Ting-Chen Tseng

An Injectable, Self‐Healing Hydrogel to Repair the Central Nervous System

An injectable, self-healing hydrogel (≈1.5 kPa) is developed for healing nerve-system deficits. Neurosphere-like progenitors proliferate in the hydrogel and differentiate into neuron-like cells. In the zebrafish injury model, the central nervous system function is partially rescued by injection of the hydrogel and significantly rescued by injection of the neurosphere-laden hydrogel. The self-healing hydrogel may thus potentially repair the central nervous system.

Nanoparticle uptake and gene transfer efficiency for MSCs on chitosan and chitosan-hyaluronan substrates.

Nanoparticles (NPs) are usually surface modified to increase endocytosis for applications in cellular imaging and gene delivery. The influence of cell culture substrates on endocytosis remains relatively unexplored. This study investigated the substrate-mediated effects on the uptake of NPs by mesenchymal stem cells (MSCs). Two types of NPs were employed, negatively charged paramagnetic iron oxide (Fe(3)O(4)) NPs (~5 nm) and bare plasmid DNA pTRE-Tight-DsRED2 (3.3 kb, ~5 nm), each of which were poorly endocytosed by the adipose-derived MSCs grown on tissue culture polystyrene (TCPS). When cells were cultured on chitosan or hyaluronan-modified chitosan (chitosan-HA) membranes, significant increases (>5-fold) in the intracellular uptake of Fe(3)O(4) NPs as well as transfectability of plasmid DNA were demonstrated. The enhancement in transgene expression was more pronounced than that using the transfection agent. The beneficial effects were not caused by elevated proliferation or a change in the differentiation state of interacting MSCs. On chitosan and chitosan-HA, cells moved fast and formed spheroids. The cytoskeletal arrangement associated with the up-regulated RhoA activity during spheroid formation may have accounted for the increased endocytosis. Using different inhibitors, the endocytosis pathways were further clarified. Both Fe(3)O(4) NPs and plasmid DNA were taken up primarily by clathrin-mediated endocytosis on chitosan (~50%). The caveolae-mediated endocytosis on chitosan-HA was more evident (~30-40%) than that on chitosan (<25%). For plasmid DNA but not Fe(3)O(4) NPs, macropinocytosis also occurred on both substrates. Chitosan and chitosan-HA as cell culture substrates may activate different endocytic pathways of MSCs to increase NP internalization or plasmid transfection. The substrate-mediated endocytosis described here may represent a new and potentially attractive approach to facilitate stem cell labeling or to improve gene delivery efficiency without altering cell viability and differentiation.

Substrate-mediated nanoparticle/gene delivery to MSC spheroids and their applications in peripheral nerve regeneration

Substrate-derived mesenchymal stem cell (MSC) spheroids show greater differentiation capacities than dispersed single cells in vitro. During spheroid formation, nanoparticles (NPs)/genes may be delivered into the cells. In this study, MSCs were conveniently labeled with superparamagnetic Fe3O4 NPs, or transfected with brain-derived neurotrophic factor (BDNF) gene, by the substrate-mediated NP/gene uptake. With the promising in vitro data showing the beneficial effect on neural development and neurotrophic factor expression, MSCs were combined with a polymeric nerve conduit to bridge a 10 mm transection gap of rat sciatic nerve. High-resolution (7-T) magnetic resonance imaging (MRI) was used to track the transplanted cells. Nerve regeneration was assessed by functional recovery and histology. Results revealed that Fe3O4 NP-labeled MSCs were successfully visualized by MRI in vivo. Animals receiving BDNF-transfected MSC spheroids demonstrated the shortest gap bridging time (<21 days), the largest regenerated nerve, and the thickest myelin sheath at 31 days. Compared to MSC single cells, the pristine or BDNF-transfected MSC spheroids significantly promoted the functional recovery of animals, especially for the BDNF-transfected MSC spheroids. The transplanted MSCs were incorporated in the regenerated nerve and differentiated into non-myelinating Schwann cells after 31 days. This study suggests that the substrate-mediated gene delivery and NP labeling may provide extra values for MSC spheroids to carry therapeutic/diagnostic agents in cell-based therapy.

