Related Publication

  • 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.

    Background: Mechanical stimulation of bone increases bone mass and fracture healing, at least in part, throughincreases in proliferation of osteoblasts and osteoprogenitor cells. Researchers have previously performedin vitrostudies of ultrasound-induced osteoblast proliferation but mostly used fixed ultrasound settings and have reportedwidely varying and inconclusive results. Here we critically investigated the effects of the excitation parameters oflow-intensity pulsed ultrasound (LIPUS) stimulation on proliferation of MC3T3-E1 preosteoblastic cells in monolayercultures.

    Methods:We used a custom-designed ultrasound exposure system to vary the key ultrasound parameters—intensity,frequency and excitation duration. MC3T3-E1 cells were seeded in 12-well cell culture plates. Unless otherwise specified,treated cells, in groups of three, were excited twice for 10 min with an interval of 24 h in between after cell seeding.Proliferation rates of these cells were determined using BrdU and MTS assays 24 h after the last LIPUS excitation.All data are presented as the mean ± standard error. The statistical significance was determined using Student'stwo-sample two-tailedttests.

    Results:Using discrete LIPUS intensities ranging from 1 to 500 mW/cm2(SATA, spatial average-temporal average), wefound that approximately 75 mW/cm2produced the greatest increase in osteoblast proliferation. Ultrasound exposuresat higher intensity (approximately 465 mW/cm2) significantly reduced proliferation in MC3T3-E1 cells, suggesting thathigh-intensity pulsed ultrasound may increase apoptosis or loss of adhesion in these cells.Variation in LIPUS frequency from 0.5 MHz to 5 MHz indicated that osteoblast proliferation rate was not frequencydependent. We found no difference in the increase in proliferation rate if LIPUS was applied for 30 min/day or 10 min/day, indicating a habituation response.

    Conclusion:This study concludes that a short-term stimulation with optimum intensity can enhance proliferation ofpreosteoblast-like bone cells that plays an important role in bone formation and accelerated fracture healing, alsosuggesting a possible therapeutic treatment for reduced bone mass.

  • 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 bone marrow mesenchymal stem cell osteogenesis in 3D bioprinted tissue scaffolds with low intensity pulsed ultrasound stimulation,” Nature Scientific Report, 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,” Nature Scientific Report, 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, 2018Acoustic 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, 2018Acoustic characterization of 3D printed PEGDA 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 2018 “Ultrasound and microbubbles enhance osteogenic differentiation of human mesenchymal stem cells 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, 2018Acoustic characterization of 3D printed PEGDA 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.

    Background: Mechanical stimulation of bone increases bone mass and fracture healing, at least in part, throughincreases in proliferation of osteoblasts and osteoprogenitor cells. Researchers have previously performedin vitrostudies of ultrasound-induced osteoblast proliferation but mostly used fixed ultrasound settings and have reportedwidely varying and inconclusive results. Here we critically investigated the effects of the excitation parameters oflow-intensity pulsed ultrasound (LIPUS) stimulation on proliferation of MC3T3-E1 preosteoblastic cells in monolayercultures.

    Methods:We used a custom-designed ultrasound exposure system to vary the key ultrasound parameters—intensity,frequency and excitation duration. MC3T3-E1 cells were seeded in 12-well cell culture plates. Unless otherwise specified,treated cells, in groups of three, were excited twice for 10 min with an interval of 24 h in between after cell seeding.Proliferation rates of these cells were determined using BrdU and MTS assays 24 h after the last LIPUS excitation.All data are presented as the mean ± standard error. The statistical significance was determined using Student'stwo-sample two-tailedttests.

    Results:Using discrete LIPUS intensities ranging from 1 to 500 mW/cm2(SATA, spatial average-temporal average), wefound that approximately 75 mW/cm2produced the greatest increase in osteoblast proliferation. Ultrasound exposuresat higher intensity (approximately 465 mW/cm2) significantly reduced proliferation in MC3T3-E1 cells, suggesting thathigh-intensity pulsed ultrasound may increase apoptosis or loss of adhesion in these cells.Variation in LIPUS frequency from 0.5 MHz to 5 MHz indicated that osteoblast proliferation rate was not frequencydependent. We found no difference in the increase in proliferation rate if LIPUS was applied for 30 min/day or 10 min/day, indicating a habituation response.

    Conclusion:This study concludes that a short-term stimulation with optimum intensity can enhance proliferation ofpreosteoblast-like bone cells that plays an important role in bone formation and accelerated fracture healing, alsosuggesting a possible therapeutic treatment for reduced bone mass.

  • 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 bone marrow mesenchymal stem cell osteogenesis in 3D bioprinted tissue scaffolds with low intensity pulsed ultrasound stimulation,” Nature Scientific Report, 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,” Nature Scientific Report, 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, 2018Acoustic 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, 2018Acoustic characterization of 3D printed PEGDA 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 2018 “Ultrasound and microbubbles enhance osteogenic differentiation of human mesenchymal stem cells 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, 2018Acoustic 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, 2018Acoustic characterization of 3D printed PEGDA 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 2018 “Ultrasound and microbubbles enhance osteogenic differentiation of human mesenchymal stem cells 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,” Nature Scientific Report, 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 bone marrow mesenchymal stem cell osteogenesis in 3D bioprinted tissue scaffolds with low intensity pulsed ultrasound stimulation,” Nature Scientific Report, 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.

