**1. Introduction**

The surface of dental- or bone-implanted objects must commonly be modified to yield a particular surface roughness in order to increase their surface area for osteoblast attachment, and to enhance the bioactive and osteoconductive properties of the underlying substrate. Effective surface treatment methods include sand- or grit-blasting using abrasives, chemical treatments, and the deposition of calcium phosphate (CaP) coatings.

Technological developments have enabled the preparation of nano-scale structures, including anodized aluminum with neat arrays of holes known as porous alumina, which is a biomedical material. Biomedical engineering involves cell culture, biomedical materials, and surface modification. The cell growth is improved by a biomaterial with an effective structure. Numerous scholars are interested in the scale, micro-structure, and nano-structure of biomaterials.

The powder blasting method for hydroxyapatite (HA) was utilized to blast on a pure titanium (Ti) substrate. They found that the content and crystal structure of Ti substrate after blasting were the same as those of pure HA. The bonding strength of Ti substrate after powder blasting was larger than that by the dip coating, electrolysis deposition, and electrochemical deposition [1]. An animal test was performed for pure Ti after surface modification by blasting. The results demonstrated that the thickness of new bone on Ti substrate after being HA blasted exceeded that of pure Ti substrate [2]. A new method was developed for blasting a Ti surface that involved aluminum oxide (Al2O3) and a dopant (HA, fluoro apatite (FA), magnesium apatite (MgA) and carbonate apatite (CO3A)), and a cell culture was performed on that surface. The results indicated the greatest proliferation of cells on the Ti substrate that was blasted by Al2O3 and CO3A particles [3]. The biocompatibility of Ti substrate was discussed on the condition of being treated by HA blasting alone and by HA blasting with Al2O3. The results revealed that the surface roughness of the Ti substrate was greater following Al2O3 treatment and HA blasting. A cell culture revealed that the viability of cells on Ti substrate that was treated with Al2O3 followed by HA blasting exceeded that of the substrate that had undergone only HA blasting. The results also revealed that the growth of laminate bone has good situation on the Ti substrate that was treated with Al2O3 and HA blasting [4]. The antibacterial effectiveness of Ti substrate treated with pure HA particles or HA combined with zinc apatite (ZnA), silver apatite (AgA), or strontium apatite (SrA) particles were evaluated, and it was found that the substrate that was treated with HA and AgA performed best in this respect [5]. The wear and friction of a TiAl6V4 substrate that was combined with Al2O3 and teflon, silicon carbide (SiC), or boron carbide (B4C) by blasting method was investigated [6]. An MG63 cell culture was conducted on a Ti substrate after blasting with HA and sintered CaP particles. The results revealed that surface modification increased cell proliferation on the Ti substrate [7]. A MG63 cell culture was carried out in vitro on the TiAl6V4 following surface modification (using different co-blasting methods). Their results demonstrated that co-blasting with bioglass and HA particles improved the osteoconduction and growth of cells on TiAl6V4. Their results also indicated that co-blasting of the TiAl6V4 substrate yielded a better alkaline phosphatase (ALP) value than the plasma spray method [8,9]. The researchers reviewed 348 papers on MG63 proliferation on Ti and TiAl6V4 substrates that had undergone various methods of surface modification [10]. The nanostructure of substrate affected the adhesion and proliferation of cells in vitro. The results showed that the moderately rough substrates with large fractal dimension could boost cell proliferation [11,12]. The morphology and biocompatibility of polished nitinol (NiTi) and Ti material surfaces treated with a mixed solution of three acids (HCl–HF–H3PO4) were evaluated. The results showed that surfaces treated with HCl–HF–H3PO4 had higher roughness, lower cytotoxicity, and better biocompatibility than controls [13,14]. MG63 cells were seeded on machined pretreated, nano-modified pretreated, sandblasted/acid-etched, and nano-modified sandblasted/acid-etched Ti disks. The results revealed that the nanoscale structures in combination with micro-/submicro-scale roughness improved osteoblast differentiation and local factor production, which indicated the potential for improved implant osseointegration [15,16].

The oxidation of anodic aluminum oxide (AAO) in sulfuric acid, phosphoric acid, or oxalic acid yields anodized porous alumina. Generally, AAO has a highly porous array structure and straight uniform pores. The diameter of pores varies with anodic reaction conditions. Straight nano-channels of AAO are often used to provide a framework for the formation of highly regular nano-structured materials. Porous anodic alumina membranes are formed from metal aluminum in acidic solution by two-step anodization [17–23]. The most commonly used electrolytes are sulfuric acid, oxalic acid, and phosphoric acid solution. In the anodizing process, aluminum is the anode, an electric field is applied, and the surface of the aluminum forms an oxide film. The extent of electrolytic oxidation increases with the voltage. Varying the electrolyte and the electrolysis time yields alumina membranes with pore diameters up to several hundred nanometers, or as small as 5 nm. The hole density up to 1011 holes/cm [24–27] and film thickness from 10 to 100 μm can be obtained. The porous alumina template is by far the most widely used template because it has monodispersed characteristic, it can resist high temperatures, and has high strength. The resulting nanotopography combines ordered nanostructures with widely varying surface energies, providing a unique platform for studying cell–substrate interactions. Human dermal fibroblasts were cultured on these substrates. Surface patterning with nanoscale pillars markedly affected cell morphology, which was independent of surface energy. Cell spreading was significantly reduced on both hydrophobic and hydrophilic surfaces with nanopillars. This analytical result shows that surfaces which resist cell spreading can be fabricated by generating suitable nanoscale topography, without concern for the effect of surface chemistry on hydrophilicity [28–31]. Popat et al. [32] established that the cell activity on AAO exceeded that on pure aluminum. Hoess et al. [33] showed that the filopodia of a HepG2 cell passed through nanoholes with a diameter of 263 nm, favoring cell adhesion and proliferation.

The motivation of this study is to study the cell culture on aluminum templates with various structures for application on dental- or bone-implanted objects. The purpose of this study is to discuss the behaviors of cell culture on the various structures of Al template by different surface modification methods. The authors have developed the mini screw on prosthodontics in Taipei Medical University. The mini screw was used as the aluminum material. The research emphasizes that the surface property of the mini screw (as the implanted object) influences the osseointegration. This investigation concerns cell culture on aluminum templates with flat structures, micro-structures, nano-structures, and micro/nano-structures. This study focuses on the various structures of Al templates for osteoblast-like cells (MG63, human osteosarcoma cell), because these cells (MG63) have different effects on aluminum templates of micro-sized structures formed by micro-powder blasting and on aluminum templates of anodized nanometer-sized structures and on aluminum templates of micro/nano-structures by micro-powder blasting and anodized process. This study emphasizes the surface roughness and surface property (hydrophilic or hydrophobic) on aluminum templates with various structures for cell culture. The purpose of this study is to apply the implanted object for bone or teeth to osseointegration. This can improve the stability of bone or dental implanted objects and decrease the repair time of bone or teeth. The null hypothesis is that the surface modification methods (micro-powder blasting, anodized process, micro-powder blasting + anodized process) only have an effect on the surface properties of the Al template.
