Mimetic Matrix Research Articles

Cardiac ultrastructure inspired matrix induces advanced metabolic and functional maturation of differentiated human cardiomyocytes.

The vast potential of human induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs) in preclinical models of cardiac pathologies, precision medicine, and drug screening remains to be fully realized because hiPSC-CMs are immature without adult-like characteristics. Here, we present a method to accelerate hiPSC-CM maturation on a substrate, cardiac mimetic matrix (CMM), mimicking adult human heart matrix ligand chemistry, rigidity, and submicron ultrastructure, which synergistically mature hiPSC-CMs rapidly within 30days. hiPSC-CMs matured on CMM exhibit systemic transcriptomic maturation toward an adult heart state, are aligned with high strain energy, metabolically rely on oxidative phosphorylation and fatty acid oxidation, and display enhanced redox handling capability, efficient calcium handling, and electrophysiological features of ventricular myocytes. Endothelin-1-induced pathological hypertrophy is mitigated on CMM, highlighting the role of a native cardiac microenvironment in withstanding hypertrophy progression. CMM is a convenient model for accelerated development of ventricular myocytes manifesting highly specialized cardiac-specific functions.

'Educated' Osteoblasts Reduce Osteoclastogenesis in a Bone-Tumor Mimetic Microenvironment.

Simple SummaryPatients with late-stage bone metastatic breast cancer experience skeletal related events, including osteolytic lesion formation, due to overactive osteoclast bone resorption. It is well-known that osteoclast function is altered by breast cancer cells in bone. Breast cancer cells stimulate osteoblasts to secrete factors that initiate osteoclast differentiation and activation. Our lab has previously identified a novel subpopulation of osteoblasts in the bone-tumor microenvironment called “educated” osteoblasts (EOs) that alter breast cancer cell proliferation. The aim of this study was to identify how osteoclasts are affected by EOs during metastatic breast cancer progression in bone. Our results demonstrated that pre-osteoclast interaction with EOs reduces osteoclast formation and bone resorption in a bone-tumor mimetic microenvironment. Furthermore, we identified that altered osteoclast formation can be modulated, in part, by tumor necrosis factor alpha (TNFα). Overall, our data demonstrate osteoclastogenesis is reduced by EO cells, suggesting EO cells have a protective effect in bone and exert an inhibitory effect on tumor progression.Breast cancer (BC) metastases to bone disrupt the balance between osteoblasts and osteoclasts, leading to excessive bone resorption. We identified a novel subpopulation of osteoblasts with tumor-inhibitory properties, called educated osteoblasts (EOs). Here we sought to examine the effect of EOs on osteoclastogenesis during tumor progression. We hypothesized that EOs affect osteoclast development in the bone-tumor niche, leading to suppressed pre-osteoclast fusion and bone resorption. Conditioned media (CM) was analyzed for protein expression of osteoclast factors receptor activator of nuclear factor kappa-β ligand (RANKL), osteoprotegerin (OPG), and tumor necrosis factor alpha (TNFα) via ELISA. EOs were co-cultured with pre-osteoclasts on a bone mimetic matrix to assess osteoclast resorption. Pre-osteoclasts were tri-cultured with EOs plus metastatic BC cells and assessed for tartrate-resistance acid phosphatase (TRAP)-positive, multinucleated (≥3 nuclei), mature osteoclasts. Tumor-bearing murine tibias were stained for TRAP to determine osteoclast number in-vivo. EO CM expressed reduced amounts of soluble TNFα and OPG compared to naïve osteoblast CM. Osteoclasts formed in the presence of EOs were smaller and less in number. Upon co-culture on a mimetic bone matrix, a 50% reduction in the number of TRAP-positive osteoclasts formed in the presence of EOs was observed. The tibia of mice inoculated with BC cells had less osteoclasts per bone surface in bones with increased numbers of EO cells. These data suggest EOs reduce osteoclastogenesis and bone resorption. The data imply EOs provide a protective effect against bone resorption in bone metastatic BC.

Tropoelastin-Coated Tendon Biomimetic Scaffolds Promote Stem Cell Tenogenic Commitment and Deposition of Elastin-Rich Matrix.

