Brown Adipose Tissue as a Unique Niche for Islet Organoid Transplantation: Insights From In Vivo Imaging.

Background: Transplantation of human-induced pluripotent stem cell (hiPSC)-derived islet organoids is a promising cell replacement therapy for type 1 diabetes (T1D). It is important to improve the efficacy of islet organoids transplantation by identifying new transplantation sites with high vascularization and sufficient accommodation to support graft survival with a high capacity for oxygen delivery. Methods: A human-induced pluripotent stem cell line (hiPSCs-L1) was generated constitutively expressing luciferase. Luciferase-expressing hiPSCs were differentiated into islet organoids. The islet organoids were transplanted into the scapular brown adipose tissue (BAT) of nonobese diabetic/severe combined immunodeficiency disease (NOD/SCID) mice as the BAT group and under the left kidney capsule (KC) of NOD/SCID mice as a control group, respectively. Bioluminescence imaging (BLI) of the organoid grafts was performed on days 1, 7, 14, 28, 35, 42, 49, 56, and 63 posttransplantation. Results: BLI signals were detected in all recipients, including both the BAT and control groups. The BLI signal gradually decreased in both BAT and KC groups. However, the graft BLI signal intensity under the left KC decreased substantially faster than that of the BAT. Furthermore, our data show that islet organoids transplanted into streptozotocin-induced diabetic mice restored normoglycemia. Positron emission tomography/MRI verified that the islet organoids were transplanted at the intended location in these diabetic mice. Immunofluorescence staining revealed the presence of functional organoid grafts, as confirmed by insulin and glucagon staining. Conclusions: Our results demonstrate that BAT is a potentially desirable site for islet organoid transplantation for T1D therapy.

Metabolic-Glycoengineering-Enabled Molecularly Specific Acoustic Tweezing Cytometry for Targeted Mechanical Stimulation of Cell Surface Sialoglycans.

In this study, we developed a novel type of dibenzocyclooctyne (DBCO)-functionalized microbubbles (MBs) and validated their attachment to azide-labelled sialoglycans on human pluripotent stem cells (hPSCs) generated by metabolic glycoengineering (MGE). This enabled the application of mechanical forces to sialoglycans on hPSCs through molecularly specific acoustic tweezing cytometry (mATC), that is, displacing sialoglycan-anchored MBs using ultrasound (US). It was shown that subjected to the acoustic radiation forces of US pulses, sialoglycan-anchored MBs exhibited significantly larger displacements and faster, more complete recovery after each pulse than integrin-anchored MBs, indicating that sialoglycans are more stretchable and elastic than integrins on hPSCs in response to mechanical force. Furthermore, stimulating sialoglycans on hPSCs using mATC reduced stage-specific embryonic antigen-3 (SSEA-3) and GD3 expression but not OCT4 and SOX2 nuclear localization. Conversely, stimulating integrins decreased OCT4 nuclear localization but not SSEA-3 and GD3 expression, suggesting that mechanically stimulating sialoglycans and integrins initiated distinctive mechanoresponses during the early stages of hPSC differentiation. Taken together, these results demonstrated that MGE-enabled mATC uncovered not only different mechanical properties of sialoglycans on hPSCs and integrins but also their different mechanoregulatory impacts on hPSC differentiation, validating MGE-based mATC as a new, powerful tool for investigating the roles of glycans and other cell surface biomolecules in mechanotransduction.

Multichannel resonant acoustic rheometry system for quantification of coagulation of multiple human plasma samples.

