Effect of topographical control by a micro-molding process on the activity of human Mesenchymal Stem Cells on alumina ceramics
© Kim et al. 2015
Received: 16 August 2015
Accepted: 27 October 2015
Published: 4 November 2015
Numerous studies have reported that microgrooves on metal and polymer materials can affect cell adhesion, proliferation, differentiation and guidance. However, our knowledge of the cell activity associated with microgrooves on ceramics, such as alumina, zirconia, hydroxyapatite and etc, is very incomplete, owing to difficulties in the engraving of microgrooves on the hard surface of the base material. In this study, microgrooves on alumina were fabricated by a casting process using a polydimethylsiloxane micro-mold. The cell responses of Human Mesenchymal Stem Cells on the alumina microgrooves were then evaluated.
Microgrooves on an alumina surface by micro-mold casting can enhance the adhesion, differentiation of osteoblasts as well as gene expression related to osteoblast differentiation. The ALP activity and calcium concentration of the cells on alumina microgrooves were increased by more than twice compared to a non-microgrooved alumina surface. Moreover, regarding the osteoblast differentiation of hMSCs, the expression of ALP, RUNX2, OSX, OC and OPN on the microgrooved alumina were all significantly increased by 1.5 ~ 2.5 fold compared with the non-microgrooved alumina.
Altering the topography on alumina by creating microgrooves using a micro-molding process has an important impact on the behavior of hMSCs, including the adhesion, differentiation of osteoblasts and osteoblast-specific gene expression. The significant increase in hMSC activity is explained by the increasing of material transportation in parallel direction and by the extending of spreading distance in perpendicular direction.
Microgrooves have been reported to be effective for altering cell shape, changing cell adhesion and proliferation on polystyrene , titanium  and silicon . Alumina is a bio-inert ceramic that is used in dental and orthopedic applications due to its chemical stability, mechanical properties and biocompatibility . However, applying micropatterns to alumina by using conventional microfabrication techniques such as micromachining, laser cutting and etching techniques  is very difficult, due to its brittleness, hardness and inertness [6, 7]. Danish Nadeem et al., reported on the fabrication of Mesenchymal Stem Cells (MSCs) on alumina micropatterns with widths of 50 μm and cell alignment on 10 μm widths using an embossing technique . A few related reports have reviewed cell guidance and cell differentiation on microgrooved alumina. In addition, information concerning hMSC behavior, including gene expression related to osteoblastic differentiation on microgrooved alumina in a wide range to 180 μm, which approaches the width of microgrooves for a micromachined titanium implant, is insufficient.
In this study, microgrooved alumina was fabricated using a micro-molding process. Such a process involved fabricating a microgrooved alumina substrate by casting slurry on a polydimethylsiloxane (PDMS) replica, drying to a green body and subsequently sintering to produce a dense body. The procedure is capable of transferring geometries and surface details of a titanium mother model which was micropatterned by photolithography . The activity of human Mesenchymal Stem Cells (hMSCs) as well as gene expression on the microgrooved surfaces of alumina was investigated. Furthermore, we compared the surface chemistry, surface hydrophilicity, cell adhesion, activity and maturation on titanium with those on alumina. The effect of surface chemistry and material properties on cell response was also evaluated.
Fabrication of microgrooved surfaces on alumina ceramic
Titanium and alumina substrate with various surface topographies used in this study (μm)
Ridge width [μm]
Groove width [μm]
The surface morphology and roughness ratio (r; relative value of measured surface area to unit area) of specimens was characterized by a confocal laser scanning microscopy (OLS3000-300 mm autostage, Olympus, Japan) with a 408-nm argon laser excitation source. The contrast in the confocal laser-scanning microscope is based on the reflection of the laser beam from the specimen surface. Thus, point-by-point images were reconstructed under small point scans in the X-Y direction. The percentage of area in the spectra of C1s and O1s bonding on the titanium and the alumina surface was characterized using X-ray photoelectron spectroscopy (XPS, PHI5000, Physical Electronics, U. S. A). XPS experiments were performed in an ultra-high vacuum using Al Ka (hv = 1486.6 eV) radiation. High resolution XPS analysis of the C1s and O1s peaks was performed in the range of 280–295 eV and 526–536 eV with a step of 0.05 eV, respectively. The scan spectra were analyzed by skewed Gauss-Lorenz Line shapes using the XPS peak 4.0 software associated with the XPS instrumentation. A drop shape analysis system (EasyDrop standard, KRUSS GmbH, Germany) was used for the water contact angle (WCA) measurements. A 6 μl drop of distilled water was used as a probe, and measurements were taken for each drop. The drop images were captured serially five times every 5 s by a video camera. The water contact angle of each surface was calculated using an image analysis software program. WCA analyses are performed separate in parallel and perpendicular directions to the microgrooves.
