O2 variant chip to simulate site-specific skeletogenesis from hypoxic bone marrow.

The stemness of bone marrow mesenchymal stem cells (BMSCs) is maintained by hypoxia. The oxygen level increases from vessel-free cartilage to hypoxic bone marrow and, furthermore, to vascularized bone, which might direct the chondrogenesis to osteogenesis and regenerate the skeletal system. Hence, oxygen was diffused from relatively low to high levels throughout a three-dimensional chip. When we cultured BMSCs in the chip and implanted them into the rabbit defect models of low-oxygen cartilage and high-oxygen calvaria bone, (i) the low oxygen level (base) promoted stemness and chondrogenesis of BMSCs with robust antioxidative potential; (ii) the middle level (two times ≥ low) pushed BMSCs to quiescence; and (iii) the high level (four times ≥ low) promoted osteogenesis by disturbing the redox balance and stemness. Last, endochondral or intramembranous osteogenesis upon transition from low to high oxygen in vivo suggests a developmental mechanism-driven solution to promote chondrogenesis to osteogenesis in the skeletal system by regulating the oxygen environment.

Tissue Niche Miniature of Glioblastoma Patient Treated with Nano-Awakeners to Induce Suicide of Cancer Stem Cells.

Patient-specific cancer therapies can evolve by vitalizing the mother tissue-like cancer niche, cellular profile, genetic signature, and drug responsiveness. This evolution has enabled the elucidation of a key mechanism along with development of the mechanism-driven therapy. After surgical treatment, glioblastoma (GBM) patients require prompt therapy within 14 days in a patient-specific manner. Hence, this study approaches direct culture of GBM patient tissue (1 mm diameter) in a microchannel network chip. Cancer vasculature-mimetic perfusion can support the preservation of the mother tissue-like characteristic signatures and microenvironment. When temozolomide and radiation are administered within 1 day, the responsiveness of the tissue in the chip reflected the clinical outcomes, thereby overcoming the time-consuming process of cell and organoid culture. When the tissue chip culture is continued, the intact GBM signature gets lost, and the outward migration of stem cells from the tissue origin increases, indicating a leaving-home effect on the family dismantle. Nanovesicle production using GBM stem cells enables self-chasing of the cells that escape the temozolomide effect owing to quiescence. The anti-PTPRZ1 peptide display and temozolomide loading to nanovesicles awakes cancer stem cells from the quiescent stage to death. This study suggests a GBM clinic-driven avatar platform and mechanism-learned nanotherapy for translation.

Sprayable nanomicelle hydrogels and inflammatory bowel disease patient cell chips for development of intestinal lesion-specific therapy.

All-in-one treatments represent a paradigm shift in future medicine. For example, inflammatory bowel disease (IBD) is mainly diagnosed by endoscopy, which could be applied for not only on-site monitoring but also the intestinal lesion-targeted spray of injectable hydrogels. Furthermore, molecular conjugation to the hydrogels would program both lesion-specific adhesion and drug-free therapy. This study validated this concept of all-in-one treatment by first utilizing a well-known injectable hydrogel that underwent efficient solution-to-gel transition and nanomicelle formation as a translatable component. These properties enabled spraying of the hydrogel onto the intestinal walls during endoscopy. Next, peptide conjugation to the hydrogel guided endoscopic monitoring of IBD progress upon adhesive gelation with subsequent moisturization of inflammatory lesions, specifically by nanomicelles. The peptide was designed to mimic the major component that mediates intestinal interaction with Bacillus subtilis flagellin during IBD initiation. Hence, the peptide-guided efficient adhesion of the hydrogel nanomicelles onto Toll-like receptor 5 (TLR5) as the main target of flagellin binding and Notch-1. The peptide binding potently suppressed inflammatory signaling without drug loading, where TLR5 and Notch-1 operated collaboratively through downstream actions of tumor necrosis factor-alpha. The results were produced using a human colorectal cell line, clinical IBD patient cells, gut-on-a-chip, a mouse IBD model, and pig experiments to validate the translational utility.

