Microengineered human blood-brain barrier platform for understanding nanoparticle transport mechanisms

Challenges in drug development of neurological diseases remain mainly ascribed to the blood-brain barrier (BBB). Despite the valuable contribution of animal models to drug discovery, it remains difficult to conduct mechanistic studies on the barrier function and interactions with drugs at molecular and cellular levels. Here we present a microphysiological platform that recapitulates the key structure and function of the human BBB and enables 3D mapping of nanoparticle distributions in the vascular and perivascular regions. We demonstrate on-chip mimicry of the BBB structure and function by cellular interactions, key gene expressions, low permeability, and 3D astrocytic network with reduced reactive gliosis and polarized aquaporin-4 (AQP4) distribution. Moreover, our model precisely captures 3D nanoparticle distributions at cellular levels and demonstrates the distinct cellular uptakes and BBB penetrations through receptor-mediated transcytosis. Our BBB platform may present a complementary in vitro model to animal models for prescreening drug candidates for the treatment of neurological diseases.ope

Body-Shaping Membrane to Regenerate Breast Fat by Elastic Structural Holding

Tissue regeneration requires structural holding and movement support using tissue-type-specific aids such as bone casts, skin bandages, and joint protectors. Currently, an unmet need exists in aiding breast fat regeneration as the breast moves following continuous body motion by exposing the breast fat to dynamic stresses. Here, the concept of elastic structural holding is applied to develop a shape-fitting moldable membrane for breast fat regeneration ("adipoconductive") after surgical defects are made. The membrane has the following key characteristics: (a) It contains a panel of honeycomb structures, thereby efficiently handling motion stress through the entire membrane; (b) a strut is added into each honeycomb in a direction perpendicular to gravity, thereby suppressing the deformation and stress concentration upon lying and standing; and (c) thermo-responsive moldable elastomers are used to support structural holding by suppressing large deviations of movement that occur sporadically. The elastomer became moldable upon a temperature shift above Tm. The structure can then be fixed as the temperature decreases. As result, the membrane promotes adipogenesis by activating mechanotransduction in a fat miniature model with pre-adipocyte spheroids under continuous shaking in vitro and in a subcutaneous implant placed on the motion-prone back areas of rodents in vivo.ope

Hydrogel cross-linking-programmed release of nitric oxide regulates source-dependent angiogenic behaviors of human mesenchymal stem cell

Angiogenesis is stimulated by nitric oxide (NO) production in endothelial cells (ECs). Although proangiogenic actions of human mesenchymal stem cells (hMSCs) have been extensively studied, the mechanistic role of NO in this action remains obscure. Here, we used a gelatin hydrogel that releases NO upon crosslinking by a transglutaminase reaction ("NO gel"). Then, the source-specific behaviors of bone marrow versus adipose tissue-derived hMSCs (BMSCs versus ADSCs) were monitored in the NO gels. NO inhibition resulted in significant decreases in their angiogenic activities. The NO gel induced pericyte-like characteristics in BMSCs in contrast to EC differentiation in ADSCs, as evidenced by tube stabilization versus tube formation, 3D colocalization versus 2D coformation with EC tube networks, pericyte-like wound healing versus EC-like vasculogenesis in gel plugs, and pericyte versus EC marker production. These results provide previously unidentified insights into the effects of NO in regulating hMSC source-specific angiogenic mechanisms and their therapeutic applications. Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).ope

Aging Donor-Derived Human Mesenchymal Stem Cells Exhibit Reduced Reactive Oxygen Species Loads and Increased Differentiation Potential Following Serial Expansion on a PEG-PCL Copolymer Substrate

Human mesenchymal stem cells (hMSCs) have been widely studied for therapeutic development in tissue engineering and regenerative medicine. They can be harvested from human donors via tissue biopsies, such as bone marrow aspiration, and cultured to reach clinically relevant cell numbers. However, an unmet issue lies in the fact that the hMSC donors for regenerative therapies are more likely to be of advanced age. Their stem cells are not as potent compared to those of young donors, and continue to lose healthy, stemness-related activities when the hMSCs are serially passaged in tissue culture plates. Here, we have developed a cheap, scalable, and effective copolymer film to culture hMSCs obtained from aged human donors over several passages without loss of reactive oxygen species (ROS) handling or differentiation capacity. Assays of cell morphology, reactive oxygen species load, and differentiation potential demonstrate the effectiveness of copolymer culture on reduction in senescence-related activities of aging donor-derived hMSCs that could hinder the therapeutic potential of autologous stem cell therapies.ope