Resonant Acoustic Rheometry (RAR), a newly developed ultrasound-based technique for non-contact characterization of soft viscoelastic materials, has shown promise for quantitative viscoelastic assessment of temporally changing soft biomaterials in real time, and may be used to monitor blood coagulation process. Here, we report the development of a novel, multichannel RAR (mRAR) system for simultaneous measurements of multiple temporally evolving samples and demonstration of its use for monitoring the coagulation of multiple small-volume plasma samples. The mRAR system was constructed using an array of 4 custom-designed ultrasound transducers at 5.0 MHz and a novel electronic driving system that controlled the generation of synchronized ultrasound pulses for real time assessment of multiple samples simultaneously. As a proof-of-concept of the operation of the mRAR system, we performed tests using pooled normal human plasma samples and anti-coagulated plasma samples from patients treated with warfarin with a range of International Normalized Ratio (INR) values as well-characterized samples with different coagulation kinetics. Our results show that simultaneous tracking of dynamic changes in 4 plasma samples triggered by either kaolin or tissue factor was achieved for the entire duration of coagulation. The mRAR system captured distinct changes in the samples and identified parameters including the clotting start time and parameters associated with the stiffness of the final clots that were consistent with INR levels. Data from this study demonstrate the feasibility of the mRAR system for efficient characterization of the kinetic coagulation processes of multiple plasma samples.

Rapid responses of human pluripotent stem cells to cyclic mechanical strains applied to integrin by acoustic tweezing cytometry.

Acoustic tweezing cytometry (ATC) is an ultrasound-based biophysical technique that has shown the capability to promote differentiation of human pluripotent stem cells (hPSCs). This study systematically examined how hPSCs respond to cyclic mechanical strains applied by ATC via displacement of integrin-bound microbubbles (averaged diameter of 4.3 µm) using ultrasound pulses (acoustic pressure 0.034 MPa, center frequency 1.24 MHz and pulse repetition frequency 1 Hz). Our data show downregulation of pluripotency marker Octamer-binding transcription factor 4 (OCT4) by at least 10% and increased nuclear localization of Yes-associated protein (YAP) by almost 100% in hPSCs immediately after ATC application for as short as 1 min and 5 min respectively. Analysis of the movements of integrin-anchored microbubbles under ATC stimulations reveals different stages of viscoelastic characteristic behavior and increasing deformation of the integrin-cytoskeleton (CSK) linkage. The peak displacement of integrin-bound microbubbles increased from 1.45 ± 0.16 to 4.74 ± 0.67 µm as the duty cycle of ultrasound pulses increased from 5% to 50% or the duration of each ultrasound pulse increased from 0.05 to 0.5 s. Real-time tracking of integrin-bound microbubbles during ATC application detects high correlation of microbubble displacements with OCT4 downregulation in hPSCs. Together, our data showing fast downregulation of OCT4 in hPSCs in respond to ATC stimulations highlight the unique mechanosensitivity of hPSCs to integrin-targeted cyclic force/strain dependent on the pulse duration or duty cycle of ultrasound pulses, providing insights into the mechanism of ATC-induced accelerated differentiation of hPSCs.

Crossover of surface waves and capillary-viscous-elastic transition in soft biomaterials detected by resonant acoustic rheometry.

Viscoelastic properties of hydrogels are important for their application in science and industry. However, rheological assessment of soft hydrogel biomaterials is challenging due to their complex, rapid, and often time-dependent behaviors. Resonant acoustic rheometry (RAR) is a newly developed technique capable of inducing and measuring resonant surface waves in samples in a non-contact fashion. By applying RAR at high temporal resolution during thrombin-induced fibrin gelation and ultraviolet-initiated polyethylene glycol (PEG) polymerization, we observed distinct changes in both frequency and amplitude of the resonant surface waves as the materials changed over time. RAR detected a series of capillary-elastic, capillary-viscous, and visco-elastic transitions that are uniquely manifested as crossover of different types of surface waves in the temporally evolving materials. These results reveal the dynamic interplay of surface tension, viscosity, and elasticity that is controlled by the kinetics of polymerization and crosslinking during hydrogel formation. RAR overcomes many limitations of conventional rheological approaches by offering a new way to comprehensively and longitudinally characterize soft materials during dynamic processes.

Multichannel Resonant Acoustic Rheometry System for Rapid and Efficient Quantification of Human Plasma Coagulation.