Fabrication of bioactive conduits containing the fibroblast growth factor 1 and neural stem cells for peripheral nerve regeneration across a 15?mm critical gap

Nerve conduits are often used in combination with bioactive molecules and stem cells to enhance peripheral nerve regeneration. In this study, the acidic fibroblast growth factor 1 (FGF1) was immobilized onto the microporous/micropatterned poly (D, L-lactic acid) (PLA) nerve conduits after open air plasma treatment. PLA substrates grafted with chitosan in the presence of a small amount of gold nanoparticles (nano Au) showed a protective effect on the activity of the immobilized FGF1 in vitro. Different conduits were tested for their ability to bridge a 15 mm critical gap defect in a rat sciatic nerve injury model. Axon regeneration and functional recovery were evaluated by histology, walking track analysis and electrophysiology. Among different conduits, PLA conduits grafted with chitosan-nano Au and the FGF1 after plasma activation had the greatest regeneration capacity and functional recovery in the experimental animals. When the above conduit was seeded with aligned neural stem cells, the efficacy was further enhanced and it approached that of the autograft group. This work suggested that microporous/micropatterned nerve conduits containing bioactive growth factors may be successfully fabricated by micropatterning techniques, open plasma activation, and immobilization, which, combined with aligned stem cells, may synergistically contribute to the regeneration of the severely damaged peripheral nerve.

Self-healing hydrogel for tissue repair in the central nervous system

Neurological disorders are diseases of the central and peripheral nervous systems. These disorders include Alzheimers disease, epilepsy, brain tumor, and cerebrovascular diseases (stroke, migraine and other headache disorders, multiple sclerosis, Parkinsons disease, and neuroinfections). Hundreds of millions of people worldwide are affected by neurological disorders. Approximately 6.2 million people die because of stroke each year; over 80% of deaths take place in low- and middle-income countries. More than 50 million people worldwide have epilepsy. It is estimated that there are globally 35.6 million people with dementia with 7.7 million new cases every year. Alzheimers disease is the most common cause of dementia and may contribute to 60–70% of cases. The prevalence of migraine is more than 10% worldwide. Therefore, repairing the damaged nervous system is one of the greatest challenges in medicine. Damage to the peripheral nervous system (PNS) can lead to the loss of sensation, motor function, and muscle weakness, but the PNS is capable of significant spontaneous regeneration and in many cases some function can be restored. In contrast, neuronal regeneration following damage to the central nervous system (CNS) is generally unsuccessful and the injuries of CNS can cause permanent paralysis and loss of sensation. Therefore, CNS injuries present significant therapeutic challenges. Current clinical options for CNS disorder treatment, including drug delivery and rehabilitation therapy, are limited and these treatment options do not fully restore the original functions. Recently, cellular therapy has emerged as a treatment option for repair and regeneration of nerve injuries. However, transplantation of stem cells to the injured sites showed poor cell survival and engraftment (Wu et al., 2011). In this regard, the combination of functional biomaterials and cell delivery is a favorable and promising strategy for CNS repair.

