anatase TiO2 nanodots. Fibronectin which is subsequently adsorbed on anatase TiO2 nanodots then retains a more active conformation for osteoblast adhesion and mineralization.

Mineralized collagen coatings formed by electrochemical deposition

Understanding and controlling the process of electrochemical deposition (ECD) of a mineralized collagen coating on metallic orthopedic implants is important for engineering highly bioactive coatings. In this work, the influence of different ECD parameters was investigated. The results showed that the mineralization degree of the coatings increased with deposition time, voltage potential and H2O2 addition, while chitosan addition led to weakening of mineralization, heavy mineralization resulted in a porous coating morphology. Furthermore, two typical coatings, dense and porous, were analyzed to investigate their microstructure and evaluated for their cytocompatibility; the dense coating showed better osteoblast adhesion and proliferation. Based on our understanding of how the different coating parameters influenced the coating, we proposed an ECD process in which the pH gradient near the cathode and the collagen isoelectric point were suggested to play crucial roles in controlling the mineralization and morphology of the coatings. The proposed ECD process may offer a guide for controlled deposition of a desired bioactive coating.

Enhanced loading and controlled release of rhBMP-2 in thin mineralized collagen coatings with the aid of chitosan nanospheres and its biological evaluations

Loading of an appropriate amount of rhBMP-2 and avoiding its burst-release are key challenges for upgrading the biological performance of thin bioactive coatings on metal implants. In this study, we adopted incorporation of chitosan nanospheres into thin mineralized collagen coatings to enhance rhBMP-2 loading and improve releasing behavior based on the good affinity of chitosan for proteins and the large surface area of nanospheres. We realized the incorporation process by electrophoretic injection. Using chitosan nanospheres, we were able to increase the rhBMP-2 loading amount in the thin coating by 2.7-fold (from 446 ng cm-2 to 1186 ng cm-2), and showed that the rhBMP-2 exhibited sustained release behavior. MC3T3-E1 cells cultured on the rhBMP-2/chitosan nanosphere-incorporated thin coatings (Col/Cs/BMP) showed good cell attachment and proliferative behavior, and high levels of differentiation and mineralization. In in vivo tests, spiral CT analysis and histological observations demonstrated that Col/Cs/BMP coatings on metal implants were able to increase bone density and accelerate new bone growth after implantation for 4 to 8 weeks, and the boundary between host bones and new bones disappeared after implantation for 8 weeks. The pull-out tests further confirmed that the Col/Cs/BMP coatings could significantly enhance osseointegration. The present results indicate that incorporation of chitosan nanospheres into thin coatings is an effective way to enhance the loading amount properly, improve the release behavior of rhBMP-2 and finally accelerate the osseointegration process.

Improvement of drug elution in thin mineralized collagen coatings with PLGA‐PEG‐PLGA micelles

A mineralized collagen (MC) coating on metallic implants has shown great potential as orthopedic material due to high biological responses. However, their drug delivery capacity remains unsatisfactory since a serious burst release may occur and long-term release is hard to be achieved. Aiming to improve the drug-eluting capability, we incorporated drug-loaded PLGA-PEG-PLGA (PPP) micelles into the thin coating. The in vitro release profiles showed that the burst release in the initial 8 h of the modified coating decreased from 81% to 58% compared to MC coating alone; meanwhile, the release duration was prolonged from 3 days to 1 week. Additionally, the release kinetics of vancomycin hydrochloride (VH, the model drug) could be adjusted by changing the size and concentration of PPP micelles. Interestingly, less initial release of VH caused by micelle immobilization did not affect the antibacterial activity in the early stage of implantation according to the antimicrobial test. The cytocompatibility assay demonstrated that the VH-loaded PPP micelles did not have negative effect on the bioactivity of coating which greatly enhanced cell activity compared to bare Ti substrates. Thus, the MC coatings with PPP micelles could be an effective implant route for bone repair.

Modulation of protein behavior through light responses of TiO2 nanodots films.

In this work, the behavior of protein molecules adsorbed on TiO2 nanodots films are modulated through the light responses of the nanodots. TiO2 nanodots films are first prepared through phase separation induced self assembly. Then, bovine serum albumin (BSA) is adsorbed on TiO2 nanodots films and exposed to ultraviolet (365u2009nm) illumination. It is found the conformation of surface-bound BSA molecules changes with ultraviolet illumination. Moreover, the BSA molecules conjugate to the surface-bound molecules, which are in the overlayer, are released. The reason is ascribed to that TiO2 nanodots absorb ultraviolet and result in the increase of surface hydroxyl groups on nanodots. Such increase further leads to intensified attraction of -NH3 groups in the surface-bound BSA molecules. That not only changes the conformation of the surface-bound BSA molecules, but also weaken the conjugation between surface-bound molecules and other BSA molecules in the overlayer. Eventually, the overlayer of BSA molecules is released. It is believed that such protein conformation variation and release behavior induced through light responses of TiO2 nanodots are crucial in understanding the biomedical performance of TiO2 nanostructures. Also, it could be widely utilized in tailoring of the materials-protein interactions.