Tissue Engineering

Katiyar A, Duncan R L, Sarkar K 2014 “Ultrasound stimulation increases proliferation of MC3T3-E1 preosteoblast-like cells,” Journal of Therapeutic Ultrasound, 2, 1, 1-10.

Srivastava P, Malipeddi Reddy A, Sarkar K 2016 “Steady shear rheology of a viscous emulsion in the presence of finite inertia at moderate volume fractions: sign reversal of normal stress differences,” Journal of Fluid Mechanics, 85, 494-522.

The shear rheology of an emulsion of viscous drops in the presence of finite inertiais investigated using direct numerical simulation. In the absence of inertia, emulsionsdisplay a non-Newtonian rheology with positive first and negative second normalstress differences. However, recently it was discovered that a small amount ofdrop-level inertia alters their signs – the first normal stress difference becomesnegative and the second one becomes positive, each in a small range of capillarynumbers (Li & Sarkar,J. Rheol., vol. 49, 2005, pp. 1377–1394). Sign reversal wasshown numerically and analytically, but only in the limit of a dilute emulsion wheredrop–drop interactions were neglected. Here, we compute the rheology of a density-and viscosity-matched emulsion, accounting for the interactions in the volume fractionrange of 5 %–27 % and Reynolds number range of 0.1–10. The computed rheologicalproperties (effective shear viscosity and first and second normal stress differences) inthe Stokes limit match well with previous theoretical (Choi–Schowalter in the dilutelimit) and simulated results (for concentrated systems) using the boundary elementmethod. The two distinct components of the rheology arising from the interfacialstresses at the drop surface and the perturbative Reynolds stresses are investigated asfunctions of the drop Reynolds number, capillary number and volume fraction. Thesign change is caused by the increasing drop inclination in the presence of inertia,which in turn directly affects the interfacial stresses. Increase of the volume fractionor capillary number increases the critical Reynolds number for sign reversals due toenhanced alignment of the drops with the flow directions. The effect of increasingthe volume fraction on the rheology is explained by relating it to interactions andspecifically to the contact pair-distribution function computed from the simulation.The excess stresses are seen to show an approximately linear behaviour with theReynolds number in the range of 0.1–5, while with the capillary number and volumefraction, the variation is weakly quadratic.

Zhou X, Castro NJ, Zhu W, Cui H, Aliabouzar M, Sarkar K, Zhang LG 2016 “Improved human bone marrow mesenchymal stem cell osteogenesis in 3D bioprinted tissue scaffolds with low intensity pulsed ultrasound stimulation,” Scientific Reports, 6, 32876.

3D printing and ultrasound techniques are showing great promise in the evolution of human musculoskeletal tissue repair and regeneration medicine. The uniqueness of the present study was to combine low intensity pulsed ultrasound (LIPUS) and advanced 3D printing techniques to synergistically improve growth and osteogenic differentiation of human mesenchymal stem cells (MSC). Specifically, polyethylene glycol diacrylate bioinks containing cell adhesive Arginine-Glycine-Aspartic acid-Serene (RGDS) peptide and/or nanocrystalline hydroxyapatite (nHA) were used to fabricate 3D scaffolds with different geometric patterns via novel table-top stereolithography 3D printer. The resultant scaffolds provide a highly porous and interconnected 3D environment to support cell proliferation. Scaffolds with small square pores were determined to be the optimal geometric pattern for MSC attachment and growth. The optimal LIPUS working parameters were determined to be 1.5 MHz, 20% duty cycle with 150 mW/cm2 intensity. Results demonstrated that RGDS peptide and nHA containing 3D printed scaffolds under LIPUS treatment can greatly promote MSC proliferation, alkaline phosphatase activity, calcium deposition and total protein content. These results illustrate the effectiveness of the combination of LIPUS and biomimetic 3D printing scaffolds as a valuable combinatorial tool for improved MSC function, thus make them promising for future clinical and various regenerative medicine application.

Aliabouzar M, Zhang LG, Sarkar K, 2016 “Lipid coated microbubbles and low intensity pulsed ultrasound enhance chondrogenesis of human mesenchymal stem cells in 3D printed scaffolds,” Scientific Reports, 6, 37728.

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.

Miao S, Castro NJ, Nowicki M, Xia L, Cui H, Zhou X, Zhu W, Lee S, Sarkar K, Vozzi G, Tabata Y, Fisher J, Zhang LG 2017 “4D printing of polymeric materials for tissue and organ regeneration,” Materials Today, 20, 577-591.

Four dimensional (4D) printing is an emerging technology with great capacity for fabricating complex,stimuli-responsive 3D structures, providing great potential for tissue and organ engineering applica-tions. Although the 4D concept wasfirst highlighted in 2013, extensive research has rapidly developed,along with more-in-depth understanding and assertions regarding the definition of 4D. In this review,we begin by establishing the criteria of 4D printing, followed by an extensive summary of state-of-the-art technological advances in thefield. Both transformation-preprogrammed 4D printing and 4Dprinting of shape memory polymers are intensively surveyed. Afterwards we will explore and discussthe applications of 4D printing in tissue and organ regeneration, such as developing synthetic tissuesand implantable scaffolds, as well as future perspectives and conclusions.

Aliabouzar M, Zhang LG, Sarkar K, 2017 “Effects of scaffold microstructure and low intensity pulsed ultrasound on chondrogenic differentiation of human mesenchymal stem cells,” Biotechnology & Bioengineering, 115, 495-506.

The effects of low intensity pulsed ultrasound (LIPUS) on proliferation andchondrogenic differentiation of human mesenchymal stem cells (hMSCs) seeded on3D printed poly-(ethylene glycol)-diacrylate (PEG-DA) scaffolds with varying poregeometries (square and hexagonal channels) were investigated. The scaffold withsquare pores resulted in higher hMSC growth and chondrogenic differentiation than asolid or a hexagonally porous scaffold. The optimal LIPUS parameters at 1.5 MHz werefound to be 100 mW/cm2and 20% duty cycle. LIPUS stimulation increasedproliferation by up to 60% after 24 hr. For chondrogenesis, we evaluated key cartilagebiomarkers abundant in cartilage tissue; glycosaminoglycan (GAG), type II collagen andtotal collagen. LIPUS stimulation enhanced GAG synthesis up to 16% and 11% forscaffoldswithsquareandhexagonalpatterns,respectively,after2weeks.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 LIPUSstimulation, 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 printingand hMSCs to regenerate damaged cartilage tissues.

Aliabouzar M, Kumar KN, Sarkar K, 2018 “Acoustic vaporization threshold of lipid coated perfluoropentane droplets,” Journal of the Acoustical Society of America, 143, 2001-2012.

Phase shift droplets vaporizable by acoustic stimulation offer the advantages of producing micro-bubbles as contrast agentsin situas well as higher stability and the possibility of achieving smallersizes. Here, the acoustic droplet vaporization (ADV) threshold of a suspension of droplets with aperfluoropentane (PFP) core (diameter 400–3000 nm) is acoustically measured as a function of theexcitation 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 deter-mine the ADV phenomenon, and findings are discussed. The average threshold obtained using threedifferent scattered components increases with frequency—1.0560.28 MPa at 2.25 MHz,1.8960.57 MPa at 5 MHz, and 2.3460.014 MPa at 10 MHz. The scattered response from vapor-ized 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 thresh-old value.

Aliabouzar M, Zhang LG, Sarkar K, 2018 “Acoustic and mechanical characterization of 3D-printed scaffolds for tissue engineering applications,” Biomedical Materials, 13,055013.

The acoustic and mechanical properties of 3D-printed porous poly-(ethylene glycol)-diacrylate(PEGDA)hydrogel scaffolds were investigated using an ultrasound pulse echo technique on differentscaffold microstructures(solid, hexagonal and square pores). Acoustic parameters such as speed ofsound, acoustic impedance and attenuation coefficient as well as physical parameters such as the porestructure, effective density and elastic moduli were determined. The results show that microstructure(porosity and pore geometry)plays a crucial role in defining properties of 3D-printed scaffolds,achieving the highest attenuation for the scaffold with hexagonal pores and showing a decrease insound speed and elastic moduli with increasing porosity. The properties were also found to be similarto those of soft tissues, making PEGDA scaffolds a suitable candidate for tissue engineeringapplications. To evaluate their cellular performance, adhesion and proliferation of humanmesenchymal stem cells(hMSCs)in these scaffolds were investigated. The porous scaffolds performedbetter than the solid one, recording the highest cell attachment and growth for the scaffold with thesquare pores.

Osborn J, Aliabouzar A, Zhou X, Rao R, Zhang LG, Sarkar K 2019 “Enhanced Osteogenic Differentiation of Human Mesenchymal Stem Cells Using Microbubbles and LowIntensity Pulsed Ultrasound on 3D Printed Scaffolds,” Advanced Biosystems, 2, 1800257.

Lipid-coated microbubbles, clinically approved as contrast enhancing agents for ultrasound imaging, are investigated for the first time for their possible applications in bone tissue engineering. Effects of microbubbles (average diameter 1.1 μm) coated by a mixture of lipids (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], and 1,2-dipalmitoyl-3-trimethylmmonium-propane) in the presence of low intensity pulsed ultrasound (LIPUS) on human mesenchymal stem cells seeded on 3D printed poly(lactic acid) porous scaffolds are investigated. LIPUS stimulation (30 mW cm−2, 1.5 MHz, 20% duty cycle) for 3 min a day with 0.5% v/v microbubbles results in a significant increase in proliferation (up to 19.3%) when compared to control after 1, 3, and 5 d. A 3-week osteogenic differentiation study shows a significant increase in total protein content (up to 27.5%), calcium deposition (up to 4.3%), and alkaline phosphatase activity (up to 43.1%) initiated by LIPUS with and without the presence of microbubbles. The microbubbles are found to remain stable during exposure, and their sustained oscillations demonstrably help focus the LIPUS energy toward enhanced cellular response. Integrating LIPUS and microbubbles promises to be a novel and effective strategy for bone tissue engineering and regeneration therapies.

Aliabouzar M, Zhang LG, Sarkar K, 2018 “Acoustic and mechanical characterization of 3D-printed scaffolds for tissue engineering applications,” Biomedical Materials, 13,055013.

The acoustic and mechanical properties of 3D-printed porous poly-(ethylene glycol)-diacrylate(PEGDA)hydrogel scaffolds were investigated using an ultrasound pulse echo technique on differentscaffold microstructures(solid, hexagonal and square pores). Acoustic parameters such as speed ofsound, acoustic impedance and attenuation coefficient as well as physical parameters such as the porestructure, effective density and elastic moduli were determined. The results show that microstructure(porosity and pore geometry)plays a crucial role in defining properties of 3D-printed scaffolds,achieving the highest attenuation for the scaffold with hexagonal pores and showing a decrease insound speed and elastic moduli with increasing porosity. The properties were also found to be similarto those of soft tissues, making PEGDA scaffolds a suitable candidate for tissue engineeringapplications. To evaluate their cellular performance, adhesion and proliferation of humanmesenchymal stem cells(hMSCs)in these scaffolds were investigated. The porous scaffolds performedbetter than the solid one, recording the highest cell attachment and growth for the scaffold with thesquare pores.

Katiyar A, Duncan R L, Sarkar K 2014 “Ultrasound stimulation increases proliferation of MC3T3-E1 preosteoblast-like cells,” Journal of Therapeutic Ultrasound, 2, 1, 1-10.

Srivastava P, Malipeddi Reddy A, Sarkar K 2016 “Steady shear rheology of a viscous emulsion in the presence of finite inertia at moderate volume fractions: sign reversal of normal stress differences,” Journal of Fluid Mechanics, 85, 494-522.

The shear rheology of an emulsion of viscous drops in the presence of finite inertiais investigated using direct numerical simulation. In the absence of inertia, emulsionsdisplay a non-Newtonian rheology with positive first and negative second normalstress differences. However, recently it was discovered that a small amount ofdrop-level inertia alters their signs – the first normal stress difference becomesnegative and the second one becomes positive, each in a small range of capillarynumbers (Li & Sarkar,J. Rheol., vol. 49, 2005, pp. 1377–1394). Sign reversal wasshown numerically and analytically, but only in the limit of a dilute emulsion wheredrop–drop interactions were neglected. Here, we compute the rheology of a density-and viscosity-matched emulsion, accounting for the interactions in the volume fractionrange of 5 %–27 % and Reynolds number range of 0.1–10. The computed rheologicalproperties (effective shear viscosity and first and second normal stress differences) inthe Stokes limit match well with previous theoretical (Choi–Schowalter in the dilutelimit) and simulated results (for concentrated systems) using the boundary elementmethod. The two distinct components of the rheology arising from the interfacialstresses at the drop surface and the perturbative Reynolds stresses are investigated asfunctions of the drop Reynolds number, capillary number and volume fraction. Thesign change is caused by the increasing drop inclination in the presence of inertia,which in turn directly affects the interfacial stresses. Increase of the volume fractionor capillary number increases the critical Reynolds number for sign reversals due toenhanced alignment of the drops with the flow directions. The effect of increasingthe volume fraction on the rheology is explained by relating it to interactions andspecifically to the contact pair-distribution function computed from the simulation.The excess stresses are seen to show an approximately linear behaviour with theReynolds number in the range of 0.1–5, while with the capillary number and volumefraction, the variation is weakly quadratic.

Zhou X, Castro NJ, Zhu W, Cui H, Aliabouzar M, Sarkar K, Zhang LG 2016 “Improved human bone marrow mesenchymal stem cell osteogenesis in 3D bioprinted tissue scaffolds with low intensity pulsed ultrasound stimulation,” Scientific Reports, 6, 32876.

