Ultrasensitive, Multiplexed Buoyant Sensor for Monitoring Cytokines in Biofluids.

Multiplexed quantification of low-abundance protein biomarkers in complex biofluids is important for biomedical research and clinical diagnostics. However, in situ sampling without perturbing biological systems remains challenging. In this work, we report a buoyant biosensor that enables in situ monitoring of protein analytes at attomolar concentrations with a 15 min temporal resolution. The buoyant biosensor implemented with fluorescent nanolabels enabled the ultrasensitive and multiplexed detection and quantification of cytokines. Implementing the biosensor in a digital manner (i.e., counting the individual nanolabels) further improves the low detection limit. We demonstrate that the biosensor enables the detection and quantification of the time-varying concentrations of cytokines (e.g., IL-6 and TNF-α) in macrophage culture media without perturbing the live cells. The easy-to-apply biosensor with attomolar sensitivity and multiplexing capability can enable an in situ analysis of protein biomarkers in various biofluids and tissues to aid in understanding biological processes and diagnosing and treating diverse diseases.

Detergent-Based Decellularization for Anisotropic Cardiac-Specific Extracellular Matrix Scaffold Generation.

Cell-derived extracellular matrix (ECM) has become increasingly popular in tissue engineering applications due to its ability to provide tailored signals for desirable cellular responses. Anisotropic cardiac-specific ECM scaffold decellularized from human induced pluripotent stem cell (hiPSC)-derived cardiac fibroblasts (hiPSC-CFs) mimics the native cardiac microenvironment and provides essential biochemical and signaling cues to hiPSC-derived cardiomyocytes (hiPSC-CMs). The objective of this study was to assess the efficacy of two detergent-based decellularization methods: (1) a combination of ethylenediaminetetraacetic acid and sodium dodecyl sulfate (EDTA + SDS) and (2) a combination of sodium deoxycholate and deoxyribonuclease (SD + DNase), in preserving the composition and bioactive substances within the aligned ECM scaffold while maximumly removing cellular components. The decellularization effects were evaluated by characterizing the ECM morphology, quantifying key structural biomacromolecules, and measuring preserved growth factors. Results showed that both treatments met the standard of cell removal (less than 50 ng/mg ECM dry weight) and substantially preserved major ECM biomacromolecules and growth factors. The EDTA + SDS treatment was more time-efficient and has been determined to be a more efficient method for generating an anisotropic ECM scaffold from aligned hiPSC-CFs. Moreover, this cardiac-specific ECM has demonstrated effectiveness in supporting the alignment of hiPSC-CMs and their expression of mature structural and functional proteins in in vitro cultures, which is crucial for cardiac tissue engineering.

Perfusability and immunogenicity of implantable pre-vascularized tissues recapitulating features of native capillary network.

Vascularization is a key pre-requisite to engineered anatomical scale three dimensional (3-D) constructs to ensure their nutrient and oxygen supply upon implantation. Presently, engineered pre-vascularized 3-D tissues are limited to only micro-scale hydrogels, which meet neither the anatomical scale needs nor the complexity of natural extracellular matrix (ECM) environments. Anatomical scale perfusable constructs are critically needed for translational applications. To overcome this challenge, we previously developed pre-vascularized ECM sheets with long and oriented dense microvascular networks. The present study further evaluated the patency, perfusability and innate immune response toward these pre-vascularized constructs. Macrophage-co-cultured pre-vascularized constructs were evaluated in vitro to confirm micro-vessel patency and perturbations in macrophage metabolism. Subcutaneously implanted pre-vascularized constructs remained viable and formed a functional anastomosis with host vasculature within 3 days of implantation. This completely biological pre-vascularized construct holds great potential as a building block to engineer perfusable anatomical scale tissues.

Comparison of phasor analysis and biexponential decay curve fitting of autofluorescence lifetime imaging data for machine learning prediction of cellular phenotypes.

