Gene Therapy Using Efficient Direct Lineage Reprogramming Technology for Neurological Diseases.

Gene therapy is an innovative approach in the field of regenerative medicine. This therapy entails the transfer of genetic material into a patient's cells to treat diseases. In particular, gene therapy for neurological diseases has recently achieved significant progress, with numerous studies investigating the use of adeno-associated viruses for the targeted delivery of therapeutic genetic fragments. This approach has potential applications for treating incurable diseases, including paralysis and motor impairment caused by spinal cord injury and Parkinson's disease, and it is characterized by dopaminergic neuron degeneration. Recently, several studies have explored the potential of direct lineage reprogramming (DLR) for treating incurable diseases, and highlighted the advantages of DLR over conventional stem cell therapy. However, application of DLR technology in clinical practice is hindered by its low efficiency compared with cell therapy using stem cell differentiation. To overcome this limitation, researchers have explored various strategies such as the efficiency of DLR. In this study, we focused on innovative strategies, including the use of a nanoporous particle-based gene delivery system to improve the reprogramming efficiency of DLR-induced neurons. We believe that discussing these approaches can facilitate the development of more effective gene therapies for neurological disorders.

Three-dimensional bioprinting of polysaccharide-based self-healing hydrogels with dual cross-linking.

Three-dimensional (3D) bioprinting technique is useful to fabricate constructs with functional and biological structures for various biomedical applications. Oxidized hyaluronate (OHA) and glycol chitosan (GC) can form autonomous self-healing hydrogels when adipic acid dihydrazide (ADH) is used. We demonstrate that hyaluronate-alginate hybrid (HAH) polymers can be used for secondary physical cross-linking of OHA/GC/ADH hydrogel with calcium ions after 3D printing. The molecular weight of hyaluronate can be varied while keeping the molecular weight of alginate in HAH. The mechanical stiffness and stability of gels after 3D printing are strongly dependent on the molecular weight of HAH at the same cross-linking density. In vitro chondrogenic differentiation of ATDC5 cells encapsulated in 3D-printed constructs is dependent on the molecular weight of HAH in gels. This dual cross-linking system consisting of naturally occurring biocompatible polysaccharides may have potential in the 3D bioprinting of custom-made scaffolds for tissue engineering applications.

3D Printing of Polysaccharide-Based Self-Healing Hydrogel Reinforced with Alginate for Secondary Cross-Linking.

Three-dimensional (3D) bioprinting has been attractive for tissue and organ regeneration with the possibility of constructing biologically functional structures useful in many biomedical applications. Autonomous healing of hydrogels composed of oxidized hyaluronate (OHA), glycol chitosan (GC), and adipic acid dihydrazide (ADH) was achieved after damage. Interestingly, the addition of alginate (ALG) to the OHA/GC/ADH self-healing hydrogels was useful for the dual cross-linking system, which enhanced the structural stability of the gels without the loss of their self-healing capability. Various characteristics of OHA/GC/ADH/ALG hydrogels, including viscoelastic properties, cytotoxicity, and 3D printability, were investigated. Additionally, potential applications of 3D bioprinting of OHA/GC/ADH/ALG hydrogels for cartilage regeneration were investigated in vitro. This hydrogel system may have potential for bioprinting of a custom-made scaffold in various tissue engineering applications.

One-step harvest and delivery of micropatterned cell sheets mimicking the multi-cellular microenvironment of vascularized tissue.

Techniques for harvest and delivery of cell sheets have been improving for decades. However, cell sheets with complicated patterns closely related to natural tissue architecture were hardly achieved. Here, we developed an efficient method to culture and harvest cell sheets with complex shape (noted as microtissues) using temperature-responsive hydrogel consisting of expandable polyethylene oxide polymer at low temperature. Firstly, a temperature-responsive hydrogel surface with honeycomb patterns (50 and 100 µm in width) were developed through microcontact printing of polydopamine (PD). The human dermal fibroblasts (HDFBs) and human umbilical vein endothelial cells (HUVECs) spontaneously formed honeycomb-shaped microtissues on the patterned hydrogel surface. The microtissues on the hydrogel were able to be harvested and directly delivered to the desired target through thermal expansion of the hydrogel at 4 °C with an efficiency close to 80% within 10 min which is faster than conventional method based on poly(N-isopropylacrylamide). The microtissues maintained their original honeycomb network and intact structures. Honeycomb-patterned cell sheets also were fabricated through serial seeding of various cell lines, including HDFBs, HUVECs, and human adipose-derived stem cells, in which cells were attached along the honeycomb pattern. The underlying honeycomb patterns in the cell sheets were successfully maintained for 3 days, even after delivery. In addition, patterned cell sheets were successfully delivered in vivo while maintaining an intact structure for 7 days. Together, our findings demonstrate that micropatterned temperature-responsive hydrogel is an efficient method of one-step culturing and delivery of complex microtissues and should prove useful in various tissue engineering applications. STATEMENT OF SIGNIFICANCE: Scaffold-free cell delivery techniques, including cell sheet engineering, have been developed for decades. However, there is limited research regarding culture and delivery of microtissues with complex architecture mimicking natural tissue. Herein, we developed a micro-patterned hydrogel platform for the culture and delivery of honeycomb-shaped microtissues. Honeycomb patterns were chemically engineered on the temperature-responsive hydrogel through microcontact printing of polydopamine to selectively allow for human dermal fibroblast or human umbilical vein endothelial cell adhesion. They spontaneously formed honeycomb-shaped microtissues within 24 hr upon cell seeding and directly delivered to various target area including in vivo via thermal expansion of the hydrogel at 4 °C, suggesting that the micro-patterned hydrogel can be an efficient tool for culture and delivery of complex microtissue.

Controlling the porous structure of alginate ferrogel for anticancer drug delivery under magnetic stimulation.

Stimulus-responsive drug delivery systems have been widely used for many biomedical applications. Magnetic stimulation may serve as an important external stimulus for drug delivery. In this study, we hypothesized that the on-demand release of anticancer drugs could be achieved with a macroporous alginate ferrogel under the influence of magnetic stimulation to enhance therapeutic efficacy in a tumor-bearing mouse model. A ferrogel containing alginate, iron oxide nanoparticle, and gelatin particle was prepared by ionic crosslinking with calcium ions and dissolving the gelatin particle at 37 °C. We investigated the influence of porosity on the degree of deformation of alginate ferrogel and evaluated the release behavior of doxorubicin (DOX) by applying magnetic field to the ferrogel. In vitro viability of cancer cells cultured with DOX-releasing macroporous alginate ferrogel after magnetic stimulation was greatly decreased compared to that of cells cultured with alginate ferrogel. The therapeutic efficacy of DOX-releasing macroporous alginate ferrogel also increased in tumor-bearing mice following magnetic stimulation. Thus, this approach to design a ferrogel responsive to magnetic stimulation may prove useful for the development of smart drug delivery systems.