Translating scientific discoveries in tissue engineering and regenerative medicine (TE/RM) into clinically adopted therapies is hindered by fragmented development pipelines, regulatory and manufacturing challenges, and limited funding. Despite substantial investment by the U.S. National Institutes of Health (NIH), few NIH-funded TE/RM projects achieve commercialization or regulatory approval by the US Food and Drug Administration. The gap between academic innovation and clinical implementation is particularly evident in the dental, oral, and craniofacial (DOC) domain, where market and reimbursement constraints further restrict translation. To address these barriers, the National Institute of Dental and Craniofacial Research established the Dental, Oral and Craniofacial Tissue Regeneration Consortium (DOCTRC), comprising two nationwide Resource Centers tasked with guiding promising technologies from universities and small businesses through preclinical validation toward clinical adoption. This translational science case study outlines DOCTRC's translational model, highlighting lessons learned from five cohorts of interdisciplinary translational project teams, strategies for navigating manufacturing and regulatory pathways, and approaches for aligning academic innovation with clinical and market needs. The unique impact of the DOCTRC framework demonstrates how disciplined product development activities, non-dilutive funding mechanisms, and a comprehensive support ecosystem can accelerate technology translation, offering a scalable model for other biomedical fields.

Background and Purpose:Cardiotoxicity is a major cause for drug failure throughout the drug development process, with particular concern for action potential prolongation and arrhythmia. Hence, such liabilities are heavily considered during the early phases of drug design to prevent dangerous compounds from progressing. New approach methodologies (NAMs) that efficiently examine this risk early in the discovery pipeline should help streamline drug development programs. We developed a cardiac NAM, a 384-well open bath platform consisting of cardiac tissue derived from human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes, enabling high-throughput drug screening while maintaining the structural and functional complexity of 3D cardiac micromuscles. Methods:We dramatically increased throughput without compromising physiological relevance provided by the 3D micromuscle structure. Our 384-well open bath high-throughput platform allowed evaluation of multiple compounds at a time, enabling us to study the CiPA (comprehensive in vitro proarrhythmia assay) drug panel for proarrhythmia screening. We obtained phenotypic fingerprints of all 28 compounds (9 low, 11 intermediate, and 8 high arrhythmia risk; https://cipaproject.org) in dose-escalation studies around their respective clinical concentrations. The analysis was augmented with an in silico pipeline that used phenotypic biomarkers to invert data into a mathematical model of cellular currents to infer which ion channels were affected upon drug exposure, and then trained a ML model to predict channel block. Results and Conclusions:We found accurate detection of arrhythmic potential for most of the compounds, and the in silico model inversions were consistent with published values of compound channel block. All the high risk compounds showed action potential duration (APD) prolongation coupled with either action potential abnormalities, early afterdepolarizations (EADs), or beat cessation. For the intermediate risk group, 9 out of 11 compounds caused APD prolongation alone or in combination with EADs while 2 others showed either beat cessation or beat rate change. Augmentation of APD analysis with detailed biophysical modeling and ML tools provided meaningful insight into the mechanisms involved in APD changes. Overall, our cardiac NAM allowed for fast and relevant screening for mechanistic understanding of APD prolongation and proarrhythmic activity, at massively increased throughput compared to other 3D micromuscle models.