Introduction: Autofluorescence imaging of the coenzymes reduced nicotinamide (phosphate) dinucleotide (NAD(P)H) and oxidized flavin adenine dinucleotide (FAD) provides a label-free method to detect cellular metabolism and phenotypes. Time-domain fluorescence lifetime data can be analyzed by exponential decay fitting to extract fluorescence lifetimes or by a fit-free phasor transformation to compute phasor coordinates. Methods: Here, fluorescence lifetime data analysis by biexponential decay curve fitting is compared with phasor coordinate analysis as input data to machine learning models to predict cell phenotypes. Glycolysis and oxidative phosphorylation of MCF7 breast cancer cells were chemically inhibited with 2-deoxy-d-glucose and sodium cyanide, respectively; and fluorescence lifetime images of NAD(P)H and FAD were obtained using a multiphoton microscope. Results: Machine learning algorithms built from either the extracted lifetime values or phasor coordinates predict MCF7 metabolism with a high accuracy (∼88%). Similarly, fluorescence lifetime images of M0, M1, and M2 macrophages were acquired and analyzed by decay fitting and phasor analysis. Machine learning models trained with features from curve fitting discriminate different macrophage phenotypes with improved performance over models trained using only phasor coordinates. Discussion: Altogether, the results demonstrate that both curve fitting and phasor analysis of autofluorescence lifetime images can be used in machine learning models for classification of cell phenotype from the lifetime data.

Updates on Recent Clinical Assessment of Commercial Chronic Wound Care Products.

Impaired wound healing after trauma, disorders, and surgeries impact millions of people globally every year. Dysregulation in orchestrated healing mechanisms and underlying medical complications make chronic wound management extremely challenging. Besides standard-of-care treatments including broad spectrum antibiotics and wound-debridement, novel adjuvant therapies are clinically tested and commercialized. These include topical agents, skin substitutes, growth factor delivery, and stem cell therapies. With a goal to overcome factors playing pivotal role in delayed wound healing, researchers are exploring novel approaches to elicit desirable healing outcomes in chronic wounds. Although recent innovations in wound care products, therapies, and devices are extensively reviewed in past, a comprehensive review summarizing their clinical outcomes is surprisingly lacking. Herein, this work reviews the commercially available wound care products and their performance in clinical trials to provide a statistically comprehensive understanding of their safety and efficacy. The performance and suitability of various commercial wound care platforms, including xenogeneic and allogenic products, wound care devices, and novel biomaterials, are discussed for chronic wounds. The current clinical evaluation will provide a comprehensive understanding of the benefits and drawbacks of the most-recent approaches and will enable researchers and healthcare providers to develop next-generation technologies for chronic wound management.

Tissue Engineering-Based Strategies for Diabetic Foot Ulcer Management.

Significance: Diabetic foot ulcers (DFU) are a mounting problem with the increasingly frail population. Injuries that would otherwise heal are kept open by risk factors such as diabetes, obesity, and age-related conditions, which interferes with the natural wound healing processes. Recent Advances: This review summarizes recent advancements in the field of tissue engineering for the treatment of DFUs. FDA-approved approaches, including signaling-based therapies, stem cell therapies, and skin substitutes are summarized and cutting-edge experimental technologies that have the potential to manage chronic wounds, such as skin printing, skin organogenesis, skin self-assembly, and prevascularization, are discussed. Critical Issues: The standard of care for chronic wounds involves wound debridement, wound dressings, and resolving the underlying cause such as lowering the glycemic index and reducing wound pressure. Current DFU treatments are limited by low wound closure rates and poor regrown skin quality. New adjuvant therapies that facilitate wound closure in place of or in conjunction with standard care are critically needed. Future Directions: Tissue engineering strategies are limited by the plasticity of adult human cells. In addition to traditional techniques, genetic modification, although currently an emerging technology, has the potential to unlock human regeneration and can be incorporated in future therapeutics.

Fabrication of a Completely Biological and Anisotropic Human Mesenchymal Stem Cell-Based Vascular Graft.

Tissue-engineered small-diameter vascular grafts are required to match mechanical properties as well as cellular and extracellular architecture of native blood vessels. Although various engineering technologies have been developed, the most reliable strategy highlights the needs for incorporating completely biological components and anisotropic cellular and biomolecular organization into the tissue-engineered vascular graft (TEVG). Based on the antithrombogenic, immunoregulatory, and regenerative properties of human mesenchymal stem cells (hMSCs), this chapter provides a step-by-step protocol for generating a completely biological and anisotropic TEVG that comprises of hMSCs and highly aligned extracellular matrix (ECM) nanofibers. The hMSCs were grown on an aligned nanofibrous ECM scaffold derived from an oriented human dermal fibroblast (hDF) sheet and then wrapped around a temporary mandrel to form a tubular assembly, followed by a maturation process in a rotating wall vessel (RWV) bioreactor. The resulting TEVG demonstrates anisotropic structural and mechanical properties similar to that of native blood vessels. A completely biological, anisotropic, and mechanically strong TEVG that incorporates immunoregulatory hMSCs is promising to meet the urgent needs of a surgical intervention for bypass grafting.

