Abstract
Human osteoblasts react to external biophysical stimulation by several mechanisms, including propagating an external signal by electrical currents via a cellular membrane and intracellular biochemical secondary pathways. The resultant effect is the modulation of proliferation and phenotypic activity. Therefore, a series of research projects were carried out according to the hypothesis that there are specific alternating high-frequency biophysical parameters for the induction of the phenotypic cell function of human osteoblasts in vitro and that by using the specific biophysical parameters for osteoblast stimulation, viable bone tissue can be generated in vitro that is safe and effective for use as a bone graft in vivo. For this purpose, specially designed experimental modalities were used for the external application of mechanical vibration, alternating LED irradiance, and electromagnetic fields in the 20-60 Hz range of frequencies to monolayer cultures of human osteoblasts.By using these methods, effective biophysical parameters for cell stimulation were defined. These parameters are different and distinctive for phenotypic cell function vs. synthetic activities.
The results indicate a low-intensity threshold of photobiomodulation of osteoblasts in vitro by 40 Hz pulsed irradiance. A narrow spectrum of low intensity pulsed (40 Hz) LED light irradiance caused photobiomodulation in the osteoblast by a significant increase in the number of cells and cell death, measured by lactate dehydrogenase content in culture media, (p<0.05) in the green spectrum range with diffuse transmittance 560-650 nm and maximal cell irradiance of 0.04 W/m2, and a significant decrease (p<0.05) in osteoblast maturation by a blue range of spectrum irradiance (alkaline phosphatase cellular specific activity decrease following irradiance with diffuse transmittance 420-580 nm, maximal cell irradiance 0.05 W/m2).
Cell exposure to pulsed electromagnetic field (PEMF) at a distinct range of 5-15 kHz of basic frequency in pulses of 20-30 Hz caused a shift of the cell cycle towards the G1 phase. External mechanical stimulation of cells by vibration showed different sub-ranges of effective vibration parameters for osteoblast proliferation and phenotypic cell function, i.e., 20 Hz frequency vibration significantly increased (p<0.05) DNA content in cells, indicating increased cell proliferation. In contrast, the maturation state (expressed by alkaline phosphatase specific activity in cells) increased significantly (p<0.05) following the 60 Hz stimulation.
Thus, these experiments indicate that the application of alternating biophysical energy (light, electromagnetic field, mechanical vibration) in the frequency range of 20-60 Hz causes proliferative and phenotypic effects in human osteoblasts.
Subsequentially, by applying biophysical stimulation (mechanical), viable bone-like tissue was generated in vitro that is safe and effective for bridging critical bone gaps in vivo (investigated using small animal models).
Through this series of research projects, high-frequency ranges of alternating biophysical stimulation for phenotypic cell function and proliferation of human osteoblasts in vitro were determined.
These findings reveal the ability to implement in vitro tissue engineering techniques of osteoblast manipulation in culture by external biophysical methods for clinical use to treat critical bone loss by autologous bone grafting. Accordingly, by using mechanical stimulation of cells in a high-frequency range, I have developed a way to generate bonelike tissue in vitro for the potential clinical implementation as autologous bone graft in orthopedic, neuro, maxillofacial and dental surgery. Additional preclinical studies on the generated bone-like material's ability to bridge critical bone gaps should be done on a large animal model for the subsequential regulatory approval to proceed to the Phase IIA clinical studies, essential for large-scale clinical use.
| Date of Award | Oct 2021 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Marta Roldo (Supervisor) |