Bioethical implications of organ-on-a-chip on modernizing drug development.

Organ-on-chips are three-dimensional microdevices that emulate the structure, functionality, and behavior of specific tissues or organs using human cells. Combining organoids with microfabricated fluidic channels and microelectronics, these systems offer a promising platform for studying disease mechanisms, drug responses, and tissue performance. By replicating the in vivo microenvironment, these devices can recreate complex cell interactions in controlled conditions and facilitate research in various fields, including drug toxicity and efficacy studies, biochemical analysis, and disease pathogenesis. Integrating human induced pluripotent stem cells further enhances their applicability, thereby enabling patient-specific disease modeling for precision medicine. Although challenges like economy-of-scale, multichip integration, and regulatory compliance exist, advances in this modular technology show promise for lowering drug development costs, improving reproducibility, and reducing the reliance on animal testing. The ethical landscape surrounding organ-on-chip usage presents both benefits and concerns. While these chips offer an alternative to animal testing and potential cost savings, they raise ethical considerations related to community engagement, informed consent, and the need for standardized guidelines. Ensuring public acceptance and involvement in decision-making is vital to address misinformation and mistrust. Furthermore, personalized medicine models using patient-derived cells demand careful consideration of potential ethical dilemmas, such as modeling physiological functions of fetuses or brains and determining the extent of protection for these models. To achieve the full potential of organ-on-a-chip models, collaboration between scientists, ethicists, and regulators is essential to fulfil the promise of transforming drug development, advancing personalized medicine, and contributing to a more ethical and efficient biomedical research landscape.

Clinical trials for stem cell therapies.

In recent years, clinical trials with stem cells have taken the emerging field in many new directions. While numerous teams continue to refine and expand the role of bone marrow and cord blood stem cells for their vanguard uses in blood and immune disorders, many others are looking to expand the uses of the various types of stem cells found in bone marrow and cord blood, in particular mesenchymal stem cells, to uses beyond those that could be corrected by replacing cells in their own lineage. Early results from these trials have produced mixed results often showing minor or transitory improvements that may be attributed to extracellular factors. More research teams are accelerating the use of other types of adult stem cells, in particular neural stem cells for diseases where beneficial outcome could result from either in-lineage cell replacement or extracellular factors. At the same time, the first three trials using cells derived from pluripotent cells have begun.

Biophysical mechanisms of single-cell interactions with microtopographical cues.

Biophysical cues encoded in the extracellular matrix (ECM) are increasingly being explored to control cell behavior in tissue engineering applications. Recently, we showed that cell adhesion to microtopographical structures ("micropegs") can suppress proliferation in a manner that may be blunted by inhibiting cellular contractility, suggesting that this effect is related to altered cell-scaffold mechanotransduction. We now directly investigate this possibility at the microscale through a combination of live-cell imaging, single-cell mechanics methods, and analysis of gene expression. Using time-lapse imaging, we show that when cells break adhesive contacts with micropegs, they form F-actin-filled tethers that extend and then rupture at a maximum, critical length that is greater than trailing-edge tethers observed on topographically flat substrates. This critical tether length depends on myosin activation, with inhibition of Rho-associated kinase abolishing topography-dependent differences in tether length. Using cellular de-adhesion and atomic force microscopy indentation measurements, we show that the micropegs enhance cell-scaffold adhesive interactions without changing whole-cell elasticity. Moreover, micropeg adhesion increases expression of specific mechanotransductive genes, including RhoA GTPase and myosin heavy chain II, and, in myoblasts, the functional marker connexin 43. Together, our data support a model in which microtopographical cues alter the local mechanical microenvironment of cells by modulating adhesion and adhesion-dependent mechanotransductive signaling.

Cell-shape regulation of smooth muscle cell proliferation.