Quenching Epigenetic Drug Resistance Using Antihypoxic Microparticles in Glioblastoma Patient-Derived Chips.

Glioblastoma (GBM) is one of the most intractable tumor types due to the progressive drug resistance upon tumor mass expansion. Incremental hypoxia inside the growing tumor mass drives epigenetic drug resistance by activating nongenetic repair of antiapoptotic DNA, which could be impaired by drug treatment. Hence, rescuing intertumor hypoxia by oxygen-generating microparticles may promote susceptibility to antitumor drugs. Moreover, a tumor-on-a-chip model enables user-specified alternation of clinic-derived samples. This study utilizes patient-derived glioblastoma tissue to generate cell spheroids with size variations in a 3D microchannel network chip (GBM chip). As the spheroid size increases, epigenetic drug resistance is promoted with inward hypoxia severance, as supported by the spheroid size-proportional expression of hypoxia-inducible factor-1a in the chip. Loading antihypoxia microparticles onto the spheroid surface significantly reduces drug resistance by silencing the expression of critical epigenetic factor, resulting in significantly decreased cell invasiveness. The results are confirmed in vitro using cell line and patient samples in the chip as well as chip implantation into a hypoxic hindlimb ischemia model in mice, which is an unprecedented approach in the field.

Cancer Patient Tissueoid with Self-Homing Nano-Targeting of Metabolic Inhibitor.

The current paradigm of cancer medicine focuses on patient- and/or cancer-specific treatments, which has led to continuous progress in the development of patient representatives (e.g., organoids) and cancer-targeting carriers for drug screening. As breakthrough concepts, i) living cancer tissues convey intact profiles of patient-specific microenvironmental signatures. ii) The growth mechanisms of cancer mass with intense cell-cell interactions can be harnessed to develop self-homing nano-targeting by using cancer cell-derived nanovesicles (CaNVs). Hence, a tissueoid model of ovarian cancer (OC) is developed by culturing OC patient tissues in a 3D gel chip, whose microchannel networks enable perfusion to maintain tissue viability. A novel model of systemic cancer responses is approached by xenografting OC tissueoids into ischaemic hindlimbs in nude mice. CaNVs are produced to carry general chemotherapeutics or new drugs under pre/clinical studies that target the BRCA mutation or energy metabolism, thereby increasing the test scope. This pioneer study cross-validates drug responses from the OC clinic, tissueoid, and animal model by demonstrating the alignment of results in drug type-specific efficiency, BRCA mutation-dependent drug efficiency, and metabolism inhibition-based anti-cancer effects. Hence, this study provides a directional foundation to accelerate the discovery of patient-specific drugs with CaNV application towards future precision medicine.

Cell-Membrane-Derived Nanoparticles with Notch-1 Suppressor Delivery Promote Hypoxic Cell-Cell Packing and Inhibit Angiogenesis Acting as a Two-Edged Sword.

Cell-cell interactions regulate intracellular signaling via reciprocal contacts of cell membranes in tissue regeneration and cancer growth, indicating a critical need of membrane-derived tools in studying these processes. Hence, cell-membrane-derived nanoparticles (CMNPs) are produced using tonsil-derived mesenchymal stem cells (TMSCs) from children owing to their short doubling time. As target cell types, laryngeal cancer cells are compared to bone-marrow-derived MSCs (BMSCs) because of their cartilage damaging and chondrogenic characteristics, respectively. Treating spheroids of these cell types with CMNPs exacerbates interspheroid hypoxia with robust maintenance of the cell-cell interaction signature for 7 days. Both cell types prefer a hypoxic environment, as opposed to blood vessel formation that is absent in cartilage but is required for cancer growth. Hence, angiogenesis is inhibited by displaying the Notch-1 aptamer on CMNPs. Consequently, laryngeal cancer growth is suppressed efficiently in contrast to improved chondroprotection observed in a series of cell and animal experiments using a xenograft mouse model of laryngeal cancer. Altogether, CMNPs execute a two-edged sword function of inducing hypoxic cell-cell packing, followed by suppressing angiogenesis to promote laryngeal cancer death and chondrogenesis simultaneously. This study presents a previously unexplored therapeutic strategy for anti-cancer and chondroprotective treatment using CMNPs.