Resonant Acoustic Rheometry (RAR), a newly developed ultrasound-based technique for non-contact characterization of soft viscoelastic materials, has shown promise for quantitative assessment of plasma coagulation by monitoring the entire dynamic process in real time. Here, we report the development of a multichannel RAR (mRAR) system for simultaneous monitoring of the coagulation of multiple small-volume plasma samples, a capability that is critical to efficiently provide improved assessment of coagulation. The mRAR system was constructed using an array of 4 custom-designed ultrasound transducers at 5.0 MHz and an electronic driving system that controlled the generation of synchronized ultrasound pulses for real time monitoring of multiple samples simultaneously. The mRAR system was tested using Coumadin-treated plasma samples with a range of International Normalized Ratio (INR) values, as well as normal pooled plasma samples. Tracking of dynamic changes in clotting of plasma samples triggered by either kaolin or tissue factor was performed for the entire duration of coagulation. The mRAR system captured distinct changes in the samples and identified parameters including clotting time, clotting speed, and the mechanical properties of the clots that were consistent with Coumadin dose and INR levels Data from this study demonstrate the feasibility of the mRAR system for the rapid, efficient, and accurate characterization of plasma coagulation.

Resonant Acoustic Rheometry to Measure Coagulation Kinetics in Hemophilia A and Healthy Plasma: A Novel Viscoelastic Method.

Compared with conventional coagulation tests and factor-specific assays, viscoelastic hemostatic assays (VHAs) can provide a more thorough evaluation of clot formation and lysis but have several limitations including clot deformation. In this proof-of-concept study, we test a noncontact technique, termed resonant acoustic rheometry (RAR), for measuring the kinetics of human plasma coagulation. Specifically, RAR utilizes a dual-mode ultrasound technique to induce and detect surface oscillation of blood samples without direct physical contact and measures the resonant frequency of the surface oscillation over time, which is reflective of the viscoelasticity of the sample. Analysis of RAR results of normal plasma allowed defining a set of parameters for quantifying coagulation. RAR detected a flat-line tracing of resonant frequency in hemophilia A plasma that was corrected with the addition of tissue factor. Our RAR results captured the kinetics of plasma coagulation and the newly defined RAR parameters correlated with increasing tissue factor concentration in both healthy and hemophilia A plasma. These findings demonstrate the feasibility of RAR as a novel approach for VHA, providing the foundation for future studies to compare RAR parameters to conventional coagulation tests, factor-specific assays, and VHA parameters.

Resonant acoustic rheometry for non-contact characterization of viscoelastic biomaterials.

Resonant Acoustic Rheometry (RAR) is a new, non-contact technique to characterize the mechanical properties of soft and viscoelastic biomaterials, such as hydrogels, that are used to mimic the extracellular matrix in tissue engineering. RAR uses a focused ultrasound pulse to generate a microscale perturbation at the sample surface and tracks the ensuing surface wave using pulse-echo ultrasound. The frequency spectrum of the resonant surface waves is analyzed to extract viscoelastic material properties. In this study, RAR was used to characterize fibrin, gelatin, and agarose hydrogels. Single time point measurements of gelled samples with static mechanical properties showed that RAR provided consistent quantitative data and measured intrinsic material characteristics independent of ultrasound parameters. RAR was also used to longitudinally track dynamic changes in viscoelastic properties over the course of fibrin gelation, revealing distinct phase and material property transitions. Application of RAR was verified using finite element modeling and the results were validated against rotational shear rheometry. Importantly, RAR circumvents some limitations of conventional rheology methods and can be performed in a high-throughput manner using conventional labware. Overall, these studies demonstrate that RAR can be a valuable tool to noninvasively quantify the viscoelastic mechanical properties of soft hydrogel biomaterials.

Visualization and quantification of dynamic intercellular coupling in human embryonic stem cells using single cell sonoporation.

