MOLECULAR & CELLULAR TOXICOLOGY, Vol. 3, No. 1, 36-45, March 2007 Gene Expression of Osteosarcoma Cells on Various Coated Titanium Materials Sung-Hwa Sohn1, Jae Bun Lee2, Ki-Nam Kim1, are made with Ti alloys. However, the exact effect of In Kyoung Kim1, Seung Ho Lee1, Hye Won Kim1, Ti on osteoblasts is still unknown2-4. Successful ap- Sang-Hui Seo1, Yu-Ri Kim1, Sang-Wan Shin2, plication of such materials for bone regeneration Jae-Jun Ryu2 & Meyoung-Kon Kim1 often involved mixing with autogenous bone, a sour- ce of osteoblastic cells and precursors2. 1Department of Biochemistry & Molecular Biology Surface topography may affect the formation of a 2Department of Dentistry, College of Medicine, Korea University, fibrous capsule around implants, inflammatory res- Seoul 136-705, Korea ponse at tissue-implant interface, fibroblast attach- Correspondence and requests for materials should be addressed ment, angiogenesis, epithelial down-growth around to M-K. Kim ([email protected]) percutaneous devices, and many cellular processes such as cellular differentiation, transcription, cell Accepted 8 March 2007 metabolism, protein production, and phenotypic ex- pression1,3,5-7. Diverse implant surface may contribute to the regulation of osteoblast differentiation by infl- Abstract uencing the level of gene expression of key osteo- genic factors7,8. Morphometric analyses had shown Several features of the implant surface, such as differences in bone-implant contact percentages with topography, roughness, and composition play a the varying of surface characteristics, as well as a relevant role in implant integration with bone. This sensitivity of cells to surface topography9,10. Gene ex- study was conducted in order to determine the pression in response to the placement of implants effects of different-coatings on Ti surfaces on the with different surface topographies11-16. Biomaterial biological responses of a human osteoblast-like cell composition and surface topography regulate cell line (MG63). MG63 cells were cultured on HA (Hyd- attachment, focal contact formation and cytoskeletal roxyapatite coating on Titanium), Ano (HA coating on organization with long-term effects on osteoblastic anodized surface Titanium), Zr (zirconium-coating on 17 Titanium), and control (non-coating on Titanium). cell maturation, and subsequent mineralization . The morphology of these cells was assessed by We hypothesized that different-coatings on Ti sur- SEM. The cDNAs prepared from the total RNAs of face conditions would be associated with differential the MG63 were hybridized into a human cDNA bone-matrix gene expression and interfacial streng- microarray (1,152 elements). The appearances of the ths, which may lead to the development of more ad- surfaces observed by SEM were different on each of vanced therapeutic prosthetic interventions associated the three dental substrate types. MG63 cells cultur- with dental implant therapy and tissue-engineering ed on HA, Ano, Zr, and control exhibited cell-matrix biological applications. interactions. In the expression of several genes were Scanning electron microscopic (SEM) examina- up-, and down-regulated on the different surfaces. tions revealed morphologic differences on Ti surfaces The attachment and expression of key osteogenic (Table 1). Figure 1 is scatter plot for comparing the regulatory genes were enhanced by the surface expression profiles of different-coatings on Ti and morphology of the dental materials used. control. Regeneration analysis of Z scores from two independent samples of different ceramic-coatings on Keywords: Titanium, HA coating, Gene expression Ti and control were performed and Z scores of profiling, cDNA microarray individual genes were plotted. To obtain a molecular portrait of the relationships between the metabolisms Titanium (Ti) and its alloys have been widely used associated with experimental group, we used a hie- for implants that interact with bone cells in vitro and rarchical clustering algorithm to group genes on the in vivo1. For decades, maxillofacial, and orthopedic basis of similar expression patterns and the data is surgeons have placed implants, screws and plates, presented in a matrix format (Figure 2). Each row of and prostheses to substitute lost teeth, to fix bone Figure 2 represents the hybridization results for a fragments, and to replace joints, respectively. Also, single DNA element of the array and each column many surgical instruments, such as drills and saws, represents the expression levels for all genes in a Biological Profiles in HA, Ano, and Zr Titanium 37 Table 1. The surface morphology & characteristics. 