3D printing and ultrasound techniques are showing great promise in the evolution of human musculoskeletal tissue repair and regeneration medicine. The uniqueness of the present study was to combine low intensity pulsed ultrasound (LIPUS) and advanced 3D printing techniques to synergistically improve growth and osteogenic differentiation of human mesenchymal stem cells (MSC). Specifically, polyethylene glycol diacrylate bioinks containing cell adhesive Arginine-Glycine-Aspartic acid-Serene (RGDS) peptide and/or nanocrystalline hydroxyapatite (nHA) were used to fabricate 3D scaffolds with different geometric patterns via novel table-top stereolithography 3D printer. The resultant scaffolds provide a highly porous and interconnected 3D environment to support cell proliferation. Scaffolds with small square pores were determined to be the optimal geometric pattern for MSC attachment and growth. The optimal LIPUS working parameters were determined to be 1.5 MHz, 20% duty cycle with 150 mW/cm2 intensity. Results demonstrated that RGDS peptide and nHA containing 3D printed scaffolds under LIPUS treatment can greatly promote MSC proliferation, alkaline phosphatase activity, calcium deposition and total protein content. These results illustrate the effectiveness of the combination of LIPUS and biomimetic 3D printing scaffolds as a valuable combinatorial tool for improved MSC function, thus make them promising for future clinical and various regenerative medicine application.

Aliabouzar M, Zhang LG, Sarkar K, 2016 “Lipid coated microbubbles and low intensity pulsed ultrasound enhance chondrogenesis of human mesenchymal stem cells in 3D printed scaffolds,” Scientific Reports, 6, 37728.

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.

Miao S, Castro NJ, Nowicki M, Xia L, Cui H, Zhou X, Zhu W, Lee S, Sarkar K, Vozzi G, Tabata Y, Fisher J, Zhang LG 2017 “4D printing of polymeric materials for tissue and organ regeneration,” Materials Today, 20, 577-591.

Four dimensional (4D) printing is an emerging technology with great capacity for fabricating complex,stimuli-responsive 3D structures, providing great potential for tissue and organ engineering applica-tions. Although the 4D concept wasfirst highlighted in 2013, extensive research has rapidly developed,along with more-in-depth understanding and assertions regarding the definition of 4D. In this review,we begin by establishing the criteria of 4D printing, followed by an extensive summary of state-of-the-art technological advances in thefield. Both transformation-preprogrammed 4D printing and 4Dprinting of shape memory polymers are intensively surveyed. Afterwards we will explore and discussthe applications of 4D printing in tissue and organ regeneration, such as developing synthetic tissuesand implantable scaffolds, as well as future perspectives and conclusions.

Aliabouzar M, Zhang LG, Sarkar K, 2017 “Effects of scaffold microstructure and low intensity pulsed ultrasound on chondrogenic differentiation of human mesenchymal stem cells,” Biotechnology & Bioengineering, 115, 495-506.

The effects of low intensity pulsed ultrasound (LIPUS) on proliferation andchondrogenic differentiation of human mesenchymal stem cells (hMSCs) seeded on3D printed poly-(ethylene glycol)-diacrylate (PEG-DA) scaffolds with varying poregeometries (square and hexagonal channels) were investigated. The scaffold withsquare pores resulted in higher hMSC growth and chondrogenic differentiation than asolid or a hexagonally porous scaffold. The optimal LIPUS parameters at 1.5 MHz werefound to be 100 mW/cm2and 20% duty cycle. LIPUS stimulation increasedproliferation by up to 60% after 24 hr. For chondrogenesis, we evaluated key cartilagebiomarkers abundant in cartilage tissue; glycosaminoglycan (GAG), type II collagen andtotal collagen. LIPUS stimulation enhanced GAG synthesis up to 16% and 11% forscaffoldswithsquareandhexagonalpatterns,respectively,after2weeks.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 LIPUSstimulation, 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 printingand hMSCs to regenerate damaged cartilage tissues.

Aliabouzar M, Kumar KN, Sarkar K, 2018 “Acoustic vaporization threshold of lipid coated perfluoropentane droplets,” Journal of the Acoustical Society of America, 143, 2001-2012.

Phase shift droplets vaporizable by acoustic stimulation offer the advantages of producing micro-bubbles as contrast agentsin situas well as higher stability and the possibility of achieving smallersizes. Here, the acoustic droplet vaporization (ADV) threshold of a suspension of droplets with aperfluoropentane (PFP) core (diameter 400–3000 nm) is acoustically measured as a function of theexcitation 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 deter-mine the ADV phenomenon, and findings are discussed. The average threshold obtained using threedifferent scattered components increases with frequency—1.0560.28 MPa at 2.25 MHz,1.8960.57 MPa at 5 MHz, and 2.3460.014 MPa at 10 MHz. The scattered response from vapor-ized 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 thresh-old value.

Aliabouzar M, Zhang LG, Sarkar K, 2018 “Acoustic and mechanical characterization of 3D-printed scaffolds for tissue engineering applications,” Biomedical Materials, 13,055013.

The acoustic and mechanical properties of 3D-printed porous poly-(ethylene glycol)-diacrylate(PEGDA)hydrogel scaffolds were investigated using an ultrasound pulse echo technique on differentscaffold microstructures(solid, hexagonal and square pores). Acoustic parameters such as speed ofsound, acoustic impedance and attenuation coefficient as well as physical parameters such as the porestructure, effective density and elastic moduli were determined. The results show that microstructure(porosity and pore geometry)plays a crucial role in defining properties of 3D-printed scaffolds,achieving the highest attenuation for the scaffold with hexagonal pores and showing a decrease insound speed and elastic moduli with increasing porosity. The properties were also found to be similarto those of soft tissues, making PEGDA scaffolds a suitable candidate for tissue engineeringapplications. To evaluate their cellular performance, adhesion and proliferation of humanmesenchymal stem cells(hMSCs)in these scaffolds were investigated. The porous scaffolds performedbetter than the solid one, recording the highest cell attachment and growth for the scaffold with thesquare pores.

Osborn J, Aliabouzar A, Zhou X, Rao R, Zhang LG, Sarkar K 2019 “Enhanced Osteogenic Differentiation of Human Mesenchymal Stem Cells Using Microbubbles and LowIntensity Pulsed Ultrasound on 3D Printed Scaffolds,” Advanced Biosystems, 2, 1800257.

Lipid-coated microbubbles, clinically approved as contrast enhancing agents for ultrasound imaging, are investigated for the first time for their possible applications in bone tissue engineering. Effects of microbubbles (average diameter 1.1 μm) coated by a mixture of lipids (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], and 1,2-dipalmitoyl-3-trimethylmmonium-propane) in the presence of low intensity pulsed ultrasound (LIPUS) on human mesenchymal stem cells seeded on 3D printed poly(lactic acid) porous scaffolds are investigated. LIPUS stimulation (30 mW cm−2, 1.5 MHz, 20% duty cycle) for 3 min a day with 0.5% v/v microbubbles results in a significant increase in proliferation (up to 19.3%) when compared to control after 1, 3, and 5 d. A 3-week osteogenic differentiation study shows a significant increase in total protein content (up to 27.5%), calcium deposition (up to 4.3%), and alkaline phosphatase activity (up to 43.1%) initiated by LIPUS with and without the presence of microbubbles. The microbubbles are found to remain stable during exposure, and their sustained oscillations demonstrably help focus the LIPUS energy toward enhanced cellular response. Integrating LIPUS and microbubbles promises to be a novel and effective strategy for bone tissue engineering and regeneration therapies.

Aliabouzar M, Kumar KN, Sarkar K, 2018 “Acoustic vaporization threshold of lipid coated perfluoropentane droplets,” Journal of the Acoustical Society of America, 143, 2001-2012.

Phase shift droplets vaporizable by acoustic stimulation offer the advantages of producing micro-bubbles as contrast agentsin situas well as higher stability and the possibility of achieving smallersizes. Here, the acoustic droplet vaporization (ADV) threshold of a suspension of droplets with aperfluoropentane (PFP) core (diameter 400–3000 nm) is acoustically measured as a function of theexcitation 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 deter-mine the ADV phenomenon, and findings are discussed. The average threshold obtained using threedifferent scattered components increases with frequency—1.0560.28 MPa at 2.25 MHz,1.8960.57 MPa at 5 MHz, and 2.3460.014 MPa at 10 MHz. The scattered response from vapor-ized 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 thresh-old value.

Aliabouzar M, Zhang LG, Sarkar K, 2018 “Acoustic and mechanical characterization of 3D-printed scaffolds for tissue engineering applications,” Biomedical Materials, 13,055013.

The acoustic and mechanical properties of 3D-printed porous poly-(ethylene glycol)-diacrylate(PEGDA)hydrogel scaffolds were investigated using an ultrasound pulse echo technique on differentscaffold microstructures(solid, hexagonal and square pores). Acoustic parameters such as speed ofsound, acoustic impedance and attenuation coefficient as well as physical parameters such as the porestructure, effective density and elastic moduli were determined. The results show that microstructure(porosity and pore geometry)plays a crucial role in defining properties of 3D-printed scaffolds,achieving the highest attenuation for the scaffold with hexagonal pores and showing a decrease insound speed and elastic moduli with increasing porosity. The properties were also found to be similarto those of soft tissues, making PEGDA scaffolds a suitable candidate for tissue engineeringapplications. To evaluate their cellular performance, adhesion and proliferation of humanmesenchymal stem cells(hMSCs)in these scaffolds were investigated. The porous scaffolds performedbetter than the solid one, recording the highest cell attachment and growth for the scaffold with thesquare pores.

Osborn J, Aliabouzar A, Zhou X, Rao R, Zhang LG, Sarkar K 2019 “Enhanced Osteogenic Differentiation of Human Mesenchymal Stem Cells Using Microbubbles and LowIntensity Pulsed Ultrasound on 3D Printed Scaffolds,” Advanced Biosystems, 2, 1800257.

Lipid-coated microbubbles, clinically approved as contrast enhancing agents for ultrasound imaging, are investigated for the first time for their possible applications in bone tissue engineering. Effects of microbubbles (average diameter 1.1 μm) coated by a mixture of lipids (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], and 1,2-dipalmitoyl-3-trimethylmmonium-propane) in the presence of low intensity pulsed ultrasound (LIPUS) on human mesenchymal stem cells seeded on 3D printed poly(lactic acid) porous scaffolds are investigated. LIPUS stimulation (30 mW cm−2, 1.5 MHz, 20% duty cycle) for 3 min a day with 0.5% v/v microbubbles results in a significant increase in proliferation (up to 19.3%) when compared to control after 1, 3, and 5 d. A 3-week osteogenic differentiation study shows a significant increase in total protein content (up to 27.5%), calcium deposition (up to 4.3%), and alkaline phosphatase activity (up to 43.1%) initiated by LIPUS with and without the presence of microbubbles. The microbubbles are found to remain stable during exposure, and their sustained oscillations demonstrably help focus the LIPUS energy toward enhanced cellular response. Integrating LIPUS and microbubbles promises to be a novel and effective strategy for bone tissue engineering and regeneration therapies.

Aliabouzar M, Zhang LG, Sarkar K, 2017 “Effects of scaffold microstructure and low intensity pulsed ultrasound on chondrogenic differentiation of human mesenchymal stem cells,” Biotechnology & Bioengineering, 115, 495-506.

The effects of low intensity pulsed ultrasound (LIPUS) on proliferation andchondrogenic differentiation of human mesenchymal stem cells (hMSCs) seeded on3D printed poly-(ethylene glycol)-diacrylate (PEG-DA) scaffolds with varying poregeometries (square and hexagonal channels) were investigated. The scaffold withsquare pores resulted in higher hMSC growth and chondrogenic differentiation than asolid or a hexagonally porous scaffold. The optimal LIPUS parameters at 1.5 MHz werefound to be 100 mW/cm2and 20% duty cycle. LIPUS stimulation increasedproliferation by up to 60% after 24 hr. For chondrogenesis, we evaluated key cartilagebiomarkers abundant in cartilage tissue; glycosaminoglycan (GAG), type II collagen andtotal collagen. LIPUS stimulation enhanced GAG synthesis up to 16% and 11% forscaffoldswithsquareandhexagonalpatterns,respectively,after2weeks.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 LIPUSstimulation, 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 printingand hMSCs to regenerate damaged cartilage tissues.

Miao S, Castro NJ, Nowicki M, Xia L, Cui H, Zhou X, Zhu W, Lee S, Sarkar K, Vozzi G, Tabata Y, Fisher J, Zhang LG 2017 “4D printing of polymeric materials for tissue and organ regeneration,” Materials Today, 20, 577-591.

Four dimensional (4D) printing is an emerging technology with great capacity for fabricating complex,stimuli-responsive 3D structures, providing great potential for tissue and organ engineering applica-tions. Although the 4D concept wasfirst highlighted in 2013, extensive research has rapidly developed,along with more-in-depth understanding and assertions regarding the definition of 4D. In this review,we begin by establishing the criteria of 4D printing, followed by an extensive summary of state-of-the-art technological advances in thefield. Both transformation-preprogrammed 4D printing and 4Dprinting of shape memory polymers are intensively surveyed. Afterwards we will explore and discussthe applications of 4D printing in tissue and organ regeneration, such as developing synthetic tissuesand implantable scaffolds, as well as future perspectives and conclusions.

