3D printed multi-growth factor delivery patches fabricated using dual-crosslinked decellularized extracellular matrix-based hybrid inks to promote cerebral angiogenesis.

Generally, brain angiogenesis is a tightly regulated process, which scarcely occurred in the absence of specific pathological conditions. Delivery of exogenous angiogenic factors enables the induction of desired angiogenesis by stimulating neovasculature formation. However, effective strategies of mimicking the angiogenesis process with exogenous factors have not yet been fully explored. Herein, we develop a 3D printed spatiotemporally compartmentalized cerebral angiogenesis inducing (SCAI) hydrogel patch, releasing dual angiogenic growth factors (GFs), using extracellular matrix-based hybrid inks. We introduce a new hybrid biomaterial-based ink for printing patches through dual crosslinking mechanisms: Chemical crosslinking with aza-Michael addition reaction with combining methacrylated hyaluronic acid (HAMA) and vascular-tissue-derived decellularized extracellular matrix (VdECM), and thermal crosslinking of VdECM. 3D printing technology, a useful approach with fabrication versatility with customizable systems and multiple biomaterials, is adopted to print three-layered hydrogel patch with spatially separated dual GFs as outer- and inner-layers that provide tunable release profiles of multiple GFs and fabrication versatility. Consequently, these layers of the patch spatiotemporally separated with dual GFs induce excellent neovascularization in the brain area, monitored by label-free photoacoustic microscopy in vivo. The developed multi-GFs releasing patch may offer a promising therapeutic approach of spatiotemporal drugs releasing such as cerebral ischemia, ischemic heart diseases, diabetes, and even use as vaccines. STATEMENT OF SIGNIFICANCE: Effective strategies of mimicking the angiogenesis process with exogenous factors have not yet been fully explored. In this study, we develop a 3D printed spatiotemporally compartmentalized cerebral angiogenesis inducing (SCAI) hydrogel patch, releasing dual angiogenic growth factors (GFs) using extracellular matrix-based hybrid inks. We introduce a new hybrid biomaterial-based ink through dual crosslinking mechanisms: Chemical crosslinking with aza-Michael addition, and thermal crosslinking. 3D printing technology is adopted to print three-layered hydrogel patch with spatially separated dual GFs as outer- and inner-layers that provide tunable release profiles of multiple GFs and fabrication versatility. Consequently, these layers of the patch spatiotemporally separated with dual GFs induce excellent neovascularization in the brain area, monitored by photoacoustic microscopy in vivo.

Tumor Spheroids of an Aggressive Form of Central Neurocytoma Have Transit-Amplifying Progenitor Characteristics with Enhanced EGFR and Tumor Stem Cell Signaling.

Central neurocytoma (CN) has been known as a benign neuronal tumor. In rare cases, CN undergoes malignant transformation to glioblastomas (GBM). Here we examined its cellular origin by characterizing differentiation potential and gene expression of CN-spheroids. First, we demonstrate that both CN tissue and cultured primary cells recapitulate the hierarchal cellular composition of subventricular zone (SVZ), which is comprised of neural stem cells (NSCs), transit amplifying progenitors (TAPs), and neuroblasts. We then derived spheroids from CN which displayed EGFR+/ MASH+ TAP and BLBP+ radial glial cell (RGC) characteristic, and mitotic neurogenesis and gliogenesis by single spheroids were observed with cycling multipotential cells. CN-spheroids expressed increased levels of pluripotency and tumor stem cell genes such as KLF4 and TPD5L1, when compared to their differentiated cells and human NSCs. Importantly, Gene Set Enrichment Analysis showed that gene sets of GBM-Spheroids, EGFR Signaling, and Packaging of Telomere Ends are enriched in CN-spheroids in comparison with their differentiated cells. We speculate that CN tumor stem cells have TAP and RGC characteristics, and upregulation of EGFR signaling as well as downregulation of eph-ephrin signaling have critical roles in tumorigenesis of CN. And their ephemeral nature of TAPs destined to neuroblasts, might reflect benign nature of CN.

Inhibition of HIF1α and PDK Induces Cell Death of Glioblastoma Multiforme.

Glioblastoma multiforme (GBM) is the most common and aggressive form of brain tumors. GBMs, like other tumors, rely relatively less on mitochondrial oxidative phosphorylation (OXPHOS) and utilize more aerobic glycolysis, and this metabolic shift becomes augmented under hypoxia. In the present study, we investigated the physiological significance of altered glucose metabolism and hypoxic adaptation in the GBM cell line U251 and two newly established primary GBMs (GBM28 and GBM37). We found that these three GBMs exhibited differential growth rates under hypoxia compared to those under normoxia. Under normoxia, the basal expressions of HIF1α and the glycolysis-associated genes, PDK1, PDK3, and GLUT1, were relatively low in U251 and GBM28, while their basal expressions were high in GBM37. Under hypoxia, the expressions of these genes were enhanced further in all three GBMs. Treatment with dichloroacetate (DCA), an inhibitor of pyruvate dehydrogenase kinase (PDK), induced cell death in GBM28 and GBM37 maintained under normoxia, whereas DCA effects disappeared under hypoxia, suggesting that hypoxic adaptation dominated DCA effects in these GBMs. In contrast, the inhibition of HIF1α with chrysin suppressed the expression of PDK1, PDK3, and GLUT1 and markedly promoted cell death of all GBMs under both normoxia and hypoxia. Interestingly, however, GBMs treated with chrysin under hypoxia still sustained higher viability than those under normoxia, and chrysin and DCA co-treatment was unable to eliminate this hypoxia-dependent resistance. Together, these results suggest that hypoxic adaptation is critical for maintaining viability of GBMs, and targeting hypoxic adaptation can be an important treatment option for GBMs.

Fully Implantable Deep Brain Stimulation System with Wireless Power Transmission for Long-term Use in Rodent Models of Parkinson's Disease.

OBJECTIVE: The purpose of this study to develop new deep-brain stimulation system for long-term use in animals, in order to develop a variety of neural prostheses. METHODS: Our system has two distinguished features, which are the fully implanted system having wearable wireless power transfer and ability to change the parameter of stimulus parameter. It is useful for obtaining a variety of data from a long-term experiment. RESULTS: To validate our system, we performed pre-clinical test in Parkinson's disease-rat models for 4 weeks. Through the in vivo test, we observed the possibility of not only long-term implantation and stability, but also free movement of animals. We confirmed that the electrical stimulation neither caused any side effect nor damaged the electrodes. CONCLUSION: We proved possibility of our system to conduct the long-term pre-clinical test in variety of parameter, which is available for development of neural prostheses.