Updates on clinical trials evaluating the regenerative potential of allogenic mesenchymal stem cells in COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected nearly 118 million people and caused ~2.6 million deaths worldwide by early 2021, during the coronavirus disease 2019 (COVID-19) pandemic. Although the majority of infected patients show mild-to-moderate symptoms, a small fraction of patients develops severe symptoms. Uncontrolled cytokine production and the lack of substantive adaptive immune response result in hypoxia, acute respiratory distress syndrome (ARDS), or multiple organ failure in severe COVID-19 patients. Since the current standard of care treatment is insufficient to alleviate severe COVID-19 symptoms, many clinics have been prompted to perform clinical trials involving the infusion of mesenchymal stem cells (MSCs) due to their immunomodulatory and therapeutic properties. Several phases I/II clinical trials involving the infusion of allogenic MSCs have been performed last year. The focus of this review is to critically evaluate the safety and efficacy outcomes of the most recent, placebo-controlled phase I/II clinical studies that enrolled a larger number of patients, in order to provide a statistically relevant and comprehensive understanding of MSC's therapeutic potential in severe COVID-19 patients. Clinical outcomes obtained from these studies clearly indicate that: (i) allogenic MSC infusion in COVID-19 patients with ARDS is safe and effective enough to decreases a set of inflammatory cytokines that may drive COVID-19 associated cytokine storm, and (ii) MSC infusion efficiently improves COVID-19 patient survival and reduces recovery time. These findings strongly support further investigation into MSC-infusion in larger clinical trials for COVID-19 patients with ARDS, who currently have a nearly 50% of mortality rate.

Preservation of microvascular integrity and immunomodulatory property of prevascularized human mesenchymal stem cell sheets.

Prevascularization is essential to ensure the viability, functionality, and successful integration of tissue-engineered three-dimensional (3D) constructs with surrounding host tissues after transplantation. Human mesenchymal stem cell (hMSC) sheet can be prevascularized by coculturing with endothelial cells (ECs), and then be further used as building blocks for engineering 3D complex tissues. In addition, predifferentiation of hMSCs into a tissue-specific lineage in vitro has been proven to promote graft engraftment and regeneration. However, it is unclear if the prevascularized hMSC sheets can still maintain their microvascular integrity as well as the immune-regulatory properties after their tissue-specific differentiation. The objective of this study was to investigate the effects of differentiation cues on the microvascular structure, angiogenic factor secretion, and immunogenic responses of prevascularized hMSC sheets. The results showed that upon coculturing with ECs, hMSC sheets successfully formed microvascular network, while maintaining hMSCs' multi-lineage differentiation capability. The next step, osteogenic and adipogenic induction, damaged the preformed microvascular structures and compromised the angiogenic factor secretion ability of hMSCs. Nonetheless, this effect was mitigated by adjusting the concentration of differentiation factors. The subcutaneous transplantation in an immunocompetent rat model demonstrated that the osteogenic differentiated prevascularized hMSC sheet preserved its microvascular structure and immunomodulatory properties comparable to the undifferentiated prevascularized hMSC sheets. This study suggested that a balanced and optimal differentiation condition can effectively promote the tissue-specific predifferentiation of prevascularized hMSC sheet while maintaining its immunomodulatory and tissue integration properties.

A step-by-step protocol for generating human fibroblast cell-derived completely biological extracellular matrix scaffolds.

