The impact of lutein-loaded poly(lactic-co-glycolic acid) nanoparticles following topical application: An in vitro and in vivo study.

Antioxidant therapies are of interest in the prevention and management of ocular disorders such as cataracts. Although an active area of interest, topical therapy with antioxidants for the treatment of cataracts is complicated by multiple ocular anatomical barriers, product stability, and solubility. Entrapment and delivery of antioxidants with poly(lactic-co-glycolic acid) nanoparticles is a possible solution to these challenges, however, little is known regarding their effects in vitro or in vivo. Our first aim was to investigate the impact of blank and lutein loaded PLGA nanoparticles on viability and development of reactive oxygen species in lens epithelial cells in vitro. Photo-oxidative stress was induced by ultraviolet light exposure with cell viability and reactive oxygen species monitored. Next, an in vivo, selenite model was utilized to induce cataract formation in rodents. Eyes were treated topically with both free lutein and lutein loaded nanoparticles (LNP) at varying concentrations. Eyes were monitored for the development of anterior segment changes and cataract formation. The ability of nanodelivered lutein to reach the anterior segment of the eye was evaluated by liquid chromatography coupled to mass spectrometry of aqueous humor samples and liquid chromatography coupled to tandem mass spectrometry (targeted LC-MS/MS) of lenses. LNP had a minimal impact on the viability of lens epithelial cells during the short exposure timeframe (24 h) and at concentrations < 0.2 µg LNP/µl. A significant reduction in the development of reactive oxygen species was also noted. Animals treated with LNPs at an equivalent lutein concentration of 1,278 µg /mL showed the greatest reduction in cataract scores. Lutein delivery to the anterior segment was confirmed through evaluation of aqueous humor and lens sample evaluation. Topical treatment was not associated with the development of secondary keratitis or anterior uveitis when applied once daily for one week. LNPs may be an effective in the treatment of cataracts.

Injectable Antioxidant and Oxygen-Releasing Lignin Composites to Promote Wound Healing.

The application of engineered biomaterials for wound healing has been pursued since the beginning of tissue engineering. Here, we attempt to apply functionalized lignin to confer antioxidation to the extracellular microenvironments of wounds and to deliver oxygen from the dissociation of calcium peroxide for enhanced vascularization and healing responses without eliciting inflammatory responses. Elemental analysis showed 17 times higher quantity of calcium in the oxygen-releasing nanoparticles. Lignin composites including the oxygen-generating nanoparticles released around 700 ppm oxygen per day at least for 7 days. By modulating the concentration of the methacrylated gelatin, we were able to maintain the injectability of lignin composite precursors and the stiffness of lignin composites suitable for wound healing after photo-cross-linking. In situ formation of lignin composites with the oxygen-releasing nanoparticles enhanced the rate of tissue granulation, the formation of blood vessels, and the infiltration of α-smooth muscle actin+ fibroblasts into the wounds over 7 days. At 28 days after surgery, the lignin composite with oxygen-generating nanoparticles remodeled the collagen architecture, resembling the basket-weave pattern of unwounded collagen with minimal scar formation. Thus, our study shows the potential of functionalized lignin for wound-healing applications requiring balanced antioxidation and controlled release of oxygen for enhanced tissue granulation, vascularization, and maturation of collagen.

A modular surgical simulator for microlaryngoscopy using standard instruments and the carbon dioxide laser.

Objective: Build a microlaryngoscopy surgical simulator for endoscopic laryngeal surgery using standard microsurgical instruments and a CO2 laser. Study design: Anatomical modeling, CAD design and 3D printed manufacturing. Subjects and methods: We created a modular design for a microlaryngoscopy simulator in CAD software. Components include plastic and stainless-steel models of a standard operating laryngoscope and a cassette system for mounting porcine or synthetic models of the vocal folds. All simulator parts, including the metallic laryngoscope model, were manufactured using 3D printing technology. Tumors were simulated in porcine tissue models by injecting a soy protein-based tumor phantom. Residents and faculty in the Louisiana State University otolaryngology department evaluated the system. Each participant performed microlaryngoscopy with laser resection on a porcine larynx and cold instrument procedures on synthetic vocal folds. Participants scored the simulator using a 5-point Likert scale. Results: The microlaryngeal surgical simulator demonstrated in this project is realistic, economical, and easily assembled. We have included 3D printed parts files and detailed assembly instructions that will enable educators interested in surgical simulation to build the device.Participants in the simulator evaluation session felt that the simulator faithfully represented the procedure to resect vocal fold lesions using a CO2 laser. The synthetic model allows the trainee to develop hand-eye coordination while using standard laryngeal instruments. Conclusions: The simulator described herein will enable surgeons to acquire the surgical skills necessary to perform operative microlaryngoscopy prior to operating on live patients.

