Construction of multiplexed transcriptome NGS libraries of microdissected tissue samples based on combinational DNA barcode microbeads.

The combination of single-cell RNA sequencing and microdissection techniques that preserves positional information has become a major tool for spatial transcriptome analyses. However, high costs and time requirements, especially for experiments at the single cell scale, make it challenging for this approach to meet the demand for increased throughput. Therefore, we proposed combinational DNA barcode (CDB)-seq as a medium-throughput, multiplexed approach combining Smart-3SEQ and CDB magnetic microbeads for transcriptome analyses of microdissected tissue samples. We conducted a comprehensive comparison of conditions for CDB microbead preparation and related factors and then applied CDB-seq to RNA extracts, fresh frozen (FF) and formalin-fixed paraffin-embedded (FFPE) mouse brain tissue samples. CDB-seq transcriptomic profiles of tens of microdissected samples could be obtained in a simple, cost-effective way, providing a promising method for future spatial transcriptomics.

Single-Nucleus RNA-Seq: Open the Era of Great Navigation for FFPE Tissue.

Single-cell sequencing (scRNA-seq) has revolutionized our ability to explore heterogeneity and genetic variations at the single-cell level, opening up new avenues for understanding disease mechanisms and cell-cell interactions. Single-nucleus RNA-sequencing (snRNA-seq) is emerging as a promising solution to scRNA-seq due to its reduced ionized transcription bias and compatibility with richer samples. This approach will provide an exciting opportunity for in-depth exploration of billions of formalin-fixed paraffin-embedded (FFPE) tissues. Recent advancements in single-cell/nucleus gene expression workflows tailored for FFPE tissues have demonstrated their feasibility and provided crucial guidance for future studies utilizing FFPE specimens. In this review, we provide a broad overview of the nuclear preparation strategies, the latest technologies of snRNA-seq applicable to FFPE samples. Finally, the limitations and potential technical developments of snRNA-seq in FFPE samples are summarized. The development of snRNA-seq technologies for FFPE samples will lay a foundation for transcriptomic studies of valuable samples in clinical medicine and human sample banks.

Defining Specific Cell States of MPTP-Induced Parkinson's Disease by Single-Nucleus RNA Sequencing.

Parkinson's disease (PD) is a neurodegenerative disease with an impairment of movement execution that is related to age and genetic and environmental factors. 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a neurotoxin widely used to induce PD models, but the effect of MPTP on the cells and genes of PD has not been fully elucidated. By single-nucleus RNA sequencing, we uncovered the PD-specific cells and revealed the changes in their cellular states, including astrocytosis and endothelial cells' absence, as well as a cluster of medium spiny neuron cells unique to PD. Furthermore, trajectory analysis of astrocyte and endothelial cell populations predicted candidate target gene sets that might be associated with PD. Notably, the detailed regulatory roles of astrocyte-specific transcription factors Dbx2 and Sox13 in PD were revealed in our work. Finally, we characterized the cell-cell communications of PD-specific cells and found that the overall communication strength was enhanced in PD compared with a matched control, especially the signaling pathways of NRXN and NEGR. Our work provides an overview of the changes in cellular states of the MPTP-induced mouse brain.

Electrospun Nanofibers for Tissue Engineering with Drug Loading and Release.

Electrospinning technologies have been applied in the field of tissue engineering as materials, with nanoscale-structures and high porosity, can be easily prepared via this method to bio-mimic the natural extracellular matrix (ECM). Tissue engineering aims to fabricate functional biomaterials for the repairment and regeneration of defective tissue. In addition to the structural simulation for accelerating the repair process and achieving a high-quality regeneration, the combination of biomaterials and bioactive molecules is required for an ideal tissue-engineering scaffold. Due to the diversity in materials and method selection for electrospinning, a great flexibility in drug delivery systems can be achieved. Various drugs including antibiotic agents, vitamins, peptides, and proteins can be incorporated into electrospun scaffolds using different electrospinning techniques and drug-loading methods. This is a review of recent research on electrospun nanofibrous scaffolds for tissue-engineering applications, the development of preparation methods, and the delivery of various bioactive molecules. These studies are based on the fabrication of electrospun biomaterials for the repair of blood vessels, nerve tissues, cartilage, bone defects, and the treatment of aneurysms and skin wounds, as well as their applications related to oral mucosa and dental fields. In these studies, due to the optimal selection of drugs and loading methods based on electrospinning, in vitro and in vivo experiments demonstrated that these scaffolds exhibited desirable effects for the repair and treatment of damaged tissue and, thus, have excellent potential for clinical application.