Aliabouzar M, Zhang LG, Sarkar K, 2016 “Lipid coated microbubbles and low intensity pulsed ultrasound enhance chondrogenesis of human mesenchymal stem cells in 3D printed scaffolds,” Scientific Reports, 6, 37728.

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.

Srivastava P, Malipeddi Reddy A, Sarkar K 2016 “Steady shear rheology of a viscous emulsion in the presence of finite inertia at moderate volume fractions: sign reversal of normal stress differences,” Journal of Fluid Mechanics, 85, 494-522.

The shear rheology of an emulsion of viscous drops in the presence of finite inertiais investigated using direct numerical simulation. In the absence of inertia, emulsionsdisplay a non-Newtonian rheology with positive first and negative second normalstress differences. However, recently it was discovered that a small amount ofdrop-level inertia alters their signs – the first normal stress difference becomesnegative and the second one becomes positive, each in a small range of capillarynumbers (Li & Sarkar,J. Rheol., vol. 49, 2005, pp. 1377–1394). Sign reversal wasshown numerically and analytically, but only in the limit of a dilute emulsion wheredrop–drop interactions were neglected. Here, we compute the rheology of a density-and viscosity-matched emulsion, accounting for the interactions in the volume fractionrange of 5 %–27 % and Reynolds number range of 0.1–10. The computed rheologicalproperties (effective shear viscosity and first and second normal stress differences) inthe Stokes limit match well with previous theoretical (Choi–Schowalter in the dilutelimit) and simulated results (for concentrated systems) using the boundary elementmethod. The two distinct components of the rheology arising from the interfacialstresses at the drop surface and the perturbative Reynolds stresses are investigated asfunctions of the drop Reynolds number, capillary number and volume fraction. Thesign change is caused by the increasing drop inclination in the presence of inertia,which in turn directly affects the interfacial stresses. Increase of the volume fractionor capillary number increases the critical Reynolds number for sign reversals due toenhanced alignment of the drops with the flow directions. The effect of increasingthe volume fraction on the rheology is explained by relating it to interactions andspecifically to the contact pair-distribution function computed from the simulation.The excess stresses are seen to show an approximately linear behaviour with theReynolds number in the range of 0.1–5, while with the capillary number and volumefraction, the variation is weakly quadratic.

Zhou X, Castro NJ, Zhu W, Cui H, Aliabouzar M, Sarkar K, Zhang LG 2016 “Improved human bone marrow mesenchymal stem cell osteogenesis in 3D bioprinted tissue scaffolds with low intensity pulsed ultrasound stimulation,” Scientific Reports, 6, 32876.

3D printing and ultrasound techniques are showing great promise in the evolution of human musculoskeletal tissue repair and regeneration medicine. The uniqueness of the present study was to combine low intensity pulsed ultrasound (LIPUS) and advanced 3D printing techniques to synergistically improve growth and osteogenic differentiation of human mesenchymal stem cells (MSC). Specifically, polyethylene glycol diacrylate bioinks containing cell adhesive Arginine-Glycine-Aspartic acid-Serene (RGDS) peptide and/or nanocrystalline hydroxyapatite (nHA) were used to fabricate 3D scaffolds with different geometric patterns via novel table-top stereolithography 3D printer. The resultant scaffolds provide a highly porous and interconnected 3D environment to support cell proliferation. Scaffolds with small square pores were determined to be the optimal geometric pattern for MSC attachment and growth. The optimal LIPUS working parameters were determined to be 1.5 MHz, 20% duty cycle with 150 mW/cm2 intensity. Results demonstrated that RGDS peptide and nHA containing 3D printed scaffolds under LIPUS treatment can greatly promote MSC proliferation, alkaline phosphatase activity, calcium deposition and total protein content. These results illustrate the effectiveness of the combination of LIPUS and biomimetic 3D printing scaffolds as a valuable combinatorial tool for improved MSC function, thus make them promising for future clinical and various regenerative medicine application.

Katiyar A, Duncan R L, Sarkar K 2014 “Ultrasound stimulation increases proliferation of MC3T3-E1 preosteoblast-like cells,” Journal of Therapeutic Ultrasound, 2, 1, 1-10.

Aliabouzar M, Kumar KN, Sarkar K, 2018 “Acoustic vaporization threshold of lipid coated perfluoropentane droplets,” Journal of the Acoustical Society of America, 143, 2001-2012.

Phase shift droplets vaporizable by acoustic stimulation offer the advantages of producing micro-bubbles as contrast agentsin situas well as higher stability and the possibility of achieving smallersizes. Here, the acoustic droplet vaporization (ADV) threshold of a suspension of droplets with aperfluoropentane (PFP) core (diameter 400–3000 nm) is acoustically measured as a function of theexcitation 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 deter-mine the ADV phenomenon, and findings are discussed. The average threshold obtained using threedifferent scattered components increases with frequency—1.0560.28 MPa at 2.25 MHz,1.8960.57 MPa at 5 MHz, and 2.3460.014 MPa at 10 MHz. The scattered response from vapor-ized 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 thresh-old value.

Aliabouzar M, Zhang LG, Sarkar K, 2018 “Acoustic and mechanical characterization of 3D-printed scaffolds for tissue engineering applications,” Biomedical Materials, 13,055013.

The acoustic and mechanical properties of 3D-printed porous poly-(ethylene glycol)-diacrylate(PEGDA)hydrogel scaffolds were investigated using an ultrasound pulse echo technique on differentscaffold microstructures(solid, hexagonal and square pores). Acoustic parameters such as speed ofsound, acoustic impedance and attenuation coefficient as well as physical parameters such as the porestructure, effective density and elastic moduli were determined. The results show that microstructure(porosity and pore geometry)plays a crucial role in defining properties of 3D-printed scaffolds,achieving the highest attenuation for the scaffold with hexagonal pores and showing a decrease insound speed and elastic moduli with increasing porosity. The properties were also found to be similarto those of soft tissues, making PEGDA scaffolds a suitable candidate for tissue engineeringapplications. To evaluate their cellular performance, adhesion and proliferation of humanmesenchymal stem cells(hMSCs)in these scaffolds were investigated. The porous scaffolds performedbetter than the solid one, recording the highest cell attachment and growth for the scaffold with thesquare pores.

Osborn J, Aliabouzar A, Zhou X, Rao R, Zhang LG, Sarkar K 2019 “Enhanced Osteogenic Differentiation of Human Mesenchymal Stem Cells Using Microbubbles and LowIntensity Pulsed Ultrasound on 3D Printed Scaffolds,” Advanced Biosystems, 2, 1800257.

Lipid-coated microbubbles, clinically approved as contrast enhancing agents for ultrasound imaging, are investigated for the first time for their possible applications in bone tissue engineering. Effects of microbubbles (average diameter 1.1 μm) coated by a mixture of lipids (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], and 1,2-dipalmitoyl-3-trimethylmmonium-propane) in the presence of low intensity pulsed ultrasound (LIPUS) on human mesenchymal stem cells seeded on 3D printed poly(lactic acid) porous scaffolds are investigated. LIPUS stimulation (30 mW cm−2, 1.5 MHz, 20% duty cycle) for 3 min a day with 0.5% v/v microbubbles results in a significant increase in proliferation (up to 19.3%) when compared to control after 1, 3, and 5 d. A 3-week osteogenic differentiation study shows a significant increase in total protein content (up to 27.5%), calcium deposition (up to 4.3%), and alkaline phosphatase activity (up to 43.1%) initiated by LIPUS with and without the presence of microbubbles. The microbubbles are found to remain stable during exposure, and their sustained oscillations demonstrably help focus the LIPUS energy toward enhanced cellular response. Integrating LIPUS and microbubbles promises to be a novel and effective strategy for bone tissue engineering and regeneration therapies.

Aliabouzar M, Kumar KN, Sarkar K, 2018 “Acoustic vaporization threshold of lipid coated perfluoropentane droplets,” Journal of the Acoustical Society of America, 143, 2001-2012.

Phase shift droplets vaporizable by acoustic stimulation offer the advantages of producing micro-bubbles as contrast agentsin situas well as higher stability and the possibility of achieving smallersizes. Here, the acoustic droplet vaporization (ADV) threshold of a suspension of droplets with aperfluoropentane (PFP) core (diameter 400–3000 nm) is acoustically measured as a function of theexcitation 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 deter-mine the ADV phenomenon, and findings are discussed. The average threshold obtained using threedifferent scattered components increases with frequency—1.0560.28 MPa at 2.25 MHz,1.8960.57 MPa at 5 MHz, and 2.3460.014 MPa at 10 MHz. The scattered response from vapor-ized 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 thresh-old value.

Miao S, Castro NJ, Nowicki M, Xia L, Cui H, Zhou X, Zhu W, Lee S, Sarkar K, Vozzi G, Tabata Y, Fisher J, Zhang LG 2017 “4D printing of polymeric materials for tissue and organ regeneration,” Materials Today, 20, 577-591.

Four dimensional (4D) printing is an emerging technology with great capacity for fabricating complex,stimuli-responsive 3D structures, providing great potential for tissue and organ engineering applica-tions. Although the 4D concept wasfirst highlighted in 2013, extensive research has rapidly developed,along with more-in-depth understanding and assertions regarding the definition of 4D. In this review,we begin by establishing the criteria of 4D printing, followed by an extensive summary of state-of-the-art technological advances in thefield. Both transformation-preprogrammed 4D printing and 4Dprinting of shape memory polymers are intensively surveyed. Afterwards we will explore and discussthe applications of 4D printing in tissue and organ regeneration, such as developing synthetic tissuesand implantable scaffolds, as well as future perspectives and conclusions.

Aliabouzar M, Zhang LG, Sarkar K, 2018 “Acoustic and mechanical characterization of 3D-printed scaffolds for tissue engineering applications,” Biomedical Materials, 13,055013.

The acoustic and mechanical properties of 3D-printed porous poly-(ethylene glycol)-diacrylate(PEGDA)hydrogel scaffolds were investigated using an ultrasound pulse echo technique on differentscaffold microstructures(solid, hexagonal and square pores). Acoustic parameters such as speed ofsound, acoustic impedance and attenuation coefficient as well as physical parameters such as the porestructure, effective density and elastic moduli were determined. The results show that microstructure(porosity and pore geometry)plays a crucial role in defining properties of 3D-printed scaffolds,achieving the highest attenuation for the scaffold with hexagonal pores and showing a decrease insound speed and elastic moduli with increasing porosity. The properties were also found to be similarto those of soft tissues, making PEGDA scaffolds a suitable candidate for tissue engineeringapplications. To evaluate their cellular performance, adhesion and proliferation of humanmesenchymal stem cells(hMSCs)in these scaffolds were investigated. The porous scaffolds performedbetter than the solid one, recording the highest cell attachment and growth for the scaffold with thesquare pores.

Aliabouzar M, Zhang LG, Sarkar K, 2017 “Effects of scaffold microstructure and low intensity pulsed ultrasound on chondrogenic differentiation of human mesenchymal stem cells,” Biotechnology & Bioengineering, 115, 495-506.

The effects of low intensity pulsed ultrasound (LIPUS) on proliferation andchondrogenic differentiation of human mesenchymal stem cells (hMSCs) seeded on3D printed poly-(ethylene glycol)-diacrylate (PEG-DA) scaffolds with varying poregeometries (square and hexagonal channels) were investigated. The scaffold withsquare pores resulted in higher hMSC growth and chondrogenic differentiation than asolid or a hexagonally porous scaffold. The optimal LIPUS parameters at 1.5 MHz werefound to be 100 mW/cm2and 20% duty cycle. LIPUS stimulation increasedproliferation by up to 60% after 24 hr. For chondrogenesis, we evaluated key cartilagebiomarkers abundant in cartilage tissue; glycosaminoglycan (GAG), type II collagen andtotal collagen. LIPUS stimulation enhanced GAG synthesis up to 16% and 11% forscaffoldswithsquareandhexagonalpatterns,respectively,after2weeks.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 LIPUSstimulation, 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 printingand hMSCs to regenerate damaged cartilage tissues.

Srivastava P, Malipeddi Reddy A, Sarkar K 2016 “Steady shear rheology of a viscous emulsion in the presence of finite inertia at moderate volume fractions: sign reversal of normal stress differences,” Journal of Fluid Mechanics, 85, 494-522.

The shear rheology of an emulsion of viscous drops in the presence of finite inertiais investigated using direct numerical simulation. In the absence of inertia, emulsionsdisplay a non-Newtonian rheology with positive first and negative second normalstress differences. However, recently it was discovered that a small amount ofdrop-level inertia alters their signs – the first normal stress difference becomesnegative and the second one becomes positive, each in a small range of capillarynumbers (Li & Sarkar,J. Rheol., vol. 49, 2005, pp. 1377–1394). Sign reversal wasshown numerically and analytically, but only in the limit of a dilute emulsion wheredrop–drop interactions were neglected. Here, we compute the rheology of a density-and viscosity-matched emulsion, accounting for the interactions in the volume fractionrange of 5 %–27 % and Reynolds number range of 0.1–10. The computed rheologicalproperties (effective shear viscosity and first and second normal stress differences) inthe Stokes limit match well with previous theoretical (Choi–Schowalter in the dilutelimit) and simulated results (for concentrated systems) using the boundary elementmethod. The two distinct components of the rheology arising from the interfacialstresses at the drop surface and the perturbative Reynolds stresses are investigated asfunctions of the drop Reynolds number, capillary number and volume fraction. Thesign change is caused by the increasing drop inclination in the presence of inertia,which in turn directly affects the interfacial stresses. Increase of the volume fractionor capillary number increases the critical Reynolds number for sign reversals due toenhanced alignment of the drops with the flow directions. The effect of increasingthe volume fraction on the rheology is explained by relating it to interactions andspecifically to the contact pair-distribution function computed from the simulation.The excess stresses are seen to show an approximately linear behaviour with theReynolds number in the range of 0.1–5, while with the capillary number and volumefraction, the variation is weakly quadratic.