Volumetric muscle loss (VML) is characterized as the loss of muscle tissue that exceeds the muscle's self-repair mechanism, resulting in incomplete restoration of muscle mass and function. Existing treatment modalities, including muscle grafts or autologous muscle transfers, are limited by constraints such as tissue availability and donor site morbidity. Moreover, the inadequate recovery of muscle may lead to fibrosis within the VML site, impeding the process of muscle regeneration and resulting in permanent deficits. Emerging therapeutics, such as hydrogels, show promise in addressing the limitations of current therapeutics and have the potential to significantly reduce fibrosis and facilitate the restoration of muscle form and function following VML injury and repair. This study evaluated the therapeutic potential of repairing a 30% VML injury in the rat tibialis anterior muscle with engineered skeletal muscle units (SMUs), alone, and in combination with a hyaluronic acid-based hydrogel (HyA-HG). Following 1- or 3-months post-implantation, muscle structure and function were assessed. The results indicated that the incorporation of HyA-HG in combination with our SMUs resulted in improvements in force production for VML injuries repaired for 1 month. However, over extended recovery periods (3 months), sustained superior improvements in muscle function with the combination therapy were not observed compared with the repair with just an SMU. Moreover, histological analyses revealed that muscle treated with SMUs and HyA-HG exhibited a greater cross-sectional area and force production in the early stages of recovery (1-month post-surgery) compared with untreated VML sites or those treated with HyA-HG only. However, after 3 months, muscle mass and force production in all experimental groups reached comparable levels, suggesting a transient benefit of the combination therapy. Our findings highlight the potential of HyA-HG and SMU combination therapy to enhance early functional recovery following VML.

Endothelial cell damage after cold preservation and reperfusion injury causes deterioration of the endothelial barrier and ultimately results in edema, leading to transplant failure. Here, a vascular microphysiological system (MPS) is introduced as a testbed to investigate the combinational effect of thermal and fluid perturbations (i.e., wall shear stress) on human endothelial barrier function. Two methods of organ storage are compared: isochoric supercooling (ISC) preservation, which prevents ice formation at subzero temperatures; and, the standard clinical protocol of static cold storage (SCS) at 4 °C. Integrating electrical impedance measurements on chip allow real-time monitoring and quantification of barrier function during preservation and reperfusion protocols. Isochoric supercooling preservation enables longer periods of preservation with superior recovery of barrier function during reperfusion, and has lower metabolic activities compared to static cold storage. Genomic analysis reveals injury and recovery mechanisms at the molecular level for the different preservation and reperfusion conditions. The multifunctional vascular microphysiological system provides a physiologically relevant in vitro model recapitulating ischemia-reperfusion injury to the endothelium. The vascular MPS has potential for optimizing organ preservation protocols, ultimately improving organ transplant viability.

New approach methodologies (NAMs), including microphysiological systems (MPSs), can recapitulate structural and functional complexities of organs. Vanoxerine was reported to induce cardiac adverse events, including torsade de points (TdP), in a Phase III clinical trial. Despite earlier nonclinical animal models and Phase I–II clinical trials, events of QT prolongation or proarrhythmia were not observed. Here, we utilized cardiac NAMs to evaluate the functional consequences of vanoxerine treatment on human cardiac excitation–contraction coupling. The cardiac MPS used in this study was a microfabricated fluidic culture platform with human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) capable of evaluating voltage, intracellular calcium handling, and contractility. Likewise, the hiPSC-CM comprehensive in vitro proarrhythmia assay (CiPA) was employed based on multielectrode array (MEA). Vanoxerine treatment delayed repolarization in a concentration-dependent manner and induced proarrhythmic events in both NAM platforms. The complex cardiac MPS displayed a frequency-dependent vanoxerine response such that EADs were eliminated at a faster pacing rate (1.5 Hz). Moreover, exposure analysis revealed a 99% vanoxerine loss in the cardiac MPS. TdP risk analysis demonstrated high to intermediate TdP risk at clinically relevant concentrations of vanoxerine and frequency-independent EAD events in the hiPSC-CM CiPA model. These findings demonstrate that nonclinical cardiac NAMs can recapitulate clinical outcomes, including detection of vanoxerine-induced delayed repolarization and proarrhythmic effects. Moreover, this work provides a foundation to evaluate the safety and efficacy of novel compounds to reduce the dependence on animal studies.