Vascular smooth muscle cells (SMCs) play an important role in vascular remodeling. Heterogeneity and phenotypic changes in SMCs are usually accompanied by a morphological difference, i.e., elongated/spindle-like versus spread-out or epithelioid/rhomboid cell shapes. However, it is not known whether the cell shape directly regulates SMC proliferation, and what the underlying mechanisms are. In this study, microgrooves and micropatterned matrix islands were used to engineer the cell shape and investigate the associated biophysical and biological mechanisms. Compared to spread-out SMCs on nonpatterned surfaces, SMCs on micropatterned surfaces demonstrated elongated morphology, significantly lower cell and nucleus shape indexes, less spreading, a lower proliferation rate, and a similar response (but to a lesser extent) to platelet-derived growth factor, transforming growth factor-beta, and mechanical stretching. DNA microarray profiling revealed a lower expression of neuron-derived orphan receptor-1 (NOR-1) in elongated SMCs. Knocking down NOR-1 suppressed DNA synthesis in SMCs, suggesting that NOR-1 is a mediator of cell elongation effects. Regulation of DNA synthesis in SMCs by the cell shape alone and a decrease in DNA synthesis in the case of small cell spreading area were achieved by micropatterning SMCs on matrix islands of different shapes and spreading areas. Changes in the cell shape also affected the nucleus shape, whereas variations in the cell spreading area modulated the nucleus volume, indicating a possible link between nucleus morphology (both shape and volume) and DNA synthesis. The findings of this investigation provide insight into cell shape effects on cell structure and proliferation, and have direct implications for vascular pathophysiology.

Contractility-dependent modulation of cell proliferation and adhesion by microscale topographical cues.

Engineering of cellular assembly on biomaterial scaffolds by utilizing microscale topographical cues has emerged as a powerful strategy in cardiovascular tissue engineering and regenerative medicine. However, the mechanisms through which these cues are processed to yield changes in canonical cell behaviors remain unclear. Previously, we showed that when mixtures of cardiomyocytes and fibroblasts were cultured on polydimethylsiloxane surfaces studded with microscale pillars (micropegs), fibroblast proliferation was dramatically suppressed, which suggests that the micropegs could be exploited to minimize fibrosis and scar formation. Here, we demonstrate that this effect relies on altered adhesive and micromechanical interactions between individual cells and micropegs. First, we show that the proliferation of a cell physically attached to a micropeg is significantly lower than that of a cell cultured on a featureless region of the substrate. Micropeg adhesion is accompanied by a marked elongation in cell and nuclear shape. When fibroblast contractility is pharmacologically attenuated through low-dose inhibition of either Rho-associated kinase or myosin light chain kinase, the potency with which micropeg adhesion suppresses cell proliferation is significantly reduced. Together, our results support a model in which cell fate decisions may be directly manipulated within tissue engineering scaffolds by the inclusion of microtopographical structures that alter cellular mechanics.

Regulation of vascular smooth muscle cells and mesenchymal stem cells by mechanical strain.

Vascular smooth muscle cells (SMCs) populate in the media of the blood vessel, and play an important role in the control of vasoactivity and the remodeling of the vessel wall. Blood vessels are constantly subjected to hemodynamic stresses, and the pulsatile nature of the blood flow results in a cyclic mechanical strain in the vessel walls. Accumulating evidence in the past two decades indicates that mechanical strain regulates vascular SMC phenotype, function and matrix remodeling. Bone marrow mesenchymal stem cell (MSC) is a potential cell source for vascular regeneration therapy, and may be used to generate SMCs to construct tissue-engineered vascular grafts for blood vessel replacements. In this review, we will focus on the effects of mechanical strain on SMCs and MSCs, e.g., cell phenotype, cell morphology, cytoskeleton organization, gene expression, signal transduction and receptor activation. We will compare the responses of SMCs and MSCs to equiaxial strain, uniaxial strain and mechanical strain in three-dimensional culture. Understanding the hemodynamic regulation of SMC and MSC functions will provide a basis for the development of new vascular therapies and for the construction of tissue-engineered vascular grafts.