Zhou X, Castro NJ, Zhu W, Cui H, Aliabouzar M, Sarkar K, Zhang LG 2016 “Improved human bone marrow mesenchymal stem cell osteogenesis in 3D bioprinted tissue scaffolds with low intensity pulsed ultrasound stimulation,” Scientific Reports, 6, 32876.

3D printing and ultrasound techniques are showing great promise in the evolution of human musculoskeletal tissue repair and regeneration medicine. The uniqueness of the present study was to combine low intensity pulsed ultrasound (LIPUS) and advanced 3D printing techniques to synergistically improve growth and osteogenic differentiation of human mesenchymal stem cells (MSC). Specifically, polyethylene glycol diacrylate bioinks containing cell adhesive Arginine-Glycine-Aspartic acid-Serene (RGDS) peptide and/or nanocrystalline hydroxyapatite (nHA) were used to fabricate 3D scaffolds with different geometric patterns via novel table-top stereolithography 3D printer. The resultant scaffolds provide a highly porous and interconnected 3D environment to support cell proliferation. Scaffolds with small square pores were determined to be the optimal geometric pattern for MSC attachment and growth. The optimal LIPUS working parameters were determined to be 1.5 MHz, 20% duty cycle with 150 mW/cm2 intensity. Results demonstrated that RGDS peptide and nHA containing 3D printed scaffolds under LIPUS treatment can greatly promote MSC proliferation, alkaline phosphatase activity, calcium deposition and total protein content. These results illustrate the effectiveness of the combination of LIPUS and biomimetic 3D printing scaffolds as a valuable combinatorial tool for improved MSC function, thus make them promising for future clinical and various regenerative medicine application.

Aliabouzar M, Zhang LG, Sarkar K, 2016 “Lipid coated microbubbles and low intensity pulsed ultrasound enhance chondrogenesis of human mesenchymal stem cells in 3D printed scaffolds,” Scientific Reports, 6, 37728.

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.

Katiyar A, Duncan R L, Sarkar K 2014 “Ultrasound stimulation increases proliferation of MC3T3-E1 preosteoblast-like cells,” Journal of Therapeutic Ultrasound, 2, 1, 1-10.

Aliabouzar M, Zhang LG, Sarkar K, 2018 “Acoustic and mechanical characterization of 3D-printed scaffolds for tissue engineering applications,” Biomedical Materials, 13,055013.

The acoustic and mechanical properties of 3D-printed porous poly-(ethylene glycol)-diacrylate(PEGDA)hydrogel scaffolds were investigated using an ultrasound pulse echo technique on differentscaffold microstructures(solid, hexagonal and square pores). Acoustic parameters such as speed ofsound, acoustic impedance and attenuation coefficient as well as physical parameters such as the porestructure, effective density and elastic moduli were determined. The results show that microstructure(porosity and pore geometry)plays a crucial role in defining properties of 3D-printed scaffolds,achieving the highest attenuation for the scaffold with hexagonal pores and showing a decrease insound speed and elastic moduli with increasing porosity. The properties were also found to be similarto those of soft tissues, making PEGDA scaffolds a suitable candidate for tissue engineeringapplications. To evaluate their cellular performance, adhesion and proliferation of humanmesenchymal stem cells(hMSCs)in these scaffolds were investigated. The porous scaffolds performedbetter than the solid one, recording the highest cell attachment and growth for the scaffold with thesquare pores.

Mobadersany N, Sarkar K 2019 “Acoustic microstreaming near a plane wall due to a pulsating free or coated bubble: velocity, vorticity and closed streamlines,” Journal of Fluid Mechanics, 875 781-806.

Acoustic microstreaming due to an oscillating microbubble, either coated or free, is analytically investigated. The detailed flow field is obtained and the closed streamlines of the ring vortex generated by microstreaming are plotted in both Eulerian and Lagrangian descriptions. Analytical expressions are found for the ring vortex showing that its length depends only on the separation of the microbubble from the wall
and the dependence is linear. The circulation as a scalar measure of the vortex is computed quantitatively identifying its spatial location. The functional dependence of circulation on bubble separation and coating parameters is shown to be similar to that of the shear stress.

Singha S, Malipeddy AR, Zurita-Gotor M, Sarkar K, Shen K, Loewenberg M, Migler KB, Blawzdziewicz J 2019 “Mechanisms of spontaneous chain formation and subsequent microstructural evolution in shear-driven strongly confined drop monolayers,” Soft Matter, 15, 4873-4889.

It was experimentally demonstrated by Migler and his collaborators [Phys. Rev. Lett., 2001, 86, 1023; Langmuir, 2003, 19, 8667] that a strongly confined drop monolayer sheared between two parallel plates can spontaneously develop a flow-oriented drop-chain morphology. Here we show that the formation of the chain-like microstructure is driven by far-field Hele-Shaw quadrupolar interactions between
drops, and that drop spacing within chains is controlled by the effective drop repulsion associated with the existence of confinement-induced reversing streamlines, i.e., the swapping trajectory effect. Using
direct numerical simulations and an accurate quasi-2D model that incorporates quadrupolar and swapping-trajectory contributions, we analyze microstructural evolution in a monodisperse drop
monolayer. Consistent with experimental observations, we find that drop spacing within individual chains is usually uniform. Further analysis shows that at low area fractions all chains have the same spacing, but at higher area fractions there is a large spacing variation from chain to chain. These findings are explained in terms of uncompressed and compressed chains. At low area fractions most chains are
uncompressed (spacing equals lst, which is the stable separation of an isolated pair). At higher area fractions compressed chains (with tighter spacing) are formed in a process of chain zipping along
y-shaped structural defects. We also discuss the relevance of our findings to other shear-driven systems, such as suspensions of spheres in non-Newtonian fluids.

Malipeddy AR, Sarkar K 2019 “Collective diffusivity in a sheared viscous emulsion: effects of viscosity ratio,” Physical Review Fluids, 4, 093603.

The shear-induced collective or gradient diffusivity in an emulsion of viscous drops,
specifically as a function of viscosity ratio, was computed using a fully resolved numerical method. An initially randomly packed layer of viscous drops spreading due to drop-drop interactions in an imposed shear has been simulated. The collective diffusivity coefficient was computed using a self-similar solution of the drop concentration profile. We also obtained the collective diffusivity (the collective diffusivity coefficient multiplied by the average drop volume fraction), computing the dynamic structure factor from the simulated drop positions—an analysis typically applied only to homogeneous systems. The two quantities computed using entirely different methods are in broad agreement, including their predictions of nonmonotonic variations with increasing capillary number and viscosity ratio. The computed values were also found to match with past experimental
measurements. The collective diffusivity coefficient computed here, as expected, is 1 order of magnitude larger than the self-diffusivity coefficient for a dilute emulsion previously computed using pairwise simulation of viscous drops in shear. The collective diffusivity coefficient computed here shows a nonmonotonic variation with viscosity ratio, in contrast to self-diffusivity computed using pairwise computation. The difference might point to an intrinsic difference in physics underlying the two diffusivities. Alternatively, it also might
arise from drops not reaching equilibrium deformation in the period after one interaction and before the next—an effect absent in the pairwise simulation used for the computation of self-diffusivity. We offer a qualitative explanation of the nonmonotonic variation by relating it to average nonmonotonic drop deformation with increasing viscosity ratio. We
also provide empirical correlations of the collective diffusivity as a function of viscosity ratio and capillary number.

Malipeddy AR, Sarkar K 2019 “Collective diffusivity in a sheared viscous emulsion: effects of viscosity ratio,” Physical Review Fluids, 4, 093603.

The shear-induced collective or gradient diffusivity in an emulsion of viscous drops,
specifically as a function of viscosity ratio, was computed using a fully resolved numerical method. An initially randomly packed layer of viscous drops spreading due to drop-drop interactions in an imposed shear has been simulated. The collective diffusivity coefficient was computed using a self-similar solution of the drop concentration profile. We also obtained the collective diffusivity (the collective diffusivity coefficient multiplied by the average drop volume fraction), computing the dynamic structure factor from the simulated drop positions—an analysis typically applied only to homogeneous systems. The two quantities computed using entirely different methods are in broad agreement, including their predictions of nonmonotonic variations with increasing capillary number and viscosity ratio. The computed values were also found to match with past experimental
measurements. The collective diffusivity coefficient computed here, as expected, is 1 order of magnitude larger than the self-diffusivity coefficient for a dilute emulsion previously computed using pairwise simulation of viscous drops in shear. The collective diffusivity coefficient computed here shows a nonmonotonic variation with viscosity ratio, in contrast to self-diffusivity computed using pairwise computation. The difference might point to an intrinsic difference in physics underlying the two diffusivities. Alternatively, it also might
arise from drops not reaching equilibrium deformation in the period after one interaction and before the next—an effect absent in the pairwise simulation used for the computation of self-diffusivity. We offer a qualitative explanation of the nonmonotonic variation by relating it to average nonmonotonic drop deformation with increasing viscosity ratio. We
also provide empirical correlations of the collective diffusivity as a function of viscosity ratio and capillary number.

Singha S, Malipeddy AR, Zurita-Gotor M, Sarkar K, Shen K, Loewenberg M, Migler KB, Blawzdziewicz J 2019 “Mechanisms of spontaneous chain formation and subsequent microstructural evolution in shear-driven strongly confined drop monolayers,” Soft Matter, 15, 4873-4889.

It was experimentally demonstrated by Migler and his collaborators [Phys. Rev. Lett., 2001, 86, 1023; Langmuir, 2003, 19, 8667] that a strongly confined drop monolayer sheared between two parallel plates can spontaneously develop a flow-oriented drop-chain morphology. Here we show that the formation of the chain-like microstructure is driven by far-field Hele-Shaw quadrupolar interactions between
drops, and that drop spacing within chains is controlled by the effective drop repulsion associated with the existence of confinement-induced reversing streamlines, i.e., the swapping trajectory effect. Using
direct numerical simulations and an accurate quasi-2D model that incorporates quadrupolar and swapping-trajectory contributions, we analyze microstructural evolution in a monodisperse drop
monolayer. Consistent with experimental observations, we find that drop spacing within individual chains is usually uniform. Further analysis shows that at low area fractions all chains have the same spacing, but at higher area fractions there is a large spacing variation from chain to chain. These findings are explained in terms of uncompressed and compressed chains. At low area fractions most chains are
uncompressed (spacing equals lst, which is the stable separation of an isolated pair). At higher area fractions compressed chains (with tighter spacing) are formed in a process of chain zipping along
y-shaped structural defects. We also discuss the relevance of our findings to other shear-driven systems, such as suspensions of spheres in non-Newtonian fluids.

Mobadersany N, Sarkar K 2019 “Acoustic microstreaming near a plane wall due to a pulsating free or coated bubble: velocity, vorticity and closed streamlines,” Journal of Fluid Mechanics, 875 781-806.

Acoustic microstreaming due to an oscillating microbubble, either coated or free, is analytically investigated. The detailed flow field is obtained and the closed streamlines of the ring vortex generated by microstreaming are plotted in both Eulerian and Lagrangian descriptions. Analytical expressions are found for the ring vortex showing that its length depends only on the separation of the microbubble from the wall
and the dependence is linear. The circulation as a scalar measure of the vortex is computed quantitatively identifying its spatial location. The functional dependence of circulation on bubble separation and coating parameters is shown to be similar to that of the shear stress.

Aliabouzar M, Kumar KN, Sarkar K, 2018 “Acoustic vaporization threshold of lipid coated perfluoropentane droplets,” Journal of the Acoustical Society of America, 143, 2001-2012.

Phase shift droplets vaporizable by acoustic stimulation offer the advantages of producing micro-bubbles as contrast agentsin situas well as higher stability and the possibility of achieving smallersizes. Here, the acoustic droplet vaporization (ADV) threshold of a suspension of droplets with aperfluoropentane (PFP) core (diameter 400–3000 nm) is acoustically measured as a function of theexcitation 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 deter-mine the ADV phenomenon, and findings are discussed. The average threshold obtained using threedifferent scattered components increases with frequency—1.0560.28 MPa at 2.25 MHz,1.8960.57 MPa at 5 MHz, and 2.3460.014 MPa at 10 MHz. The scattered response from vapor-ized 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 thresh-old value.

Aliabouzar M, Zhang LG, Sarkar K, 2018 “Acoustic and mechanical characterization of 3D-printed scaffolds for tissue engineering applications,” Biomedical Materials, 13,055013.

The acoustic and mechanical properties of 3D-printed porous poly-(ethylene glycol)-diacrylate(PEGDA)hydrogel scaffolds were investigated using an ultrasound pulse echo technique on differentscaffold microstructures(solid, hexagonal and square pores). Acoustic parameters such as speed ofsound, acoustic impedance and attenuation coefficient as well as physical parameters such as the porestructure, effective density and elastic moduli were determined. The results show that microstructure(porosity and pore geometry)plays a crucial role in defining properties of 3D-printed scaffolds,achieving the highest attenuation for the scaffold with hexagonal pores and showing a decrease insound speed and elastic moduli with increasing porosity. The properties were also found to be similarto those of soft tissues, making PEGDA scaffolds a suitable candidate for tissue engineeringapplications. To evaluate their cellular performance, adhesion and proliferation of humanmesenchymal stem cells(hMSCs)in these scaffolds were investigated. The porous scaffolds performedbetter than the solid one, recording the highest cell attachment and growth for the scaffold with thesquare pores.

