Engineered tissue geometry and Plakophilin-2 regulate electrophysiology of human iPSC-derived cardiomyocytes.

Engineered heart tissues have been created to study cardiac biology and disease in a setting that more closely mimics in vivo heart muscle than 2D monolayer culture. Previously published studies suggest that geometrically anisotropic micro-environments are crucial for inducing "in vivo like" physiology from immature cardiomyocytes. We hypothesized that the degree of cardiomyocyte alignment and prestress within engineered tissues is regulated by tissue geometry and, subsequently, drives electrophysiological development. Thus, we studied the effects of tissue geometry on electrophysiology of micro-heart muscle arrays (µHM) engineered from human induced pluripotent stem cells (iPSCs). Elongated tissue geometries elicited cardiomyocyte shape and electrophysiology changes led to adaptations that yielded increased calcium intake during each contraction cycle. Strikingly, pharmacologic studies revealed that a threshold of prestress and/or cellular alignment is required for sodium channel function, whereas L-type calcium and rapidly rectifying potassium channels were largely insensitive to these changes. Concurrently, tissue elongation upregulated sodium channel (NaV1.5) and gap junction (Connexin 43, Cx43) protein expression. Based on these observations, we leveraged elongated µHM to study the impact of loss-of-function mutation in Plakophilin 2 (PKP2), a desmosome protein implicated in arrhythmogenic disease. Within µHM, PKP2 knockout cardiomyocytes had cellular morphology similar to what was observed in isogenic controls. However, PKP2-/- tissues exhibited lower conduction velocity and no functional sodium current. PKP2 knockout µHM exhibited geometrically linked upregulation of sodium channel but not Cx43, suggesting that post-translational mechanisms, including a lack of ion channel-gap junction communication, may underlie the lower conduction velocity observed in tissues harboring this genetic defect. Altogether, these observations demonstrate that simple, scalable micro-tissue systems can provide the physiologic stresses necessary to induce electrical remodeling of iPS-CM to enable studies on the electrophysiologic consequences of disease-associated genomic variants.

Exudative Retinal Detachment and Ciliochoroidal Effusion in Preeclampsia.

Purpose: To describe a novel case of focal exudative retinal detachment, choroidal effusion, and acute angle closure in preeclampsia. Methods: A case report is presented. Results: A 37-year-old woman at 38 weeks gestation presented with 2 weeks of progressive blurred vision in the left eye. She had a visual acuity (VA) of 20/800 and an intraocular pressure (IOP) of 26 mm Hg in the left eye (compared with 17 mm Hg in the right eye). Examination showed subretinal fluid in the posterior pole, ciliochoroidal effusion, and angle closure in the left eye without pathology in the right eye. She was found to have hypertension and proteinuria consistent with preeclampsia. The visual symptoms resolved after delivery. At the 1-month follow-up, she had a VA of 20/60 OS, symmetric IOPs, and resolved subretinal and choroidal effusions. Conclusions: To our knowledge, this is the first reported case of ciliochoroidal effusion in the setting of preeclampsia. It may aid in the diagnosis of preeclampsia's ocular manifestations and broaden pathophysiological understanding.

A normative database of wide-field swept-source optical coherence tomography angiography quantitative metrics in a large cohort of healthy adults.

PURPOSE: Data from healthy eyes is needed to interpret optical coherence tomography angiography (OCTA) findings. However, very little normative data is available for wide-field swept-source OCTA (WF SS-OCTA), particularly 12 × 12-mm and disc-centered angiograms. Therefore, we aim to report quantitative metrics in a large sample of control eyes. METHODS: In this cross-sectional observational study, 482 eyes of 375 healthy adults were imaged on the 100 kHz Zeiss PLEX® Elite 9000 using protocols centered on the fovea (3 × 3, 6 × 6, and 12 × 12-mm) and optic disc (6 × 6 and 12 × 12-mm) between December 2018 and January 2022. The ARI Network (Zeiss Portal v5.4) was used to calculate vessel density (VD) and vessel skeletonized density (VSD) in the superficial capillary plexus, deep capillary plexus, and whole retina, as well as foveal avascular zone (FAZ) parameters. Mixed-effect multiple linear regression models were used for statistical analysis. RESULTS: The subjects' median age was 55 (38-63) years, and 201 (53.6%) were female. Greater age and worse best-corrected visual acuity (BCVA) were associated with significantly lower VD and VSD (p < 0.05). VD and VSD differed based on race and cataract status, but not sex, on some scan protocols (p < 0.05). FAZ circularity decreased with age, and FAZ dimensions differed based on race and ethnicity in certain scan protocols. CONCLUSIONS: We report a large database of parafoveal and peripapillary vascular metrics in several angiogram sizes. In referencing these values, researchers must consider characteristics such as age, race, and BCVA, but will have a valuable point of comparison for OCTA measurements in pathologic settings.