    Background: Mechanical stimulation of bone increases bone mass and fracture healing, at least in part, throughincreases in proliferation of osteoblasts and osteoprogenitor cells. Researchers have previously performedin vitrostudies of ultrasound-induced osteoblast proliferation but mostly used fixed ultrasound settings and have reportedwidely varying and inconclusive results. Here we critically investigated the effects of the excitation parameters oflow-intensity pulsed ultrasound (LIPUS) stimulation on proliferation of MC3T3-E1 preosteoblastic cells in monolayercultures.

    Methods:We used a custom-designed ultrasound exposure system to vary the key ultrasound parameters—intensity,frequency and excitation duration. MC3T3-E1 cells were seeded in 12-well cell culture plates. Unless otherwise specified,treated cells, in groups of three, were excited twice for 10 min with an interval of 24 h in between after cell seeding.Proliferation rates of these cells were determined using BrdU and MTS assays 24 h after the last LIPUS excitation.All data are presented as the mean ± standard error. The statistical significance was determined using Student'stwo-sample two-tailedttests.

    Results:Using discrete LIPUS intensities ranging from 1 to 500 mW/cm2(SATA, spatial average-temporal average), wefound that approximately 75 mW/cm2produced the greatest increase in osteoblast proliferation. Ultrasound exposuresat higher intensity (approximately 465 mW/cm2) significantly reduced proliferation in MC3T3-E1 cells, suggesting thathigh-intensity pulsed ultrasound may increase apoptosis or loss of adhesion in these cells.Variation in LIPUS frequency from 0.5 MHz to 5 MHz indicated that osteoblast proliferation rate was not frequencydependent. We found no difference in the increase in proliferation rate if LIPUS was applied for 30 min/day or 10 min/day, indicating a habituation response.

    Conclusion:This study concludes that a short-term stimulation with optimum intensity can enhance proliferation ofpreosteoblast-like bone cells that plays an important role in bone formation and accelerated fracture healing, alsosuggesting a possible therapeutic treatment for reduced bone mass.

  • Aliabouzar M, Kumar KN, Sarkar K, 2018Acoustic 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, 2018Acoustic characterization of 3D printed PEGDA 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 2018 “Ultrasound and microbubbles enhance osteogenic differentiation of human mesenchymal stem cells 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, 2018Acoustic 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, 2018Acoustic characterization of 3D printed PEGDA 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 bone marrow mesenchymal stem cell osteogenesis in 3D bioprinted tissue scaffolds with low intensity pulsed ultrasound stimulation,” Nature Scientific Report, 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,” Nature Scientific Report, 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.

    Background: Mechanical stimulation of bone increases bone mass and fracture healing, at least in part, throughincreases in proliferation of osteoblasts and osteoprogenitor cells. Researchers have previously performedin vitrostudies of ultrasound-induced osteoblast proliferation but mostly used fixed ultrasound settings and have reportedwidely varying and inconclusive results. Here we critically investigated the effects of the excitation parameters oflow-intensity pulsed ultrasound (LIPUS) stimulation on proliferation of MC3T3-E1 preosteoblastic cells in monolayercultures.

    Methods:We used a custom-designed ultrasound exposure system to vary the key ultrasound parameters—intensity,frequency and excitation duration. MC3T3-E1 cells were seeded in 12-well cell culture plates. Unless otherwise specified,treated cells, in groups of three, were excited twice for 10 min with an interval of 24 h in between after cell seeding.Proliferation rates of these cells were determined using BrdU and MTS assays 24 h after the last LIPUS excitation.All data are presented as the mean ± standard error. The statistical significance was determined using Student'stwo-sample two-tailedttests.

    Results:Using discrete LIPUS intensities ranging from 1 to 500 mW/cm2(SATA, spatial average-temporal average), wefound that approximately 75 mW/cm2produced the greatest increase in osteoblast proliferation. Ultrasound exposuresat higher intensity (approximately 465 mW/cm2) significantly reduced proliferation in MC3T3-E1 cells, suggesting thathigh-intensity pulsed ultrasound may increase apoptosis or loss of adhesion in these cells.Variation in LIPUS frequency from 0.5 MHz to 5 MHz indicated that osteoblast proliferation rate was not frequencydependent. We found no difference in the increase in proliferation rate if LIPUS was applied for 30 min/day or 10 min/day, indicating a habituation response.

    Conclusion:This study concludes that a short-term stimulation with optimum intensity can enhance proliferation ofpreosteoblast-like bone cells that plays an important role in bone formation and accelerated fracture healing, alsosuggesting a possible therapeutic treatment for reduced bone mass.

  • Aliabouzar M, Zhang LG, Sarkar K, 2018Acoustic characterization of 3D printed PEGDA 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.

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