Hormone autocrination by vascularized hydrogel delivery of ovary spheroids to rescue ovarian dysfunctions.

The regeneration potential of implantable organ model hydrogels is applied to treat a loss of ovarian endocrine function in women experiencing menopause and/or cancer therapy. A rat ovariectomy model is used to harvest autologous ovary cells while subsequently producing a layer-by-layer form of follicle spheroids. Implantation of a microchannel network hydrogel with cell spheroids [vascularized hydrogel with ovarian spheroids (VHOS)] into an ischemic hindlimb of ovariectomized rats significantly aids the recovery of endocrine function with hormone release, leading to full endometrium regeneration. The VHOS implantation effectively suppresses the side effects observed with synthetic hormone treatment (i.e., tissue overgrowth, hyperplasia, cancer progression, deep vein thrombosis) to the normal levels, while effectively preventing the representative aftereffects of menopause (i.e., gaining fatty weight, inducing osteoporosis). These results highlight the unprecedented therapeutic potential of an implantable VHOS against menopause and suggest that it may be used as an alternative approach to standard hormone therapy.

Immunomodulatory Scaffolds Derived from Lymph Node Extracellular Matrices.

Immunomodulation in the local tissue microenvironment is pivotal for the determination of macrophage phenotypes and regulation of functions necessary for pro-healing effects. Herein, we demonstrate that a lymph node extracellular matrix (LNEM) prepared by the decellularization of lymph node tissues can mimic lymph node microenvironments for immunomodulation in two-dimensional (2D) and three-dimensional (3D) formats. The LNEM exhibits strengthened immunomodulatory effects in comparison to conventional collagen-based platforms. A 3D LNEM hydrogel is more effective than the 2D LNEM coating in inducing M2 macrophage polarization. The 3D LNEM induces macrophage elongation and enhances the M2-type marker expression and the secretion of anti-inflammatory cytokines. Additionally, the phagocytic function of macrophages is improved upon exposure to the intricate 3D LNEM environment. We demonstrate the reduced susceptibility of liver organoids to a hepatotoxic drug when co-cultured with macrophages in a 3D LNEM. This effect could be attributed to the enhanced anti-inflammatory functions and indicates its potential as a drug-testing platform that enables drug responses similar to those observed in vivo. Finally, the implantation of an LNEM hydrogel in a mouse volumetric muscle loss model facilitates the recruitment of host macrophages to the site of injury and enhances macrophage polarization toward the M2 phenotype for tissue healing in vivo. Therefore, 3D immune system-mimicking biomaterials could serve as useful platforms for tissue modeling and regenerative medicine development.

Serum glucose excretion after Roux-en-Y gastric bypass: a potential target for diabetes treatment.

OBJECTIVE: The mechanisms underlying type 2 diabetes resolution after Roux-en-Y gastric bypass (RYGB) are unclear. We suspected that glucose excretion may occur in the small bowel based on observations in humans. The aim of this study was to evaluate the mechanisms underlying serum glucose excretion in the small intestine and its contribution to glucose homeostasis after bariatric surgery. DESIGN: 2-Deoxy-2-[18F]-fluoro-D-glucose (FDG) was measured in RYGB-operated or sham-operated obese diabetic rats. Altered glucose metabolism was targeted and RNA sequencing was performed in areas of high or low FDG uptake in the ileum or common limb. Intestinal glucose metabolism and excretion were confirmed using 14C-glucose and FDG. Increased glucose metabolism was evaluated in IEC-18 cells and mouse intestinal organoids. Obese or ob/ob mice were treated with amphiregulin (AREG) to correlate intestinal glycolysis changes with changes in serum glucose homeostasis. RESULTS: The AREG/EGFR/mTOR/AKT/GLUT1 signal transduction pathway was activated in areas of increased glycolysis and intestinal glucose excretion in RYGB-operated rats. Intraluminal GLUT1 inhibitor administration offset improved glucose homeostasis in RYGB-operated rats. AREG-induced signal transduction pathway was confirmed using IEC-18 cells and mouse organoids, resulting in a greater capacity for glucose uptake via GLUT1 overexpression and sequestration in apical and basolateral membranes. Systemic and local AREG administration increased GLUT1 expression and small intestinal membrane translocation and prevented hyperglycaemic exacerbation. CONCLUSION: Bariatric surgery or AREG administration induces apical and basolateral membrane GLUT1 expression in the small intestinal enterocytes, resulting in increased serum glucose excretion in the gut lumen. Our findings suggest a novel, potentially targetable glucose homeostatic mechanism in the small intestine.