Gap junctions (GJs), which are proteinaceous channels, couple adjacent cells by permitting direct exchange of intracellular molecules with low molecular weights. GJ intercellular communication (GJIC) plays a critical role in regulating behaviors of human embryonic stem cells (hESCs), affecting their proliferation and differentiation. Here we report a novel use of sonoporation that enables single cell intracellular dye loading and dynamic visualization/quantification of GJIC in hESC colonies. By applying a short ultrasound pulse to excite single microbubbles tethered to cell membranes, a transient pore on the cell membrane (sonoporation) is generated which allows intracellular loading of dye molecules and influx of Ca2+ into single hESCs. We employ live imaging for continuous visualization of intercellular dye transfer and Ca2+ diffusion in hESC colonies. We quantify cell-cell permeability based on dye diffusion using mass transport models. Our results reveal heterogeneous intercellular connectivity and a variety of spatiotemporal characteristics of intercellular Ca2+ waves in hESC colonies induced by sonoporation of single cells.

Injectable pre-cultured tissue modules catalyze the formation of extensive functional microvasculature in vivo.

Revascularization of ischemic tissues is a major barrier to restoring tissue function in many pathologies. Delivery of pro-angiogenic factors has shown some benefit, but it is difficult to recapitulate the complex set of factors required to form stable vasculature. Cell-based therapies and pre-vascularized tissues have shown promise, but the former require time for vascular assembly in situ while the latter require invasive surgery to implant vascularized scaffolds. Here, we developed cell-laden fibrin microbeads that can be pre-cultured to form primitive vascular networks within the modular structures. These microbeads can be delivered in a minimally invasive manner and form functional microvasculature in vivo. Microbeads containing endothelial cells and stromal fibroblasts were pre-cultured for 3 days in vitro and then injected within a fibrin matrix into subcutaneous pockets on the dorsal flanks of SCID mice. Vessels deployed from these pre-cultured microbeads formed functional connections to host vasculature within 3 days and exhibited extensive, mature vessel coverage after 7 days in vivo. Cellular microbeads showed vascularization potential comparable to bulk cellular hydrogels in this pilot study. Furthermore, our findings highlight some potentially advantageous characteristics of pre-cultured microbeads, such as volume preservation and vascular network distribution, which may be beneficial for treating ischemic diseases.

High Frequency Spectral Ultrasound Imaging to Detect Metastasis in Implanted Biomaterial Scaffolds.

For most cancers, metastasis is the point at which disease is no longer curable. Earlier detection of metastasis, when it is undetectable by current clinical methods, may enable better outcomes. We have developed a biomaterial implant that recruits metastatic cancer cells in mouse models of breast cancer. Here, we investigate spectral ultrasound imaging (SUSI) as a non-invasive strategy for detecting metastasis to the implanted biomaterial scaffolds. Our results show that SUSI, which detects parameters related to tissue composition and structure, identified changes at an early time point when tumor cells were recruited to scaffolds in orthotopic breast cancer mouse models. These changes were not associated with acellular components in the scaffolds but were reflected in the cellular composition in the scaffold microenvironment, including an increase in CD31 + CD45-endothelial cell number in tumor bearing mice. In addition, we built a classification model based on changes in SUSI parameters from scaffold measurements to stratify tumor free and tumor bearing status. Combination of a linear discriminant analysis and bagged decision trees model resulted in an area under the curve of 0.92 for receiver operating characteristics analysis. With the potential for early non-invasive detection, SUSI could facilitate clinical translation of the scaffolds for monitoring metastatic disease.

Acoustic tweezing cytometry for mechanical phenotyping of macrophages and mechanopharmaceutical cytotripsy.

