Patterned Arteriole-Scale Vessels Enhance Engraftment, Perfusion, and Vessel Branching Hierarchy of Engineered Human Myocardium for Heart Regeneration.

Heart regeneration after myocardial infarction (MI) using human stem cell-derived cardiomyocytes (CMs) is rapidly accelerating with large animal and human clinical trials. However, vascularization methods to support the engraftment, survival, and development of implanted CMs in the ischemic environment of the infarcted heart remain a key and timely challenge. To this end, we developed a dual remuscularization-revascularization therapy that is evaluated in a rat model of ischemia-reperfusion MI. This study details the differentiation of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) for engineering cardiac tissue containing patterned engineered vessels 400 µm in diameter. Vascularized engineered human myocardial tissues (vEHMs) are cultured in static conditions or perfused in vitro prior to implantation and evaluated after two weeks. Immunohistochemical staining indicates improved engraftment of hiPSC-CMs in in vitro-perfused vEHMs with greater expression of SMA+ vessels and evidence of inosculation. Three-dimensional vascular reconstructions reveal less tortuous and larger intra-implant vessels, as well as an improved branching hierarchy in in vitro-perfused vEHMs relative to non-perfused controls. Exploratory RNA sequencing of explanted vEHMs supports the hypothesis that co-revascularization impacts hiPSC-CM development in vivo. Our approach provides a strong foundation to enhance vEHM integration, develop hierarchical vascular perfusion, and maximize hiPSC-CM engraftment for future regenerative therapy.

Design and optimization of line-field optical coherence tomography at visible wavebands.

Parallel line-field Fourier-domain optical coherence tomography (LF-FDOCT) has emerged to enable relatively higher speeds than the conventional FDOCT system. In the LF-FDOCT, one B-scan is captured at a time instead of scanning the beam to acquire hundreds of A-scans. On the other hand, spectroscopic OCT using the visible waveband provides absorption information over multiple wavelengths at each voxel. This information of spectral absorption enables quantitative measurement of blood oxygenation, voxel by voxel. Here, we presented the design and optimization of a LF-FDOCT system at the visible waveband (520-620 nm), especially using a generic Camera Link area sensor (2048 × 1088 pixels). To optimize the axial resolution and depth of imaging volume, we simulated various parameters and found that two Nyquist optima can exist, the origin and implication of which has been discussed. As a result, our system acquired 1088 A-scans in parallel at the camera's frame rate of 281 frame per second, achieving an equivalent rate of over 300,000 A-scan/s, while minimizing sacrifice in the point spread function (2.8 × 3.1 × 3.2 µm3, x × y × z) and the field of view (750 × 750 × 750 µm3). As an example of application, we presented high-speed imaging of blood oxygenation in the rodent brain cortex.

Assessing the Angiogenic Efficacy of Pleiotrophin Released from Injectable Heparin-Alginate Gels.

With this work, we design alginate-based hydrogels for therapeutically directing revascularization and repair processes in vivo. We immobilize pleiotrophin (PTN) in injectable hydrogel formulations as the target factor to stimulate proangiogenic responses in endothelial cells. The optimized heparin-alginate/chitosan hydrogels, produced by internal crosslinking with calcium carbonate, show good biocompatibility and injectability and allow controlling the release of immobilized proteins in the subcutaneous tissue over a period of 7 days. In vitro assays, performed with translational human induced pluripotent stem cell-derived endothelial cells, and the in vivo Matrigel plug assay are conducted to demonstrate the angiogenic effects of PTN on endothelial cells. Our results indicate that PTN stimulates endothelial cell morphogenesis in vitro and the migration of endothelial cells and macrophages as soon as 4 days after injections of the developed hydrogels, promoting the formation of structures similar to the healthy granulation tissue, which is an indicator of healing in ischemic wounds. These studies provide the rationale for further investigating this novel therapeutic for pursuing increased vascular density for efficient regeneration of ischemic tissues, by leveraging the host endothelial cell population to initiate angiogenic and reparative processes in vivo. Impact statement Localized, sustained, and controlled delivery of angiogenic factors is crucial for enabling the formation of novel vascular networks in ischemic tissues. This study describes the development of an injectable heparin-alginate/collagen hydrogel for controlling the in vivo release and bioactivity of pleiotrophin (PTN), a heparin-binding factor with significant angiogenic activity. We demonstrate that PTN promotes angiogenesis in an in vitro model of hypoxia and in preclinical subcutaneous models. These results advance our understanding of PTN function in guiding therapeutic angiogenesis and are critical to inform the development of novel translational strategies for ischemic tissue repair and regeneration.

Engineering Immunomodulatory Biomaterials for Regenerating the Infarcted Myocardium.

