Potential of propagation-based synchrotron X-ray phase-contrast computed tomography for cardiac tissue engineering.

Hydrogel-based cardiac tissue engineering offers great promise for myocardial infarction repair. The ability to visualize engineered systems in vivo in animal models is desired to monitor the performance of cardiac constructs. However, due to the low density and weak X-ray attenuation of hydrogels, conventional radiography and micro-computed tomography are unable to visualize the hydrogel cardiac constructs upon their implantation, thus limiting their use in animal systems. This paper presents a study on the optimization of synchrotron X-ray propagation-based phase-contrast imaging computed tomography (PCI-CT) for three-dimensional (3D) visualization and assessment of the hydrogel cardiac patches. First, alginate hydrogel was 3D-printed into cardiac patches, with the pores filled by fibrin. The hydrogel patches were then surgically implanted on rat hearts. A week after surgery, the hearts including patches were excised and embedded in a soft-tissue-mimicking gel for imaging by using PCI-CT at an X-ray energy of 25 keV. During imaging, the sample-to-detector distances, CT-scan time and the region of interest (ROI) were varied and examined for their effects on both imaging quality and radiation dose. The results showed that phase-retrieved PCI-CT images provided edge-enhancement fringes at a sample-to-detector distance of 147 cm that enabled visualization of anatomical and microstructural features of the myocardium and the implanted patch in the tissue-mimicking gel. For visualization of these features, PCI-CT offered a significantly higher performance than the dual absorption-phase and clinical magnetic resonance (3 T) imaging techniques. Furthermore, by reducing the total CT-scan time and ROI, PCI-CT was examined for lowering the effective dose, meanwhile without much loss of imaging quality. In effect, the higher soft tissue contrast and low-dose potential of PCI-CT has been used along with an acceptable overall animal dose to achieve the high spatial resolution needed for cardiac implant visualization. As a result, PCI-CT at the identified imaging parameters offers great potential for 3D assessment of microstructural features of hydrogel cardiac patches.

Rate-programming of nano-particulate delivery systems for smart bioactive scaffolds in tissue engineering.

Development of smart bioactive scaffolds is of importance in tissue engineering, where cell proliferation, differentiation and migration within scaffolds can be regulated by the interactions between cells and scaffold through the use of growth factors (GFs) and extra cellular matrix peptides. One challenge in this area is to spatiotemporally control the dose, sequence and profile of release of GFs so as to regulate cellular fates during tissue regeneration. This challenge would be addressed by rate-programming of nano-particulate delivery systems, where the release of GFs via polymeric nanoparticles is controlled by means of the methods of, such as externally-controlled and physicochemically/architecturally-modulated so as to mimic the profile of physiological GFs. Identifying and understanding such factors as the desired release profiles, mechanisms of release, physicochemical characteristics of polymeric nanoparticles, and externally-triggering stimuli are essential for designing and optimizing such delivery systems. This review surveys the recent studies on the desired release profiles of GFs in various tissue engineering applications, elucidates the major release mechanisms and critical factors affecting release profiles, and overviews the role played by the mathematical models for optimizing nano-particulate delivery systems. Potentials of stimuli responsive nanoparticles for spatiotemporal control of GF release are also presented, along with the recent advances in strategies for spatiotemporal control of GF delivery within tissue engineered scaffolds. The recommendation for the future studies to overcome challenges for developing sophisticated particulate delivery systems in tissue engineering is discussed prior to the presentation of conclusions drawn from this paper.

Optimization of nanoparticles for cardiovascular tissue engineering.

Nano-particulate delivery systems have increasingly been playing important roles in cardiovascular tissue engineering. Properties of nanoparticles (e.g. size, polydispersity, loading capacity, zeta potential, morphology) are essential to system functions. Notably, these characteristics are regulated by fabrication variables, but in a complicated manner. This raises a great need to optimize fabrication process variables to ensure the desired nanoparticle characteristics. This paper presents a comprehensive experimental study on this matter, along with a novel method, the so-called Geno-Neural approach, to analyze, predict and optimize fabrication variables for desired nanoparticle characteristics. Specifically, ovalbumin was used as a protein model of growth factors used in cardiovascular tissue regeneration, and six fabrication variables were examined with regard to their influence on the characteristics of nanoparticles made from high molecular weight poly(lactide-co-glycolide). The six-factor five-level central composite rotatable design was applied to the conduction of experiments, and based on the experimental results, a geno-neural model was developed to determine the optimum fabrication conditions. For desired particle sizes of 150, 200, 250 and 300 nm, respectively, the optimum conditions to achieve the low polydispersity index, higher negative zeta potential and higher loading capacity were identified based on the developed geno-neural model and then evaluated experimentally. The experimental results revealed that the polymer and the external aqueous phase concentrations and their interactions with other fabrication variables were the most significant variables to affect the size, polydispersity index, zeta potential, loading capacity and initial burst release of the nanoparticles, while the electron microscopy images of the nanoparticles showed their spherical geometries with no sign of large pores or cracks on their surfaces. The release study revealed that the onset of the third phase of release can be affected by the polymer concentration. Circular dichroism spectroscopy indicated that ovalbumin structural integrity is preserved during the encapsulation process. Findings from this study would greatly contribute to the design of high molecular weight poly(lactide-co-glycolide) nanoparticles for prolonged release patterns in cardiovascular engineering.