Aliabouzar M, Zhang LG, Sarkar K, 2018 “Acoustic and mechanical characterization of 3D-printed scaffolds for tissue engineering applications,” Biomedical Materials, 13,055013.

The acoustic and mechanical properties of 3D-printed porous poly-(ethylene glycol)-diacrylate(PEGDA)hydrogel scaffolds were investigated using an ultrasound pulse echo technique on differentscaffold microstructures(solid, hexagonal and square pores). Acoustic parameters such as speed ofsound, acoustic impedance and attenuation coefficient as well as physical parameters such as the porestructure, effective density and elastic moduli were determined. The results show that microstructure(porosity and pore geometry)plays a crucial role in defining properties of 3D-printed scaffolds,achieving the highest attenuation for the scaffold with hexagonal pores and showing a decrease insound speed and elastic moduli with increasing porosity. The properties were also found to be similarto those of soft tissues, making PEGDA scaffolds a suitable candidate for tissue engineeringapplications. To evaluate their cellular performance, adhesion and proliferation of humanmesenchymal stem cells(hMSCs)in these scaffolds were investigated. The porous scaffolds performedbetter than the solid one, recording the highest cell attachment and growth for the scaffold with thesquare pores.

Aliabouzar M, Kumar KN, Sarkar K, 2018 “Acoustic vaporization threshold of lipid coated perfluoropentane droplets,” Journal of the Acoustical Society of America, 143, 2001-2012.

Phase shift droplets vaporizable by acoustic stimulation offer the advantages of producing micro-bubbles as contrast agentsin situas well as higher stability and the possibility of achieving smallersizes. Here, the acoustic droplet vaporization (ADV) threshold of a suspension of droplets with aperfluoropentane (PFP) core (diameter 400–3000 nm) is acoustically measured as a function of theexcitation 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 deter-mine the ADV phenomenon, and findings are discussed. The average threshold obtained using threedifferent scattered components increases with frequency—1.0560.28 MPa at 2.25 MHz,1.8960.57 MPa at 5 MHz, and 2.3460.014 MPa at 10 MHz. The scattered response from vapor-ized 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 thresh-old value.

Osborn J, Aliabouzar A, Zhou X, Rao R, Zhang LG, Sarkar K 2019 “Enhanced Osteogenic Differentiation of Human Mesenchymal Stem Cells Using Microbubbles and LowIntensity Pulsed Ultrasound on 3D Printed Scaffolds,” Advanced Biosystems, 2, 1800257.

Lipid-coated microbubbles, clinically approved as contrast enhancing agents for ultrasound imaging, are investigated for the first time for their possible applications in bone tissue engineering. Effects of microbubbles (average diameter 1.1 μm) coated by a mixture of lipids (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], and 1,2-dipalmitoyl-3-trimethylmmonium-propane) in the presence of low intensity pulsed ultrasound (LIPUS) on human mesenchymal stem cells seeded on 3D printed poly(lactic acid) porous scaffolds are investigated. LIPUS stimulation (30 mW cm−2, 1.5 MHz, 20% duty cycle) for 3 min a day with 0.5% v/v microbubbles results in a significant increase in proliferation (up to 19.3%) when compared to control after 1, 3, and 5 d. A 3-week osteogenic differentiation study shows a significant increase in total protein content (up to 27.5%), calcium deposition (up to 4.3%), and alkaline phosphatase activity (up to 43.1%) initiated by LIPUS with and without the presence of microbubbles. The microbubbles are found to remain stable during exposure, and their sustained oscillations demonstrably help focus the LIPUS energy toward enhanced cellular response. Integrating LIPUS and microbubbles promises to be a novel and effective strategy for bone tissue engineering and regeneration therapies.

Aliabouzar M, Zhang LG, Sarkar K, 2017 “Effects of scaffold microstructure and low intensity pulsed ultrasound on chondrogenic differentiation of human mesenchymal stem cells,” Biotechnology & Bioengineering, 115, 495-506.

The effects of low intensity pulsed ultrasound (LIPUS) on proliferation andchondrogenic differentiation of human mesenchymal stem cells (hMSCs) seeded on3D printed poly-(ethylene glycol)-diacrylate (PEG-DA) scaffolds with varying poregeometries (square and hexagonal channels) were investigated. The scaffold withsquare pores resulted in higher hMSC growth and chondrogenic differentiation than asolid or a hexagonally porous scaffold. The optimal LIPUS parameters at 1.5 MHz werefound to be 100 mW/cm2and 20% duty cycle. LIPUS stimulation increasedproliferation by up to 60% after 24 hr. For chondrogenesis, we evaluated key cartilagebiomarkers abundant in cartilage tissue; glycosaminoglycan (GAG), type II collagen andtotal collagen. LIPUS stimulation enhanced GAG synthesis up to 16% and 11% forscaffoldswithsquareandhexagonalpatterns,respectively,after2weeks.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 LIPUSstimulation, 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 printingand hMSCs to regenerate damaged cartilage tissues.

Miao S, Castro NJ, Nowicki M, Xia L, Cui H, Zhou X, Zhu W, Lee S, Sarkar K, Vozzi G, Tabata Y, Fisher J, Zhang LG 2017 “4D printing of polymeric materials for tissue and organ regeneration,” Materials Today, 20, 577-591.

Four dimensional (4D) printing is an emerging technology with great capacity for fabricating complex,stimuli-responsive 3D structures, providing great potential for tissue and organ engineering applica-tions. Although the 4D concept wasfirst highlighted in 2013, extensive research has rapidly developed,along with more-in-depth understanding and assertions regarding the definition of 4D. In this review,we begin by establishing the criteria of 4D printing, followed by an extensive summary of state-of-the-art technological advances in thefield. Both transformation-preprogrammed 4D printing and 4Dprinting of shape memory polymers are intensively surveyed. Afterwards we will explore and discussthe applications of 4D printing in tissue and organ regeneration, such as developing synthetic tissuesand implantable scaffolds, as well as future perspectives and conclusions.

Aliabouzar M, Zhang LG, Sarkar K, 2016 “Lipid coated microbubbles and low intensity pulsed ultrasound enhance chondrogenesis of human mesenchymal stem cells in 3D printed scaffolds,” Scientific Reports, 6, 37728.

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.

Zhou X, Castro NJ, Zhu W, Cui H, Aliabouzar M, Sarkar K, Zhang LG 2016 “Improved human bone marrow mesenchymal stem cell osteogenesis in 3D bioprinted tissue scaffolds with low intensity pulsed ultrasound stimulation,” Scientific Reports, 6, 32876.

3D printing and ultrasound techniques are showing great promise in the evolution of human musculoskeletal tissue repair and regeneration medicine. The uniqueness of the present study was to combine low intensity pulsed ultrasound (LIPUS) and advanced 3D printing techniques to synergistically improve growth and osteogenic differentiation of human mesenchymal stem cells (MSC). Specifically, polyethylene glycol diacrylate bioinks containing cell adhesive Arginine-Glycine-Aspartic acid-Serene (RGDS) peptide and/or nanocrystalline hydroxyapatite (nHA) were used to fabricate 3D scaffolds with different geometric patterns via novel table-top stereolithography 3D printer. The resultant scaffolds provide a highly porous and interconnected 3D environment to support cell proliferation. Scaffolds with small square pores were determined to be the optimal geometric pattern for MSC attachment and growth. The optimal LIPUS working parameters were determined to be 1.5 MHz, 20% duty cycle with 150 mW/cm2 intensity. Results demonstrated that RGDS peptide and nHA containing 3D printed scaffolds under LIPUS treatment can greatly promote MSC proliferation, alkaline phosphatase activity, calcium deposition and total protein content. These results illustrate the effectiveness of the combination of LIPUS and biomimetic 3D printing scaffolds as a valuable combinatorial tool for improved MSC function, thus make them promising for future clinical and various regenerative medicine application.

Srivastava P, Malipeddi Reddy A, Sarkar K 2016 “Steady shear rheology of a viscous emulsion in the presence of finite inertia at moderate volume fractions: sign reversal of normal stress differences,” Journal of Fluid Mechanics, 85, 494-522.

The shear rheology of an emulsion of viscous drops in the presence of finite inertiais investigated using direct numerical simulation. In the absence of inertia, emulsionsdisplay a non-Newtonian rheology with positive first and negative second normalstress differences. However, recently it was discovered that a small amount ofdrop-level inertia alters their signs – the first normal stress difference becomesnegative and the second one becomes positive, each in a small range of capillarynumbers (Li & Sarkar,J. Rheol., vol. 49, 2005, pp. 1377–1394). Sign reversal wasshown numerically and analytically, but only in the limit of a dilute emulsion wheredrop–drop interactions were neglected. Here, we compute the rheology of a density-and viscosity-matched emulsion, accounting for the interactions in the volume fractionrange of 5 %–27 % and Reynolds number range of 0.1–10. The computed rheologicalproperties (effective shear viscosity and first and second normal stress differences) inthe Stokes limit match well with previous theoretical (Choi–Schowalter in the dilutelimit) and simulated results (for concentrated systems) using the boundary elementmethod. The two distinct components of the rheology arising from the interfacialstresses at the drop surface and the perturbative Reynolds stresses are investigated asfunctions of the drop Reynolds number, capillary number and volume fraction. Thesign change is caused by the increasing drop inclination in the presence of inertia,which in turn directly affects the interfacial stresses. Increase of the volume fractionor capillary number increases the critical Reynolds number for sign reversals due toenhanced alignment of the drops with the flow directions. The effect of increasingthe volume fraction on the rheology is explained by relating it to interactions andspecifically to the contact pair-distribution function computed from the simulation.The excess stresses are seen to show an approximately linear behaviour with theReynolds number in the range of 0.1–5, while with the capillary number and volumefraction, the variation is weakly quadratic.

Srivastava P, Malipeddi Reddy A, Sarkar K 2016 “Steady shear rheology of a viscous emulsion in the presence of finite inertia at moderate volume fractions: sign reversal of normal stress differences,” Journal of Fluid Mechanics, 85, 494-522.

The shear rheology of an emulsion of viscous drops in the presence of finite inertiais investigated using direct numerical simulation. In the absence of inertia, emulsionsdisplay a non-Newtonian rheology with positive first and negative second normalstress differences. However, recently it was discovered that a small amount ofdrop-level inertia alters their signs – the first normal stress difference becomesnegative and the second one becomes positive, each in a small range of capillarynumbers (Li & Sarkar,J. Rheol., vol. 49, 2005, pp. 1377–1394). Sign reversal wasshown numerically and analytically, but only in the limit of a dilute emulsion wheredrop–drop interactions were neglected. Here, we compute the rheology of a density-and viscosity-matched emulsion, accounting for the interactions in the volume fractionrange of 5 %–27 % and Reynolds number range of 0.1–10. The computed rheologicalproperties (effective shear viscosity and first and second normal stress differences) inthe Stokes limit match well with previous theoretical (Choi–Schowalter in the dilutelimit) and simulated results (for concentrated systems) using the boundary elementmethod. The two distinct components of the rheology arising from the interfacialstresses at the drop surface and the perturbative Reynolds stresses are investigated asfunctions of the drop Reynolds number, capillary number and volume fraction. Thesign change is caused by the increasing drop inclination in the presence of inertia,which in turn directly affects the interfacial stresses. Increase of the volume fractionor capillary number increases the critical Reynolds number for sign reversals due toenhanced alignment of the drops with the flow directions. The effect of increasingthe volume fraction on the rheology is explained by relating it to interactions andspecifically to the contact pair-distribution function computed from the simulation.The excess stresses are seen to show an approximately linear behaviour with theReynolds number in the range of 0.1–5, while with the capillary number and volumefraction, the variation is weakly quadratic.

Katiyar A, Duncan R L, Sarkar K 2014 “Ultrasound stimulation increases proliferation of MC3T3-E1 preosteoblast-like cells,” Journal of Therapeutic Ultrasound, 2, 1, 1-10.

Aliabouzar M, Zhang LG, Sarkar K, 2018 “Acoustic and mechanical characterization of 3D-printed scaffolds for tissue engineering applications,” Biomedical Materials, 13,055013.

The acoustic and mechanical properties of 3D-printed porous poly-(ethylene glycol)-diacrylate(PEGDA)hydrogel scaffolds were investigated using an ultrasound pulse echo technique on differentscaffold microstructures(solid, hexagonal and square pores). Acoustic parameters such as speed ofsound, acoustic impedance and attenuation coefficient as well as physical parameters such as the porestructure, effective density and elastic moduli were determined. The results show that microstructure(porosity and pore geometry)plays a crucial role in defining properties of 3D-printed scaffolds,achieving the highest attenuation for the scaffold with hexagonal pores and showing a decrease insound speed and elastic moduli with increasing porosity. The properties were also found to be similarto those of soft tissues, making PEGDA scaffolds a suitable candidate for tissue engineeringapplications. To evaluate their cellular performance, adhesion and proliferation of humanmesenchymal stem cells(hMSCs)in these scaffolds were investigated. The porous scaffolds performedbetter than the solid one, recording the highest cell attachment and growth for the scaffold with thesquare pores.