The utility of procalcitonin for diagnosing bacteremia and bacterial pneumonia in hospitalized oncology patients.

PURPOSE: Procalcitonin (PCT) is an inflammatory marker elevated in bacteremia and bacterial pneumonia. We aimed to assess the real-world diagnostic accuracy of PCT in hospitalized patients with malignancy. METHODS: A retrospective cohort of 715 patients with cancer who had PCT measured during 750 admissions was analyzed. Diagnosis of bacteremia was determined using blood culture data. Diagnosis of bacterial pneumonia was based on radiographic infiltrate and/or sputum culture. PCT's performance was assessed using receiver operating characteristic (ROC) curves, sensitivity, and specificity. RESULTS: Patients had bacteremia, bacterial pneumonia, or both during 210 admissions (28%). PCT elevation above 0.5 ng/mL was significantly associated with diagnosed infection in the overall population (p < 0.0001) and in subgroups with solid tumor malignancies (p < 0.0001) and hematologic malignancies (p = 0.008). PCT was associated with infectious status in patients with any metastases, but not those with primary lung cancer, lung metastases, neuroendocrine tumors, febrile neutropenia, or history of bone marrow transplant (BMT). The area under the ROC curve for PCT in the overall population was 0.655. An ideal cutoff of 0.21 ng/mL led to a sensitivity of 60% and specificity of 59%. At cutoffs of 0.5 ng/mL and 0.05 ng/mL, PCT's sensitivity was 39% and 94%, while specificity was 79% and 17%, respectively. CONCLUSION: In this large cohort of hospitalized oncology patients, PCT elevation was associated with diagnosed bacteremia and/or bacterial pneumonia. However, specificity was limited, and PCT elevation was not associated with diagnosed infection in some subpopulations. While PCT may have some diagnostic utility for hospitalized oncology patients, values must be interpreted cautiously and considering clinical context.

Elastomer-Grafted iPSC-Derived Micro Heart Muscles to Investigate Effects of Mechanical Loading on Physiology.

Mechanical loading plays a critical role in cardiac pathophysiology. Engineered heart tissues derived from human induced pluripotent stem cells (iPSCs) allow rigorous investigations of the molecular and pathophysiological consequences of mechanical cues. However, many engineered heart muscle models have complex fabrication processes and require large cell numbers, making it difficult to use them together with iPSC-derived cardiomyocytes to study the influence of mechanical loading on pharmacology and genotype-phenotype relationships. To address this challenge, simple and scalable iPSC-derived micro-heart-muscle arrays (µHM) have been developed. "Dog-bone-shaped" molds define the boundary conditions for tissue formation. Here, we extend the µHM model by forming these tissues on elastomeric substrates with stiffnesses spanning from 5 to 30 kPa. Tissue assembly was achieved by covalently grafting fibronectin to the substrate. Compared to µHM formed on plastic, elastomer-grafted µHM exhibited a similar gross morphology, sarcomere assembly, and tissue alignment. When these tissues were formed on substrates with different elasticity, we observed marked shifts in contractility. Increased contractility was correlated with increases in calcium flux and a slight increase in cell size. This afterload-enhanced µHM system enables mechanical control of µHM and real-time tissue traction force microscopy for cardiac physiology measurements, providing a dynamic tool for studying pathophysiology and pharmacology.

IL-10 lentivirus-laden hydrogel tubes increase spinal progenitor survival and neuronal differentiation after spinal cord injury.