Human bone marrow-derived mesenchymal stem cells (hMSC) were purchased from Lonza (Lonza, MD, USA) and grown in MSC Growth Medium (MSCGM; Lonza, MD, USA) at 37 °C with 5 % CO2 and 95 % humidity. Tissues were obtained from subjects following informed consent guidelines as described by the Institutional Review Board protocol of Kyung Hee University Hospital at Gangdong. Cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM: Welgene, Daegu, Korea) containing 10 % FBS (fetal bovine serum, Sigma-Aldrich, St. Louis, MO, USA), 100U/ml penicillin, and 100 μg/ml streptomycin at 37 °C under 5 % CO2. MSCs at passages 3–5 were used in this study.
Cell Count Kit (CCK) cell adhesion assay
MSCs were seeded on the 24-well titanium and alumina surfaces at a concentration of 1 × 104 cells/well and cultured for 16 h at 37 °C under 5 % CO2. Twenty microliters of Cell Counting Kit reagent (50 ml, CCK-8; Dojindo, Kumamoto, Japan) were added to each well followed by incubation for 2 h. The reaction products were transferred to 96-well plates and monitored using a microplate reader (Bio-Rad, Hercules, CA, USA) at 450 nm.
Alkaline phosphatase activity assay
MSCs were seeded on the 24-well titanium and alumina surface at 4 × 104 cells/well and cultured for 2 days to achieve confluence. The cells were then cultured in osteogenic media [DMEM supplemented with 10 % FBS (Invitrogen), 50 μg/ml α-ascorbic acid (Sigma-Aldrich, St. Louis, MO, USA), 10 mM β-glycerophosphate (Sigma-Aldrich), 100 mM dexametasone (Sigma-Aldrich)] at 37 °C under 5 % CO2 for 14 days to investigate ALP activity. The reaction products were transferred to 96-well plates and monitored using a microplate reader (Bio-Rad, Hercules, CA, USA) at 405 nm, and measurements were compared using pnitrophenol standards and normalized to total protein levels.
Extracellular calcium deposition assay
Extracellular calcium deposition on the titanium and alumina surface was quantified to investigate the steps of osteoblast differentiation. MSCs was seeded on 24-well titanium and alumina surfaces at a concentration of 4 × 104 cells/well and cultured for 2 days to achieve confluence. Cells were then cultured in osteogenic media at 37 °C under 5 % CO2 for 21 days. Cells were rinsed in a phosphate buffered solution (PBS, Gibco BRL, Grand Island, NY, USA) and the surface with remaining calcium deposits were incubated with 0.5 N HCl at 4.8 °C overnight. Following centrifugation, the amount of calcium present in the acidic supernatant was quantified using Calcium Liquicolor (Stanbio Laboratory, Boerne, TX, USA). Light absorbance was measured using a microplatereader (Bio-Rad) at 650 nm. Total calcium (μg/well) was calculated from standard curves derived from absorbance values versus the calcium levels of controls measured in parallel with the experimental surfaces.
Osteoblast-related gene expression
The relative mRNA expression of five osteo-related genes was analyzed in the hMSCs using quantitative real-time PCR. The five genes analysed were RUNX2 (run-related trascription factor2), OSX (osterix), ALP (alkaline phosphatase), OCN (osteoclacin), OPN (osteopontin). MSCs was seeded on 24-well alumina surface at a density of 4 × 104 cells/well and incubated for 2 days until reaching confluence. Cells were then cultured for 14 days in osteogenic media at 37 °C under 5 % CO2. Total RNA was extracted using Trizol (Invitrogen). RNA concentration was determined using a NanoDrop 1000 (Nano-Drop Technologies, Wilmington, DE, USA). An iScript cDNA Synthesis Kit (Bio-Rad) with 1 mg of total RNA was used to reverse-transcribe cDNA. The mRNA expression levels of the five osteo-related genes were determined relative to the internal GAPDH control using a TaqMan1 Gene Expression Assay Kit (Applied Biosystems, Foster City, CA, USA). Chromo4 Reverse Transcription-Polymerase Chain Reactions (Bio-Rad Laboratories, Hemel Hempstead, UK) were performed using IQ Supermix (Bio-Rad). The MJ Opticon Monitor Analysis Software (Bio-Rad) was used to quantify gene expression levels. Experimental values were normalized to GAPDH in order to obtain relative expression levels.