Cardiac organs-on-a-chip (OoCs) or microphysiological systems have the potential to predict cardiac effects of new drug candidates, including unanticipated cardiac outcomes, which are among the main causes for drug attrition. This protocol describes how to prepare and use a cardiac OoC containing cardiomyocytes differentiated from human induced pluripotent stem cells (hiPS cells). The use of cells derived from hiPS cells as reliable sources of human cells from diverse genetic backgrounds also holds great potential, especially when cultured in OoCs that are physiologically relevant culture platforms. To promote the broad adoption of hiPS cell-derived cardiac OoCs in the drug development field, there is a need to first ensure reproducibility in their preparation and use. This protocol aims to provide key information on how to reduce sources of variability during hiPS cell maintenance, differentiation, loading and maturation in OoCs. Variability in these procedures can lead to inconsistent purity after differentiation and variable function between batches of microtissues formed in OoCs. This protocol also focuses on describing the handling and functional assessment of cardiac microtissues using live-cell microscopy approaches to quantify parameters of cellular electrophysiology, calcium transients and contractility. The protocol consists of five stages: (1) thaw and maintain hiPS cells, (2) differentiate hiPS cell cardiomyocytes, (3) load differentiated cells into OoCs, (4) maintain and characterize loaded cells, and (5) evaluate and utilize cardiac OoCs. Execution of the entire protocol takes ~40 days. The required skills to carry out the protocol are experience with sterile techniques, mammalian cell culture and maintaining hiPS cells in a pluripotent state. Using human induced pluripotent stem cell-derived cardiomyocytes in an organ-on-a-chip model to assay cardiac function: electrophysiology, calcium and contractility. This protocol aims to provide key information on how to reduce variability.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are increasingly important in preclinical drug assessments, particularly for identifying potential cardiotoxicity. In this study, we utilize data from microphysiological systems of hiPSC-CMs to evaluate cellular characteristics, such as action potential duration, beat rate, conduction velocity and mechanical displacement. Based on these data, high-fidelity mathematical models facilitate precise assessments of critical biophysical parameters of the cells, including membrane ion channel conductances, cross-bridge cycle transition rates and cell-to-cell conductance. We emphasize the distinction between synchronized transients and travelling waves, highlighting their implications for deducing the biophysical properties of hiPSC-CMs. In this study, we analyse the effects of the drug compounds flecainide, quinidine, nifedipine, verapamil, blebbistatin and omecamtiv. Our findings show that for drug-induced changes in parameters describing membrane currents and contractile machinery close to ranges reported in the literature, the computed biomarkers align well with measured biomarkers. This study is the first to apply spatially resolved, cell-based models to identify drug effects through measurements of transmembrane potential and mechanical displacement, marking a significant step forward in using computational models for evaluating drug safety and offering a new approach to early identification of adverse drug reactions.

Decreased contractility of the border zone (BZ) myocardium after MI is associated with oxidative stress mediated contractile protein dysfunction. We hypothesized that injection of a thermo-responsive hydrogel with enhanced reactive oxygen species (ROS) scavenging would improve the in vivo contractility of BZ myocardium. To that end, 14 adult male sheep underwent MI. Of those, 6 sheep had a comb-like copolymer synthesized from N-isopropyl acrylamide and 4000 MW methoxy poly(ethylene glycol) methacrylate (NIPAAm-co-PEG4000) injected into the MI zone (MI+Hydrogel). In vivo cardiac magnetic resonance imaging (CMR), including cine DENSE (Displacement encoding with stimulated echoes), was performed before and 6 weeks after MI to measure LV geometry and regional displacement. Tissue ROS and in vitro muscle strip developed force were measured in the BZ and remote regions. Compared to the MI group 6 weeks after MI, MI+Hydrogel exhibited: 1) a reduction in indexed LV end-diastolic (ED) volume; 2) an increase in average wall thickness at ED; and, 3) an increase in average peak circumferential strain in the BZ near the MI border. ROS was significantly lower in the MI+Hydrogel group and muscle strip developed force was significantly increased in the BZ. These observations support the hypothesis that injection of a hydrogel with enhanced ROS scavenging improves in vivo BZ contractility and attenuates ventricular remodeling after MI in sheep. STATEMENT OF SIGNIFICANCE: This work supports our hypothesis that intramyocardial injection of hydrogel with enhanced reactive oxygen species (ROS) scavenging improves in vivo contractility of myocardium bordering the infarct and attenuates pathological ventricular remodeling after myocardial infarction. We believe this multidisciplinary research would be of interest by researchers in the fields of hydrogel biomaterials, finite element biomechanical analysis, and cardiovascular diseases.