In vitro and in vivo studies of electroactive reduced graphene oxide-modified nanofiber scaffolds for peripheral nerve regeneration.

Graphene, as a promising biomaterial, has received great attention in biomedical fields due to its intriguing properties, especially the conductivity and biocompatibility. Given limited studies on the effects of graphene-based scaffolds on peripheral nerve regeneration in vitro and in vivo under electrical stimulation (ES), the present study was intended to systematically investigate how conductive graphene-based nanofibrous scaffolds regulate Schwann cell (SC) behavior including migration, proliferation and myelination, and PC12 cell differentiation in vitro via ES, and whether these conductive scaffolds could guide SC migration and promote nerve regeneration in vivo. Briefly, the reduced graphene oxide (RGO) was coated onto ApF/PLCL nanofibrous scaffolds via in situ redox reaction of the graphene oxide (GO). In vitro, RGO-coated ApF/PLCL (AP/RGO) scaffolds significantly enhanced SC migration, proliferation, and myelination including myelin-specific gene expression and neurotrophic factor secretion. The conditioned media of SCs cultured on AP/RGO scaffolds under ES could induce the differentiation of PC12 cells in a separate culture. In addition, PC12 cells cultured on the conductive AP/RGO scaffolds also showed elevated differentiation upon ES. In vivo implantation of the conductive AP/RGO nerve guidance conduits into rat sciatic nerve defects exhibited a similar healing capacity to autograft, which is the current gold standard in peripheral nerve regeneration. In view of the performance of AP/RGO scaffolds in modulating cell functions in vitro and promoting nerve regeneration in vivo, it is expected that the graphene-based conductive nanofibrous scaffolds would exhibit their potential in peripheral nerve repair and regeneration. STATEMENT OF SIGNIFICANCE: Despite the demonstrated capability of bridging the distal and proximal peripheral nerves, it remains a significant challenge with current artificial nerve conduits to achieve the desired physiological functions, e.g., the transmission of electrical stimuli. Herein, we explored the possibility of combining the conductive properties of graphene with electrospun nanofiber to create the electroactive biomimetic scaffolds for nerve tissue regeneration. In vitro and in vivo studies were carried out: (1) In vitro, the conductive nanofibrous scaffolds significantly promoted SC migration, proliferation and myelination including myelin specific gene expression and neurotrophicfactor secretion, and induced PC12 cell differentiation with electrical stimulation. (2) In vivo, the conductive nerve guidance conduit exhibited similar effects with the gold standard autograft. In view of the performance of this conductive scaffold in modulating the cell functions in vitro and promoting nerve regeneration in vivo, it is expected that the graphene-modified nanofibrous scaffolds will exhibit their potential in peripheral nerve repair and regeneration.

Three-dimensional electrospun nanofibrous scaffolds displaying bone morphogenetic protein-2-derived peptides for the promotion of osteogenic differentiation of stem cells and bone regeneration.

Tissue scaffolds with three-dimensional (3D) nanofibrous biomimetic structures have attracted attention in the field of bone regeneration. In recent years, emerging strategies based on electrospinning technologies have facilitated the preparation of 3D nanofibrous scaffolds. Based on these developments, in this study, 3D scaffolds possessing both nanofibrous morphologies and interconnected pores were fabricated for their potential in bone tissue engineering. By combining homogenizing, freeze-drying, and thermal crosslinking techniques, nano-hydroxyapatite/PLLA/gelatin (nHA/PLA/GEL) 3D nanofibrous scaffolds were prepared using pre-fabricated electrospun nanofibers. Then, utilizing a polydopamine (pDA)-assisted coating strategy, bone morphogenetic protein-2 (BMP-2)-derived peptides were further immobilized onto the 3D scaffolds to obtain the resulting nano-hydroxyapatite/PLLA/gelatin-peptide (nHA/PLA/GEL-PEP) 3D nanofibrous scaffolds capable of sustained release. Bone mesenchymal stem cells (BMSCs) were cultured on the 3D nanofibrous scaffolds, then relative cell viability, alkaline phosphatase (ALP) activity, and gene expression assays were performed to study the effects of the scaffolds on cell growth and osteogenic differentiation in vitro. Furthermore, the ability of bone formation in vivo was evaluated using a rat cranial bone defect model. In vitro and in vivo results demonstrated that the 3D nanofibrous scaffolds incorporated with nHA and BMP-2 peptides exhibited favorable biocompatibility and osteoinductivity. Therefore, these nanofibrous scaffolds have excellent potential in bone regenerative medicine.