Macrophages are immune cells responsible for tissue debridement and fighting infection. Clofazimine, an FDA-approved antibiotic, accumulates and precipitates as rod-shaped, crystal-like drug inclusions within macrophage lysosomes. Drug treatment as well as pathophysiological states could induce changes in macrophage mechanical property which in turn impact their phenotype and function. Here we report the use of acoustic tweezing cytometry as a new approach for in situ mechanical phenotyping of macrophages and for targeted macrophage cytotripsy. Acoustic tweezing cytometry applies ultrasound pulses to exert controlled forces to individual cells via integrin-bound microbubbles, enabling a creep test for measuring cellular mechanical property or inducing irreversible changes to the cells. Our results revealed that macrophages with crystal-like drug inclusions became significantly softer with higher cell compliance, and behaved more elastic with faster creep and recovery time constants. On the contrary, phagocytosis of solid polyethylene microbeads or treatment with soluble clofazimine rendered macrophages stiffer. Most notably, application of ultrasound pulses of longer duration and higher amplitude in ATC actuated the integrin-bound microbubbles to mobilize the crystal-like drug inclusions inside macrophages, turning the rod-shaped drug inclusions into intracellular microblender that effectively destructed the cells. This phenomenon of acoustic mechanopharmaceutical cytotripsy may be exploited for ultrasound activated, macrophage-directed drug release and delivery.

Rapid translocation of pluripotency-related transcription factors by external uniaxial forces.

Human embryonic stem cells subjected to a one-time uniaxial stretch for as short as 30-min on a flexible substrate coated with Matrigel experienced rapid and irreversible nuclear-to-cytoplasmic translocation of NANOG and OCT4, but not Sox2. Translocations were directed by intracellular transmission of biophysical signals from cell surface integrins to nuclear CRM1 and were independent of exogenous soluble factors. On E-CADHERIN-coated substrates, presumably with minimal integrin engagement, mechanical strain-induced rapid nuclear-to-cytoplasmic translocation of the three transcription factors. These findings might provide fundamental insights into early developmental processes and may facilitate mechanotransduction-mediated bioengineering approaches to influencing stem cell fate determination.

Integrin-Targeted Cyclic Forces Accelerate Neural Tube-Like Rosette Formation from Human Embryonic Stem Cells.

Mechanical forces play important roles in human embryonic stem cell (hESC) differentiation. To investigate the impact of dynamic mechanical forces on neural induction of hESCs, this study employs acoustic tweezing cytometry (ATC) to apply cyclic forces/strains to hESCs by actuating integrin-bound microbubbles using ultrasound pulses. Accelerated neural induction of hESCs is demonstrated as the result of combined action of ATC and neural induction medium (NIM). Specifically, application of ATC for 30 min followed by culture in NIM upregulates neuroecdoderm markers Pax6 and Sox1 as early as 6 h after ATC, and induces neural tube-like rosette formation at 48 h after ATC. In contrast, no changes are observed in hESCs cultured in NIM without ATC treatment. In the absence of NIM, ATC application decreases Oct4, but does not increase Pax6 and Sox1 expression, nor does it induce neural rossette formation. The effects of ATC are abolished by inhibition of FAK, myosin activity, and RhoA/ROCK signaling. Taken together, the results reveal a synergistic action of ATC and NIM as an integrated mechanobiology mechanism that requires both integrin-targeted cyclic forces and chemical factors for accelerated neural induction of hESCs.

Acoustic Actuation of Integrin-Bound Microbubbles for Mechanical Phenotyping during Differentiation and Morphogenesis of Human Embryonic Stem Cells.

Early human embryogenesis is a dynamic developmental process, involving continuous and concomitant changes in gene expression, structural reorganization, and cellular mechanics. However, the lack of investigation methods has limited the understanding of how cellular mechanical properties change during early human embryogenesis. In this study, ultrasound actuation of functionalized microbubbles targeted to integrin (acoustic tweezing cytometry, ATC) is employed for in situ measurement of cell stiffness during human embryonic stem cell (hESC) differentiation and morphogenesis. Cell stiffness, which is regulated by cytoskeleton structure, remains unchanged in undifferentiated hESCs, but significantly increases during neural differentiation. Further, using the recently established in vitro 3D embryogenesis models, ATC measurements reveal that cells continue to stiffen while maintaining pluripotency during epiblast cyst formation. In contrast, during amniotic cyst formation, cells first become stiffer during luminal cavity formation, but softens significantly when cells differentiate to form amniotic cysts. These results suggest that cell stiffness changes not only due to 3D spatial organization, but also with cell fate change. ATC therefore provides a versatile platform for in situ measurement of cellular mechanical property, and cell stiffness may be used as a mechanical biomarker for cell lineage diversification and cell fate specification during embryogenesis.