A complex cellular cascade characterizes the pathophysiological response following spinal cord injury (SCI) limiting regeneration. Biomaterial and stem cell combination therapies together have shown synergistic effects, compared to the independent benefits of each intervention, and represent a promising approach towards regaining function after injury. In this study, we combine our polyethylene glycol (PEG) cell delivery platform with lentiviral-mediated overexpression of the anti-inflammatory cytokine interleukin (IL)-10 to improve mouse embryonic Day 14 (E14) spinal progenitor transplant survival. Immediately following injury in a mouse SCI hemisection model, five PEG tubes were implanted followed by direct injection into the tubes of lentivirus encoding for IL-10. Two weeks after tube implantation, mouse E14 spinal progenitors were injected directly into the integrated tubes, which served as a soft substrate for cell transplantation. Together, the tubes with the IL-10 encoding lentivirus improved E14 spinal progenitor survival, assessed at 2 weeks posttransplantation (4 weeks postinjury). On average, 8.1% of E14 spinal progenitors survived in mice receiving IL-10 lentivirus-laden tubes compared with 0.7% in mice receiving transplants without tubes, an 11.5-fold difference. Surviving E14 spinal progenitors gave rise to neurons when injected into tubes. Axon elongation and remyelination were observed, in addition to a significant increase in functional recovery in mice receiving IL-10 lentivirus-laden tubes with E14 spinal progenitor delivery compared to the injury only control by 4 weeks postinjury. All other conditions did not exhibit increased stepping until 8 or 12 weeks postinjury. This system affords increased control over the transplantation microenvironment, offering the potential to improve stem cell-mediated tissue regeneration.

Acute Implantation of Aligned Hydrogel Tubes Supports Delayed Spinal Progenitor Implantation.

An important role of neural stem cell transplantation is repopulating neural and glial cells that actively promote repair following spinal cord injury (SCI). However, stem cell survival after transplantation is severely hampered by the inflammatory environment that arises after SCI. Biomaterials have a demonstrated history of managing post-SCI inflammation and can serve as a vehicle for stem cell delivery. In this study, we utilize macroporous polyethylene glycol (PEG) tubes, which were previously found to modulate the post-SCI microenvironment, to serve as a viable, soft substrate for injecting mouse embryonic day 14 (E14) spinal progenitors 2 weeks after tube implantation into a mouse SCI model. At 2 weeks after transplantation (4 weeks after injury), 4.3% of transplanted E14 spinal progenitors survived when transplanted directly into tubes compared to 0.7% when transplanted into the injury alone. Surviving E14 spinal progenitors exhibited a commitment to the neuronal lineage at 4 weeks post-injury, as assessed by both early and late phenotypic markers. Mice receiving tubes with E14 spinal progenitor transplantations had on average 21 ± 4 axons/mm2 regenerated compared to 8 ± 1 axons/mm2 for the injury only control, which corresponded with a significant increase in remyelination compared to the injury only control, while all conditions exhibited improved forelimb control 4 weeks after injury compared to the injury only. Collectively, we have demonstrated the feasibility of using PEG tubes to modify the implantation site and improve survival of transplanted E14 spinal progenitors.

Aligned hydrogel tubes guide regeneration following spinal cord injury.

Directing the organization of cells into a tissue with defined architectures is one use of biomaterials for regenerative medicine. To this end, hydrogels are widely investigated as they have mechanical properties similar to native soft tissues and can be formed in situ to conform to a defect. Herein, we describe the development of porous hydrogel tubes fabricated through a two-step polymerization process with an intermediate microsphere phase that provides macroscale porosity (66.5%) for cell infiltration. These tubes were investigated in a spinal cord injury model, with the tubes assembled to conform to the injury and to provide an orientation that guides axons through the injury. Implanted tubes had good apposition and were integrated with the host tissue due to cell infiltration, with a transient increase in immune cell infiltration at 1 week that resolved by 2 weeks post injury compared to a gelfoam control. The glial scar was significantly reduced relative to control, which enabled robust axon growth along the inner and outer surface of the tubes. Axon density within the hydrogel tubes (1744 axons/mm2) was significantly increased more than 3-fold compared to the control (456 axons/mm2), with approximately 30% of axons within the tube myelinated. Furthermore, implantation of hydrogel tubes enhanced functional recovery relative to control. This modular assembly of porous tubes to fill a defect and directionally orient tissue growth could be extended beyond spinal cord injury to other tissues, such as vascular or musculoskeletal tissue. STATEMENT OF SIGNIFICANCE: Tissue engineering approaches that mimic the native architecture of healthy tissue are needed following injury. Traditionally, pre-molded scaffolds have been implemented but require a priori knowledge of wound geometries. Conversely, hydrogels can conform to any injury, but do not guide bi-directional regeneration. In this work, we investigate the feasibility of a system of modular hydrogel tubes to promote bi-directional regeneration after spinal cord injury. This system allows for tubes to be cut to size during surgery and implanted one-by-one to fill any injury, while providing bi-directional guidance. Moreover, this system of tubes can be broadly applied to tissue engineering approaches that require a modular guidance system, such as repair to vascular or musculoskeletal tissues.