Aliabouzar M, Kumar KN, Sarkar K, 2018 “Acoustic vaporization threshold of lipid coated perfluoropentane droplets,” Journal of the Acoustical Society of America, 143, 2001-2012.

Phase shift droplets vaporizable by acoustic stimulation offer the advantages of producing micro-bubbles as contrast agentsin situas well as higher stability and the possibility of achieving smallersizes. Here, the acoustic droplet vaporization (ADV) threshold of a suspension of droplets with aperfluoropentane (PFP) core (diameter 400–3000 nm) is acoustically measured as a function of theexcitation 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 deter-mine the ADV phenomenon, and findings are discussed. The average threshold obtained using threedifferent scattered components increases with frequency—1.0560.28 MPa at 2.25 MHz,1.8960.57 MPa at 5 MHz, and 2.3460.014 MPa at 10 MHz. The scattered response from vapor-ized 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 thresh-old value.

Zhou X, Castro NJ, Zhu W, Cui H, Aliabouzar M, Sarkar K, Zhang LG 2016 “Improved human bone marrow mesenchymal stem cell osteogenesis in 3D bioprinted tissue scaffolds with low intensity pulsed ultrasound stimulation,” Scientific Reports, 6, 32876.

3D printing and ultrasound techniques are showing great promise in the evolution of human musculoskeletal tissue repair and regeneration medicine. The uniqueness of the present study was to combine low intensity pulsed ultrasound (LIPUS) and advanced 3D printing techniques to synergistically improve growth and osteogenic differentiation of human mesenchymal stem cells (MSC). Specifically, polyethylene glycol diacrylate bioinks containing cell adhesive Arginine-Glycine-Aspartic acid-Serene (RGDS) peptide and/or nanocrystalline hydroxyapatite (nHA) were used to fabricate 3D scaffolds with different geometric patterns via novel table-top stereolithography 3D printer. The resultant scaffolds provide a highly porous and interconnected 3D environment to support cell proliferation. Scaffolds with small square pores were determined to be the optimal geometric pattern for MSC attachment and growth. The optimal LIPUS working parameters were determined to be 1.5 MHz, 20% duty cycle with 150 mW/cm2 intensity. Results demonstrated that RGDS peptide and nHA containing 3D printed scaffolds under LIPUS treatment can greatly promote MSC proliferation, alkaline phosphatase activity, calcium deposition and total protein content. These results illustrate the effectiveness of the combination of LIPUS and biomimetic 3D printing scaffolds as a valuable combinatorial tool for improved MSC function, thus make them promising for future clinical and various regenerative medicine application.

Aliabouzar M, Zhang LG, Sarkar K, 2016 “Lipid coated microbubbles and low intensity pulsed ultrasound enhance chondrogenesis of human mesenchymal stem cells in 3D printed scaffolds,” Scientific Reports, 6, 37728.

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.

Aliabouzar M, Zhang LG, Sarkar K, 2018 “Acoustic and mechanical characterization of 3D-printed scaffolds for tissue engineering applications,” Biomedical Materials, 13,055013.

The acoustic and mechanical properties of 3D-printed porous poly-(ethylene glycol)-diacrylate(PEGDA)hydrogel scaffolds were investigated using an ultrasound pulse echo technique on differentscaffold microstructures(solid, hexagonal and square pores). Acoustic parameters such as speed ofsound, acoustic impedance and attenuation coefficient as well as physical parameters such as the porestructure, effective density and elastic moduli were determined. The results show that microstructure(porosity and pore geometry)plays a crucial role in defining properties of 3D-printed scaffolds,achieving the highest attenuation for the scaffold with hexagonal pores and showing a decrease insound speed and elastic moduli with increasing porosity. The properties were also found to be similarto those of soft tissues, making PEGDA scaffolds a suitable candidate for tissue engineeringapplications. To evaluate their cellular performance, adhesion and proliferation of humanmesenchymal stem cells(hMSCs)in these scaffolds were investigated. The porous scaffolds performedbetter than the solid one, recording the highest cell attachment and growth for the scaffold with thesquare pores.

Osborn J, Aliabouzar A, Zhou X, Rao R, Zhang LG, Sarkar K 2019 “Enhanced Osteogenic Differentiation of Human Mesenchymal Stem Cells Using Microbubbles and LowIntensity Pulsed Ultrasound on 3D Printed Scaffolds,” Advanced Biosystems, 2, 1800257.

Lipid-coated microbubbles, clinically approved as contrast enhancing agents for ultrasound imaging, are investigated for the first time for their possible applications in bone tissue engineering. Effects of microbubbles (average diameter 1.1 μm) coated by a mixture of lipids (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], and 1,2-dipalmitoyl-3-trimethylmmonium-propane) in the presence of low intensity pulsed ultrasound (LIPUS) on human mesenchymal stem cells seeded on 3D printed poly(lactic acid) porous scaffolds are investigated. LIPUS stimulation (30 mW cm−2, 1.5 MHz, 20% duty cycle) for 3 min a day with 0.5% v/v microbubbles results in a significant increase in proliferation (up to 19.3%) when compared to control after 1, 3, and 5 d. A 3-week osteogenic differentiation study shows a significant increase in total protein content (up to 27.5%), calcium deposition (up to 4.3%), and alkaline phosphatase activity (up to 43.1%) initiated by LIPUS with and without the presence of microbubbles. The microbubbles are found to remain stable during exposure, and their sustained oscillations demonstrably help focus the LIPUS energy toward enhanced cellular response. Integrating LIPUS and microbubbles promises to be a novel and effective strategy for bone tissue engineering and regeneration therapies.

Osborn J, Aliabouzar A, Zhou X, Rao R, Zhang LG, Sarkar K 2019 “Enhanced Osteogenic Differentiation of Human Mesenchymal Stem Cells Using Microbubbles and LowIntensity Pulsed Ultrasound on 3D Printed Scaffolds,” Advanced Biosystems, 2, 1800257.

Lipid-coated microbubbles, clinically approved as contrast enhancing agents for ultrasound imaging, are investigated for the first time for their possible applications in bone tissue engineering. Effects of microbubbles (average diameter 1.1 μm) coated by a mixture of lipids (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], and 1,2-dipalmitoyl-3-trimethylmmonium-propane) in the presence of low intensity pulsed ultrasound (LIPUS) on human mesenchymal stem cells seeded on 3D printed poly(lactic acid) porous scaffolds are investigated. LIPUS stimulation (30 mW cm−2, 1.5 MHz, 20% duty cycle) for 3 min a day with 0.5% v/v microbubbles results in a significant increase in proliferation (up to 19.3%) when compared to control after 1, 3, and 5 d. A 3-week osteogenic differentiation study shows a significant increase in total protein content (up to 27.5%), calcium deposition (up to 4.3%), and alkaline phosphatase activity (up to 43.1%) initiated by LIPUS with and without the presence of microbubbles. The microbubbles are found to remain stable during exposure, and their sustained oscillations demonstrably help focus the LIPUS energy toward enhanced cellular response. Integrating LIPUS and microbubbles promises to be a novel and effective strategy for bone tissue engineering and regeneration therapies.

Osborn J, Aliabouzar A, Zhou X, Rao R, Zhang LG, Sarkar K 2019 “Enhanced Osteogenic Differentiation of Human Mesenchymal Stem Cells Using Microbubbles and LowIntensity Pulsed Ultrasound on 3D Printed Scaffolds,” Advanced Biosystems, 2, 1800257.

Lipid-coated microbubbles, clinically approved as contrast enhancing agents for ultrasound imaging, are investigated for the first time for their possible applications in bone tissue engineering. Effects of microbubbles (average diameter 1.1 μm) coated by a mixture of lipids (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], and 1,2-dipalmitoyl-3-trimethylmmonium-propane) in the presence of low intensity pulsed ultrasound (LIPUS) on human mesenchymal stem cells seeded on 3D printed poly(lactic acid) porous scaffolds are investigated. LIPUS stimulation (30 mW cm−2, 1.5 MHz, 20% duty cycle) for 3 min a day with 0.5% v/v microbubbles results in a significant increase in proliferation (up to 19.3%) when compared to control after 1, 3, and 5 d. A 3-week osteogenic differentiation study shows a significant increase in total protein content (up to 27.5%), calcium deposition (up to 4.3%), and alkaline phosphatase activity (up to 43.1%) initiated by LIPUS with and without the presence of microbubbles. The microbubbles are found to remain stable during exposure, and their sustained oscillations demonstrably help focus the LIPUS energy toward enhanced cellular response. Integrating LIPUS and microbubbles promises to be a novel and effective strategy for bone tissue engineering and regeneration therapies.

Osborn J, Aliabouzar A, Zhou X, Rao R, Zhang LG, Sarkar K 2019 “Enhanced Osteogenic Differentiation of Human Mesenchymal Stem Cells Using Microbubbles and LowIntensity Pulsed Ultrasound on 3D Printed Scaffolds,” Advanced Biosystems, 2, 1800257.

Lipid-coated microbubbles, clinically approved as contrast enhancing agents for ultrasound imaging, are investigated for the first time for their possible applications in bone tissue engineering. Effects of microbubbles (average diameter 1.1 μm) coated by a mixture of lipids (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], and 1,2-dipalmitoyl-3-trimethylmmonium-propane) in the presence of low intensity pulsed ultrasound (LIPUS) on human mesenchymal stem cells seeded on 3D printed poly(lactic acid) porous scaffolds are investigated. LIPUS stimulation (30 mW cm−2, 1.5 MHz, 20% duty cycle) for 3 min a day with 0.5% v/v microbubbles results in a significant increase in proliferation (up to 19.3%) when compared to control after 1, 3, and 5 d. A 3-week osteogenic differentiation study shows a significant increase in total protein content (up to 27.5%), calcium deposition (up to 4.3%), and alkaline phosphatase activity (up to 43.1%) initiated by LIPUS with and without the presence of microbubbles. The microbubbles are found to remain stable during exposure, and their sustained oscillations demonstrably help focus the LIPUS energy toward enhanced cellular response. Integrating LIPUS and microbubbles promises to be a novel and effective strategy for bone tissue engineering and regeneration therapies.

Mobadersany N, Sarkar K 2019 “Acoustic microstreaming near a plane wall due to a pulsating free or coated bubble: velocity, vorticity and closed streamlines,” Journal of Fluid Mechanics, 875 781-806.

Acoustic microstreaming due to an oscillating microbubble, either coated or free, is analytically investigated. The detailed flow field is obtained and the closed streamlines of the ring vortex generated by microstreaming are plotted in both Eulerian and Lagrangian descriptions. Analytical expressions are found for the ring vortex showing that its length depends only on the separation of the microbubble from the wall
and the dependence is linear. The circulation as a scalar measure of the vortex is computed quantitatively identifying its spatial location. The functional dependence of circulation on bubble separation and coating parameters is shown to be similar to that of the shear stress.

Singha S, Malipeddy AR, Zurita-Gotor M, Sarkar K, Shen K, Loewenberg M, Migler KB, Blawzdziewicz J 2019 “Mechanisms of spontaneous chain formation and subsequent microstructural evolution in shear-driven strongly confined drop monolayers,” Soft Matter, 15, 4873-4889.

It was experimentally demonstrated by Migler and his collaborators [Phys. Rev. Lett., 2001, 86, 1023; Langmuir, 2003, 19, 8667] that a strongly confined drop monolayer sheared between two parallel plates can spontaneously develop a flow-oriented drop-chain morphology. Here we show that the formation of the chain-like microstructure is driven by far-field Hele-Shaw quadrupolar interactions between
drops, and that drop spacing within chains is controlled by the effective drop repulsion associated with the existence of confinement-induced reversing streamlines, i.e., the swapping trajectory effect. Using
direct numerical simulations and an accurate quasi-2D model that incorporates quadrupolar and swapping-trajectory contributions, we analyze microstructural evolution in a monodisperse drop
monolayer. Consistent with experimental observations, we find that drop spacing within individual chains is usually uniform. Further analysis shows that at low area fractions all chains have the same spacing, but at higher area fractions there is a large spacing variation from chain to chain. These findings are explained in terms of uncompressed and compressed chains. At low area fractions most chains are
uncompressed (spacing equals lst, which is the stable separation of an isolated pair). At higher area fractions compressed chains (with tighter spacing) are formed in a process of chain zipping along
y-shaped structural defects. We also discuss the relevance of our findings to other shear-driven systems, such as suspensions of spheres in non-Newtonian fluids.

Singha S, Malipeddy AR, Zurita-Gotor M, Sarkar K, Shen K, Loewenberg M, Migler KB, Blawzdziewicz J 2019 “Mechanisms of spontaneous chain formation and subsequent microstructural evolution in shear-driven strongly confined drop monolayers,” Soft Matter, 15, 4873-4889.

It was experimentally demonstrated by Migler and his collaborators [Phys. Rev. Lett., 2001, 86, 1023; Langmuir, 2003, 19, 8667] that a strongly confined drop monolayer sheared between two parallel plates can spontaneously develop a flow-oriented drop-chain morphology. Here we show that the formation of the chain-like microstructure is driven by far-field Hele-Shaw quadrupolar interactions between
drops, and that drop spacing within chains is controlled by the effective drop repulsion associated with the existence of confinement-induced reversing streamlines, i.e., the swapping trajectory effect. Using
direct numerical simulations and an accurate quasi-2D model that incorporates quadrupolar and swapping-trajectory contributions, we analyze microstructural evolution in a monodisperse drop
monolayer. Consistent with experimental observations, we find that drop spacing within individual chains is usually uniform. Further analysis shows that at low area fractions all chains have the same spacing, but at higher area fractions there is a large spacing variation from chain to chain. These findings are explained in terms of uncompressed and compressed chains. At low area fractions most chains are
uncompressed (spacing equals lst, which is the stable separation of an isolated pair). At higher area fractions compressed chains (with tighter spacing) are formed in a process of chain zipping along
y-shaped structural defects. We also discuss the relevance of our findings to other shear-driven systems, such as suspensions of spheres in non-Newtonian fluids.

