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Use of molecular beacons to image effects of titanium surface microstructure on beta1 integrin expression in live osteoblast-like cells.

ABSTRACT: This study used molecular beacon technology to examine substrate-dependent changes in integrin subunit expression in living cells. Molecular beacons are oligonucleotide probes that can be delivered into live cells to allow for real-time imaging of mRNA. They have a stem-loop hairpin structure with a fluorophore-quencher pair, which opens when bound to the target mRNA sequence, resulting in a fluorescent signal upon excitation. A novel molecular beacon that is specific to the beta1 integrin subunit mRNA was developed and used to image osteoblast-like MG63 cells in vitro on both glass and titanium surfaces of varying roughness. Specificity was verified by comparing the molecular beacon signal intensities to real-time PCR results in both wild-type cells and cells with shRNA knockdown of beta1 integrin mRNA. The molecular beacon was able to detect changes due to both surface microtopography and silencing of the mRNA target. The results showed that effects of the substrate on beta1 mRNA noted previously in confluent cultures were evident in pre-confluent cells as well, supporting the hypothesis that beta1 integrin pairs are important in proliferation as well as differentiation of osteoblasts. This technique overcomes the limitations of traditional gene assays (PCR, immunofluorescence) by allowing for the real-time measurement and tracking of specific mRNAs in individual live cells prior to confluence.

SUBMITTER: Lennon FE

PROVIDER: S-EPMC3350792 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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Use of molecular beacons to image effects of titanium surface microstructure on beta1 integrin expression in live osteoblast-like cells.

Lennon Frances E FE Hermann Christopher D CD Olivares-Navarrete Rene R Rhee Won Jong WJ Schwartz Zvi Z Bao Gang G Boyan Barbara D BD

Biomaterials 20100731 30

This study used molecular beacon technology to examine substrate-dependent changes in integrin subunit expression in living cells. Molecular beacons are oligonucleotide probes that can be delivered into live cells to allow for real-time imaging of mRNA. They have a stem-loop hairpin structure with a fluorophore-quencher pair, which opens when bound to the target mRNA sequence, resulting in a fluorescent signal upon excitation. A novel molecular beacon that is specific to the beta1 integrin subun ...[more]

PMID: 20674005

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Project description:A critical stage during osseointegration of a titanium (Ti) implant is primary bone remodeling, which involves cross talk among osteoclast precursors, osteoclasts, mesenchymal stem cells (MSCs), and osteoblasts. This phase couples the processes of bone formation and resorption. During remodeling, osteoclasts produce factors capable of regulating MSC migration and osteogenesis. Furthermore, they degrade primary bone, creating a foundation with a specific chemistry, stiffness, and morphology for osteoblasts to synthesize and calcify their matrix. MSCs and osteoblasts receiving cues from the implant surface produce factors capable of regulating osteoclasts in order to promote net new bone formation. The purpose of this study was to determine the effects Ti implant surfaces have on bone remodeling. Human MSCs and normal human osteoblasts (NHOsts) were cultured separately on 15 mm grade 2 smooth PT, hydrophobic-microrough SLA, hydrophilic-microrough Ti (mSLA) (Institut Straumann AG, Basel, Switzerland), or tissue culture polystyrene (TCPS). After 7d, conditioned media from surface cultures were used to treat human osteoclasts for 2d. Activity was measured by fluorescence of released collagen followed by mRNA quantification. This study demonstrates that MSC and NHOst cultures are able to suppress osteoclast activity in a surface dependent manner and osteoclast mRNA levels are selectively regulated by surface treatments. The substrate-dependent regulatory effect was mitigated when MSCs were silenced for integrin subunits and when conditioned media were denatured. These results indicate that MSCs and NHOsts regulate at least two aspects of remodeling: reduced fusion of new osteoclasts and reduced activity of existing osteoclasts. STATEMENT OF SIGNIFICANCE:In this study, we developed a novel in vitro model to study how microstructured and hydrophilic titanium implants impact bone remodeling for dental and orthopaedic applications. Our approach intersects biomaterials and systems physiology, revealing for the first time that implant surface properties are capable of regulating the communication among the cells involved in remodeling of primary bone during osseointegration. We believe that the basic research presented in our manuscript will provide important knowledge in our understanding of factors that impact implant success. Furthermore, it provides a solid foundation for the development of materials that enable rapid osseointegration and earlier loading times for implants in bone that has been compromised by trauma or disease.

Dataset Information

Use of molecular beacons to image effects of titanium surface microstructure on beta1 integrin expression in live osteoblast-like cells.

Publications

Use of molecular beacons to image effects of titanium surface microstructure on beta1 integrin expression in live osteoblast-like cells.

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