6 (a) Coating Surface morphology 5 4 3 Non-coating HA 2 Control on Titanium 1 0 -20 2 4 6 -1 -2 HA coating on Control HA Titanium 8 (b) 7 6 5 ANO HA coating on anodized 4 surface Titamium 3 Ano 2 1 0 -20-1 2 4 6 Zr Zrconium coating -2 on Titanium Control 7 *Magnification 3000×, Ti surface morphology using a scanning (c) 6 electron microscopy (SEM). 5 4 3 single hybridization sample. The expression level of Zr each gene was visualized in color, relative to its me- 2 dian expression level across all samples. Red repre- 1 sented expression greater than the mean, green re- 0 -20 2 4 6 presents expression less than the mean and color -1 intensity denotes the degree of deviation from the -2 mean. Gray represented the median expression level. Control Distinct samples representing similar gene patterns from control cells were aligned in adjacent rows. The Figure 1. Scatter plots for comparison of expression profile between control and different coating on Ti surface. Ex- cells included in this map were samples from right pression profiles of control (Non-coating on Titanium) versus experimental and control group. Coordinately expre- different coating on Ti surface on MG63 cells. (a) HA (Hyd- ssed genes were grouped into clusters, which were roxyapatite coating on Titanium) on MG63 cells versus named on the basis of the cellular process in which control; (b) Ano (HA coating on anodized surface Titamium) the component genes participated in. The clustergram on MG63 cells versus control; (c) Zr (Zrconium coating on revealed that clusters of genes related to progression Titanium) on MG63 cells versus control are shown as bi- variate scatter plots of 1,152 genes from the microarray. The were up- and down-regulated in experimental group, values are corrected intensities relative to control, represent- as compared to control group (Figure 2). Table 2 ing levels of expression for the DNA elements of the micro- shows the expression of various genes in different Ti arrays. surfaces compared with the control group. effects of different metals require in vitro and in vivo Discussion biological tests of any medical or dental device be- fore its definite use in humans. Biological testing of The frequently observed unwanted biological medical and dental devices is necessary in order to 38 Mol. Cell. Toxicol. Vol. 3(1), 36-45, 2007 Figure 2. Molecular portrait of different Ti surfaces on MG63 cells. Cells were treated with different Ti for 72 h. Microarray data from controls (Non-coating on Titanium) and HA (Hydroxyapatite coating on Titanium), Ano (HA coating on anodized surface Titamium), or Zr (Zrconium coating on Titanium) treated cells were combined and clustered. Cluster analysis was performed on Z-transformed microarray data using two separate programs available as shareware from Michael Eisens’ lab. Each gene is represented by a single row of colored boxes; each experimental sample is represented by a single column. The entire clustered image is shown on the left. Full gene names are shown for coordinately expressed clusters, containing genes involved in osteointegration. evaluate the biological behaviour of biomaterials. cultures, several genes were up-regulated or down- Osteoblastic cells began to secrete several extracellu- regulated (Figure 2). These genes were categorized lar matrix (ECM) proteins. They will also attach on depending on their functions, as follows (Table 2). the implant surface and are necessary for adhesion The bone morphogenetic proteins (BMPs) are a due to their specific binding to cell surface receptors. family of secreted signaling molecules that can in- The formation of cell attachment to the alloys seems duce ectopic bone growth. Many BMPs are part of to be slower than on pure Ti. A reasonable explana- the transforming growth factor-beta (TGFB) super- tion for this observation is that the formation of cell- family. BMPs were originally identified by an ability implant contacts may be hampered on rough sur- of demineralized bone extract to induce endochondral faces18,19 while Carinci et al.20 and Lossdorfer et al.21 osteogenesis in vivo in an extraskeletal site. Based on demonstrated that surface roughness affects prolifera- its expression early in embryogenesis, the BMP en- tion, differentiation, local factor production. And coded by this gene has a proposed role in early devel- alkaline phosphatase, osteocalcin, and Transforming opment. In addition, the fact that this BMP is closely Growth Factor beta were increased on the rougher related to BMP5 and BMP7 has led to speculation of surfaces. We also attempted to determine the effects possible bone inductive activity22,23. In our experi- of different implant surfaces on the phenotype and ment, we noted the up-regulation of BMP8 and gene expression of MG63 cells. In the experimental BMPR2 on HA surfaces, and an up-regulation of Biological Profiles in HA, Ano, and Zr Titanium 39 Table 2.