Conserved pathway activation following xenogeneic, heterotypic fusion.

Fusion between cells of different organisms (i.e., xenogeneic hybrids) can occur, and for humans this may occur in the course of tissue transplantation, animal handling, and food production. Previous work shows that conferred advantages are rare in xenogeneic hybrids, whereas risks of cellular dysregulation are high. Here, we explore the transcriptome of individual xenogeneic hybrids of human mesenchymal stem cells and murine cardiomyocytes soon after fusion and ask whether the process is stochastic or involves conserved pathway activation. Toward this end, single-cell RNA sequencing was used to analyze the transcriptomes of hybrid cells with respect to the human and mouse genomes. Consistent with previous work, hybrids possessed a unique transcriptome distinct from either fusion partner but were dominated by the cardiomyocyte transcriptome. New in this work is the documentation that a few genes that were latent in both fusion partners were consistently expressed in hybrids. Specifically, human growth hormone 1, murine ribosomal protein S27, and murine ATP synthase H+ transporting, mitochondrial Fo complex subunit C2 were expressed in nearly all hybrids. The consistent activation of latent genes between hybrids suggests conserved signaling mechanisms that either cause or are the consequence of fusion of these 2 cell types and might serve as a target for limiting unwanted xenogeneic fusion in the future.-Yuan, C., Freeman, B. T., McArdle, T. J., Jung, J. P., Ogle, B. M. Conserved pathway activation following xenogeneic, heterotypic fusion.

Engineering Breast Cancer Microenvironments and 3D Bioprinting.

The extracellular matrix (ECM) is a critical cue to direct tumorigenesis and metastasis. Although two-dimensional (2D) culture models have been widely employed to understand breast cancer microenvironments over the past several decades, the 2D models still exhibit limited success. Overwhelming evidence supports that three dimensional (3D), physiologically relevant culture models are required to better understand cancer progression and develop more effective treatment. Such platforms should include cancer-specific architectures, relevant physicochemical signals, stromal-cancer cell interactions, immune components, vascular components, and cell-ECM interactions found in patient tumors. This review briefly summarizes how cancer microenvironments (stromal component, cell-ECM interactions, and molecular modulators) are defined and what emerging technologies (perfusable scaffold, tumor stiffness, supporting cells within tumors and complex patterning) can be utilized to better mimic native-like breast cancer microenvironments. Furthermore, this review emphasizes biophysical properties that differ between primary tumor ECM and tissue sites of metastatic lesions with a focus on matrix modulation of cancer stem cells, providing a rationale for investigation of underexplored ECM proteins that could alter patient prognosis. To engineer breast cancer microenvironments, we categorized technologies into two groups: (1) biochemical factors modulating breast cancer cell-ECM interactions and (2) 3D bioprinting methods and its applications to model breast cancer microenvironments. Biochemical factors include matrix-associated proteins, soluble factors, ECMs, and synthetic biomaterials. For the application of 3D bioprinting, we discuss the transition of 2D patterning to 3D scaffolding with various bioprinting technologies to implement biophysical cues to model breast cancer microenvironments.

Single-cell RNA-seq reveals activation of unique gene groups as a consequence of stem cell-parenchymal cell fusion.

