Effects of calcium ion incorporation on osteoblast gene expression in MC3T3‐E1 cells cultured on microstructured titanium surfaces

Abstract

The surface characteristics of a calcium ion (Ca)-incorporated titanium (Ti) surface, produced by hydrothermal treatment using an alkaline Ca-containing solution, and its effects on osteoblastic differentiation were investigated. MC3T3-E1 pre-osteoblastic cells were cultured on machined or grit-blasted Ti surfaces with and without Ca incorporation. The MTT assay was used to determine cell proliferation, and real-time PCR was used for quantitative analysis of osteoblastic gene expression. Hydrothermal treatment with a Ca-containing solution produced a crystalline CaTiO(3) nanostructure of approximately 100 nm in dimension, preserving original micron-scaled surface topographies and microroughness caused by machining, blasting, or blasting and etching treatments. After immersion in Hank's balanced salt solution, considerable apatite formation was observed on all surfaces of the Ca-incorporated samples. Significantly more cell proliferation was found on Ca-incorporated Ti surfaces than on untreated Ti surfaces (p < 0.001). Quantitative real-time PCR analysis showed notably higher alkaline phosphatase, osteopontin, and osteocalcin mRNA levels in cells grown on Ca-incorporated blasted surfaces than on other surfaces at an early time point. Thus, Ca incorporation may have a beneficial effect on osseointegration of microstructured Ti implants by accelerating osteoblast proliferation and differentiation during the early healing phase following implantation.

Nanofibrous yet injectable polycaprolactone-collagen bone tissue scaffold with osteoprogenitor cells and controlled release of bone morphogenetic protein-2

“…Real-time quantitative polymerase chain reaction (qPCR) was performed with SYBR Select PCR master mix (Applied Biosystems, Life Technologies) for ALP, osterix (OSX), osteopontin (OP), and osteocalcin (OCN), with hydroxymethylbilane synthase (HMBS) as the normalizing gene. The primer sequences (Table 1) obtained from published literature [35][36][37][38][39] were verified using OligoAnalyzer and purchased from IDT (Integrated DNA Technologies). PCR amplification was performed in iCycleriQ detection system (Biorad) with thermocycling performed for 10 min at 95°C followed by 40 cycles at 95°C for 15 s and 56°C for 60 s. Expression of each gene was normalized to the gene expression level of the day 0 samples for each condition.…”

Analysis of Osteoblast Differentiation on Polymer Thin Films Embedded with Carbon Nanotubes

“…Total RNA was extracted using Trizol reagent (Gibco BRL Life Technologies) after the appropriate incubation time for cultures, and RNA samples were quantified. To synthesize first-strand complementary DNA, reverse transcription was performed as described previously [22]. We then conducted qPCR assays as described previously [22] using the oligonucleotide primers shown in Table 1.…”

“…To synthesize first-strand complementary DNA, reverse transcription was performed as described previously [22]. We then conducted qPCR assays as described previously [22] using the oligonucleotide primers shown in Table 1. The reference gene Gapdh was used as an internal control gene.…”

Chemical Analysis of a Novel Coating Material, CaTiO3-aC

“…Presence of Ca 2+ has been reported to be effective for proliferation of osteogenic cells 7,8,10,17,18) .…”