Microphysiological systems (MPS) provide a highly controlled environment for the development and testing of human-induced pluripotent stem cell-based cardiac microtissues, with promising applications in disease modeling and drug development. Through optical measurements in such systems, we can quantify mechanical features such as motion and velocity during contraction. While these are useful for evaluating relative changes in muscle twitch, it remains challenging to quantify and characterize the actual active tension driving the contraction. Here, we aimed to quantify the active tension over time and space by solving an inverse problem in cardiac mechanics expressed by partial differential equations (PDEs). We formulated this as a PDE-constrained optimization problem based on a mechanical model defined for two-dimensional representations of the microtissues. Our optimization predicts active tension generated by the tissue as well as the fiber direction angle distribution. We used synthetic as well as experimental data to investigate the performance of our inversion protocol. Next, we employed the procedure to evaluate active tension changes in drug escalation studies of the inotropes omecamtiv mecarbil and Bay K8644. For both drug compounds, we observed a comparable increase in displacement, strain, and model-predicted active strain values upon higher drug doses. The estimated active tension was observed to be highest in the middle part of the tissue, and the fiber direction was mostly aligned with the longitudinal direction of the tissue. The computational framework presented here allows for spatiotemporal estimation of active tension in cardiac microtissues based on optical measurements. In the future, such methodologies might develop into valuable tools in drug development protocols.

Rotator cuff tears are common musculotendinous injuries with a high risk of permanent functional disability. Following surgical repair, sub-optimal patient outcomes are directly correlated with poor muscle quality; namely, injury site fatty infiltration, fibrosis, and muscle atrophy. Muscle resident fibro-adipogenic progenitor cells (FAPs) have been identified as key regulators of post-injury skeletal muscle regeneration and repair by maintaining a pro-myogenic environment. In this work, human FAPs (hFAPs) were encapsulated into hyaluronic acid (HyA)-based hydrogels functionalized with bsp-RGD(15) cell adhesion peptide, heparin, and a matrix metalloproteinase (MMP)-cleavable crosslinker. Hydrogel-encapsulated hFAPs increased expression of the pro-myogenic marker UCP1 and production of the anti-inflammatory cytokine IL-10 while downregulating the expression of the fibrotic marker αSMA over time. A murine model of unilateral rotator cuff transection, denervation, and delayed repair was treated with the HyA hydrogel or PBS and compared to a contralateral, non-injured control limb. Muscle histology 6 weeks post-repair revealed that the hydrogel reduced fibrosis, FI, and muscle atrophy while supporting vascularization of the injured tissue region. Collectively, these results suggest that the HyA hydrogel alone can promote muscle regeneration in a clinically relevant delayed repair model of rotator cuff tear, which is hypothesized due to controlled FAP differentiation into pro-myogenic lineages.