Fusion of donor mesenchymal stem cells with parenchymal cells of the recipient can occur in the brain, liver, intestine and heart following transplantation. The therapeutic benefit or detriment of resultant hybrids is unknown. Here we sought a global view of phenotypic diversification of mesenchymal stem cell-cardiomyocyte hybrids and associated time course. Using single-cell RNA-seq, we found hybrids consistently increase ribosome components and decrease genes associated with the cell cycle suggesting an increase in protein production and decrease in proliferation to accommodate the fused state. But in the case of most other gene groups, hybrids were individually distinct. In fact, though hybrids can express a transcriptome similar to individual fusion partners, approximately one-third acquired distinct expression profiles in a single day. Some hybrids underwent reprogramming, expressing pluripotency and cardiac precursor genes latent in parental cells and associated with developmental and morphogenic gene groups. Other hybrids expressed genes associated with ontologic cancer sets and two hybrids of separate experimental replicates clustered with breast cancer cells, expressing critical oncogenes and lacking tumor suppressor genes. Rapid transcriptional diversification of this type garners consideration in the context of cellular transplantation to damaged tissues, those with viral infection or other microenvironmental conditions that might promote fusion.

Heterogeneous Differentiation of Human Mesenchymal Stem Cells in 3D Extracellular Matrix Composites.

Extracellular matrix (ECM) proteins are structural elements of tissue and also potent signaling molecules. Previously, our laboratory showed that ECM of 2D coatings can trigger differentiation of bone marrow-derived mesenchymal stem cells (MSCs) into mesodermal lineages in an ECM-specific manner over 14 days, in some cases comparable to chemical induction. To test whether a similar effect was possible in a 3D, tissue-like environment, we designed a synthetic-natural biomaterial composite. The composite can present whole-molecule ECM proteins to cells, even those that do not spontaneously form hydrogels ex vivo, in 3D. To this end, we entrapped collagen type I, laminin-111, or fibronectin in ECM composites with MSCs and directly compared markers of mesodermal differentiation including cardiomyogenic (ACTC1), osteogenic (SPP1), adipogenic (PPARG), and chondrogenic (SOX9) in 2D versus 3D. We found the 3D condition largely mimicked the 2D condition such that the addition of type I collagen was the most potent inducer of differentiation to all lineages tested. One notable difference between 2D and 3D was pronounced adipogenic differentiation in 3D especially in the presence of exogenous collagen type I. In particular, PPARG gene expression was significantly increased ∼16-fold relative to chemical induction, in 3D and not in 2D. Unexpectedly, 3D engagement of ECM proteins also altered immunomodulatory function of MSCs in that expression of IL-6 gene was elevated relative to basal levels in 2D. In fact, levels of IL-6 gene expression in 3D composites containing exogenously supplied collagen type I or fibronectin were statistically similar to levels attained in 2D with tumor necrosis factor-α (TNF-α) stimulation and these levels were sustained over a 2-week period. Thus, this novel biomaterial platform allowed us to compare the biochemical impact of whole-molecule ECM proteins in 2D versus 3D indicating enhanced adipogenic differentiation and IL-6 expression of MSC in the 3D context. Exploiting the biochemical impact of ECM proteins on MSC differentiation and immunomodulation could augment the therapeutic utility of MSCs.

Single-Cell RNA-Seq of Bone Marrow-Derived Mesenchymal Stem Cells Reveals Unique Profiles of Lineage Priming.

The plasticity and immunomodulatory capacity of mesenchymal stem cells (MSCs) have spurred clinical use in recent years. However, clinical outcomes vary and many ascribe inconsistency to the tissue source of MSCs. Yet unconsidered is the extent of heterogeneity of individual MSCs from a given tissue source with respect to differentiation potential and immune regulatory function. Here we use single-cell RNA-seq to assess the transcriptional diversity of murine mesenchymal stem cells derived from bone marrow. We found genes associated with MSC multipotency were expressed at a high level and with consistency between individual cells. However, genes associated with osteogenic, chondrogenic, adipogenic, neurogenic and vascular smooth muscle differentiation were expressed at widely varying levels between individual cells. Further, certain genes associated with immunomodulation were also inconsistent between individual cells. Differences could not be ascribed to cycles of proliferation, culture bias or other cellular process, which might alter transcript expression in a regular or cyclic pattern. These results support and extend the concept of lineage priming of MSCs and emphasize caution for in vivo or clinical use of MSCs, even when immunomodulation is the goal, since multiple mesodermal (and even perhaps ectodermal) outcomes are a possibility. Purification might enable shifting of the probability of a certain outcome, but is unlikely to remove multilineage potential altogether.

ECM-incorporated hydrogels cross-linked via native chemical ligation to engineer stem cell microenvironments.

