In-Vivo Induced CAR-T Cell for the Potential Breakthrough to Overcome the Barriers of Current CAR-T Cell Therapy.

Chimeric antigen receptor T cell (CAR-T cell) therapy has shown impressive success in the treatment of hematological malignancies, but the systemic toxicity and complex manufacturing process of current autologous CAR-T cell therapy hinder its broader applications. Universal CAR-T cells have been developed to simplify the production process through isolation and editing of allogeneic T cells from healthy persons, but the allogeneic CAR-T cells have recently encountered safety concerns, and clinical trials have been halted by the FDA. Thus, there is an urgent need to seek new ways to overcome the barriers of current CAR-T cell therapy. In-vivo CAR-T cells induced by nanocarriers loaded with CAR-genes and gene-editing tools have shown efficiency for regressing leukemia and reducing systemic toxicity in a mouse model. The in-situ programming of autologous T-cells avoids the safety concerns of allogeneic T cells, and the manufacture of nanocarriers can be easily standardized. Therefore, the in-vivo induced CAR-T cells can potentially overcome the abovementioned limitations of current CAR-T cell therapy. Here, we provide a review on CAR structures, gene-editing tools, and gene delivery techniques applied in immunotherapy to help design and develop new in-vivo induced CAR-T cells.

N-acetyl cysteine-loaded graphene oxide-collagen hybrid membrane for scarless wound healing.

Wound dressings composed of natural polymers, such as type I collagen, possess good biocompatibility, water holding capacity, air permeability, and degradability, and can be used in wound repair. However, due to the persistent oxidative stress in the wound area, the migration and proliferation of fibroblasts might be suppressed, leading to poor healing. Thus, collagen-containing scaffolds are not suitable for accelerated wound healing. Antioxidant N-acetyl cysteine (NAC) is known to reduce the reactive oxygen species (ROS) and has been widely used in the clinic. Theoretically, the carboxyl group of NAC allows loading of graphene oxide (GO) for sustained release and may also enhance the mechanical properties of the collagen scaffold, making it a better wound-dressing material. Herein, we demonstrated an innovative approach for a potential skin-regenerating hybrid membrane using GO incorporated with collagen I and NAC (N-Col-GO) capable of continuously releasing antioxidant NAC. Methods: The mechanical stability, water holding capacity, and biocompatibility of the N-Col-GO hybrid membrane were measured in vitro. A 20 mm rat full-skin defect model was created to evaluate the repair efficiency of the N-Col-GO hybrid membrane. The vascularization and scar-related genes in the wound area were also examined. Results: Compared to the Col only scaffold, N-Col-GO hybrid membrane exhibited a better mechanical property, stronger water retention capacity, and slower NAC release ability, which likely promote fibroblast migration and proliferation. Treatment with the N-Col-GO hybrid membrane in the rat wound model showed complete healing 14 days after application which was 22% faster than the control group. HE and Masson staining confirmed faster collagen deposition and better epithelization, while CD31 staining revealed a noticeable increase of vascularization. Furthermore, Rt-PCR demonstrated decreased mRNA expression of profibrotic and overexpression of anti-fibrotic factors indicative of the anti-scar effect. Conclusion: These findings suggest that N-Col-GO drug release hybrid membrane serves as a better platform for scarless skin regeneration.

Biomimetic open porous structured core-shell microtissue with enhanced mechanical properties for bottom-up bone tissue engineering.

Background: Microtissues constructed with hydrogels promote cell expansion and specific differentiation by mimicking the microarchitecture of native tissues. However, the suboptimal mechanical property and osteogenic activity of microtissues fabricated by natural polymers need further improvement for bone reconstruction application. Core-shell designed structures are composed of an inner core part and an outer part shell, combining the characteristics of different materials, which improve the mechanical property of microtissues. Methods: A micro-stencil array chip was used to fabricate an open porous core-shell micro-scaffold consisting of gelatin as shell and demineralized bone matrix particles modified with bone morphogenetic protein-2 (BMP-2) as core. Single gelatin micro-scaffold was fabricated as a control. Rat bone marrow mesenchymal stem cells (BMSCs) were seeded on the micro-scaffolds, after which they were dynamic cultured and osteo-induced in mini-capsule bioreactors to fabricate microtissues. The physical characteristics, biocompatibility, osteo-inducing and controlled release ability of the core-shell microtissue were evaluated in vitro respectively. Then microtissues were tested in vivo via ectopic implantation and orthotopic bone implantation in rat model. Results: The Young's modulus of core-shell micro-scaffold was nearly triple that of gelatin micro-scaffold, which means the core-shell micro-scaffolds have better mechanical property. BMSCs rapidly proliferated and retained the highest viability on core-shell microtissues. The improved osteogenic potential of core-shell microtissues was evidenced by the increased calcification based on von kossa staining and osteo-relative gene expression. At 3months after transplantation, core-shell microtissue group formed the highest number of mineralized tissues in rat ectopic subcutaneous model, and displayed the largest amount of new bony tissue deposition in rat orthotopic cranial defect. Conclusion: The novel core-shell microtissue construction strategy developed may become a promising cell delivery platform for bone regeneration.

Collagen Functionalized With Graphene Oxide Enhanced Biomimetic Mineralization and in Situ Bone Defect Repair.

Biomimetic mineralization using simulated body fluid (SBF) can form a bonelike apatite (Ap) on the natural polymers and enhance osteoconductivity and biocompatibility, and reduce immunological rejection. Nevertheless, the coating efficiency of the bonelike apatite layer on natural polymers still needs to be improved. Graphene oxide (GO) is rich in functional groups, such as carbonyls (-COOH) and hydroxyls (-OH), which can provide more active sites for biomimetic mineralization and improve the proliferation of the rat bone marrow stromal cells (r-BMSCs). In this study, we introduced 0%, 0.05%, 0.1%, and 0.2% w/v concentrations of GO into collagen (Col) scaffolds and immersed the fabricated scaffolds into SBF for 1, 7, and 14 days. In vitro environment scanning electron microscopy (ESEM), energy-dispersive spectrometry (EDS), thermogravimetric analysis (TGA), micro-CT, calcium quantitative analysis, and cellular analysis were used to evaluate the formation of bonelike apatite on the scaffolds. In vivo implantation of the scaffolds into the rat cranial defect was used to analyze the bone regeneration ability. The resulting GO-Col-Ap scaffolds exhibited a porous and interconnected structure coated with a homogeneous distribution of bonelike apatite on their surfaces. The Ca/P ratio of 0.1% GO-Col-Ap group was equal to that of natural bone tissue on the basis of EDS analysis. More apatites were observed in the 0.1% GO-Col-Ap group through TGA analysis, micro-CT evaluation, and calcium quantitative analysis. Furthermore, the 0.1% GO-Col-Ap group showed significantly higher r-BMSCs adhesion and proliferation in vitro and more than 2-fold higher bone formation than the Col-Ap group in vivo. Our study provides a new approach of introducing graphene oxide into bone tissue engineering scaffolds to enhance biomimetic mineralization.