Speckle-Tracking Strain Analysis for Mapping Spatiotemporal Contractility of Induced Pluripotent Stem Cell (iPSC)-Derived Cardiomyocytes.

Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hiPSC-CMs) hold tremendous potential for cardiovascular disease modeling, drug screening, personalized medicine, and pathophysiology studies. The availability of a robust protocol and functional assay for studying phenotypic behavior of hiPSC-CMs is essential for establishing an in vitro disease model. Many heart diseases manifest due to changes in the mechanical strain of cardiac tissue. Therefore, non-invasive evaluation of the contractility properties of hiPSC-CMs remains crucial to gain an insight into the pathogenesis of cardiac diseases. Speckle tracking-based strain analysis is an efficient non-invasive method that uses video microscopy and image analysis of beating hiPSC-CMs for quantitative evaluation of mechanical contractility properties. This article presents step-by-step protocols for extracting quantitative contractility properties of an hiPSC-CM system obtained from five members of a family, of whom three were affected by DiGeorge syndrome, using speckle tracking-based strain analysis. The hiPSCs from the family members were differentiated and purified into hiPSC-CMs using metabolic selection. Time-lapse images of hiPSC-CMs were acquired using high-spatial-resolution and high-time-resolution phase-contrast video microscopy. Speckled images were characterized by evaluating the cross-correlation coefficient, speckle size, speckle contrast, and speckle quality of the images. The optimum parameters of the speckle tracking algorithm were determined by performing sensitivity analysis concerning computation time, effective mapping area, average contraction velocity, and strain. Furthermore, the hiPSC-CM response to adrenaline was evaluated to validate the sensitivity of the strain analysis algorithm. Then, we applied speckle tracking-based strain analysis to characterize the dynamic behavior of patient-specific hiPSC-CMs from the family members affected/unaffected by DiGeorge syndrome. Here, we report an efficient and manipulation-free method to analyze the contraction displacement vector and velocity field, contraction-relaxation strain rate, and contractile cycles. Implementation of this method allows for quantitative analysis of the contractile phenotype characteristics of hiPSC-CMs to distinguish possible cardiac manifestation of DiGeorge syndrome. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Differentiation of iPSCs into iPSC-derived cardiomyocytes (iPSC-CMs) and metabolic selection of differentiated iPSC-CMs Support Protocol 1: Culture, maintenance, and expansion of human iPSCs Support Protocol 2: Immunohistochemistry of iPSC-CMs Basic Protocol 2: Time-lapse speckle imaging of iPSC-CMs and speckle quality characterization Support Protocol 3: Enhancement of local contrast of videos by applying contrast limited adaptive histogram equalization (CLAHE) to all frames Support Protocol 4: Evaluation of average speckle size Support Protocol 5: Evaluation of average speckle contrast Support Protocol 6: Determination of relative peak height, Pc(x), of consecutive images acquired from video microscopy of iPSC-CMs Basic Protocol 3: Speckle tracking-based analysis of beating iPSC-CMs Support Protocol 7: Validation of sensitivity of the speckle tracking analysis for mapping the contractility of iPSC-CMs Basic Protocol 4: Data extraction, visualization, and mapping of contractile cycles of iPSC-CMs.

Immune-privileged tissues formed from immunologically cloaked mouse embryonic stem cells survive long term in allogeneic hosts.