Electrospun Bilayer Composite Vascular Graft with an Inner Layer Modified by Polyethylene Glycol and Haparin to Regenerate the Blood Vessel.

In this study, we prepared a composite vascular graft with two layers. The inner layer, which was comprised of degradable Poly(lactic-co-glycolic acid) (PLGA)/Collagen (PC) nanofibers modified by mesoporous silica nanoparticles (MSN), was grafted with polyethylene glycol (PEG) and heparin to promote cell proliferation and to improve blood compatibility. The outer layer was comprised of polyurethane (PU) nanofibers in order to provide mechanical support. The growth and proliferation of human umbilical vein endothelial cells (HUVECs) in the inner layer was significant, and blood compatibility testing showed that the inner layer had good blood compatibility. The MSN-PEG-Heparin on the fiber surface was observed in vitro during the degradation of the inner layer. After 60 days, the weight of fiber membrane decreased by 92.4%. The inner layer did not cause an inflammatory reaction during the degradation process in vivo and there was uniform cellular growth on the PC/MSN-PEG-Heparin fiber membrane. Composite grafts implanted into the rabbit carotid artery were evaluated for 8 weeks by H&E and immunohistochemical staining, demonstrating that a monolayer of endothelium (CD31-labeled) and smooth muscle (αSMA-labeled) regenerated on the composite graft. Our results demonstrate that the composite graft, with a functional inner layer, has potential to be used for small-caliber blood vessels with long-term patency.

Dual-layer aligned-random nanofibrous scaffolds for improving gradient microstructure of tendon-to-bone healing in a rabbit extra-articular model.

BACKGROUND: Tendon/ligament injuries are common sports injuries. Clinically, the repair of a ruptured tendon or ligament to its bony insertion is needed, but the enthesis structure is not well reestablished following surgical repair. Herein, we fabricated dual-layer aligned-random scaffold (ARS) by electrospinning and aimed to investigate the effect of the scaffold on tendon-to-bone healing in vivo. MATERIALS AND METHODS: The random and dual-layer aligned-random silk fbroin poly(L-lactic acid-co-e-caprolactone) (P(LLA-CL)) nanofibrous scaffolds were successfully fabricated by electrospinning methods. Ninety New Zealand white rabbits were randomly divided into three groups (random scaffold [RS], ARS, and control groups), and they were subjected to surgery to establish an extra-articular tendon-to-bone healing model with autologous Achilles tendon. RESULTS: Histological assessment showed that the ARS significantly increased the area of metachromasia, decreased the interface width, and improved collagen maturation and organization at the tendon-bone interface compared with the RS and control groups. Microcomputed tomography analysis showed that the bone tunnel area of RS and ARS groups was significantly smaller than those of the control group. Real-time polymerase chain reaction showed that BMP-2 and osteopontin expression levels of the tissue at the interface between the bone and graft in the RS and ARS groups were higher than those of the control group at 6 weeks. Collagen I expression level of the ARS group was significantly higher than those of the RS and control groups at 6 and 12 weeks. Moreover, the ARS groups had a better ultimate load-to-failure and stiffness than the RS and control groups. CONCLUSION: ARS could effectively augment the tendon-to-bone integration and improve gradient microstructure in a rabbit extra-articular model by inducing the new bone formation, increasing the area of fibrocartilage, and improving collagen organization and maturation. The dual-layer aligned-random silk fibroin/P(LLA-CL) nanofibrous scaffold is proved to be a promising biomaterial for tendon-to-bone healing.