Tendon tissue engineering strategies that recreate the biophysical and biochemical native microenvironment have a greater potential to achieve regeneration. Here, we developed tendon biomimetic scaffolds using mechanically competent yarns of poly-ε-caprolactone, chitosan, and cellulose nanocrystals to recreate the inherent tendon hierarchy from a nano-to-macro scale. These were then coated with tropoelastin (TROPO) through polydopamine (PDA) linking, to mimic the native extracellular matrix (ECM) composition and elasticity. Both PDA and TROPO coatings decreased surface stiffness without masking the underlying substrate. We found that human adipose-derived stem cells (hASCs) seeded onto these TROPO biomimetic scaffolds more rapidly acquired their spindle-shape morphology and high aspect ratio characteristic of tenocytes. Immunocytochemistry shows that the PDA and TROPO-coated surfaces boosted differentiation of hASCs toward the tenogenic lineage, with sustained expression of the tendon-related markers scleraxis and tenomodulin up to 21 days of culture. Furthermore, these surfaces enabled the deposition of a tendon-like ECM, supported by the expression of collagens type I and III, tenascin, and decorin. Gene expression analysis revealed a downregulation of osteogenic and fibrosis markers in the presence of TROPO when compared with the control groups, suggesting proper ECM deposition. Remarkably, differentiated cells exposed to TROPO acquired an elastogenic profile due to the evident elastin synthesis and deposition, contributing to the formation of a more mimetic matrix in comparison with the PDA-coated and uncoated conditions. In summary, our biomimetic substrates combining biophysical and biological cues modulate stem cell behavior potentiating their long-term tenogenic commitment and the production of an elastin-rich ECM.

Lipidic Cubic Phase-Induced Membrane Protein Crystallization: Interplay Between Lipid Molecular Structure, Mesophase Structure and Properties, and Crystallogenesis

Obtaining well-diffracting crystals of membrane proteins, which is crucial to the molecular-level understanding of their intrinsic three-dimensional structure, dynamics, and function, represents a fundamental bottleneck in the field of structural biology. One of the major advances in the field of membrane protein structural determination was the realization that the nanostructured lipidic cubic phase (LCP) environment constitutes a membrane mimetic matrix that promotes solubilization, stabilization, and crystallization of specific membrane proteins. Despite two decades passing since the introduction of LCP-based membrane protein crystallization, the research community’s understanding of the processes that drive protein nucleation and macromolecular assembly in the LCP generally remains limited. In the current study, we present a detailed, systematic investigation into the relationship between the chemical structure of the lipid, the physical properties of the ensuing mesophase, the translational diffusion...

Tunichrome mimetic matrix, its perspective in abatement for carcinogenic hexavalent chromium and specific coordination behavior

Inspired by naturally occurring metal chelation strategy by pyrogallol moiety of tunichromes of marine tunicates, a strong retention of carcinogenic chromium (CrVI) is described by mimicking tunichrome matrix as a proof-of-concept investigation in water treatment applications. Harvesting tunichromes tailored with the pyrogallol moiety are extremely difficult owing to aerial sensitivity and purification difficulties, thus proving a challenging synthesis problem. Despite significant interest, knowledge of specific metal coordination in tunicate’s matrix is still ambiguous, hindering study scope. We planned to fabricate a promising tunichrome matrix (TM) tailored with pyrogallol moieties from the Earth-abundant raw materials (gallic acid and chitin nanofiber) at mass scale without artificial aid. Specifically, we aimed at atomistic understanding of contaminant and pyrogallol moiety of TM by examining the structural parameters (redox change, geometrical distortion, coordination bond). Cr K-edge extended X-ray absorption fine structure (EXAFS) corresponds well to nontoxic CrIII (average four CrO bond lengths of 1.91Å) and a small fraction of CrVI (average two CrO bond lengths of 1.62Å). A coupled reductive complexation mechanism was identified by combining near edge X-ray absorption fine structure (NEXAFS) and linear combination fits. From the viewpoint of practical utility and effective performance (nearly 119mg chromium per g weight of TM), the impacts of water chemistry conditions were investigated from a variety of contaminated waters, showing a distinct improvement compared to conventional popular materials. The gained knowledge would be beneficial for utilizing a pyrogallol moiety as an alternative greener and sustainable scavenger, specifically for toxic chromium.