Gene transfection via lipid nanoparticle (LNP)−mRNA complexes have tremendous potential for treating cardiac diseases. However, the transfection efficiency is poor and there is a lack of in vitro screening systems that predict transfection efficacy. Here we demonstrate a method for identifying LNP−mRNA complexes that diffuse efficiently within 3D cardiac micromuscles and transfect cardiomyocytes with high efficiency, using a phenotypic cardiac microphysiological system (MPS) constructed from a human induced pluripotent stem cell cardiomyocytes Cre-reporter line. LNP formulations containing an acid-degradable PEG–lipid had enhanced diffusion and gene editing efficiency in the cardiac MPS. The in vivo delivery of LNP−mRNA complexes, including luciferase and CRE mRNA, into Ai6 mice confirmed the cardiac MPS screening outcomes. Acid-degradable PEG-LNPs achieved notably superior transfection in the heart with reduced off-target liver uptake compared with standard LNP formulations. The cardiac MPS showed strong LNP transfection in vitro and pinpointed a promising formulation for in vivo mRNA delivery to the heart. A cardiac microphysiological system enables the screening and discovery of lipid nanoparticle−mRNA complexes that diffuse efficiently and results in effective in vivo mRNA delivery to the heart.

We report on the design and fabrication of a circular pillar array as an interfacial barrier for microfluidic microphysiological systems (MPS). Traditional barrier interfaces, such as porous membranes and microchannel arrays, present limitations due to inconsistent pore size, complex fabrication and device assembly, and a lack of tunability using a scalable design. Our pillar array overcomes these limitations by providing precise control over pore size, porosity, and hydraulic resistance through simple modifications of pillar dimensions. Serving as an interface between microfluidic compartments, it facilitates cell aggregation for tissue formation and acts as a tunable diffusion barrier that mimics diffusion in vivo. We demonstrate the utility of barrier design to engineer physiologically relevant cardiac microtissues and a heterotypic model with vasculature within the device. The tunable properties offer significant potential for drug screening/testing and disease modeling, enabling comparisons of drug permeability and cell migration in MPS tissue with or without vasculature.

Current treatment options for craniofacial volumetric muscle loss (VML) have disadvantages and cannot fully restore normal function. Bio-inspired semisynthetic acrylated hyaluronic acid (AcHyA) hydrogel, which fills irregularly shaped defects, resembles an extracellular matrix, and induces a minimal inflammatory response, has shown promise in experimental studies of extremity VML. We therefore sought to study AcHyA hydrogel in the treatment of craniofacial VML. For this, we used a novel model of masseter VML in the rat. Following the creation of a 5 mm × 5 mm injury to the superficial masseter and administration of AcHyA to the wound, masseters were explanted between 2 and 16 weeks postoperatively and were analyzed for evidence of muscle regeneration including fibrosis, defect size, and fiber cross-sectional area (FCSA). At 8 and 16 weeks, masseters treated with AcHyA showed significantly less fibrosis than nonrepaired controls and a smaller decrease in defect size. The mean FCSA among fibers near the defect was significantly greater among hydrogel-repaired than control masseters at 8 weeks, 12 weeks, and 16 weeks. These results show that the hydrogel mitigates the fibrotic healing response and wound contracture. Our findings also suggest that hydrogel-based treatments have potential use as a treatment for the regeneration of craniofacial VML and demonstrate a system for evaluating subsequent iterations of materials in VML injuries. Impact Statement Craniofacial volumetric muscle loss (VML) is a debilitating condition for which current treatment options are unable to restore normal appearance, or function. Tissue engineering approaches, such as hydrogel implants, may be an effective strategy to fill the volumetric defects and promote de novo muscle regeneration. In this study, we describe a novel rodent model for the study of craniofacial VML and a hyaluronic acid-based hydrogel that can be used as a treatment for the regeneration of craniofacial VML.