Limiting the precise study of the biochemical impact of whole molecule extracellular matrix (ECM) proteins on stem cell differentiation is the lack of 3D in vitro models that can accommodate many different types of ECM. Here we sought to generate such a system while maintaining consistent mechanical properties and supporting stem cell survival. To this end, we used native chemical ligation to cross-link poly(ethylene glycol) macromonomers under mild conditions while entrapping ECM proteins (termed ECM composites) and stem cells. Sufficiently low concentrations of ECM were used to maintain constant storage moduli and pore size. Viability of stem cells in composites was maintained over multiple weeks. ECM of composites encompassed stem cells and directed the formation of distinct structures dependent on ECM type. Thus, we introduce a powerful approach to study the biochemical impact of multiple ECM proteins (either alone or in combination) on stem cell behavior.

A nondenatured, noncrosslinked collagen matrix to deliver stem cells to the heart.

AIMS: Stem cell transplantation holds promise as a therapeutic approach for the repair of damaged myocardial tissue. One challenge of this approach is efficient delivery and long-term retention of the stem cells. Although several synthetic and natural biomaterials have been developed for this purpose, the ideal formulation has yet to be identified. MATERIALS & METHODS: Here we investigate the utility of a nondenatured, noncrosslinked, commercially available natural biomaterial (TissueMend(®) [TEI Biosciences, Boston, MA, USA]) for delivery of human mesenchymal stem cells (MSCs) to the murine heart. RESULTS: We found that MSCs attached, proliferated and migrated within and out of the TissueMend matrix in vitro. Human MSCs delivered to damaged murine myocardium via the matrix (2.3 × 10(4) ± 0.8 × 10(4) CD73(+) cells/matrix) were maintained in vivo for 3 weeks and underwent at least three population doublings during that period (21.9 × 10(4) ± 14.4 × 10(4) CD73(+) cells/matrix). In addition, collagen within the TissueMend matrix could be remodeled by MSCs in vivo, resulting in a significant decrease in the coefficient of alignment of fibers (0.12 ± 0.12) compared with the matrix alone (0.28 ± 0.07), and the MSCs were capable of migrating out of the matrix and into the host tissue. CONCLUSION: Thus, TissueMend matrix offers a commercially available, biocompatible and malleable vehicle for the delivery and retention of stem cells to the heart.

Multifactorial optimization of endothelial cell growth using modular synthetic extracellular matrices.

Extracellular matrices (ECMs) are complex materials, containing at least dozens of different macromolecules that are assembled together, thus complicating their optimization towards applications in 3D cell culture or tissue engineering. The natural complexity of ECMs has limited cell-matrix investigations predominantly to experiments where only one matrix component is adjusted at a time, making it difficult to uncover interactions between different matrix components or to efficiently determine optimal matrix compositions for specific desired biological responses. Here we have developed modular synthetic ECMs based on peptide self-assembly whose incorporation of multiple different peptide ligands can be adjusted. The peptides can co-assemble in a wide range of combinations to form hydrogels of uniform morphology and consistent mechanical properties, but with precisely varied mixtures of peptide ligands. The modularity of this system in turn enabled multi-factorial experimental designs for investigating interactions between these ligands and for determining a multi-peptide matrix formulation that maximized endothelial cell growth. In cultures of HUVECs, we observed a previously unknown antagonistic interaction between the laminin-derived peptide YIGSR and RGDS-mediated cell attachment and growth. We also identified an optimized combination of self-assembled peptides bearing the ligands RGDS and IKVAV that led to endothelial cell growth equivalent to that on native full-length fibronectin. Both of these findings would have been challenging to uncover using more traditional one-factor-at-a-time analyses.

Immune responses to coiled coil supramolecular biomaterials.

Self-assembly has been increasingly utilized in recent years to create peptide-based biomaterials for 3D cell culture, tissue engineering, and regenerative medicine, but the molecular determinants of these materials' immunogenicity have remained largely unexplored. In this study, a set of molecules that self-assembled through coiled coil oligomerization was designed and synthesized, and immune responses against them were investigated in mice. Experimental groups spanned a range of oligomerization behaviors and included a peptide from the coiled coil region of mouse fibrin that did not form supramolecular structures, an engineered version of this peptide that formed coiled coil bundles, and a peptide-PEG-peptide triblock bioconjugate that formed coiled coil multimers and supramolecular aggregates. In mice, the native peptide and engineered peptide did not produce any detectable antibody response, and none of the materials elicited detectable peptide-specific T cell responses, as evidenced by the absence of IL-2 and interferon-gamma in cultures of peptide-challenged splenocytes or draining lymph node cells. However, specific antibody responses were elevated in mice injected with the multimerizing peptide-PEG-peptide. Minimal changes in secondary structure were observed between the engineered peptide and the triblock peptide-PEG-peptide, making it possible that the triblock's multimerization was responsible for this antibody response.

