Tarek A. Elkhooly

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.

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.

Silver nanoparticle based coatings enhance adipogenesis compared to osteogenesis in human mesenchymal stem cells through oxidative stress

Silver nanoparticle (AgNP) based antibacterial surfaces were fabricated using plasma polymerization technology and their effects on differentiation of human bone-marrow derived mesenchymal stem cells (hMSCs) were investigated in this study. The results showed that AgNP coated surfaces do not affect the initial adhesion, spreading and proliferation of hMSCs. Furthermore, the silver coated surface promoted adipogenic differentiation of hMSCs as demonstrated by more accumulation of lipid droplets and upregulation of adipogenesis-related genes such as peroxisome proliferator activated receptor gamma (PPARγ), adipocyte determination and differentiation factor (ADD1) and CCAAT/enhancer binding protein alpha (C/EBPα). In addition, silver incorporation activated the expression of antioxidant enzymes as a consequence of the accumulation of intracellular reactive oxygen species (ROS) in adipogenic induced cells, which was correlated with the enhanced adipogenic capacity of hMSCs. ROS generation was enhanced due to silver ion release and consequently reduced osteogenesis at the early stage after 7 days of osteogenic induction as a result of reducing alkaline phosphatase (ALP) activity. However, the differentiation and mineralization capacity of osteoblasts were restored after 14 days of osteogenic induction, which indicated that adipogenesis favors intracellular ROS accumulation mediated by silver coatings compared to osteogenesis. None of the osteogenic related genes was affected by ROS mediated by AgNP dissolution. The findings in this work are instructive for the use of silver as an antibacterial agent in the areas of tissue engineering, stem cell therapies and implantable biomedical devices.

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.

The osteogenic, inflammatory and osteo-immunomodulatory performances of biomedical Ti-Ta metal–metal composite with Ca- and Si-containing bioceramic coatings

It is known that good mechanical properties, low modulus to reduce stress-shielding effect, favorable osteogenic activity and limited inflammatory response are critical factors for orthopedic implants to induce excellent osteointegration. In this study, Ti-20% Ta metal-metal composite (referred as Ti-Ta) which consisted of Ti- and Ta-rich phases was fabricated via the strategy of powder metallurgy. Micro-arc oxidation (MAO) was employed to modify the surface of Ti-Ta composite. The surfaces of Ti-Ta composite after MAO treatment at an applied voltage of 250 (referred as MAO-250 V) or 300 V (referred as MAO-300 V) exhibited three distinct zones with significantly different morphological features and surface chemistry. Osteoblast-like SaOS-2 cells were found to be preferential to attach on the Ta-rich phase and its surrounding areas, exhibiting an area-dependent adhesion tendency. However, the attachment of Raw 264.7 macrophages was found to be insensitive to the surface characteristics. The proliferation and differentiation of SaOS-2 cells cultured on various surfaces basically followed the trend: MAO-modified surfaces > Ti-Ta surface > Ti surface. The Ti-Ta and MAO-modified surfaces were found to inhibit the inflammatory response and polarize macrophages to anti-inflammatory M2 phenotype compared to Ti surface. Moreover, the microenvironments created by Ti-Ta, MAO-250 V and MAO-300 V/macrophage interactions promoted the proliferation and differentiation of SaOS-2 cells compared to that created by Ti/macrophage interactions. MAO-300 V surface exhibited further enhanced positive osteo-immunomodulatory effects compared to Ti-Ta surface. Together, the Ti-20% Ta metal-metal composite modified by MAO at an applied voltage of 300 V is considered as a promising implant material for orthopedic applications.

A facile way to prepare mesoporous spherical calcites controlled by chondroitin sulfate for shape and carboxymethyl chitosan for size

Inspired by biomineralization, monodispersed spherical calcites coated with a layer of polymer were obtained with the cooperative role of two polymers: carboxymethyl chitosan for size control and chondroitin sulfate for shape control. These calcites can be regarded as mesocrystals aggregated by nanoparticles with a mesoporous structure, and therefore have potential application as drug delivery systems.

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.

Reduced inflammatory response by incorporating magnesium into porous TiO2 coating on titanium substrate

The implant materials with proper anti-inflammatory and osteogenic properties may be promising for orthopedic applications. The inflammatory response induced by biomaterials has been regarded as one of the critical factors in determining in vivo fate of implants. Therefore, a novel bone biomaterial should have inflammation regulatory effects instead of being completely bio-inert. In the present work, the inflammation regulatory effects of exogenous magnesium (Mg) ions were investigated. Under the stimulation of lipopolysaccharide (LPS), macrophages exposed to Mg2+ exhibited down-regulated gene expressions of M1 markers (CD86, CD11c and inducible nitric oxide synthase (iNOS)) and pro-inflammatory cytokines (tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-1β), up-regulated gene expression of M2 marker CD163 and decreased TNF-α release, indicating that Mg2+ could switch macrophages from M1 to M2 phenotype. Thereafter, micro-arc oxidation (MAO) technique was employed to fabricate Mg-containing ceramic coatings on titanium substrates. Macrophages grown on Mg-containing surface were switched from M1 to M2 phenotype with the stimulation of LPS, evidenced by suppressed gene expressions of M1 markers (CD86, CD11c and iNOS) and pro-inflammatory cytokines (TNF-α and IL-1β), promoted gene expression of M2 marker CD163 and decreased TNF-α release. Moreover, gene expressions of bone morphogenetic protein-2 (BMP-2), BMP-6 and vascular endothelial growth factor (VEGF) were up-regulated on Mg incorporated MAO surface without LPS stimulation. Together, Mg could be used as an anti-inflammatory agent for suppressing inflammation and mediating osteogenesis. The integration of Mg in biomaterials could endow bone biomaterials with anti-inflammatory property.