Xing Yang

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.

Surface Chemical Gradient Affects the Differentiation of Human Adipose-Derived Stem Cells via ERK1/2 Signaling Pathway

To understand the role of surface chemistry on cell behavior and the associated molecular mechanisms, we developed and utilized a surface chemical gradient of amine functional groups by carefully adjusting the gas composition of 1,7-octadiene (OD) and allylamine (AA) of the plasma phase above a moving substrate. The chemical gradient surface used in the present work shows an increasing N/C ratio and wettability from the OD side toward the AA side with no change in surface topography. Under standard culture conditions (with serum), human adipose-derived stem cells (hASCs) adhesion and spreading area increased toward the AA side of the gradient. However, there were no differences in cell behavior in the absence of serum. These results, supported by the trends in proteins adsorption on the gradient surface, demonstrated that surface chemistry affects the response of hASCs through cell-adhesive serum proteins, rather than interacting directly with the cells. The expression of p-ERK and the osteogenic differentiation increased toward the AA side of the gradient, while adipogenic differentiation decreased in the same direction; however, when the activation of ERK1/2 was blocked by PD98059, the levels of osteogenic or adipogenic differentiation on different regions of the chemical gradient were the same. This indicates that ERK1/2 may be an important downstream signaling pathway of surface chemistry directed stem cell fate.

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.

The stimulatory effect of silica nanoparticles on osteogenic differentiation of human mesenchymal stem cells

Silica-based materials with favourable biocompatibility are generally considered as excellent candidates for applications in biomedical fields. However, previous researches mainly focused on the safety of silica-based materials, their effects on osteogenic differentiation of human mesenchymal stem cells (hMSCs) still need further investigations. In this study, core-shell fluorescent silica nanoparticles (silica NPs) with three different sizes (S1 ~ 50 nm, S2 ~ 200 nm, S3 ~ 400 nm, respectively) were prepared according to the Stöber method. The silica NPs with different sizes did not affect the cell viability (even up to a concentration of 500 µg ml-1), showing size- and dose-independent cytocompatibility of silica NPs on hMSCs. Uptake of silica NPs significantly enhanced the activity of alkaline phosphatase (ALP) and the formation of bone-like nodules of hMSCs after osteogenic induction. At the concentration of 10 µg ml-1, after treating hMSCs with larger sized silica NPs (S2 and S3), higher ALP activity of hMSCs was measured and larger sized bone-like nodules were formed by hMSCs compared with that treated with smaller sized silica NPs (S1).The enhanced osteogenic potential of hMSCs treated with silica NPs may be attributed to the Si released from silica NPs due to the lysosomal degradation inside hMSCs. These results demonstrate the stimulatory effect of silica NPs on osteogenic differentiation of hMSCs and the application potential of silica NPs in bone tissue engineering.

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.

The negative effect of silica nanoparticles on adipogenic differentiation of human mesenchymal stem cells

Nanoparticles have drawn much attention for a wide variety of applications in biomedical and bioengineering fields. The combined use of nanoparticles and human mesenchymal stem cells (hMSCs) in tissue engineering and regenerative medicine requires more knowledge of the influence of nanoparticles on cell viability and differentiation potential of hMSCs. The objective of this study is to investigate the in vitro uptake of silica nanoparticles (silica NPs) and their effect on adipogenic differentiation of hMSCs. After exposure of hMSCs to silica NPs, the uptake and localization of silica NPs were assessed using transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM). The adipogenic differentiation potential of hMSCs was examined by analyzing the formation and accumulation of lipids droplets, triglyceride (TG) content and the expression of adipogenic marker genes/proteins. The results showed that silica NPs did not affect the cell viability but significantly decreased the differentiation of hMSCs to adipocytes. These findings improve the understanding of the influence of silica NPs on adipogenic differentiation of hMSCs and will provide a reference for the applications of silica NPs in biomedical and bioengineering fields.

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.

The effect of hydroxyapatite nanoparticles on adipogenic differentiation of human mesenchymal stem cells: EFFECT OF nHA ON ADIPOGENIC DIFFERENTIATION OF hMSCs

Due to its excellent biocompatibility, nanosized hydroxyapatite (nHA) has drawn much attention for various applications in biomedical fields. There are growing concerns about its biosecurity; however, little is known about its effects on adipogenesis. In the present study, nHA with three different sizes were synthesized, and the in vitro effects of nHA on cell proliferation and adipogenic differentiation of human mesenchymal stem cells (hMSCs) were investigated. The results clearly show that nHA does not affect the cell viability, the lipids droplets formation, triglyceride (TG) synthesis, and the expression of adipogenic marker genes/proteins of hMSCs at concentrations lower than 50 μg/mL. It is concluded that the adipogenic differentiation potential of hMSCs is not affected by nHA at noncytotoxic concentrations. These will provide a reference for the applications of nHA in biomedical fields.

In Vitro Uptake of Hydroxyapatite Nanoparticles and Their Effect on Osteogenic Differentiation of Human Mesenchymal Stem Cells

There have been many applications in biomedical fields based on hydroxyapatite nanoparticles (HA NPs) over the past decades. However, the biocompatibility of HANPs is affected by exposure dose, particle size, and the way of contact with cells. The objective of this study is to investigate the effect of HA NPs with different sizes on osteogenesis using human mesenchymal stem cells (hMSCs). Three different-sized HA NPs (~50, ~100, and ~150 nm, resp.) were synthesized to study the cytotoxicity, cellular uptake, and effect on osteogenic differentiation of hMSCs. The results clearly showed that each size of HA NPs had dose-dependent cytotoxicity on hMSCs. It was found that HA NPs could be uptaken into hMSCs. The osteogenic differentiation of hMSCs was evaluated through alkaline phosphatase (ALP) activity measurement, ALP staining, immunofluorescent staining for osteopontin (OPN), and real-time polymerase chain reaction (RT-PCR) examination. As expected, HA NPs of all sizes could promote the differentiation of hMSCs towards osteoblast lineage. Among the three sizes, smaller-sized HA NPs (~50 and ~100 nm) appeared to be more effective in stimulating osteogenic differentiation of hMSCs.

Incorporation of silica nanoparticles to PLGA electrospun fibers for osteogenic differentiation of human osteoblast-like cells

Abstract The development of bone tissue engineering scaffolds still remains a challenging field, although various biomaterials have been developed for this purpose. Electrospinning is a promising approach to fabricate nanofibers with an interconnected porous structure, which can support cell adhesion, guide cell proliferation and regulate cell differentiation. The aim of this study is to fabricate composite fibers composed of poly(lactic-co-glycolic acid) (PLGA) and silica nanoparticles (NPs) via electrospinning and investigate the effect of PLGA/SiO2 composite fibers on the cellular response of osteoblast-like cells (SaOS-2 cells). SEM and EDX analysis showed that silica NPs were homogenously dispersed in the composite fibers. The mechanical behavior of the fibers showed that silica NPs acted as reinforcements at concentrations of 2.5 and 5 mg/ml. The incorporation of silica NPs led to enhancement of cell attachment and spreading on PLGA/SiO2 composite fibers. SaOS-2 cells cultured on PLGA/SiO2 composite fibers exhibited increased alkaline phosphatase activity, collagen secretion and bone nodules formation. The bone nodules formation of SaOS-2 cells increased along with the amount of incorporated silica NPs. The present findings indicate that PLGA/SiO2 composite fibers can stimulate osteogenic differentiation of SaOS-2 cells and may be a promising candidate scaffold for bone tissue engineering.