In the initial hours following the application of the calcium channel blocker (CCB) nifedipine to microtissues consisting of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), we observe notable variations in the drug’s efficacy. Here, we investigate the possibility that these temporal changes in CCB effects are associated with adaptations in the expression of calcium ion channels in cardiomyocyte membranes. To explore this, we employ a recently developed mathematical model that delineates the regulation of calcium ion channel expression by intracellular calcium concentrations. According to the model, a decline in intracellular calcium levels below a certain target level triggers an upregulation of calcium ion channels. Such an upregulation, if instigated by a CCB, would then counteract the drug’s inhibitory effect on calcium currents. We assess this hypothesis using time-dependent measurements of hiPSC-CMs dynamics and by refining an existing mathematical model of myocyte action potentials incorporating the dynamic nature of the number of calcium ion channels. The revised model forecasts that the CCB-induced reduction in intracellular calcium concentrations leads to a subsequent increase in calcium ion channel expression, thereby attenuating the drug’s overall efficacy. The data and fit models suggest that dynamic changes in cardiac cells in the presence of CCBs may be explainable by induced changes in protein expression, and that this may lead to challenges in understanding calcium based drug effects on the heart unless timings of applications are carefully considered.

Efforts to identify anti-cancer therapeutics and understand tumor-immune interactions are built with in vitro models that do not match the microenvironmental characteristics of human tissues. Using in vitro models which mimic the physical properties of healthy or cancerous tissues and a physiologically relevant culture medium, we demonstrate that the chemical and physical properties of the microenvironment regulate the composition and topology of the glycocalyx. Remarkably, we find that cancer and age-related changes in the physical properties of the microenvironment are sufficient to adjust immune surveillance via the topology of the glycocalyx, a previously unknown phenomenon observable only with a physiologically relevant culture medium.

The immature physiology of cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs) limits their utility for drug screening and disease modelling. Here we show that suitable combinations of mechanical stimuli and metabolic cues can enhance the maturation of hiPSC-derived cardiomyocytes, and that the maturation-inducing cues have phenotype-dependent effects on the cells’ action-potential morphology and calcium handling. By using microfluidic chips that enhanced the alignment and extracellular-matrix production of cardiac microtissues derived from genetically distinct sources of hiPSC-derived cardiomyocytes, we identified fatty-acid-enriched maturation media that improved the cells’ mitochondrial structure and calcium handling, and observed divergent cell-source-dependent effects on action-potential duration (APD). Specifically, in the presence of maturation media, tissues with abnormally prolonged APDs exhibited shorter APDs, and tissues with aberrantly short APDs displayed prolonged APDs. Regardless of cell source, tissue maturation reduced variabilities in spontaneous beat rate and in APD, and led to converging cell phenotypes (with APDs within the 300–450 ms range characteristic of human left ventricular cardiomyocytes) that improved the modelling of the effects of pro-arrhythmic drugs on cardiac tissue. The metabolically driven maturation of geometrically aligned human cardiac microtissues on a microfabricated chip reduces cell-phenotype-dependent variabilities in the action-potential morphology and calcium handling of the cardiomyocytes.

Evaluation of arrhythmogenic drugs is required by regulatory agencies before any new compound can obtain market approval. Despite rigorous review, cardiac disorders remain the second most common cause for safety-related market withdrawal. On the other hand, false-positive preclinical findings prohibit potentially beneficial candidates from moving forward in the development pipeline. Complex in vitro models using cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CM) have been identified as a useful tool that allows for rapid and cost-efficient screening of proarrhythmic drug risk. Currently available hiPSC-CM models employ simple two-dimensional (2D) culture formats with limited structural and functional relevance to the human heart muscle. Here, we present the use of our 3D cardiac microphysiological system (MPS), composed of a hiPSC-derived heart micromuscle, as a platform for arrhythmia risk assessment. We employed two different hiPSC lines and tested seven drugs with known ion channel effects and known clinical risk: dofetilide and bepridil (high risk); amiodarone and terfenadine (intermediate risk); and nifedipine, mexiletine, and lidocaine (low risk). The cardiac MPS successfully predicted drug cardiotoxicity risks based on changes in action potential duration, beat waveform (i.e., shape), and occurrence of proarrhythmic events of healthy patient hiPSC lines in the absence of risk cofactors. We showcase examples where the cardiac MPS outperformed existing hiPSC-CM 2D models.