Malipeddy AR, Sarkar K 2019 “Collective diffusivity in a sheared viscous emulsion: effects of viscosity ratio,” Physical Review Fluids, 4, 093603.

The shear-induced collective or gradient diffusivity in an emulsion of viscous drops,
specifically as a function of viscosity ratio, was computed using a fully resolved numerical method. An initially randomly packed layer of viscous drops spreading due to drop-drop interactions in an imposed shear has been simulated. The collective diffusivity coefficient was computed using a self-similar solution of the drop concentration profile. We also obtained the collective diffusivity (the collective diffusivity coefficient multiplied by the average drop volume fraction), computing the dynamic structure factor from the simulated drop positions—an analysis typically applied only to homogeneous systems. The two quantities computed using entirely different methods are in broad agreement, including their predictions of nonmonotonic variations with increasing capillary number and viscosity ratio. The computed values were also found to match with past experimental
measurements. The collective diffusivity coefficient computed here, as expected, is 1 order of magnitude larger than the self-diffusivity coefficient for a dilute emulsion previously computed using pairwise simulation of viscous drops in shear. The collective diffusivity coefficient computed here shows a nonmonotonic variation with viscosity ratio, in contrast to self-diffusivity computed using pairwise computation. The difference might point to an intrinsic difference in physics underlying the two diffusivities. Alternatively, it also might
arise from drops not reaching equilibrium deformation in the period after one interaction and before the next—an effect absent in the pairwise simulation used for the computation of self-diffusivity. We offer a qualitative explanation of the nonmonotonic variation by relating it to average nonmonotonic drop deformation with increasing viscosity ratio. We
also provide empirical correlations of the collective diffusivity as a function of viscosity ratio and capillary number.

Aliabouzar M, Kumar KN, Sarkar K, 2018 “Acoustic vaporization threshold of lipid coated perfluoropentane droplets,” Journal of the Acoustical Society of America, 143, 2001-2012.

Phase shift droplets vaporizable by acoustic stimulation offer the advantages of producing micro-bubbles as contrast agentsin situas well as higher stability and the possibility of achieving smallersizes. Here, the acoustic droplet vaporization (ADV) threshold of a suspension of droplets with aperfluoropentane (PFP) core (diameter 400–3000 nm) is acoustically measured as a function of theexcitation 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 deter-mine the ADV phenomenon, and findings are discussed. The average threshold obtained using threedifferent scattered components increases with frequency—1.0560.28 MPa at 2.25 MHz,1.8960.57 MPa at 5 MHz, and 2.3460.014 MPa at 10 MHz. The scattered response from vapor-ized 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 thresh-old value.

Aliabouzar M, Zhang LG, Sarkar K, 2018 “Acoustic and mechanical characterization of 3D-printed scaffolds for tissue engineering applications,” Biomedical Materials, 13,055013.

The acoustic and mechanical properties of 3D-printed porous poly-(ethylene glycol)-diacrylate(PEGDA)hydrogel scaffolds were investigated using an ultrasound pulse echo technique on differentscaffold microstructures(solid, hexagonal and square pores). Acoustic parameters such as speed ofsound, acoustic impedance and attenuation coefficient as well as physical parameters such as the porestructure, effective density and elastic moduli were determined. The results show that microstructure(porosity and pore geometry)plays a crucial role in defining properties of 3D-printed scaffolds,achieving the highest attenuation for the scaffold with hexagonal pores and showing a decrease insound speed and elastic moduli with increasing porosity. The properties were also found to be similarto those of soft tissues, making PEGDA scaffolds a suitable candidate for tissue engineeringapplications. To evaluate their cellular performance, adhesion and proliferation of humanmesenchymal stem cells(hMSCs)in these scaffolds were investigated. The porous scaffolds performedbetter than the solid one, recording the highest cell attachment and growth for the scaffold with thesquare pores.

Miao S, Castro NJ, Nowicki M, Xia L, Cui H, Zhou X, Zhu W, Lee S, Sarkar K, Vozzi G, Tabata Y, Fisher J, Zhang LG 2017 “4D printing of polymeric materials for tissue and organ regeneration,” Materials Today, 20, 577-591.

Four dimensional (4D) printing is an emerging technology with great capacity for fabricating complex,stimuli-responsive 3D structures, providing great potential for tissue and organ engineering applica-tions. Although the 4D concept wasfirst highlighted in 2013, extensive research has rapidly developed,along with more-in-depth understanding and assertions regarding the definition of 4D. In this review,we begin by establishing the criteria of 4D printing, followed by an extensive summary of state-of-the-art technological advances in thefield. Both transformation-preprogrammed 4D printing and 4Dprinting of shape memory polymers are intensively surveyed. Afterwards we will explore and discussthe applications of 4D printing in tissue and organ regeneration, such as developing synthetic tissuesand implantable scaffolds, as well as future perspectives and conclusions.

Aliabouzar M, Zhang LG, Sarkar K, 2017 “Effects of scaffold microstructure and low intensity pulsed ultrasound on chondrogenic differentiation of human mesenchymal stem cells,” Biotechnology & Bioengineering, 115, 495-506.

The effects of low intensity pulsed ultrasound (LIPUS) on proliferation andchondrogenic differentiation of human mesenchymal stem cells (hMSCs) seeded on3D printed poly-(ethylene glycol)-diacrylate (PEG-DA) scaffolds with varying poregeometries (square and hexagonal channels) were investigated. The scaffold withsquare pores resulted in higher hMSC growth and chondrogenic differentiation than asolid or a hexagonally porous scaffold. The optimal LIPUS parameters at 1.5 MHz werefound to be 100 mW/cm2and 20% duty cycle. LIPUS stimulation increasedproliferation by up to 60% after 24 hr. For chondrogenesis, we evaluated key cartilagebiomarkers abundant in cartilage tissue; glycosaminoglycan (GAG), type II collagen andtotal collagen. LIPUS stimulation enhanced GAG synthesis up to 16% and 11% forscaffoldswithsquareandhexagonalpatterns,respectively,after2weeks.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 LIPUSstimulation, 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 printingand hMSCs to regenerate damaged cartilage tissues.

Srivastava P, Malipeddi Reddy A, Sarkar K 2016 “Steady shear rheology of a viscous emulsion in the presence of finite inertia at moderate volume fractions: sign reversal of normal stress differences,” Journal of Fluid Mechanics, 85, 494-522.

The shear rheology of an emulsion of viscous drops in the presence of finite inertiais investigated using direct numerical simulation. In the absence of inertia, emulsionsdisplay a non-Newtonian rheology with positive first and negative second normalstress differences. However, recently it was discovered that a small amount ofdrop-level inertia alters their signs – the first normal stress difference becomesnegative and the second one becomes positive, each in a small range of capillarynumbers (Li & Sarkar,J. Rheol., vol. 49, 2005, pp. 1377–1394). Sign reversal wasshown numerically and analytically, but only in the limit of a dilute emulsion wheredrop–drop interactions were neglected. Here, we compute the rheology of a density-and viscosity-matched emulsion, accounting for the interactions in the volume fractionrange of 5 %–27 % and Reynolds number range of 0.1–10. The computed rheologicalproperties (effective shear viscosity and first and second normal stress differences) inthe Stokes limit match well with previous theoretical (Choi–Schowalter in the dilutelimit) and simulated results (for concentrated systems) using the boundary elementmethod. The two distinct components of the rheology arising from the interfacialstresses at the drop surface and the perturbative Reynolds stresses are investigated asfunctions of the drop Reynolds number, capillary number and volume fraction. Thesign change is caused by the increasing drop inclination in the presence of inertia,which in turn directly affects the interfacial stresses. Increase of the volume fractionor capillary number increases the critical Reynolds number for sign reversals due toenhanced alignment of the drops with the flow directions. The effect of increasingthe volume fraction on the rheology is explained by relating it to interactions andspecifically to the contact pair-distribution function computed from the simulation.The excess stresses are seen to show an approximately linear behaviour with theReynolds number in the range of 0.1–5, while with the capillary number and volumefraction, the variation is weakly quadratic.

Zhou X, Castro NJ, Zhu W, Cui H, Aliabouzar M, Sarkar K, Zhang LG 2016 “Improved human bone marrow mesenchymal stem cell osteogenesis in 3D bioprinted tissue scaffolds with low intensity pulsed ultrasound stimulation,” Scientific Reports, 6, 32876.

3D printing and ultrasound techniques are showing great promise in the evolution of human musculoskeletal tissue repair and regeneration medicine. The uniqueness of the present study was to combine low intensity pulsed ultrasound (LIPUS) and advanced 3D printing techniques to synergistically improve growth and osteogenic differentiation of human mesenchymal stem cells (MSC). Specifically, polyethylene glycol diacrylate bioinks containing cell adhesive Arginine-Glycine-Aspartic acid-Serene (RGDS) peptide and/or nanocrystalline hydroxyapatite (nHA) were used to fabricate 3D scaffolds with different geometric patterns via novel table-top stereolithography 3D printer. The resultant scaffolds provide a highly porous and interconnected 3D environment to support cell proliferation. Scaffolds with small square pores were determined to be the optimal geometric pattern for MSC attachment and growth. The optimal LIPUS working parameters were determined to be 1.5 MHz, 20% duty cycle with 150 mW/cm2 intensity. Results demonstrated that RGDS peptide and nHA containing 3D printed scaffolds under LIPUS treatment can greatly promote MSC proliferation, alkaline phosphatase activity, calcium deposition and total protein content. These results illustrate the effectiveness of the combination of LIPUS and biomimetic 3D printing scaffolds as a valuable combinatorial tool for improved MSC function, thus make them promising for future clinical and various regenerative medicine application.

Aliabouzar M, Zhang LG, Sarkar K, 2016 “Lipid coated microbubbles and low intensity pulsed ultrasound enhance chondrogenesis of human mesenchymal stem cells in 3D printed scaffolds,” Scientific Reports, 6, 37728.

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.

Katiyar A, Duncan R L, Sarkar K 2014 “Ultrasound stimulation increases proliferation of MC3T3-E1 preosteoblast-like cells,” Journal of Therapeutic Ultrasound, 2, 1, 1-10.

Katiyar A, Duncan R L, Sarkar K 2014 “Ultrasound stimulation increases proliferation of MC3T3-E1 preosteoblast-like cells,” Journal of Therapeutic Ultrasound, 2, 1, 1-10.

Aliabouzar M, Zhang LG, Sarkar K, 2016 “Lipid coated microbubbles and low intensity pulsed ultrasound enhance chondrogenesis of human mesenchymal stem cells in 3D printed scaffolds,” Scientific Reports, 6, 37728.

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.

Zhou X, Castro NJ, Zhu W, Cui H, Aliabouzar M, Sarkar K, Zhang LG 2016 “Improved human bone marrow mesenchymal stem cell osteogenesis in 3D bioprinted tissue scaffolds with low intensity pulsed ultrasound stimulation,” Scientific Reports, 6, 32876.

3D printing and ultrasound techniques are showing great promise in the evolution of human musculoskeletal tissue repair and regeneration medicine. The uniqueness of the present study was to combine low intensity pulsed ultrasound (LIPUS) and advanced 3D printing techniques to synergistically improve growth and osteogenic differentiation of human mesenchymal stem cells (MSC). Specifically, polyethylene glycol diacrylate bioinks containing cell adhesive Arginine-Glycine-Aspartic acid-Serene (RGDS) peptide and/or nanocrystalline hydroxyapatite (nHA) were used to fabricate 3D scaffolds with different geometric patterns via novel table-top stereolithography 3D printer. The resultant scaffolds provide a highly porous and interconnected 3D environment to support cell proliferation. Scaffolds with small square pores were determined to be the optimal geometric pattern for MSC attachment and growth. The optimal LIPUS working parameters were determined to be 1.5 MHz, 20% duty cycle with 150 mW/cm2 intensity. Results demonstrated that RGDS peptide and nHA containing 3D printed scaffolds under LIPUS treatment can greatly promote MSC proliferation, alkaline phosphatase activity, calcium deposition and total protein content. These results illustrate the effectiveness of the combination of LIPUS and biomimetic 3D printing scaffolds as a valuable combinatorial tool for improved MSC function, thus make them promising for future clinical and various regenerative medicine application.

Aliabouzar M, Zhang LG, Sarkar K, 2017 “Effects of scaffold microstructure and low intensity pulsed ultrasound on chondrogenic differentiation of human mesenchymal stem cells,” Biotechnology & Bioengineering, 115, 495-506.

The effects of low intensity pulsed ultrasound (LIPUS) on proliferation andchondrogenic differentiation of human mesenchymal stem cells (hMSCs) seeded on3D printed poly-(ethylene glycol)-diacrylate (PEG-DA) scaffolds with varying poregeometries (square and hexagonal channels) were investigated. The scaffold withsquare pores resulted in higher hMSC growth and chondrogenic differentiation than asolid or a hexagonally porous scaffold. The optimal LIPUS parameters at 1.5 MHz werefound to be 100 mW/cm2and 20% duty cycle. LIPUS stimulation increasedproliferation by up to 60% after 24 hr. For chondrogenesis, we evaluated key cartilagebiomarkers abundant in cartilage tissue; glycosaminoglycan (GAG), type II collagen andtotal collagen. LIPUS stimulation enhanced GAG synthesis up to 16% and 11% forscaffoldswithsquareandhexagonalpatterns,respectively,after2weeks.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 LIPUSstimulation, 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 printingand hMSCs to regenerate damaged cartilage tissues.