Acoustic Tweezing Cytometry Induces Rapid Initiation of Human Embryonic Stem Cell Differentiation.

Mechanical forces play critical roles in influencing human embryonic stem cell (hESC) fate. However, it remains largely uncharacterized how local mechanical forces influence hESC behavior in vitro. Here, we used an ultrasound (US) technique, acoustic tweezing cytometry (ATC), to apply targeted cyclic subcellular forces to hESCs via integrin-bound microbubbles (MBs). We found that ATC-mediated cyclic forces applied for 30 min to hESCs near the edge of a colony induced immediate global responses throughout the colony, suggesting the importance of cell-cell connection in the mechanoresponsiveness of hESCs to ATC-applied forces. ATC application generated increased contractile force, enhanced calcium activity, as well as decreased expression of pluripotency transcription factors Oct4 and Nanog, leading to rapid initiation of hESC differentiation and characteristic epithelial-mesenchymal transition (EMT) events that depend on focal adhesion kinase (FAK) activation and cytoskeleton (CSK) tension. These results reveal a unique, rapid mechanoresponsiveness and community behavior of hESCs to integrin-targeted cyclic forces.

Multimode ultrasound viscoelastography for three-dimensional interrogation of microscale mechanical properties in heterogeneous biomaterials.

Both static and time-dependent mechanical factors can have a profound impact on cell and tissue function, but it is challenging to measure the mechanical properties of soft materials at the scale which cells sense. Multimode ultrasound viscoelastography (MUVE) uses focused ultrasound pulses to both generate and image deformations within soft hydrogels non-invasively, at sub-millimeter resolution, and in 3D. The deformation and strain over time data are used to extract quantitative parameters that describe both the elastic and viscoelastic properties of the material. MUVE was used in creep mode to characterize the viscoelastic properties of 3D agarose, collagen, and fibrin hydrogels. Quantitative comparisons were made by extracting characteristic viscoelastic parameters using Burger's lumped parameter constitutive model. Spatial resolution of the MUVE technique was found to be approximately 200 µm, while detection sensitivity, defined as the capability to differentiate between materials based on mechanical property differences, was approximately 0.2 kPa using agarose hydrogels. MUVE was superior to nanoindentation and shear rheometry in generating consistent microscale measurements of viscoelastic behavior in soft materials. These results demonstrate that MUVE is a rapid, quantitative, and accurate method to measure the viscoelastic mechanical properties of soft 3D hydrogels at the microscale, and is a promising technique to study the development of native and engineered tissues over time.

High-frequency spectral ultrasound imaging (SUSI) visualizes early post-traumatic heterotopic ossification (HO) in a mouse model.