Anti-Atherogenic Effect of Stem Cell Nanovesicles Targeting Disturbed Flow Sites.

Atherosclerosis development leads to irreversible cascades, highlighting the unmet need for improved methods of early diagnosis and prevention. Disturbed flow formation is one of the earliest atherogenic events, resulting in increased endothelial permeability and subsequent monocyte recruitment. Here, a mesenchymal stem cell (MSC)-derived nanovesicle (NV) that can target disturbed flow sites with the peptide GSPREYTSYMPH (PREY) (PMSC-NVs) is presented which is selected through phage display screening of a hundred million peptides. The PMSC-NVs are effectively produced from human MSCs (hMSCs) using plasmid DNA designed to functionalize the cell membrane with PREY. The potent anti-inflammatory and pro-endothelial recovery effects are confirmed, similar to those of hMSCs, employing mouse and porcine partial carotid artery ligation models as well as a microfluidic disturbed flow model with human carotid artery-derived endothelial cells. This nanoscale platform is expected to contribute to the development of new theragnostic strategies for preventing the progression of atherosclerosis.

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.

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.

Three-Dimensionally Printed Breast Reconstruction Devices Facilitate Nanostructure Surface-Guided Healthy Lipogenesis.

Significant fat loss is common in silicon implantation with autologous lipofilling, the most popular type of breast surgery. To overcome this, a 3D-printed fat carrier with well-defined 200 µm radial string and spoke structure was developed, followed by an electrospun nanofiber coating on the entire device surface to promote fat adhesion. This device enhanced the mechanical properties comparably to commercial acellular dermal matrix for in vitro adipogenic differentiation of adipose-derived stem cells, implantation compatibility without foreign body responses, and maintenance of healthy lipid droplet structures. These results show the promising potential of this device to facilitate surface-guided lipogenesis in composite breast reconstruction surgery.

Spatial Organization of Superparamagnetic Iron Oxide Nanoparticles in/on Nano/Microsized Carriers Modulates the Magnetic Resonance Signal.

Superparamagnetic iron oxide nanoparticles (SPIONs) are often encapsulated into drug-carrying nano/microsized particles for simultaneous magnetic resonance (MR) imaging and treatment of diseased tissues. Unfortunately, encapsulated SPIONs may have a limited ability to modulate the T2-weighted relaxation of water protons, but this insight has not been examined systematically. This study demonstrates that SPIONs immobilized on 200 nm diameter poly(lactic- co-glycolic acid) (PLGA) nanoparticles using Pickering emulsification present 18-fold higher relaxivity than encapsulated SPIONs and 1.5-fold higher relaxivity than free SPIONs. In contrast, the SPIONs immobilized on 10 µm diameter PLGA particles exhibit a minor increase in MR relaxivity. This interesting finding will significantly impact current efforts to synthesize and assemble advanced MR contrast agents.

Porcine As a Training Module for Head and Neck Microvascular Reconstruction.