Native extracellular matrix (ECM) based scaffolds are far more superior in structural and compositional complexity than other engineered scaffolding materials such as hydrogels, electrospun fibers, and three-dimensional (3D) printed substrates. Due to the presence of native structural proteins and other macromolecules, native ECM can better restore the crucial cell-ECM crosstalk and provide a highly biomimetic microenvironment to cells. Allogenic or xenogeneic tissues have been derived by decellularization to obtain native ECM scaffolds. However, their applicability is limited by batch to batch variation, risk of pathogen transfer, undesirable immune response and scarcity of donors. Human dermal fibroblasts (hDFs) can be prescreened and maintained in a pathogen-free condition. Herein, we have described a step-by-step protocol to generate a completely biological ECM scaffold by decellularization of hDF cell sheets. Decellularization was achieved by using an anionic surfactant sodium dodecyl sulfate (SDS) and ethylene diamine tetraacetate (EDTA). The resulting ECM sheet was organized into a nanofibrous scaffold, containing major ECM structural proteins as well as other macromolecules including collagens, fibronectin, laminin and elastin. This cell-derived nanofibrous ECM is a promising scaffold material for constructing highly biomimetic functional tissues.

A Critical Review of Microelectrode Arrays and Strategies for Improving Neural Interfaces.

Though neural interface systems (NISs) can provide a potential solution for mitigating the effects of limb loss and central nervous system damage, the microelectrode array (MEA) component of NISs remains a significant limiting factor to their widespread clinical applications. Several strategies can be applied to MEA designs to increase their biocompatibility. Herein, an overview of NISs and their applications is provided, along with a detailed discussion of strategies for alleviating the foreign body response (FBR) and abnormalities seen at the interface of MEAs and the brain tissue following MEA implantation. Various surface modifications, including natural/synthetic surface coatings, hydrogels, and topography alterations, have shown to be highly successful in improving neural cell adhesion, reducing gliosis, and increasing MEA longevity. Different MEA surface geometries, such as those seen in the Utah and Michigan arrays, can help alleviate the resultant FBR by reducing insertion damage, while providing new avenues for improving MEA recording performance and resolution. Increasing overall flexibility of MEAs as well as reducing their stiffness is also shown to reduce MEA induced micromotion along with FBR severity. By combining multiple different properties into a single MEA, the severity and duration of an FBR postimplantation can be reduced substantially.

Engineering stem cell cardiac patch with microvascular features representative of native myocardium.

The natural myocardium is a highly aligned tissue with an oriented vasculature. Its characteristic cellular as well as nanoscale extracellular matrix (ECM) organization along with an oriented vascular network ensures appropriate blood supply and functional performance. Although significant efforts have been made to develop anisotropic cardiac structure, currently neither an ideal biomaterial nor an effective vascularization strategy to engineer oriented and high-density capillary-like microvessels has been achieved for clinical cardiovascular therapies. A naturally derived oriented ECM nanofibrous scaffold mimics the physiological structure and components of tissue ECM and guides neovascular network formation. The objective of this study was to create an oriented and dense microvessel network with physiological myocardial microvascular features. METHODS: Highly aligned decellularized human dermal fibroblast sheets were used as ECM scaffold to regulate physiological alignment of microvascular networks by co-culturing human mesenchymal stem cells (hMSCs) and endothelial cells (ECs). The influence of topographical features on hMSC and EC interaction was investigated to understand underlying mechanisms of neovasculature formation. RESULTS: Results demonstrate that the ECM topography can be translated to ECs via CD166 tracks and significantly improved hMSC-EC crosstalk and vascular network formation. The aligned ECM nanofibers enhanced structure, length, and density of microvascular networks compared to randomly organized nanofibrous ECM. Moreover, hMSC-EC co-culture promoted secretion of pro-angiogenic growth factors and matrix remodeling via metalloprotease-2 (MMP-2) activation, which resulted in highly dense vascular network formation with intercapillary distance (20 µm) similar to the native myocardium. CONCLUSION: HMSC-EC co-culture on the highly aligned ECM generates physiologically oriented and dense microvascular network, which holds great potential for cardiac tissue engineering.

Polydopamine and collagen coated micro-grated polydimethylsiloxane for human mesenchymal stem cell culture.