Microchannel network hydrogel induced ischemic blood perfusion connection

Angiogenesis induction into damaged sites has long been an unresolved issue. Local treatment with pro-angiogenic molecules has been the most common approach. However, this approach has critical side effects including inflammatory coupling, tumorous vascular activation, and off-target circulation. Here, the concept that a structure can guide desirable biological function is applied to physically engineer three-dimensional channel networks in implant sites, without any therapeutic treatment. Microchannel networks are generated in a gelatin hydrogel to overcome the diffusion limit of nutrients and oxygen three-dimensionally. Hydrogel implantation in mouse and porcine models of hindlimb ischemia rescues severely damaged tissues by the ingrowth of neighboring host vessels with microchannel perfusion. This effect is guided by microchannel size-specific regenerative macrophage polarization with the consequent functional recovery of endothelial cells. Multiple-site implantation reveals hypoxia and neighboring vessels as major causative factors of the beneficial function. This technique may contribute to the development of therapeutics for hypoxia/inflammatory-related diseases.ope

Anti-atherosclerotic vaccination against Porphyromonas gingivalis as a potential comparator of statin in mice

Background: Porphyromonas gingivalis (Pg) is an oral anaerobe which damages teeth and periodontal tissues. Its body infection is known to cause chronic inflammation, thereby inducing an early stage of atherosclerosis through humoral immune actions. Hence, vaccination by immunizing the proteins of P. gingivalis (Pg) post sonication with heating may prevent atherosclerosis. This study aimed to compare the effect of its vaccination with statin, which effectively prevents atherosclerosis by lowering lipids. Methods: The vaccine was produced by sonicating P. gingivalis through heating, and a total of 32 male APOE-/-mice (8-week old) were subjected Western diet for 8 weeks, in order to induce atherosclerosis in a physiological manner. Then, the mice were grouped to undergo four treatment conditions (i.e., no treatment, pitavastatin, vaccine, or pitavastatin with vaccine). Vaccination was conducted through nasal immunization and confirmed by a Pg-specific humoral immune reaction. Then, half of the mice in each group were orally injected with P. gingivalis for the next 5 weeks while the other half remained uninfected, generating a total of eight groups (n = 4/group). The mice were sacrificed at 3 weeks after the last injection. After harvesting the aorta, Oil Red O staining of en face was conducted with imaging and image analysis, and plaque formation was quantitatively determined. Results: Compared to no treatment, the vaccination through nasal immunization significantly reduced the atherosclerotic plaque sizes in APOE -/- mice under Western diet to the comparable level of statin group. When both vaccine and statin were used, no clear synergistic effect was observed as opposed to expectation. Conclusions: This study revealed that nasal immunization of heat shock P. gingivalis has a significant impact on the prevention of arteriosclerosis and acts as a potential comparator of statin.ope

Cationic Nanocylinders Promote Angiogenic Activities of Endothelial Cells

Polymers have been used extensively taking forms as scaffolds, patterned surface and nanoparticle for regenerative medicine applications. Angiogenesis is an essential process for successful tissue regeneration, and endothelial cell?cell interaction plays a pivotal role in regulating their tight junction formation, a hallmark of angiogenesis. Though continuous progress has been made, strategies to promote angiogenesis still rely on small molecule delivery or nuanced scaffold fabrication. As such, the recent paradigm shift from top-down to bottom-up approaches in tissue engineering necessitates development of polymer-based modular engineering tools to control angiogenesis. Here, we developed cationic nanocylinders (NCs) as inducers of cell?cell interaction and investigated their effect on angiogenic activities of human umbilical vein endothelial cells (HUVECs) in vitro. Electrospun poly (l-lactic acid) (PLLA) fibers were aminolyzed to generate positively charged NCs. The aninolyzation time was changed to produce two different aspect ratios of NCs. When HUVECs were treated with NCs, the electrostatic interaction of cationic NCs with negatively charged plasma membranes promoted migration, permeability and tubulogenesis of HUVECs compared to no treatment. This effect was more profound when the higher aspect ratio NC was used. The results indicate these NCs can be used as a new tool for the bottom-up approach to promote angiogenesis.ope