Fibrillar peptide gels in biotechnology and biomedicine.

Peptides, peptidomimetics, and peptide derivatives that self-assemble into fibrillar gels have received increasing interest as synthetic extracellular matrices for applications in 3D cell culture and regenerative medicine. Recently, several of these fibrillizing molecules have been functionalized with bioactive components and chemical features such as cell-binding ligands, degradable sequences, drug eluting compounds, and cross-linkable groups, thereby producing gels that can reliably display multiple factors simultaneously. This capacity for incorporating precise levels of many different biological and chemical factors is advantageous given the natural complexity of cell-matrix interactions that many current biomaterial strategies seek to mimic. In this review, recent efforts in the area of fibril-forming peptide materials are described, and advantages of biomaterials containing multiple modular elements are outlined. In addition, a few hurdles and open questions surrounding fibrillar peptide gels are discussed, including issues of the materials' structural heterogeneity, challenges in fully characterizing the diversity of their self-assembled structures, and incomplete knowledge of how the materials are processed in vivo.

Co-assembling peptides as defined matrices for endothelial cells.

Self-assembling peptides and peptide derivatives bearing cell-binding ligands are increasingly being investigated as defined cell culture matrices and as scaffolds for regenerative medicine. In order to systematically refine such scaffolds to elicit specific desired cell behaviors, ligand display should ideally be achieved without inadvertently altering other physicochemical properties such as viscoelasticity. Moreover, for in vivo applications, self-assembled biomaterials must exhibit low immunogenicity. In the present study, multi-peptide co-assembling hydrogels based on the beta-sheet fibrillizing peptide Q11 (QQKFQFQFEQQ) were designed such that they presented RGDS or IKVAV ligands on their fibril surfaces. In co-assemblies of the ligand-bearing peptides with Q11, ligand incorporation levels capable of influencing the attachment, spreading, morphology, and growth of human umbilical vein endothelial cells (HUVECs) did not significantly alter the materials' fibrillization, beta-turn secondary structure, or stiffness. RGDS-Q11 specifically increased HUVEC attachment, spreading, and growth when co-assembled into Q11 gels, whereas IKVAV-Q11 exerted a more subtle influence on attachment and morphology. Additionally, Q11 and RGDS-Q11 were minimally immunogenic in mice, making Q11-based biomaterials attractive candidates for further investigation as defined, modular extracellular matrices for applications in vitro and in vivo.

Modulating the mechanical properties of self-assembled peptide hydrogels via native chemical ligation.

Hydrogels produced from self-assembling peptides and peptide derivatives are being investigated as synthetic extracellular matrices for defined cell culture substrates and scaffolds for regenerative medicine. In many cases, however, they are less stiff than the tissues and extracellular matrices they are intended to mimic, and they are prone to cohesive failure. We employed native chemical ligation to produce peptide bonds between the termini of fibrillized beta-sheet peptides to increase gel stiffness in a chemically specific manner while maintaining the morphology of the self-assembled fibrils. Polymerization, fibril structure, and mechanical properties were measured by SDS-PAGE, mass spectrometry, TEM, circular dichroism, and oscillating rheometry; and cellular responses to matrix stiffening were investigated in cultures of human umbilical vein endothelial cells (HUVECs). Ligation led to a fivefold increase in storage modulus and a significant enhancement of HUVEC proliferation and expression of CD31 on the surface of the gels. The approach was also orthogonal to the inclusion of unprotected RGD-functionalized self-assembling peptides, which further increased proliferation. This strategy broadens the utility of self-assembled peptide materials for applications that require enhancement or modulation of matrix mechanical properties by providing a chemoselective means for doing so without significantly disrupting the gels' fibrillar structure.