The immunogenicity of transplanted allogeneic cells and tissues is a major hurdle to the advancement of cell therapies. Here we show that the overexpression of eight immunomodulatory transgenes (Pdl1, Cd200, Cd47, H2-M3, Fasl, Serpinb9, Ccl21 and Mfge8) in mouse embryonic stem cells (mESCs) is sufficient to immunologically 'cloak' the cells as well as tissues derived from them, allowing their survival for months in outbred and allogeneic inbred recipients. Overexpression of the human orthologues of these genes in human ESCs abolished the activation of allogeneic human peripheral blood mononuclear cells and their inflammatory responses. Moreover, by using the previously reported FailSafe transgene system, which transcriptionally links a gene essential for cell division with an inducible and cell-proliferation-dependent kill switch, we generated cloaked tissues from mESCs that served as immune-privileged subcutaneous sites that protected uncloaked allogeneic and xenogeneic cells from rejection in immune-competent hosts. The combination of cloaking and FailSafe technologies may allow for the generation of safe and allogeneically accepted cell lines and off-the-shelf cell products.

An Optical-Flow-Based Method to Quantify Dynamic Behavior of Human Pluripotent Stem Cell-Derived Cardiomyocytes in Disease Modeling Platforms.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) hold great promise for cardiovascular disease modeling, drug screening and personalized medicine. A crucial requirement to establish an hPSC-CM-based disease model is the availability of a reliable differentiation protocol and a functional assessment of phenotypic properties of CMs in a disease context. Characterization of relative changes in contractile behavior of CMs can provide insight not only about drug effects but into the pathogenesis of cardiovascular diseases. Image-based optical-flow analysis, which applies a speckle tracking algorithm to videomicroscopy of hPSC-CMs, is a noninvasive method to quantitatively assess the dynamics of mechanical contraction of the CMs. This method offers an efficient characterization of contractile cycles. It quantifies contraction velocity field, beat rate, contractile strain and contraction-relaxation strain rate profile, which are important phenotypic characteristics of CMs.

Influence of ionic crosslinkers (Ca2+/Ba2+/Zn2+) on the mechanical and biological properties of 3D Bioplotted Hydrogel Scaffolds.

Three dimensional (3D) bioplotting requires appropriate crosslinkers to crosslink the hydrogel precursor while simultaneously maintaining the viability of embedded cells. However, the evaluation and comparison of various types of crosslinkers in bioplotting remains underexplored to date. This paper presents our study of the influence of three ionic crosslinkers-calcium chloride (CaCl2), barium chloride (BaCl2), and zinc chloride (ZnCl2)-on the mechanical and biological properties of 3D bioplotted alginate scaffolds. The scaffold mechanical properties characterized included the elastic modulus, swelling, and degradation while the biological properties considered included Schwann cell viability and surface morphology. The mechanical and biological properties of the bioplotted scaffolds were both dependent on the crosslinkers used for fabrication; specifically, crosslinking ions resulted in the elastic modulus of the hydrogels decreasing in the order BaCl2>CaCl2>ZnCl2 over 42 days while Schwann cell viability decreased in the order CaCl2>BaCl2>ZnCl2 over 7 days. Taken together, these results offer insights that are effective in terms of manipulating the 3D bioplotting process so as to tune and optimize the mechanical and biological performance of the plotted scaffolds for tissue engineering applications.

Dispensing-based bioprinting of mechanically-functional hybrid scaffolds with vessel-like channels for tissue engineering applications - A brief review.

Over the past decades, significant progress has been achieved in the field of tissue engineering (TE) to restore/repair damaged tissues or organs and, in this regard, scaffolds made from biomaterials have played a critical role. Notably, recent advances in biomaterials and three-dimensional (3D) printing have enabled the manipulation of two or more biomaterials of distinct, yet complementary, mechanical and/or biological properties to form so-called hybrid scaffolds mimicking native tissues. Among various biomaterials, hydrogels synthesized to incorporate living cells and/or biological molecules have dominated due to their hydrated tissue-like environment. Moreover, dispensing-based bioprinting has evolved to the point that it can now be used to create hybrid scaffolds with complex structures. However, the complexities associated with multi-material bioprinting and synthesis of hydrogels used for hybrid scaffolds pose many challenges for their fabrication. This paper presents a brief review of dispensing-based bioprinting of hybrid scaffolds for TE applications. The focus is on the design and fabrication of hybrid scaffolds, including imaging techniques, potential biomaterials, physical architecture, mechanical properties, cell viability, and the importance of vessel-like channels. The key issues and challenges for dispensing-based bioprinting of hybrid scaffolds are also identified and discussed along with recommendations for future research directions. Addressing these issues will significantly enhance the design and fabrication of hybrid scaffolds to and pave the way for translating them into clinical applications.

UV-Assisted 3D Bioprinting of Nanoreinforced Hybrid Cardiac Patch for Myocardial Tissue Engineering.