Natural tissues contain highly organized cellular architecture. One of the major challenges in tissue engineering is to develop engineered tissue constructs that promote cellular growth in physiological directionality. To address this issue, micro-patterned polydimethylsiloxane (PDMS) substrates have been widely used in cell sheet engineering due to their low microfabrication cost, higher stability, excellent biocompatibility, and most importantly, ability to guide cellular growth and patterning. However, the current methods for PDMS surface modification either require a complicated procedure or generate a non-uniform surface coating, leading to the production of poor-quality cell layers. A simple and efficient surface coating method is critically needed to improve the uniformity and quality of the generated cell layers. Herein, a fast, simple and inexpensive surface coating method was analyzed for its ability to uniformly coat polydopamine (PD) with or without collagen on micro-grated PDMS substrates without altering essential surface topographical features. Topographical feature, stiffness and cytotoxicity of these PD and/or collagen based surface coatings were further analyzed. Results showed that the PD-based coating method facilitated aligned and uniform cell growth, therefore holds great promise for cell sheet engineering as well as completely biological tissue biomanufacturing.

Upgrading prevascularization in tissue engineering: A review of strategies for promoting highly organized microvascular network formation.

Functional and perfusable vascular network formation is critical to ensure the long-term survival and functionality of engineered tissues after their transplantation. Although several vascularization strategies have been reviewed in past, the significance of microvessel organization in three-dimensional (3D) scaffolds has been largely ignored. Advances in high-resolution microscopy and image processing have revealed that the majority of tissues including cardiac, skeletal muscle, bone, and skin contain highly organized microvessels that orient themselves to align with tissue architecture for optimum molecular exchange and functional performance. Here, we review strategies to develop highly organized and mature vascular networks in engineered tissues, with a focus on electromechanical stimulation, surface topography, micro scaffolding, surface-patterning, microfluidics and 3D printing. This review will provide researchers with state of the art approaches to engineer vascularized functional tissues for diverse applications. STATEMENT OF SIGNIFICANCE: Vascularization is one of the critical challenges facing tissue engineering. Recent technological advances have enabled researchers to develop microvascular networks in engineered tissues. Although far from translational applications, current vascularization strategies have shown promising outcomes. This review emphasizes the most recent technological advances and future challenges for developing organized microvascular networks in vitro. The next critical step is to achieve highly perfusable, dense, mature and organized microvascular networks representative of native tissues.

Constructing Biomimetic Cardiac Tissues: A Review of Scaffold Materials for Engineering Cardiac Patches.

Engineered cardiac patches (ECPs) hold great promise to repair ischemia-induced damages to the myocardium. Recent studies have provided robust technological advances in obtaining pure cardiac cell populations as well as various novel scaffold materials to generate engineered cardiac tissues that can significantly improve electrical and contractile functions of damaged myocardium. Given the significance in understanding the cellular and extracellular structural as well as compositional details of native human heart wall, in order to fabricate most suitable scaffold material for cardiac patches, herein, we have reviewed the structure of the human pericardium and heart wall as well as the compositional details of cardiac extracellular matrix (ECM). Moreover, several strategies to obtain cardiac-specific scaffold materials have been reviewed, including natural, synthetic and hybrid hydrogels, electrospun fibers, decellularized native tissues or whole organs, and scaffolds derived from engineered cell sheets. This review provides a comprehensive analysis of different scaffold materials for engineering cardiac tissues.

Tissue Engineering at the Blood-Contacting Surface: A Review of Challenges and Strategies in Vascular Graft Development.

Tissue engineered vascular grafts (TEVGs) are beginning to achieve clinical success and hold promise as a source of grafting material when donor grafts are unsuitable or unavailable. Significant technological advances have generated small-diameter TEVGs that are mechanically stable and promote functional remodeling by regenerating host cells. However, developing a biocompatible blood-contacting surface remains a major challenge. The TEVG luminal surface must avoid negative inflammatory responses and thrombogenesis immediately upon implantation and promote endothelialization. The surface has therefore become a primary focus for research and development efforts. The current state of TEVGs is herein reviewed with an emphasis on the blood-contacting surface. General vascular physiology and developmental challenges and strategies are briefly described, followed by an overview of the materials currently employed in TEVGs. The use of biodegradable materials and stem cells requires careful control of graft composition, degradation behavior, and cell recruitment ability to ensure that a physiologically relevant vessel structure is ultimately achieved. The establishment of a stable monolayer of endothelial cells and the quiescence of smooth muscle cells are critical to the maintenance of patency. Several strategies to modify blood-contacting surfaces to resist thrombosis and control cellular recruitment are reviewed, including coatings of biomimetic peptides and heparin.