Mitra Aliabouzar

Lipid Coated Microbubbles and Low Intensity Pulsed Ultrasound Enhance Chondrogenesis of Human Mesenchymal Stem Cells in 3D Printed Scaffolds

Lipid-coated microbubbles are used to enhance ultrasound imaging and drug delivery. Here we apply these microbubbles along with low intensity pulsed ultrasound (LIPUS) for the first time to enhance proliferation and chondrogenic differentiation of human mesenchymal stem cells (hMSCs) in a 3D printed poly-(ethylene glycol)-diacrylate (PEG-DA) hydrogel scaffold. The hMSC proliferation increased up to 40% after 5 days of culture in the presence of 0.5% (v/v) microbubbles and LIPUS in contrast to 18% with LIPUS alone. We systematically varied the acoustic excitation parameters—excitation intensity, frequency and duty cycle—to find 30 mW/cm2, 1.5 MHz and 20% duty cycle to be optimal for hMSC proliferation. A 3-week chondrogenic differentiation results demonstrated that combining LIPUS with microbubbles enhanced glycosaminoglycan (GAG) production by 17% (5% with LIPUS alone), and type II collagen production by 78% (44% by LIPUS alone). Therefore, integrating LIPUS and microbubbles appears to be a promising strategy for enhanced hMSC growth and chondrogenic differentiation, which are critical components for cartilage regeneration. The results offer possibilities of novel applications of microbubbles, already clinically approved for contrast enhanced ultrasound imaging, in tissue engineering.

Effects of Scaffold Microstructure and Low Intensity Pulsed Ultrasound on Chondrogenic Differentiation of Human Mesenchymal Stem Cells

The effects of low intensity pulsed ultrasound (LIPUS) on proliferation and chondrogenic differentiation of human mesenchymal stem cells (hMSCs) seeded on 3D printed poly‐(ethylene glycol)‐diacrylate (PEG‐DA) scaffolds with varying pore geometries (square and hexagonal channels) were investigated. The scaffold with square pores resulted in higher hMSC growth and chondrogenic differentiation than a solid or a hexagonally porous scaffold. The optimal LIPUS parameters at 1.5 MHz were found to be 100 mW/cm2 and 20% duty cycle. LIPUS stimulation increased proliferation by up to 60% after 24 hr. For chondrogenesis, we evaluated key cartilage biomarkers abundant in cartilage tissue; glycosaminoglycan (GAG), type II collagen and total collagen. LIPUS stimulation enhanced GAG synthesis up to 16% and 11% for scaffolds with square and hexagonal patterns, respectively, after 2 weeks. Additionally, type II collagen production increased by 60% and 40% for the same patterns, respectively under LIPUS stimulation after 3 weeks. These results suggest that LIPUS stimulation, which has already been approved by FDA for treatment of bone fracture, could be a highly efficient tool for tissue engineering in combination with 3D printing and hMSCs to regenerate damaged cartilage tissues.

Ultrasound and lipid-coated microbubbles for osteogenic differentiation of mesenchymal stem cells in 3D printed tissue scaffolds