Angiogenic and Osteogenic Synergy of Human Mesenchymal Stem Cells and Human Umbilical Vein Endothelial Cells Cocultured on a Nanomatrix

To date, bone tissue regeneration strategies lack an approach that effectively provides an osteogenic and angiogenic environment conducive to bone growth. In the current study, we evaluated the osteogenic and angiogenic response of human mesenchymal stem cells (hMSCs) and green fluorescent protein-expressing human umbilical vein endothelial cells (GFP-HUVECs) cocultured on a self-assembled, peptide amphiphile nanomatrix functionalized with the cell adhesive ligand RGDS (PA-RGDS). Analysis of alkaline phosphatase activity, von Kossa staining, Alizarin Red quantification, and osteogenic gene expression, indicates a significant synergistic effect between the PA-RGDS nanomatrix and coculture that promoted hMSC osteogenesis. In addition, coculturing on PA-RGDS resulted in enhanced HUVEC network formation and upregulated vascular endothelial growth factor gene and protein expression. Though PA-RGDS and coculturing hMSCs with HUVECs were each previously reported to individually enhance hMSC osteogenesis, this study is the first to demonstrate a synergistic promotion of HUVEC angiogenesis and hMSC osteogenesis by integrating coculturing with the PA-RGDS nanomatrix. We believe that using the combination of hMSC/HUVEC coculture and PA-RGDS substrate is an efficient method for promoting osteogenesis and angiogenesis, which has immense potential as an efficacious, engineered platform for bone tissue regeneration.ope

Development of Poly(ɛ-caprolactone) Scaffold Loaded with Simvastatin and Beta-Cyclodextrin Modified Hydroxyapatite Inclusion Complex for Bone Tissue Engineering

In this study, we developed poly(?-caprolactone) (PCL) 3D scaffolds using a solid free form fabrication (SFF) technique. β-cyclodextrin (βCD) was grafted to hydroxyapatite (HAp) and this βCD grafted HAp was coated onto the PCL scaffold surface, followed by drug loading through an inclusion complex interaction between the βCD and adamantane (AD) or between βCD and simvastatin (SIM). The scaffold structure was characterized by scanning electron microscopy (SEM). The release profile of simvastatin in the β-CD grafted HAp was also evaluated. Osteogenic differentiation of adipose-derived stromal cells (ADSCs) was examined using an alkaline phosphatase activity (ALP) assay. The results suggest that drug loaded PCL-HAp 3-D scaffolds enhances osteogenic differentiation of ADSCs.ope

Tunable Surface Repellency Maintains Stemness and Redox Capacity of Human Mesenchymal Stem Cells

Human bone marrow derived mesenchymal stem cells (hMSCs) hold great promise for regenerative medicine due to their multipotent differentiation capacity and immunomodulatory capabilities. Substantial research has elucidated mechanisms by which extracellular cues regulate hMSC fate decisions, but considerably less work has addressed how material properties can be leveraged to maintain undifferentiated stem cells. Here, we show that synthetic culture substrates designed to exhibit moderate cell-repellency promote high stemness and low oxidative stress-two indicators of naïve, healthy stem cells-in commercial and patient-derived hMSCs. Furthermore, the material-mediated effect on cell behavior can be tuned by altering the molar percentage (mol %) and/or chain length of poly(ethylene glycol) (PEG), the repellant block linked to hydrophobic poly(ε-caprolactone) (PCL) in the copolymer backbone. Nano- and angstrom-scale characterization of the cell-material interface reveals that PEG interrupts the adhesive PCL domains in a chain-length-dependent manner; this prevents hMSCs from forming mature focal adhesions and subsequently promotes cell-cell adhesions that require connexin-43. This study is the first to demonstrate that intrinsic properties of synthetic materials can be tuned to regulate the stemness and redox capacity of hMSCs and provides new insight for designing highly scalable, programmable culture platforms for clinical translation.ope