Biofabrication of cell supportive cardiac patches that can be directly implanted on myocardial infarct is a potential solution for myocardial infarction repair. Ideally, cardiac patches should be able to mimic myocardium extracellular matrix for rapid integration with the host tissue, raising the need to develop cardiac constructs with complex features. In particular, cardiac patches should be electrically conductive, mechanically robust and elastic, biologically active and prevascularized. In this study, we aim to biofabricate a nanoreinforced hybrid cardiac patch laden with human coronary artery endothelial cells (HCAECs) with improved electrical, mechanical, and biological behavior. A safe ultraviolet (UV) exposure time with insignificant effect on cell viability was identified for methacrylated collagen (MeCol) micropatterning. The effects of carboxyl functionalized carbon nanotubes (CNTs) on MeCol and alginate matrix morphology, mechanical properties, electrical behavior, and cellular response were investigated at different CNT mass ratios. A UV-integrated 3D-bioprinting technique was implemented to create hybrid hydrogel constructs consisting of CNT-incorporated alginate framework and cell-laden MeCol. The compressive modulus, impedance, and swelling degree of hybrid constructs were assessed over 20 days of incubation in culture medium at 37°C for different CNT mass ratios. The HCAEC viability, proliferation, and differentiation in the context of the bioprinted hybrid constructs were assessed over 10 days in vitro. The functionalized CNTs provided a highly interconnected nanofibrous meshwork that significantly improved viscoelastic behavior and electrical conductivity of photo-cross-linked MeCol. Alginate-coated CNTs provided a nanofilamentous network with fiber size of ∼25-500 nm, improving not only electrical and mechanical properties but also HCAEC attachment and elongation compared to pristine alginate. The CNT-reinforced 3D-printed hybrid constructs presented significantly higher stiffness and electrical conductivity particularly in the physiologically relevant frequency range (∼5 Hz). The CNT-reinforced hybrid implants maintained a significantly higher swelling degree over 20 days of culturing compared to CNT-free hybrid constructs. For a selected CNT mass ratio, HCAECs presented significant cellular proliferation, migration, and differentiation (lumen-like formation) over 10 days of incubation in vitro. Findings from this study deliver essential steps toward developing conductive, robust, and potentially prevascularized hybrid cardiac patches.

Computational nanomedicine for mechanistic elucidation of bilayer nanoparticle-mediated release for tissue engineering.

AIM: Temporal control of growth-factor release from nanoparticles is essential to many tissue engineering applications, yet remains a challenge due to its complicated behavior. The interplay between nanoparticle characteristics and release mechanisms can be captured using computational models. This study aims to develop two novel models to represent the release of bilayer nanoparticles. MATERIALS & METHODS: Bilayer nanoparticles were prepared and characterized experimentally. 'Local volume averaging' and 'Geno-Mechanistic' models were developed and validated with experiments, and then used to identify critical release parameters and elucidate the release mechanisms. RESULTS: Models presented an agreement with experimental data and successfully estimated transport/degradation parameters, which were closely associated with nanoparticle polymer mass ratio and crystallinity. Models suggested that despite relatively rapid core degradation, shell predominantly controlled overall release patterns. CONCLUSION: The developed models and computational frameworks offer a great potential for optimizing/tuning bilayer polymeric nanoparticles for tissue engineering applications.

Evaluation of PBS Treatment and PEI Coating Effects on Surface Morphology and Cellular Response of 3D-Printed Alginate Scaffolds.

Three-dimensional (3D) printing is an emerging technology for the fabrication of scaffolds to repair/replace damaged tissue/organs in tissue engineering. This paper presents our study on 3D printed alginate scaffolds treated with phosphate buffered saline (PBS) and polyethyleneimine (PEI) coating and their impacts on the surface morphology and cellular response of the printed scaffolds. In our study, sterile alginate was prepared by means of the freeze-drying method and then, used to prepare the hydrogel for 3D printing into calcium chloride, forming 3D scaffolds. Scaffolds were treated with PBS for a time period of two days and seven days, respectively, and PEI coating; then they were seeded with Schwann cells (RSC96) for the examination of cellular response (proliferation and differentiation). In addition, swelling and stiffness (Young's modulus) of the treated scaffolds was evaluated, while their surface morphology was assessed using scanning electron microscopy (SEM). SEM images revealed significant changes in scaffold surface morphology due to degradation caused by the PBS treatment over time. Our cell proliferation assessment over seven days showed that a two-day PBS treatment could be more effective than seven-day PBS treatment for improving cell attachment and elongation. While PEI coating of alginate scaffolds seemed to contribute to cell growth, Schwann cells stayed round on the surface of alginate over the period of cell culture. In conclusion, PBS-treatment may offer the potential to induce surface physical cues due to degradation of alginate, which could improve cell attachment post cell-seeding of 3D-printed alginate scaffolds.