Myotube assembly on nanofibrous and micropatterned polymers.

Skeletal muscle consists of parallel bundles of myotubes formed by the fusion of myoblasts. We fabricated nanofibrous and micropatterned polymers as cell culture substrates to guide the morphogenesis of muscular tissue. The nanoscale and microscale topographic features regulate cell and cytoskeleton alignment, myotube assembly, myotube striation, and myoblast proliferation. This bottom-up approach from nanoscale to tissue level demonstrates the potential of nanofibrous polymers for engineering the assembly of cell and tissue structure.

Control of cell adhesion on poly(methyl methacrylate).

Keratoprostheses have been constructed from a wide variety of transparent materials, including poly(methyl methacrylate) (PMMA). However, the success of keratoprosthesis has been plagued by numerous shortcomings that include the weakening of the implant-host interface due to weak cell adhesion and opaque fibrous membrane formation over the inner surface of the implant due to fibroblast attachment. An effective solution requires a surface modification that would selectively allow enhanced cell attachment at the implant-host interface and reduced cell attachment over the interior surface of the implant. Here, we have developed a novel and simple peptide conjugation scheme to modify PMMA surfaces, which allowed for region-specific control of cell adhesion. This method uses di-amino-PEG, which can be grafted onto PMMA using hydrolysis or aminolysis method. PEG can resist cell adhesion and protein adsorption. The functionalization of grafted di-amino-PEG molecules with RGD peptide not only restored cell adhesion to the surfaces, but also enhanced cell attachment and spreading as compared to untreated PMMA surfaces. Long-term cell migration and micropatterning studies clearly indicated that PEG-PMMA surfaces with and without RGD conjugation can be used to differentiate cell adhesion and control cell attachment spatially on PMMA, which will have potential applications in the modification of keratoprostheses.

Tissue engineering of muscle on micropatterned polymer films.

Tissue engineered skeletal muscle has potential physiologically relevant environments to study myogenesis and investigate the organization, differentiation and proliferation to be used for the therapy of muscular dysfunction. In order to engineer skeletal muscle that better resemble the structured architecture in vivo, we cultured myoblasts on topographically micropatterned elastic polymer films with 10-mum wide microgrooves. The organization and differentiation of myoblasts on nonpatterned and micropatterned PDMS films were characterized. In comparison to the myoblasts on nonpatterned PDMS films, myoblasts on micropatterned PDMS films aligned themselves along the direction of the microgrooves. The myoblasts on micropatterned films formed long and unbranched myotubes that had uniform diameter and aligned in the microgroove direction, suggesting that microgrooves promote end-to end fusion of myoblasts; in contrast, myotubes formed on nonpatterned surface were short and less uniform in diameter, and oriented in various directions. This study demonstrates a new approach to engineer muscular tissues on flexible substrate, and highlights the importance of topographical cues for creating more engineer skeletal muscle.

Regulation of vascular smooth muscle cells by micropatterning.

Vascular smooth muscle cells (SMCs) undergo morphological and phenotypic changes when cultured in vitro. To investigate whether SMC morphology regulates SMC functions, bovine aortic SMCs were grown on micropatterned collagen strips (50-, 30-, and 20-microm wide). The cell shape index and proliferation rate of SMCs on 30- and 20-microm strips were significantly lower than those on non-patterned collagen (control), and the spreading area was decreased only for cells patterned on the 20-microm strips, suggesting that SMC proliferation is dependent on cell shape index. The formation of actin stress fibers and the expression of alpha-actin were decreased in SMCs on the 20- and 30-microm collagen strips. SMCs cultured on micropatterned biomaterial poly-(D,L-lactide-co-glycolide) (PLGA) with 30-microm wide grooves also showed lower proliferation rate and less stress fibers than SMCs on non-patterned PLGA. Our findings suggest that micropatterned matrix proteins and topography can be used to control SMC morphology and that elongated cell morphology decreases SMC proliferation but is not sufficient to promote contractile phenotype.