Influence of ultrasound on osteoblasts attached to titanium in vitro.

Yevlashevskaya, Olga ORCID: 0000-0002-6820-0418 (2024). Influence of ultrasound on osteoblasts attached to titanium in vitro. University of Birmingham. Ph.D.

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Abstract

Despite the growing evidence that ultrasonic stimulation can promote bone healing on titanium (Ti)-based implants, the current understanding of mechanisms of action of therapeutic ultrasound is limited. The present work aimed to produce an experimental system which would allow the delivery of a defined dosage of ultrasound to osteoblasts incubated on Ti surfaces in vitro and observe the cells responses.
Initially two osteogenic cell lines, Saos-2 and immortalised human mesenchymal stromal cells (hMSCs) were characterised before subsequent ultrasonic treatment. Osteogenic behaviour was analysed in the presence of supplements, consisting of 283 μM ascorbic acid (Asc), 9.3 mM β-glycerophosphate (β-Gly) and 10-8 M dexamethasone (Dex), for 14 days. Saos-2 cells exhibited a reduction of 63% in viable cells compared with the negative controls, which was not observed in the hMSCs although the lactate dehydrogenase (LDH) assay did not reveal any cytotoxic effects of β-Gly. However, 5- Bromo-2’-deoxy-uridine (BrdU) staining showed a distinct antiproliferative influence of β-Gly on only Saos-2 cells. Moreover, in hMSCs treated with Asc, Dex, and β-Gly, the expression of RUNX2, OCN, BSP, and ALP were upregulated, which was not observed in the absence of β-Gly. The presence of β-Gly led to an upregulation of RUNX2 and OCN expression in Saos-2 cells. It was concluded that β-Gly demonstrated a unique proliferative stimulus in Saos-2 cells, by possibly interacting with osteogenesis of relatively mature osteoblast-like cells.
The difficulties associated with cell detachment and counting in mineralising cultures were also investigated. Cells were detached using incubation with either trypsin alone or in combination with collagenase type 1. Counting with an haemocytometer and trypan blue staining produced less variable, more robust data, compared with the automated counting based on fluorescence-based images.
Additionally, cell attachment of hMSCs to Ti surfaces was studied for implementation into the in vitro ultrasonic device. Ti surfaces with roughness, Ra of ~1 μm were prepared by acid etching and demonstrated optimal cell attachment. Ti strips were then designed to attach to transducers oscillating at 20 and 40 kHz. The strips served as an incubation surface for immortalised hMSCs and resonated according to the ultrasound frequency produced by the transducers. Polydimethylsiloxane wells were created to compartmentalise cells exposed to different excitation doses modes whilst incubated in culture medium. After exposure to continuous ultrasound at either frequency for 5 min an LDH assay showed no difference in cell death relative to controls.
The presented system offers potential for studying defined dosage and delivery regimes of ultrasound to osteoblasts incubated on Ti surfaces, while under controlled temperature conditions. Future studies using this setup will include analysis of gene expression and mineral deposition to define the potential role of ultrasonic therapy in dental and orthopaedic implantology.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):