With increasing incidence of bone disorders in a rapidly aging, sedentary and overweight population, engineered bone tissues promise to be a better alternative for conventional bone grafts. However, bone tissue engineering currently suffers due to the inability to form mechanically strong porous structures that can be grown quickly. In this study, lipid-coated microbubbles (MB), traditionally used for contrast enhanced ultrasound imaging, were applied to harness the beneficial effects of ultrasound stimulation on proliferation and osteogenic differentiation of human mesenchymal stem cells (hMSCs) on 3D printed poly(lactic acid) (PLA) scaffolds. A significant increase in cell number was observed with low intensity pulsed ultrasound (LIPUS) treatment in the presence of MB after 1, 3, and 5 days of culture on scaffolds. Total protein content, alkaline phosphatase activity, and total calcium content were also found to increase with LIPUS with and without MB indicating enhancement in osteogenic differentiation. Integrating LIPUS and MB appears to be a promising strategy for bone tissue engineering and regeneration therapies. With increasing incidence of bone disorders in a rapidly aging, sedentary and overweight population, engineered bone tissues promise to be a better alternative for conventional bone grafts. However, bone tissue engineering currently suffers due to the inability to form mechanically strong porous structures that can be grown quickly. In this study, lipid-coated microbubbles (MB), traditionally used for contrast enhanced ultrasound imaging, were applied to harness the beneficial effects of ultrasound stimulation on proliferation and osteogenic differentiation of human mesenchymal stem cells (hMSCs) on 3D printed poly(lactic acid) (PLA) scaffolds. A significant increase in cell number was observed with low intensity pulsed ultrasound (LIPUS) treatment in the presence of MB after 1, 3, and 5 days of culture on scaffolds. Total protein content, alkaline phosphatase activity, and total calcium content were also found to increase with LIPUS with and without MB indicating enhancement in osteogenic differentiation...

Acoustic characterization of 3D printed micro-structured scaffolds for tissue engineering

The acoustic and mechanical properties of 3D-printed porous poly-(ethylene glycol)-diacrylate (PEGDA) hydrogel scaffolds, as a widely used biomaterial, with different geometric channels (hexagonal and square) were explored using a pulse echo technique. The measured values of attenuation and speed of sound were found to be within the range of reported values for soft tissues making PEGDA scaffolds a suitable candidate for cartilage tissue engineering. We also showed that these properties as well as Young’s modulus can be controlled and adjusted to desired values close to biological tissues by varying the 3D printing parameters. Furthermore, our 5-day proliferation as well as three-week chondrogenic differentiation results revealed that cell growth and tissue formation depend on the geometrical features of the 3D-printed scaffolds as well. Cell adhesion and proliferation greatly improved for scaffolds with square and hexagonal pore geometries compared to nonporous scaffolds. Scaffolds with square pores were...

Acoustic vaporization threshold of lipid-coated perfluoropentane droplets

Phase shift droplets vaporizable by acoustic stimulation offer the advantages of producing microbubbles as contrast agents in situ as well as higher stability and the possibility of achieving smaller sizes. Here, the acoustic droplet vaporization (ADV) threshold of a suspension of droplets with a perfluoropentane (PFP) core (diameter 400-3000 nm) is acoustically measured as a function of the excitation frequency in a tubeless setup at room temperature. The changes in scattered responses-fundamental, sub-, and second harmonic-are investigated, a quantitative criterion is used to determine the ADV phenomenon, and findings are discussed. The average threshold obtained using three different scattered components increases with frequency-1.05 ± 0.28 MPa at 2.25 MHz, 1.89 ± 0.57 MPa at 5 MHz, and 2.34 ± 0.014 MPa at 10 MHz. The scattered response from vaporized droplets was also found to qualitatively match with that from an independently prepared lipid-coated microbubble suspension in magnitude as well as trends above the determined ADV threshold value.

Study of acoustic droplet vaporization using classical nucleation theory

Lipid coated perfluorocarbon (PFC) nanodroplets can be vaporized by an external ultrasound pulse to generate bubbles in situ for tumor imaging and drug delivery. Here we employ classical nucleation theory (CNT) to investigate the acoustical droplet vaporization (ADV), specifically the threshold value of the peak negative pressure required for ADV. The theoretical analysis predicts that the ADV threshold increases with increasing surface tension of the droplet core and frequency of excitation, while it decreases with increasing temperature and droplet size. The predictions are in qualitative agreement with experimental observations. We also estimate and discuss energy required to form critical cluster to argue that nucleation occurs inside the droplet, as was also observed by high-speed camera.

