Zhijian Shen

Effects of hierarchical micro/nano-topographies on the morphology, proliferation and differentiation of osteoblast-like cells

Coating the surfaces of titanium-based implants with appropriate hierarchical micro/nano-topographies resembling the structure of natural bone significantly enhances their biological performance. However, the relationship between nanostructures surfaces and their effects on modulating cellular response is not clearly understood. Moreover, it is not clear whether the surface chemistry or topography is the main factor on modulating cellular behavior, because the commonly used surface modification techniques for titanium-based implants simultaneously modify surface topography and chemistry. The aim of this study is to investigate osteoblast-like cell adhesion, proliferation and differentiation on hierarchical micro/nano-topographies with similar surface chemistry but different nano-scale features. Micro-arc oxidation and post hydrothermal treatment were employed to fabricate micro/nano-topographies on titanium. According to the morphological features, they were classified as microcrater (micro-topography), nanoplate (hierarchical topography with nanoplates) and nanoleaf (hierarchical topography with nanoleaves). The response of osteoblast like cells (SaOS-2) was studied on each surface after sputtering with a thin layer of gold (Au) to minimize the influence of surface chemistry. The morphological evaluation after histochemical staining revealed that the adherent cells were polygonal-shaped on microcrater surface, roundish on nanoplate surface and elongated on nanoleaf surface. Additionally, compared to microcrater surface, nanoplate surface slowed down cell proliferation and exhibited no enhancement on cell differentiation. However, nanoleaf surface supported cell proliferation and promoted cell differentiation. The results indicate that tuning morphological features of nanostructures on micro-topography can serve as a promising strategy to specifically modulate cellular response, such as cell morphology, proliferation, differentiation and mineralization.

A novel titania/calcium silicate hydrate hierarchical coating on titanium.

Recently, surface micron/nano-topographical modifications have attracted a great deal of attention because it is capable of mimicking the hierarchical characteristics of bone. In the current work, a novel titania/calcium silicate hydrate (CSH) bi-layer coating with hierarchical surface topography was successfully prepared on titanium substrate through micro-arc oxidation (MAO) and subsequent hydrothermal treatment (HT). MAO treatment could lead to a micron-scale topographical surface with numerous crater-like protuberances. The subsequent HT process enables the in situ nucleation and growth of CSH nanoplates on MAO-fabricated titania surface. The nucleation of CSH nanoplates is considered to follow a dissolution-precipitation mechanism. Compared to MAO-fabricated coating with single-scale surface topography, MAO-HT-fabricated coating with hierarchical surface topography exhibits enhanced hydrophilicity, fibronectin adsorption and initial MG-63 cell attachment. The process of cell-material interactions is considered to be triggered by surface properties of the coated layer and indirectly mediated by protein adsorption on coating surface. These results suggest that MAO-HT treatment is an efficient way to prepare coatings with hierarchical surface topography on titanium surface, which is essential for altering protein adsorption and initial cell attachment.

Specific heat treatment of selective laser melted Ti–6Al–4V for biomedical applications

The ductility of as-fabricated Ti–6Al–4V falls far short of the requirements for biomedical titanium alloy implants and the heat treatment remains the only applicable option for improvement of their mechanical properties. In the present study, the decomposition of as-fabricated martensite was investigated to provide a general understanding on the kinetics of its phase transformation. The decomposition of asfabricated martensite was found to be slower than that of water-quenched martensite. It indicates that specific heat treatment strategy is needed to be explored for as-fabricated Ti–6Al–4V. Three strategies of heat treatment were proposed based on different phase transformation mechanisms and classified as subtransus treatment, supersolvus treatment and mixed treatment. These specific heat treatments were conducted on selective laser melted samples to investigate the evolutions of microstructure and mechanical properties. The subtransus treatment leaded to a basket-weave structure without changing the morphology of columnar prior β grains. The supersolvus treatment resulted in a lamellar structure and equiaxed β grains. The mixed treatment yielded a microstructure that combines both features of the subtransus treatment and supersolvus treatment. The subtransus treatment is found to be the best choice among these three strategies for as-fabricated Ti–6Al–4V to be used as biomedical implants.

Preparation and characterization of TiO2/silicate hierarchical coating on titanium surface for biomedical applications.

In the current work, TiO2/silicate hierarchical coatings with various nanostructure morphologies were successfully prepared on titanium substrates through micro-arc oxidation (MAO) and subsequent hydrothermal treatment (HT). Moreover, the nucleation mechanism and growth behavior of the nanostructures, hydrophilicity, protein adsorption and apatite-inducing ability of various coatings were also explored. The novel TiO2/silicate hierarchical coatings comprised calcium silicate hydrate (CSH) as an outer-layer and TiO2 matrix as an inner-layer. According to the morphological features, the nanostructures were classified as nanorod, nanoplate and nanoleaf. The morphology, degree of crystallinity and crystalline phases of CSH nanostructures could be controlled by optimizing the HT conditions. The nucleation of CSH nanostructures is caused by release and re-precipitation mechanism. The TiO2/CSH hierarchical coatings exhibited some enhanced physical and biological performances compared to MAO-fabricated coating. The improvement of the hydrophilicity, fibronectin adsorption and apatite-inducing ability was found to be morphological dependent according to the following trend: nanoleaf coating>nanoplate coating>nanorod coating>MAO coating. The results indicate that the tuning of physical and morphological properties of nanostructures coated on biomaterial surface could significantly influence the hydrophilicity, protein adsorption and in vitro bioactivity of biomaterial.

SaOS-2 cell response to macro-porous boron-incorporated TiO2 coating prepared by micro-arc oxidation on titanium.