Bioprinting Pattern-Dependent Electrical/Mechanical Behavior of Cardiac Alginate Implants: Characterization and Ex Vivo Phase-Contrast Microtomography Assessment.

Three-dimensional (3D)-bioprinting techniques may be used to modulate electrical/mechanical properties and porosity of hydrogel constructs for fabrication of suitable cardiac implants. Notably, characterization of these properties after implantation remains a challenge, raising the need for the development of novel quantitative imaging techniques for monitoring hydrogel implant behavior in situ. This study aims at (i) assessing the influence of hydrogel bioprinting patterns on electrical/mechanical behavior of cardiac implants based on a 3D-printing technique and (ii) investigating the potential of synchrotron X-ray phase-contrast imaging computed tomography (PCI-CT) for estimating elastic modulus/impedance/porosity and microstructural features of 3D-printed cardiac implants in situ via an ex vivo study. Alginate laden with human coronary artery endothelial cells was bioprinted layer by layer, forming cardiac constructs with varying architectures. The elastic modulus, impedance, porosity, and other structural features, along with the cell viability and degradation of printed implants were examined in vitro over 25 days. Two selected cardiac constructs were surgically implanted onto the myocardium of rats and 10 days later, the rat hearts with implants were imaged ex vivo by means of PCI-CT at varying X-ray energies and CT-scan times. The elastic modulus/impedance, porosity, and structural features of the implant were inferred from the PCI-CT images by using statistical models and compared with measured values. The printing patterns had significant effects on implant porosity, elastic modulus, and impedance. A particular 3D-printing pattern with an interstrand distance of 900 µm and strand alignment angle of 0/45/90/135° provided relatively higher stiffness and electrical conductivity with a suitable porosity, maintaining high cell viability over 7 days. The X-ray photon energy of 30-33 keV utilizing a CT-scan time of 1-1.2 h resulted in a low-dose PCI-CT, which provided a good visibility of the low-X-ray absorbent alginate implants. After 10 days postimplantation, the PCI-CT provided a reasonably accurate estimation of implant strand thickness and alignment, pore size and interconnectivity, porosity, elastic modulus, and impedance, which were consistent with our measurements. Findings from this study suggest that 3D-printing patterns can be used to modulate electrical/mechanical behavior of alginate implants, and PCI-CT can be potentially used as a 3D quantitative imaging tool for assessing structural and electrical/mechanical behavior of hydrogel cardiac implants in small animal models.

Regulation of sequential release of growth factors using bilayer polymeric nanoparticles for cardiac tissue engineering.

AIM: Cardiac tissue engineering aims to develop engineered constructs for myocardial infarction repair, where a challenge is the control of growth factor (GF) sequential release. Herein, bilayer polymeric nanoparticles composed of a GF-encapsulating core surrounded by rate-regulating shell were developed for sequential GF release. MATERIALS & METHODS: Single and bilayer polymeric nanoparticles were fabricated, characterized and biologically assessed. A novel 'Geno-Neural model' was developed and validated for rate-programming of the nanoparticles. RESULTS: The bilayer nanoparticles featured low burst effect and time-delayed release, and allowed for sequential release of PDGF following co-release of VEGFand bFGF, which promoted angiogenesis. CONCLUSION: The nanoparticulate delivery system, along with the Geno-Neural model, offers great potential for spatiotemporal control of GF release for cardiovascular regenerative medicine.

Visualization of ultrasound induced cavitation bubbles using the synchrotron x-ray Analyzer Based Imaging technique.

Observing cavitation bubbles deep within tissue is very difficult. The development of a method for probing cavitation, irrespective of its location in tissues, would improve the efficiency and application of ultrasound in the clinic. A synchrotron x-ray imaging technique, which is capable of detecting cavitation bubbles induced in water by a sonochemistry system, is reported here; this could possibly be extended to the study of therapeutic ultrasound in tissues. The two different x-ray imaging techniques of Analyzer Based Imaging (ABI) and phase contrast imaging (PCI) were examined in order to detect ultrasound induced cavitation bubbles. Cavitation was not observed by PCI, however it was detectable with ABI. Acoustic cavitation was imaged at six different acoustic power levels and six different locations through the acoustic beam in water at a fixed power level. The results indicate the potential utility of this technique for cavitation studies in tissues, but it is time consuming. This may be improved by optimizing the imaging method.