The CCK cell adhesion, ALP activity test, extracellular calcium deposition assays, and quantitative real-time PCR of hMSCs on titanium and alumina surface were performed simultaneously and independently four times, and the mean values from these experiments were compared using a one-way analysis of variance (ANOVA). SPSS 17.0 software was used for all statistical analyses.
Results & discussion
Surface chemistry and morphology of micro-patterned surfaces
WCA could be affected by not only surface chemistry but also by surface topography. WCA analyses were performed separately in both parallel and perpendicular directions to the microgrooves. Since the surface chemistry was the same in all samples except titanium (NE0), the difference in WCA can be assumed to be exclusively due to the presence of surface microgrooves.
In the observation of WCA in the perpendicular direction to the microgroove, it was anticipated that capillary pressure would be higher as the width became narrower and that this could enhance the anisotropy of a water droplet on a microgroove substrate. However, anisotropy was increased with increasing width of the microgroove in these analyzed specimen groups and was highest for the AM90 sample. Therefore the wetting angle of a water drop was evaluated as the highest in the perpendicular direction on the AM90 sample. Up to a microgroove width of 90 μm, it was observed that grooves worked as a breakwater to advance the water droplet in the perpendicular direction. Hence, we suggest that the anisotropy of the water droplet is due to, not only the penetration of water into the grooves via a capillary effect in the parallel direction  but also hindering the advancing of a water droplet in the perpendicular direction. The WCA of microgrooved alumina in the parallel direction was decreased with increasing width of the microgroove. In the parallel direction, it was observed that the water droplet spread over a longer distance, which would seem to be originated from improving wettability and transportation by the microgroove. In comparison with the spreading distance of water droplet between specimens, the differences are relatively small in the parallel direction than in the perpendicular direction. The wide microgroove acted as a transport channel for water and induced isotropic and long spreading of droplet in the tested specimen groups.
Many studies have been reported on cell–biomaterial interactions with various micropatterned surfaces [22, 23]. The presence of a micropattern on a surface can have significant effects on cellular behavior, including cell adhesion, migration, proliferation, and differentiation . A large number of studies have been carried out in attempts to examine the effect of microgrooves on different types of cells. Other investigations reported that the attachment of fibroblasts is increased on smooth surfaces and soft tissue growth, whereas the attachment of osteoblasts is increased on rough surfaces and in bone growth .
In this study, an alumina surface was found to contain more hydroxyl groups, positive charge and cell response than a titanium surface. These results indicate that the surface chemistry and charge of a material influence the response of MSCs. Microgrooves on an alumina surface were also found to decrease WCA in the parallel direction and enhanced the cell response. Mass transport could occur effectively in the parallel direction of a trench structure. From this study, it can be concluded that cell activity, i.e., differentiation and mineralization could be enhanced by increasing the wettability and transportability on wider microgrooves of a micro-molded alumina substrate. For confirmation of these results, further evaluations will be needed to identify the specific factors responsible and to optimize the width of grooves on an alumina surface in a wider range.
We evaluated the activity of human mensenchymal stem cell on titanium, non-microgrooved alumina and microgrooved alumina prepared by a micro-molding technique using a replica of a PDMS mold. The alumina surface showed superior biological responses of osteoblast cells compared to titanium. These results indicate that higher concentrations of hydroxyl groups and positive charges of the alumina surface enhance cell activity compared to a titanium surface. The main findings were that the variation in the surface topography of alumina by microgrooves has an important impact on the behavior of hMSCs including the adhesion, differentiation of osteoblasts and osteoblast-specific gene expression. The significant increase in hMSC activity can be explained by an increase in material transportation in the parallel direction by increasing the cross-sectional area of a trench and by the extending the spreading distance in the perpendicular direction. The result is that the interruption against the groove walls in microgrooves having a wider width than 90 μm is decreased. Our results demonstrate that altering the topography of alumina by a micromolding process enhanced the wettability and isotropicity of the water droplet, which are important factors that influence the transport and propagation of materials on an alumina surface, therefore the cell activity of hMSCs including adhesion, differentiation of osteoblasts and osteoblast-specific gene expression are enhanced.
“This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (NRF-2012R1A2A2A03047477).”
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