The Importance of Low Intensity Pulsed Ultrasound Parameters for Functional Bone Tissue Engineering using Adult Stem Cells

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Title: The Importance of Low Intensity Pulsed Ultrasound Parameters for Functional Bone Tissue Engineering using Adult Stem Cells
Author: Marvel, Skylar
Advisors: Dr. Greg S. McCarty, Committee Member
Dr. Paul A. Dayton, Committee Co-Chair
Dr. Elizabeth G. Loboa, Committee Chair
Abstract: Functional bone tissue engineering is a very important emerging interdisciplinary field of research. The treatments of bone loss, defects, trauma and disease often require replacement bone tissue or the use of bone substitutes. Functional bone tissue engineering attempts to create this needed replacement bone tissue using a patient’s stem cells. One small aspect of this field involves being able to control the proliferation and osteogenic differentiation of a patient’s stem cells with the eventual goal of creating an autologous bone graft in vitro to correct bone defects. Two possible types of stem cells for this procedure are human bone marrow derived mesenchymal stem cells (hMSCs) and human adipose derived adult stem cells (hASCs), both of which have been shown to be capable of osteogenic differentiation. Many different stimuli are being actively researched to control this differentiation, such as fluid shear stress, tensile strain, electric fields, chemical signals and material properties of substrates to name a few. One newly investigated stimulus is low intensity pulsed ultrasound (LIPUS). Since the mid 1990s LIPUS has been used clinically to aid fracture healing and since 2000 has also been prescribed for the treatment of non-unions. Although the mechanisms have not been elucidated, LIPUS has been shown to increase expression of cellular osteogenic markers in vitro. However, the parameters of LIPUS have not been optimized for increasing osteogenic differentiation and the use of LIPUS has been untested on hASCs, both of which could potentially benefit the field of bone tissue engineering and are the focus of this body of work. To explore the optimization of LIPUS parameters a custom ultrasound system was designed and built to give precise control over an extensive range of values for each stimulus parameter. There were several design criteria which included making the system automatable, insuring that the system can be used with standard incubators, and reducing the amount of laboratory space required to house the system. Once built, the ultrasound system was validated by testing three different pulse repetition frequency (PRF) parameter settings for inducing osteogenic differentiation of both hMSCs and hASCs. The first set of experiments investigated effects on hASCs and showed a significant increase in calcium accretion per cell with both 1 kHz and 100 Hz PRFs; however, this result was diminished by noting that the significant differences came from variations in amounts of DNA rather than increases in calcium. The effect caused by the presence of papain digest in experimental samples on the quantification of DNA was then studied and a change in the protocol for the DNA quantification assay was made. Using the modified assay a second set of experiments investigated the effects of LIPUS on hMSCs, and although the results were not significant, there was a trend of increased calcium accretion per cell with higher PRFs indicating that, of the three PRF settings tested, the 1 kHz PRF resulted in the highest calcium per cell. The findings of this body of work show that the custom ultrasound system fulfilled the design requirements, that PRF is an important parameter in ultrasound dose for LIPUS, and that LIPUS does have an effect on hASCs. Future work based on this body of work include increasing the capacity of the ultrasound system to run multiple parallel experiments and performing additional studies to optimize the parameters for increasing osteogenic differentiation of both hMSCs and hASCs.
Date: 2009-12-04
Degree: MS
Discipline: Biomedical Engineering

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