Coronary artery disease is a severe ischemic condition characterized by the reduction of blood flow in the arteries of the heart that results in the dysfunction and death of cardiac tissue. Despite research over several decades on how to reduce long-term complications and promote angiogenesis in the infarct, the medical field has yet to define effective treatments for inducing revascularization in the ischemic tissue. With this work, we have developed functional biomaterials for the controlled release of immunomodulatory cytokines to direct immune cell fate for controlling wound healing in the ischemic myocardium. The reparative effects of colony-stimulating factor (CSF-1), and anti-inflammatory interleukins 4/6/13 (IL4/6/13) have been evaluated in vitro and in a predictive in vivo model of ischemia (the skin flap model) to optimize a new immunomodulatory biomaterial that we use for treating infarcted rat hearts. Alginate hydrogels have been produced by internal gelation with calcium carbonate (CaCO3) as carriers for the immunomodulatory cues, and their stability, degradation, rheological properties and release kinetics have been evaluated in vitro. CD14 positive human peripheral blood monocytes treated with the immunomodulatory biomaterials show polarization into pro-healing macrophage phenotypes. Unloaded and CSF-1/IL4 loaded alginate gel formulations have been implanted in skin flap ischemic wounds to test the safety and efficacy of the delivery system in vivo. Faster wound healing is observed with the new therapeutic treatment, compared to the wounds treated with the unloaded controls at day 14. The optimized therapy has been evaluated in a rat model of myocardial infarct (ischemia/reperfusion). Macrophage polarization toward healing phenotypes and global cardiac function measured with echocardiography and immunohistochemistry at 4 and 15 days demonstrate the therapeutic potential of the proposed immunomodulatory treatment in a clinically relevant infarct model.

Standard-unit measurement of cellular viability using dynamic light scattering optical coherence microscopy.

Dynamic light scattering optical coherence microscopy (DLS-OCM) integrates DLS, which measures diffusion or flow of particles by analyzing fluctuations in light scattered by the particles, and OCM, which achieves single-cell resolution by combining coherence and confocal gating, integratively enabling cellular-resolution 3D mapping of the diffusion coefficient, and flow velocity. The diffusion coefficient mapping has a potential for the non-destructive measurement of cellular viability in the standard unit but has not been validated yet. Here, we present DLS-OCM imaging of intra-cellular motility (ICM) as a surrogate of cellular viability. For this purpose, we have simultaneously obtained and compared ICM-contrast DLS-OCM images and calcium fluorescence-contrast images of retinal ganglion cells, and then characterized the responses of the measured ICM to a change in cellular viability induced by environmental conditions such as temperature and pH. The diffusion-coefficient-represented ICM exhibits consistent changes with the manipulated cellular viability.

Doppler OCT clutter rejection using variance minimization and offset extrapolation.

Doppler optical coherence tomography (OCT) is widely used for high-resolution mapping of flow velocities and is based on analysis of temporal changes in the phase of an OCT signal (i.e., how fast the OCT signal rotates in the complex plane). Determination of the rate of phase change or rotation speed critically depends on the center of rotation. Here, we demonstrate the bias in high-pass filtering, the current widely used method to determine the center of rotation, and propose two advanced methods for Doppler OCT clutter rejection. The bias in the high-pass filtering method becomes increasingly significant with lower velocities or larger signal noise. Two novel methods based on variance minimization and offset extrapolation can potentially reduce this bias and thereby improve the accuracy of Doppler OCT measurements of flow velocities, even for low-velocity and/or high-noise signals. The two novel methods and the current standard method (high-pass filtering) have been tested in combination with several currently used velocity measurement algorithms: Kasai, autocorrelation function fitting, and maximum likelihood estimation. The newly proposed methods are shown to improve the accuracy in both the center of rotation and resultant velocity by up to 60 percentage points and reduce the flow conservation error by 30% when applied to in vivo cerebral blood flow imaging of the rodent brain cortex.

Determination of confocal profile and curved focal plane for OCT mapping of the attenuation coefficient.

The attenuation coefficient has proven to be a useful tool in numerous biological applications, but accurate calculation is dependent on the characterization of the confocal effect. This study presents a method to precisely determine the confocal effect and its focal plane within a sample by examining the ratio of two optical coherence tomography (OCT) images. The method can be employed to produce a single-value estimate, or a 2D map of the focal plane accounting for the curvature or tilt within the sample. Furthermore, this method is applicable to data obtained with both high numerical aperture (NA) and low-NA lenses, thereby furthering the applicability of the attenuation coefficient to high-NA OCT data. We test and validate this method using standard samples of Intralipid 20% and 5%, improving the accuracy to 99% from 65% compared to the traditional method and preliminarily show applicability to real biological data of glioblastoma acquired in vivo in a murine model.