Live models that resemble surgical conditions of humans are needed for training free-flap harvesting and anastomosis. Animal models for training purposes have been available for years in many surgical fields. We used the female (because they are easy to handle for the procedure) Yorkshire pigs for the head and neck reconstruction by harvesting the deep inferior epigastric artery perforator or the superior epigastric artery perforator flap. The anastomosis site (neck skin defect or tracheal wall defect) was prepared via the dissection of the common carotid artery and the internal jugular vein, in which 3.5× loupe magnification was used for anastomosis as we use on human cases in real life. This procedure demonstrates a new training method using a reliable learning model and provides a detailed anatomy in a live scenario. We focused on the ischemia time, harvesting, vessel anastomosis, and designing the flap to fit the defect site. This model improves tissue handling and with the use of proper instruments can be repeated many times so that the surgeon is fully confident before starting the surgery on humans.

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.

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.

Association Between Impairment of DNA Double Strand Break Repair and Decreased Ovarian Reserve in Patients With Endometriosis.

Background: Repair of DNA double strand break (DSB) is an important mechanism for maintaining genetic stability during a DNA damage event. Although, a growing body of recent evidence suggests that DNA DSBs and related repair mechanisms may be important in ovarian aging and in various cancers, there are few reports in endometriosis. We, therefore, examined expression levels of genes pertaining to DNA DSB repair in patients with endometriosis to assess the potential effects on ovarian reserves. Materials and methods: A total of 69 women undergoing laparoscopic surgery for endometriosis and other benign conditions was included; endometriosis group (n = 38) vs. controls (n = 31). DNA DSBs in endometrial and ovarian tissues of both groups were compared via immunohistochemistry, aimed at γ-H2AX expression. To gauge genotoxin-induced DNA DSBs in endometrial stromal cells, γ-H2AX expression was determined by western blot after H2O2 treatment of cultured endometrial stromal cells (endometriosis group and controls) and Ishikawa cell-line cultures. Endometrial and ovarian tissue levels of BRCA1, BRCA2, Rad51, and ATM (ataxia-telangiectasia mutated) mRNA expression were also compared. Correlations between expression levels of genes of interest and serum anti-müllerian hormone (AMH) levels were assessed as well. Results: Expression of γ-H2AX in immunostained endometrial and ovarian tissue preparations was greater in the endometriosis group, compared with controls. After H2O2 treatment, γ-H2AX expression levels were also significantly greater in cultured stromal cells of the endometriosis group and in the Ishikawa cell line than in controls. Endometrial expression of BRCA1 and Rad51 mRNA proved significantly lower in the endometriosis group (vs. controls), as did ovarian expression of BRCA1 and BRCA2 mRNA. Serum AMH concentration showed a significant correlation with ovarian BRCA1 mRNA expression in women with endometriosis (p = 0.03). Conclusions: In women with endometriosis, expression levels of various genes implicated in DSB repair are decreased and ovarian BRCA1 expression correlates with.

Heparin-functionalized polymer graft surface eluting MK2 inhibitory peptide to improve hemocompatibility and anti-neointimal activity.

The leading cause of synthetic graft failure includes thrombotic occlusion and intimal hyperplasia at the site of vascular anastomosis. Herein, we report a co-immobilization strategy of heparin and potent anti-neointimal drug (Mitogen Activated Protein Kinase II inhibitory peptide; MK2i) by using a tyrosinase-catalyzed oxidative reaction for preventing thrombotic occlusion and neointimal formation of synthetic vascular grafts. The binding of heparin-tyramine polymer (HT) onto the polycarprolactone (PCL) surface enhanced blood compatibility with significantly reduced protein absorption (64.7% decrease) and platelet adhesion (85.6% decrease) compared to bare PCL surface. When loading MK2i, 1) the HT depot surface gained high MK2i-loading efficiency through charge-charge interaction, and 2) this depot platform enabled long-term, controlled release over 4weeks (92-272µg/mL of MK2i). The released MK2i showed significant inhibitory effects on VSMC migration through down-regulated phosphorylation of target proteins (HSP27 and CREB) associated with intimal hyperplasia. In addition, it was found that the released MK2i infiltrated into the tissue with a cumulative manner in ex vivo human saphenous vein (HSV) model. This present study demonstrates that enzymatically HT-coated surface modification is an effective strategy to induce long-term MK2i release as well as hemocompatibility, thereby improving anti-neointimal activity of synthetic vascular grafts.