Acoustic and atomic force microscopy characterization of microbubbles with varying shell chemistry

Applications of microbubbles (MBs) in diagnostic and therapeutic interventions critically depend on their stability and scattering properties. The shell chemistry of MBs defines these properties. We investigated the effects of shell chemistry on the size, abundance, acoustic response, and mechanical properties of MBs by varying the poly(ethylene glycol) (PEG) molar ratio (0 to 100%) in a two-lipid (DPPC and DPPE-PEG2000) component shell formulation. Increasing PEG concentration from 0% to 10% resulted in an increase in the number of MBs by at least 10-fold, with adverse effects upon further increases. Microbubbles made with 5–10% PEG generated the strongest fundamental as well as nonlinear (subharmonic and second harmonic) components at the excitation frequency of 2.25 MHz. We used interfacial rheological models to determine the mechanical properties of MB shells as functions of PEG concentration using experimentally measured attenuation values. We also employed atomic force microscopy (AFM) to perform th...

Effects of acoustic parameters on nanodroplet vaporization

Phase shift nanodroplets offer a number of advantages over ordinary microbubbles due to their enhanced stability and smaller size distribution. These nanodroplets undergo a phase transition from liquid to highly echogenic gaseous state when activated by sufficient acoustic energy through a process termed acoustic droplet vaporization (ADV). In this study, we synthesized lipid-coated perfluoropentane (PFP) filled nanodroplets via sonication method. We investigated the ADV threshold of these nanodroplets as a function of acoustic parameters such as excitation pressure, frequency, pulse length, and pulse repetition period (PRP). Our results indicate that ADV threshold varies significantly with PRP; while at PRP of 10 ms, the ADV threshold was found to be 3.6 MPa (pk-pk), for PRP of 1 ms, 100 μs, and 500 μs, ADV was not observed even at 10 MPa. At ADV, fundamental and odd harmonics were found to be significantly higher than the background noise. The acoustic response of ordinary perfluorobutane (PFB) filled m...

Experimental and theoretical studies on acoustic droplet vaporization

Phase shift nanodroplets are better alternatives to microbubbles due to their enhanced stability and smaller size distribution. These nanodroplets undergo phase transition from liquid to highly echogenic gaseous state under acoustic excitation through a process termed acoustic droplet vaporization (ADV). In this study, we synthesized lipid-coated perfluoropentane (PFP)-filled nanodroplets via sonication and mechanical agitation methods. We investigated the ADV threshold of these nanodroplets as a function of acoustic parameters such as excitation frequency and pulse repetition period (PRP). Experiments were performed at frequencies 2.25, 5, and 10 MHz. The acoustic signature of droplet vaporization was observed to be a broadband signal at all the studied frequencies. The ADV threshold was studied by increasing the excitation pressure in steps of 200 kPa. The ADV threshold at 2.25 and 5 MHz was found to be 1.8 and 2.2 MPa, respectively. The scattered response from droplets were studied at different PRPs of...

Effects of ultrasound in presence of microbubbles on cartilage tissue regeneration in three-dimensional printed scaffolds

Gas-filled microbubbles encapsulated with lipids and other surfactants are highly responsive to ultrasound, which has led to their effective role as ultrasound contrast agents (UCA). In this study, for the first time, we used lipid-coated microbubbles (MB) prepared in-house in order to better harness the beneficial effects of ultrasound stimulation on proliferation and chondrogenic differentiation of human mesenchymal stem cells (MSCs) within a novel 3D printed poly (ethylene glycol) diacrylate (PEG-DA) hydrogel scaffolds. A significant increase in cell number (p <0.001) was observed with low intensity pulsed ultrasound (LIPUS) treatment in the presence of 0.5 % (v/v) MB after 1, 3, and 5 days of culture. MSC proliferation enhanced up to 40% after 5 days of culture in the presence of MB and LIPUS while this value was only 18% when excited with LIPUS alone. We investigated the effects of acoustic parameters such as excitation intensity, frequency, and pulse repetition period on MSC proliferation rate. Our ...