PLG Bridge Implantation in Chronic SCI Promotes Axonal Elongation and Myelination.

Spinal cord injury (SCI) is a devastating condition that may cause permanent functional loss below the level of injury, including paralysis and loss of bladder, bowel, and sexual function. Patients are rarely treated immediately, and this delay is associated with tissue loss and scar formation that can make regeneration at chronic time points more challenging. Herein, we investigated regeneration using a poly(lactide-co-glycolide) multichannel bridge implanted into a chronic SCI following surgical resection of necrotic tissue. We characterized the dynamic injury response and noted that scar formation decreased at 4 and 8 weeks postinjury (wpi), yet macrophage infiltration increased between 4 and 8 wpi. Subsequently, the scar tissue was resected and bridges were implanted at 4 and 8 wpi. We observed robust axon growth into the bridge and remyelination at 6 months after initial injury. Axon densities were increased for 8 week bridge implantation relative to 4 week bridge implantation, whereas greater myelination, particularly by Schwann cells, was observed with 4 week bridge implantation. The process of bridge implantation did not significantly decrease the postinjury function. Collectively, this chronic model follows the pathophysiology of human SCI, and bridge implantation allows for clear demarcation of the regenerated tissue. These data demonstrate that bridge implantation into chronic SCI supports regeneration and provides a platform to investigate strategies to buttress and expand regeneration of neural tissue at chronic time points.

Combinatorial lentiviral gene delivery of pro-oligodendrogenic factors for improving myelination of regenerating axons after spinal cord injury.

Spinal cord injury (SCI) results in paralysis below the injury and strategies are being developed that support axonal regrowth, yet recovery lags, in part, because many axons are not remyelinated. Herein, we investigated strategies to increase myelination of regenerating axons by overexpression of platelet-derived growth factor (PDGF)-AA and noggin either alone or in combination in a mouse SCI model. Noggin and PDGF-AA have been identified as factors that enhance recruitment and differentiation of endogenous progenitors to promote myelination. Lentivirus encoding for these factors was delivered from a multichannel bridge, which we have previously shown creates a permissive environment and supports robust axonal growth through channels. The combination of noggin+PDGF enhanced total myelination of regenerating axons relative to either factor alone, and importantly, enhanced functional recovery relative to the control condition. The increase in myelination was consistent with an increase in oligodendrocyte-derived myelin, which was also associated with a greater density of cells of an oligodendroglial lineage relative to each factor individually and control conditions. These results suggest enhanced myelination of regenerating axons by noggin+PDGF that act on oligodendrocyte-lineage cells post-SCI, which ultimately led to improved functional outcomes.

Spinal Progenitor-Laden Bridges Support Earlier Axon Regeneration Following Spinal Cord Injury.

IMPACT STATEMENT: Spinal cord injury (SCI) results in loss of tissue innervation below the injury. Spinal progenitors have a greater ability to repair the damage and can be injected into the injury, but their regenerative potential is hampered by their poor survival after transplantation. Biomaterials can create a cell delivery platform and generate a more hospitable microenvironment for the progenitors within the injury. In this work, polymeric bridges are used to deliver embryonic spinal progenitors to the injury, resulting in increased progenitor survival and subsequent regeneration and functional recovery, thus demonstrating the importance of combined therapeutic approaches for SCI.