Substrate-mediated reprogramming of human fibroblasts into neural crest stem-like cells and their applications in neural repair

Cell- and gene-based therapies have emerged as promising strategies for treating neurological diseases. The sources of neural stem cells are limited while the induced pluripotent stem (iPS) cells have risk of tumor formation. Here, we proposed the generation of self-renewable, multipotent, and neural lineage-related neural crest stem-like cells by chitosan substrate-mediated gene transfer of a single factor forkhead box D3 (FOXD3) for the use in neural repair. A simple, non-toxic, substrate-mediated method was applied to deliver the naked FOXD3 plasmid into human fibroblasts. The transfection of FOXD3 increased cell proliferation and up-regulated the neural crest marker genes (FOXD3, SOX2, and CD271), stemness marker genes (OCT4, NANOG, and SOX2), and neural lineage-related genes (Nestin, β-tubulin and GFAP). The expression levels of stemness marker genes and neural crest maker genes in the FOXD3-transfected fibroblasts were maintained until the fifth passage. The FOXD3 reprogrammed fibroblasts based on the new method significantly rescued the neural function of the impaired zebrafish. The chitosan substrate-mediated delivery of naked plasmid showed feasibility in reprogramming somatic cells. Particularly, the FOXD3 reprogrammed fibroblasts hold promise as an easily accessible cellular source with neural crest stem-like behavior for treating neural diseases in the future.

Effect of an Epineurial-Like Biohybrid Nerve Conduit on Nerve Regeneration.

A novel approach of making a biomimetic nerve conduit was established by seeding adipose-derived adult stem cells (ADSCs) on the external wall of porous poly(D, L-lactic acid) (PLA) nerve conduits. The PLA conduits were fabricated using gas foaming salt and solvent–nonsolvent phase conversion. We examined the effect of two different porous structures (GS and GL) on ADSC growth and proliferation. The GS conduits had better structural stability, permeability, and porosity, as well as better cell viability at 4, 7, and 10 days. The epineuriallike tissue was grown from ADSC-seeded conduits cultured for 7 days in vitro and then implanted into 10-mm rat sciatic nerve defects for evaluation. The regeneration capacity and functional recovery were evaluated by histological staining, electrophysiology, walking track, and functional gait analysis after 6 weeks of implantation. Experimental data indicated that the autograft and ADSC-seeded GS conduits had better functional recovery than the blank conduits and ADSC-seeded GL conduits. The area of regenerated nerve and number of myelinated axons quantified based on the histology also indicated that the autograft and AGS groups performed better than the other two groups. We suggested that ADSCs may interact with endogenous Schwann cells and release neurotrophic factors to promote peripheral nerve regeneration. The design of the conduit may be critical for producing a biohybrid nerve conduit and to provide an epineurial-like support.

Biomaterial substrate-mediated multicellular spheroid formation and their applications in tissue engineering†

Three‐dimentional (3D) multicellular aggregates (spheroids), compared to the traditional 2D monolayer cultured cells, are physiologically more similar to the cells in vivo. So far there are various techniques to generate 3D spheroids. Spheroids obtained from different methods have already been applied to regenerative medicine or cancer research. Among the cell spheroids created by different methods, the substrate‐derived spheroids and their forming mechanism are unique. This review focuses on the formation of biomaterial substrate‐mediated multicellular spheroids and their applications in tissue engineering and tumor models. First, the authors will describe the special chitosan substrate‐derived mesenchymal stem cell (MSC) spheroids and their greater regenerative capacities in various tissues. Second, the authors will describe tumor spheroids derived on chitosan and hyaluronan substrates, which serve as a simple in vitro platform to study 3D tumor models or to perform cancer drug screening. Finally, the authors will mention the self‐assembly process for substrate‐derived multiple cell spheroids (co‐spheroids), which may recapitulate the heterotypic cell–cell interaction for co‐cultured cells or crosstalk between different types of cells. These unique multicellular mono‐spheroids or co‐spheroids represent a category of 3D cell culture with advantages of biomimetic cell–cell interaction, better functionalities, and imaging possibilities.

Visualization of peripheral nerve regeneration.

Peripheral nerve injuries are often caused by trauma and they may result in a partial or total loss of motor function or sensory perception. After nerve injuries, peripheral axons have the ability to regenerate and reconnect the proximal and distal ends of severed nerve axons if the nerve gap is small. For larger nerve gaps, surgical treatments are often required to repair the injured nerves.