Miao S, Castro NJ, Nowicki M, Xia L, Cui H, Zhou X, Zhu W, Lee S, Sarkar K, Vozzi G, Tabata Y, Fisher J, Zhang LG 2017 “4D printing of polymeric materials for tissue and organ regeneration,” Materials Today, 20, 577-591.

Four dimensional (4D) printing is an emerging technology with great capacity for fabricating complex,stimuli-responsive 3D structures, providing great potential for tissue and organ engineering applica-tions. Although the 4D concept wasfirst highlighted in 2013, extensive research has rapidly developed,along with more-in-depth understanding and assertions regarding the definition of 4D. In this review,we begin by establishing the criteria of 4D printing, followed by an extensive summary of state-of-the-art technological advances in thefield. Both transformation-preprogrammed 4D printing and 4Dprinting of shape memory polymers are intensively surveyed. Afterwards we will explore and discussthe applications of 4D printing in tissue and organ regeneration, such as developing synthetic tissuesand implantable scaffolds, as well as future perspectives and conclusions.

Miao S, Cui H, Nowicki M, Xia L, Zhou X, Lee SJ, Zhu W, Sarkar K, Zhang Z, Zhang LG 2018 "Stereolithographic 4D Bioprinting of Multiresponsive Architectures for Neural Engineering" Advanced Biosystems, 2, 1800101

4D printing represents one of the most advanced fabrication techniques forprospective applications in tissue engineering, biomedical devices, and softrobotics, among others. In this study, a novel multiresponsive architecture isdeveloped through stereolithography-based 4D printing, where a universalconcept of stress-induced shape transformation is applied to achieve the4D reprogramming. The light-induced graded internal stress followed by asubsequent solvent-induced relaxation, driving an autonomous and revers-ible change of the programmed configuration after printing, is employedand investigated in depth and details. Moreover, the fabricated constructpossesses shape memory property, offering a characteristic of multipleshape change. Using this novel multiple responsive 4D technique, a proof-of-concept smart nerve guidance conduit is demonstrated on a graphenehybrid 4D construct providing outstanding multifunctional characteristicsfor nerve regeneration including physical guidance, chemical cues, dynamicself-entubulation, and seamless integration. By employing this fabricationtechnique, creating multiresponsive smart architectures, as well as demon-strating application potential, this work paves the way for truly initiation of4D printing in various high-value research fields.

Aliabouzar M, Zhang LG, Sarkar K, 2018 “Acoustic and mechanical characterization of 3D-printed scaffolds for tissue engineering applications,” Biomedical Materials, 13,055013.

The acoustic and mechanical properties of 3D-printed porous poly-(ethylene glycol)-diacrylate(PEGDA)hydrogel scaffolds were investigated using an ultrasound pulse echo technique on differentscaffold microstructures(solid, hexagonal and square pores). Acoustic parameters such as speed ofsound, acoustic impedance and attenuation coefficient as well as physical parameters such as the porestructure, effective density and elastic moduli were determined. The results show that microstructure(porosity and pore geometry)plays a crucial role in defining properties of 3D-printed scaffolds,achieving the highest attenuation for the scaffold with hexagonal pores and showing a decrease insound speed and elastic moduli with increasing porosity. The properties were also found to be similarto those of soft tissues, making PEGDA scaffolds a suitable candidate for tissue engineeringapplications. To evaluate their cellular performance, adhesion and proliferation of humanmesenchymal stem cells(hMSCs)in these scaffolds were investigated. The porous scaffolds performedbetter than the solid one, recording the highest cell attachment and growth for the scaffold with thesquare pores.

Osborn J, Aliabouzar A, Zhou X, Rao R, Zhang LG, Sarkar K 2019 “Enhanced Osteogenic Differentiation of Human Mesenchymal Stem Cells Using Microbubbles and LowIntensity Pulsed Ultrasound on 3D Printed Scaffolds,” Advanced Biosystems, 2, 1800257.

Lipid-coated microbubbles, clinically approved as contrast enhancing agents for ultrasound imaging, are investigated for the first time for their possible applications in bone tissue engineering. Effects of microbubbles (average diameter 1.1 μm) coated by a mixture of lipids (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], and 1,2-dipalmitoyl-3-trimethylmmonium-propane) in the presence of low intensity pulsed ultrasound (LIPUS) on human mesenchymal stem cells seeded on 3D printed poly(lactic acid) porous scaffolds are investigated. LIPUS stimulation (30 mW cm−2, 1.5 MHz, 20% duty cycle) for 3 min a day with 0.5% v/v microbubbles results in a significant increase in proliferation (up to 19.3%) when compared to control after 1, 3, and 5 d. A 3-week osteogenic differentiation study shows a significant increase in total protein content (up to 27.5%), calcium deposition (up to 4.3%), and alkaline phosphatase activity (up to 43.1%) initiated by LIPUS with and without the presence of microbubbles. The microbubbles are found to remain stable during exposure, and their sustained oscillations demonstrably help focus the LIPUS energy toward enhanced cellular response. Integrating LIPUS and microbubbles promises to be a novel and effective strategy for bone tissue engineering and regeneration therapies.

Nowicki M, Zhu W, Sarkar K, Rao R, Zhang LG 2020 "3D printing multiphasic osteochondral tissue constructs with nano to micro features via PCL based bioink" Bioprinting, 17, e00066

Osteochondral tissue has a complex graded structure where biological, physiological, and mechanical properties vary significantly over the full thickness spanning the subchondral bone region beneath the joint surface to the hyaline cartilage region at the joint surface. This presents a significant challenge for tissue-engineered structures addressing osteochondral defects. Advanced bioinks, together with 3D bioprinters, may present a unique solution to this problem. The objective of this research is to apply innovative bioinks, and integrate fused deposition modeling (FDM) 3D printing with a casting technique to fabricate novel osteochondral tissue constructs for improved bone marrow human mesenchymal stem cell (hMSC) functions. Specifically, a multiphasic construct with different layer geometries was designed. A polycaprolactone (PCL) based shape memory material which is comprised of polycaprolactone-triol, castor oil, and poly(hexamethylene diisocyanate) was used as the osteochondral matrix material for the first time. Nanocrystalline hydroxyapatite (nHA) was synthesized and printed into the subchondral bone layers and chondrogenic growth factors were fabricated into the cartilage layer. The results show that the 3D printed constructs with nHA and bioactive cues have improved mechanical properties and enhanced hMSC adhesion, growth, and differentiation. This study indicates that both mechanical properties and cell performance can be easily manipulated through the bioink and the investment casting process to achieve a spatially appropriate osteogenic and chondrogenic response in engineered osteochondral constructs.

Katiyar A, Osborn J, DasBanerjee M, Zhange LG, Sarkar K, Sarker KP 2020, "Inhibition of Human Breast Cancer Cell Proliferation by Low-Intensity Ultrasound Stimulation" Journal of Ultrasound in Medicine, 39, 2043-2052

Cancer is characterized by uncontrolled cell proliferation, which makes novel therapies highly desired. In this study, the effects of near-field low-intensity pulsed ultrasound (LIPUS) stimulation on T47D human breast cancer cell and healthy immortalized MCF-12A breast epithelial cell proliferation were investigated in monolayer cultures.

Katiyar A, Osborn J, DasBanerjee M, Zhange LG, Sarkar K, Sarker KP 2020, "Inhibition of Human Breast Cancer Cell Proliferation by Low-Intensity Ultrasound Stimulation" Journal of Ultrasound in Medicine, 39, 2043-2052

Cancer is characterized by uncontrolled cell proliferation, which makes novel therapies highly desired. In this study, the effects of near-field low-intensity pulsed ultrasound (LIPUS) stimulation on T47D human breast cancer cell and healthy immortalized MCF-12A breast epithelial cell proliferation were investigated in monolayer cultures.

Katiyar A, Osborn J, DasBanerjee M, Zhange LG, Sarkar K, Sarker KP 2020, "Inhibition of Human Breast Cancer Cell Proliferation by Low-Intensity Ultrasound Stimulation" Journal of Ultrasound in Medicine, 39, 2043-2052

Cancer is characterized by uncontrolled cell proliferation, which makes novel therapies highly desired. In this study, the effects of near-field low-intensity pulsed ultrasound (LIPUS) stimulation on T47D human breast cancer cell and healthy immortalized MCF-12A breast epithelial cell proliferation were investigated in monolayer cultures.

Katiyar A, Osborn J, DasBanerjee M, Zhange LG, Sarkar K, Sarker KP 2020, "Inhibition of Human Breast Cancer Cell Proliferation by Low-Intensity Ultrasound Stimulation" Journal of Ultrasound in Medicine, 39, 2043-2052

Cancer is characterized by uncontrolled cell proliferation, which makes novel therapies highly desired. In this study, the effects of near-field low-intensity pulsed ultrasound (LIPUS) stimulation on T47D human breast cancer cell and healthy immortalized MCF-12A breast epithelial cell proliferation were investigated in monolayer cultures.

Guo S, Agarwal T, Song S, Sarkar K, Zhang LG 2025, "Development of novel multi-responsive 4D printed smart nanocomposites with polypyrrole coated iron oxides for remote and adaptive transformation" Materials Horizons, 12, 3907-3917

Four-dimensional (4D) printing, a state-of-the-art additive manufacturing technology, enables the creation of objects capable of changing shape, properties, or functionality over time in response to external stimuli. However, the lack of effective remote control and reliance on a single actuation method pose significant challenges, limiting its applications in various fields. This study aims to address these limitations by developing a novel multi-responsive nanocomposite. By coating near-infrared light (NIR)-responsive polypyrrole (PPy) onto the surface of magnetic iron oxide (Fe2O3) nanoparticles (NPs), multi-responsive PPy@Fe2O3 NPs were synthesized. Doping PPy@Fe2O3 into a thermo-responsive shape memory polymer (SMP) matrix created a nanocomposite with excellent NIR and magnetic responsiveness, enabling dynamic, remote-controlled shape transformation of printed objects with precise timing and positioning using NIR and a magnetic field. Using the nanocomposite, a proof-of-concept semi-tubular construct was fabricated to evaluate its controllable transformation capability and assess the potential for modulating neural stem cell (NSC) behaviors. Furthermore, three proof-of-concept smart robots with distinct features were designed and fabricated for cargo delivery in diverse scenarios and different purposes. Importantly, all complex operations of these robots were remotely controlled using NIR illumination and an external magnetic field. This novel approach demonstrates significant progress in addressing the key challenges of remote control and actuation in 4D printing, highlighting its potential for enhanced versatility and functionality across various applications.

Sarkar K 2025, "Dynamics of bubbles and ultrasound: Diagnostic imaging to blood pressure monitoring and tissue engineering" Physical Review Fluids, 10, 030501

Coated microbubbles in conjunction with ultrasound have emerged as an important biomedical tool for diagnostic imaging and therapeutics. Here, I offer my perspective based on research spanning over two decades, highlighting the underlying physics of linear and nonlinear bubble dynamics. I will describe our effort at mathematical modeling of contrast microbubble behaviors, specifically our adoption of an interfacial rheological model for the stabilizing shell and a hierarchical approach of model building and improvement using attenuation and scattering experiments. I will describe our collaborative effort at using the sensitivity of the acoustic response of microbubbles to ambient hydrostatic pressure for a subharmonic aided pressure estimation (SHAPE) method for noninvasive organlevel blood pressure monitoring. Other collaborative projects demonstrated microbubbles together with low-intensity pulsed ultrasound (LIPUS) to be an effective tool in tissue engineering, growing bone and cartilage from human mesenchymal stem cells in a 3D printed tissue engineering scaffold. We have theoretically investigated various underlying mechanisms for such bioeffects of ultrasound-insonated microbubbles, specifically microstreaming at low acoustic excitations and cavitating jet formation at high excitations for shelled microbubbles. Finally, I briefly describe my computational research on viscous and viscoelastic emulsions and suspensions of drops, vesicles, and biological cells.

Cui H, Zhu W, Miao S, Sarkar K, Zhang LG 2024, "4D Printed Nerve Conduit with In Situ Neurogenic Guidance for Nerve Regeneration" Tissue Engineering: Part A, 30, 293-303

Nerve repair poses a significant challenge in the field of tissue regeneration. As a bioengineered therapeutic method, nerve conduits have been developed to address damaged nerve repair. However, despite their remarkable potential, it is still challenging to encompass complex physiologically microenvironmental cues (both biophysical and biochemical factors) to synergistically regulate stem cell differentiation within the implanted nerve conduits, especially in a facile manner. In this study, a neurogenic nerve conduit with self-actuated ability has been developed by in situ immobilization of neurogenic factors onto printed architectures with aligned microgrooves. One objective was to facilitate self-entubulation, ultimately enhancing nerve repairs. Our results demonstrated that the integration of topographical and in situ biological cues could accurately mimic native microenvironments, leading to a significant improvement in neural alignment and enhanced neural differentiation within the conduit. This innovative approach offers a revolutionary method for fabricating multifunctional nerve conduits, capable of modulating neural regeneration efficiently. It has the potential to accelerate the functional recovery of injured neural tissues, providing a promising avenue for advancing nerve repair therapies.