The aims of the present study were to develop boron-incorporated TiO2 coating (B-TiO2 coating) through micro-arc oxidation (MAO) and subsequently evaluate the effect of boron incorporation on the in vitro biological performance of the coatings. The physicochemical properties of B-TiO2 coating and its response to osteoblast like cells (SaOS-2) were investigated compared to the control group without boron (TiO2 coating). The morphological and X-ray diffraction results showed that both coatings exhibited similar surface topography and phase composition, respectively. However, the incorporation of B led to an enhancement in the surface hydrophilicity of B-TiO2 coating. The spreading of SaOS-2 cells on B-TiO2 coating was faster than that on TiO2 coating. The proliferation rate of SaOS-2 cells cultured on B-TiO2 decreased after 5days of culture compared to that on TiO2 coating. SaOS-2 cells cultured on B-TiO2 coating exhibited an enhanced alkaline phosphatase (ALP) activity, Collagen I synthesis and in vitro mineralization compared to those on TiO2 coating. The present findings suggest that B-TiO2 coating is a promising candidate surface for orthopedic implants.

A dual-layer macro/mesoporous structured TiO 2 surface improves the initial adhesion of osteoblast-like cells

A dual-layer TiO2 surface with hierarchical macro and mesoporous structure was prepared by a combinational approach of micro-arc oxidation followed by evaporation-induced self-assembly of nano-crystallites. The mesoporous layer contains pores with an average size of <10nm and consists of anatase TiO2 nanocrystallites. The dual-layer hierarchical macro/mesoporous structured TiO2 surface improves the hydrophilicity and fibronectin adsorption ability in comparison with the sole macroporous or smooth TiO2 surface. With the formation of an additional mesoporous layer on macroporous TiO2 surface, the attached number of human osteogenic sarcoma cells (SaOS-2) increases in the initial incubation of 4h but it does not show significant difference after 24h compared to that attached on the macroporous or smooth surfaces. Whereas, it was noticed that SaOS-2 cells have larger spread area and more stress fibers on the macro/mesoporous structured surface than those on the other surfaces. To understand the intracellular mechanism of the initial cell adhesion on the macro/mesoporous surface, the Rho/ROCK pathway was investigated to reveal the topography-induced biological functions by introducing the ROCK inhibitor Y-27632 during cell culture. In the presence of Y-27632, cells on the macroporous surface and macro/mesoporous surface both show stellate appearance, with poor assembly stress fibers and long cell membrane protrusions. Cells on the smooth surface have larger spread areas compared to the former two surfaces. And the attached cells significantly reduced but there are no differences among the three surfaces. It reveals that the ROCK inhibitor invalidates the promotion of initial cell adhesion on the macro/mesoporous structure. This study may shed light on the mechanism behind the enhancement effect of macro/mesoporous structure for initial cell adhesion.

Effects of the hierarchical macro/mesoporous structure on the osteoblast-like cell response: EFFECTS OF THE HIERARCHICAL MACRO/MESOPOROUS STRUCTURE

To improve the success of medical devices, implants with strong surface bioactivity are urgently required. Coatings with a macroporous structure produced by micro-arc oxidation possess advantages, such as strong adhesion to substrate and excellent resistance to wear and corrosion. Mesoporous structures contain pores with sizes of 2-50 nm, which can endow the biomaterials with the ability to enhance osteogenesis and to be loaded with diverse drugs. Thus, in this study, we aimed to evaluate the effects of both macroporous and mesoporous structures using a hierarchical macro/mesoporous structure to modify the titanium implant surface. The behaviors of SaOS-2 human osteosarcoma cells on the macro/mesoporous structure, including initial adhesion, proliferation, alkaline phosphatase (ALP) activity, and collagen secretion, were investigated. Cells that attached on the macro/mesoporous surface showed the highest cell numbers and greatest spreading area after incubation for 1, 2, and 4 h compared with the polished smooth substrate and macroporous surface in the presence of fetal bovine serum (FBS). However, in the absence of FBS, cell adhesion on the polished substrate, macroporous structure, and macro/mesoporous structure did not differ significantly. Cell proliferation on the macroporous and macro/mesoporous surfaces increased compared with that on the smooth substrate surface. Furthermore, ALP activity and collagen secretion were enhanced on the macro/mesoporous structure. Our findings provided important insights into the cellular responses to macro/mesoporous structures in the field of implant surface modification.

The immunomodulatory effects of Zn-incorporated micro/nanostructured coating in inducing osteogenesis

Abstract Micro/nanostructured TiO2/ZnO coating has been shown to possess multiple functions, including antibacterial activity and bioactivity. Osteoblast-like SaOS-2 cells were employed for evaluating the in vitro osteogenic capacity of this coating and positive results were obtained. However, traditional principles of osseointegration focus only on the osteogenic differentiation alone. The effects of immunomodulation on the osteogenic activity have been largely ignored. In this study, the inflammatory responses of macrophages on the micro/nanostructured TiO2/ZnO coating were investigated. The extract media of macrophage cell line RAW264.7 cultured on the TiO2/ZnO coating were collected as indirect co-culture conditioned media. The osteogenic activity of SaOS-2 cells in the conditioned media was investigated. Adhesion, ALP activity and extracellular mineralization of cells grown in the conditioned media extracted from the micro/nanostructured TiO2/ZnO coating were found to be enhanced, compared to those grown in the conditioned media extracted from the macroporous TiO2 coating. The immune microenvironment produced by the micro/nanostructured TiO2/ZnO coating showed excellent capacity to promote osteogenesis, indicating that this coating could be a promising candidate for implant surface modification in orthopaedic and dental applications. Furthermore, this work could help us understand the interplay between the host immune system and the osteoimmunomodulatory properties of the biomaterials, and optimize the design for coating biomaterials.