Enhanced antifouling performance of hybrid PVDF ultrafiltration membrane with the dual-mode SiO2-g-PDMS nanoparticles

Abstract The polydimethylsiloxane modified silica (SiO2-g-PDMS) nanoparticles with hydrophilic core and hydrophobic shell are employed for the hybridization modification of PVDF ultrafiltration membrane to improve the separation and antifouling performances. The results suggest the modified SiO2-g-PDMS nanoparticles exhibit improved dispersion and significant amphiphilic characteristics, and the wetting properties of membrane surface and mimetic matrix of hybrid PVDF membranes are distinctly different with the contact angle values at 105° and 15°, respectively. In addition, the added SiO2-g-PDMS nanoparticles significantly improve the fouling repulsion and fouling release properties of hybrid PVDF membranes, the anti-adsorption ability and water flux recovery ratio (FRR-W) of hybrid PVDF membranes to the two typical pollutants of bovine serum albumin (BSA) and humic acid (HA) have been significantly improved. This article aims to provide a simple way to improve the antifouling performance of PVDF ultrafiltration membrane with dual-mode multi-functional modifier.

Fluorescence Recovery After Photobleaching in Lipidic Cubic Phase (LCP-FRAP): A Precrystallization Assay for Membrane Proteins.

Crystallization of integral membrane proteins (MPs) is notoriously difficult, given their poor stability outside native membrane environment and due to the interference of detergent micelles with crystallization process. MP crystallization in a membrane mimetic matrix, known as lipidic cubic phase (LCP), has recently started to gain popularity, following successes in structure determination of G protein-coupled receptors (GPCRs), transporters, and enzymes. Unlike crystallization trials in aqueous solutions where protein molecules are free to move, diffusion of MPs in LCP is restricted, and, thus, a high level of protein mobility can serve as an early indication for subsequent crystallization success. Prompted by our initial observations that precipitant conditions can dramatically affect diffusion of GPCRs in LCP, we have developed a simple precrystallization assay, based on measuring protein diffusion at a number of different conditions by fluorescence recovery after photobleaching (LCP-FRAP). Over the last few years, the LCP-FRAP assay was incorporated in our GPCR structure determination pipeline and proved as a powerful technique allowing for a faster identification of crystallization conditions for many different receptors. The assay is used to screen for the best protein constructs, ligands, LCP host lipids, precipitants, and additives, thereby focusing subsequent crystallization trials on the most promising parts of the multidimensional crystallization phase diagram, substantially increasing the likelihood of finding the right crystallization condition. Here, we describe our LCP-FRAP protocols for guiding GPCR crystallization, which can be adapted to any other MP, and discuss some of the critical considerations related to application of this assay.

Characterization of lipid matrices for membrane protein crystallization by high-throughput small angle X-ray scattering

The lipidic cubic phase (LCP) has repeatedly proven to serve as a successful membrane–mimetic matrix for a variety of difficult-to-crystallize membrane proteins. While monoolein has been the predominant lipid of choice, there is a growing need for the characterization and use of other LCP host lipids, allowing exploration of a range of structural parameters such as bilayer thickness and curvature for optimal insertion, stability and crystallogenesis of membrane proteins. Here, we describe the development of a high-throughput (HT) pipeline to employ small angle X-ray scattering (SAXS) – the most direct technique to identify lipid mesophases and measure their structural parameters – to interrogate rapidly a large number of lipid samples under a variety of conditions, similar to those encountered during crystallization. Leveraging the identical setup format for LCP crystallization trials, this method allows the quickly assessment of lipid matrices for their utility in membrane protein crystallization, and could inform the tailoring of lipid and precipitant conditions to overcome specific crystallization challenges. As proof of concept, we present HT LCP–SAXS analysis of lipid samples made of monoolein with and without cholesterol, and of monovaccenin, equilibrated with solutions used for crystallization trials and LCP fluorescence recovery after photobleaching (FRAP) experiments.