PURPOSE: Early treatment of heterotopic ossification (HO) is currently limited by delayed diagnosis due to limited visualization at early time points. In this study, we validate the use of spectral ultrasound imaging (SUSI) in an animal model to detect HO as early as one week after burn tenotomy. METHODS: Concurrent SUSI, micro CT, and histology at 1, 2, 4, and 9weeks post-injury were used to follow the progression of HO after an Achilles tenotomy and 30% total body surface area burn (n=3-5 limbs per time point). To compare the use of SUSI in different types of injury models, mice (n=5 per group) underwent either burn/tenotomy or skin incision injury and were imaged using a 55MHz probe on VisualSonics VEVO 770 system at one week post injury to evaluate the ability of SUSI to distinguish between edema and HO. Average acoustic concentration (AAC) and average scatterer diameter (ASD) were calculated for each ultrasound image frame. Micro CT was used to calculate the total volume of HO. Histology was used to confirm bone formation. RESULTS: Using SUSI, HO was visualized as early as 1week after injury. HO was visualized earliest by 4weeks after injury by micro CT. The average acoustic concentration of HO was 33% more than that of the control limb (n=5). Spectroscopic foci of HO present at 1week that persisted throughout all time points correlated with the HO present at 9weeks on micro CT imaging. CONCLUSION: SUSI visualizes HO as early as one week after injury in an animal model. SUSI represents a new imaging modality with promise for early diagnosis of HO.

Acoustic tweezing cytometry enhances osteogenesis of human mesenchymal stem cells through cytoskeletal contractility and YAP activation.

Human mesenchymal stem cells (hMSCs) have great potential for cell-based therapies for treating degenerative bone diseases. It is known that mechanical cues in the cell microenvironment play an important role in regulating osteogenic (bone) differentiation of hMSCs. However, mechanoregulation of lineage commitment of hMSCs in conventional two-dimensional (2D) monocultures or bioengineered three-dimensional (3D) tissue constructs remains suboptimal due to complex biomaterial design criteria for hMSC culture. In this study, we demonstrate the feasibility of a novel cell mechanics and mechanobiology tool, acoustic tweezing cytometry (ATC), for mechanical stimulation of hMSCs. ATC utilizes ultrasound (US) pulses to actuate functionalized lipid microbubbles (MBs) which are covalently attached to hMSCs via integrin binding to exert forces to the cells. ATC stimulation increases cytoskeletal contractility of hMSCs regardless of the cell area. Furthermore, ATC application rescues osteogenic differentiation of hMSCs in culture conditions that are intrinsically repressive for hMSC osteogenesis (e.g., soft cell culture surfaces). ATC application activates transcriptional regulator YAP to enhance hMSC osteogenesis. Our data further show that F-actin, myosin II, and RhoA/ROCK signaling functions upstream of YAP activity in mediating ATC-stimulated hMSC osteogenesis. With the capability of applying controlled dynamic mechanical stimuli to cells, ATC provides a powerful tool for mechanoregulation of stem cell behaviors in tissue engineering and regenerative medicine applications.

Microscale characterization of the viscoelastic properties of hydrogel biomaterials using dual-mode ultrasound elastography.

Characterization of the microscale mechanical properties of biomaterials is a key challenge in the field of mechanobiology. Dual-mode ultrasound elastography (DUE) uses high frequency focused ultrasound to induce compression in a sample, combined with interleaved ultrasound imaging to measure the resulting deformation. This technique can be used to non-invasively perform creep testing on hydrogel biomaterials to characterize their viscoelastic properties. DUE was applied to a range of hydrogel constructs consisting of either hydroxyapatite (HA)-doped agarose, HA-collagen, HA-fibrin, or preosteoblast-seeded collagen constructs. DUE provided spatial and temporal mapping of local and bulk displacements and strains at high resolution. Hydrogel materials exhibited characteristic creep behavior, and the maximum strain and residual strain were both material- and concentration-dependent. Burger's viscoelastic model was used to extract characteristic parameters describing material behavior. Increased protein concentration resulted in greater stiffness and viscosity, but did not affect the viscoelastic time constant of acellular constructs. Collagen constructs exhibited significantly higher modulus and viscosity than fibrin constructs. Cell-seeded collagen constructs became stiffer with altered mechanical behavior as they developed over time. Importantly, DUE also provides insight into the spatial variation of viscoelastic properties at sub-millimeter resolution, allowing interrogation of the interior of constructs. DUE presents a novel technique for non-invasively characterizing hydrogel materials at the microscale, and therefore may have unique utility in the study of mechanobiology and the characterization of hydrogel biomaterials.