Advances in tissue engineering of vasculature through three-dimensional bioprinting.

BACKGROUND: A significant challenge facing tissue engineering is the fabrication of vasculature constructs which contains vascularized tissue constructs to recapitulate viable, complex and functional organs or tissues, and free-standing vascular structures potentially providing clinical applications in the future. Three-dimensional (3D) bioprinting has emerged as a promising technology, possessing a number of merits that other conventional biofabrication methods do not have. Over the last decade, 3D bioprinting has contributed a variety of techniques and strategies to generate both vascularized tissue constructs and free-standing vascular structures. RESULTS: This review focuses on different strategies to print two kinds of vasculature constructs, namely vascularized tissue constructs and vessel-like tubular structures, highlighting the feasibility and shortcoming of the current methods for vasculature constructs fabrication. Generally, both direct printing and indirect printing can be employed in vascularized tissue engineering. Direct printing allows for structural fabrication with synchronous cell seeding, while indirect printing is more effective in generating complex architecture. During the fabrication process, 3D bioprinting techniques including extrusion bioprinting, inkjet bioprinting and light-assisted bioprinting should be selectively implemented to exert advantages and obtain the desirable tissue structure. Also, appropriate cells and biomaterials matter a lot to match various bioprinting techniques and thus achieve successful fabrication of specific vasculature constructs. CONCLUSION: The 3D bioprinting has been developed to help provide various fabrication techniques, devoting to producing structurally stable, physiologically relevant, and biologically appealing constructs. However, although the optimization of biomaterials and innovation of printing strategies may improve the fabricated vessel-like structures, 3D bioprinting is still in the infant period and has a great gap between in vitro trials and in vivo applications. The article reviews the present achievement of 3D bioprinting in generating vasculature constructs and also provides perspectives on future directions of advanced vasculature constructs fabrication.

Rapid Self-Assembly Mini-Livers Protect Mice Against Severe Hepatectomy-Induced Liver Failure.

The construction of bioartificial livers, such as liver organoids, offers significant promise for disease modeling, drug development, and regenerative medicine. However, existing methods for generating liver organoids have limitations, including lengthy and complex processes (taking 6-8 weeks or longer), safety concerns associated with pluripotency, limited functionality of pluripotent stem cell-derived hepatocytes, and small, highly variable sizes (typically ≈50-500 µm in diameter). Prolonged culture also leads to the formation of necrotic cores, further restricting size and function. In this study, a straightforward and time-efficient approach is developed for creating rapid self-assembly mini-livers (RSALs) within 12 h. Additionally, primary hepatocytes are significantly expanded in vitro for use as seeding cells. RSALs exhibit consistent larger sizes (5.5 mm in diameter), improved cell viability (99%), and enhanced liver functionality. Notably, RSALs are functionally vascularized within 2 weeks post-transplantation into the mesentery of mice. These authentic hepatocyte-based RSALs effectively protect mice from 90%-hepatectomy-induced liver failure, demonstrating the potential of bioartificial liver-based therapy.

Drug-Delivery Nanoplatform with Synergistic Regulation of Angiogenesis-Osteogenesis Coupling for Promoting Vascularized Bone Regeneration.

It has been confirmed that substantial vascularization is an effective strategy to heal large-scale bone defects in the field of bone tissue engineering. The local application of deferoxamine (DFO) is among the most common and effective methods for promoting the formation of blood vessels, although its short half-life in plasma, rapid clearance, and poor biocompatibility limit its therapeutic suitability. Herein, zeolitic imidazolate framework-8 (ZIF-8) was selected as a vehicle to extend the half-life of DFO. In the present study, a nano DFO-loaded ZIF-8 (DFO@ZIF-8) drug delivery system was established to promote angiogenesis-osteogenesis coupling. The nanoparticles were characterized, and their drug loading efficiency was examined to confirm the successful synthesis of nano DFO@ZIF-8. Additionally, due to the sustained release of DFO and Zn2+, DFO@ZIF-8 NPs were able to promote angiogenesis in human umbilical vein endothelial cells (HUVECs) culture and osteogenesis in bone marrow stem cells (BMSCs) in vitro. Furthermore, the DFO@ZIF-8 NPs promoted vascularization by enhancing the expression of type H vessels and a vascular network. The DFO@ZIF-8 NPs promoted bone regeneration in vivo by increasing the expression of OCN and BMP-2. RNA sequencing analysis revealed that the PI3K-AKT-MMP-2/9 and HIF-1α pathways were upregulated by DFO@ZIF-8 NPs in HUVECs, ultimately leading to the formation of new blood vessels. In addition, the mechanism by which DFO@ZIF-8 NPs promoted bone regeneration was potentially related to the synergistic effect of angiogenesis-osteogenesis coupling and Zn2+-mediation of the MAPK pathway. Taken together, DFO@ZIF-8 NPs, which were demonstrated to have low cytotoxicity and excellent coupling of angiogenesis and osteogenesis, represent a promising strategy for the reconstruction of critical-sized bone defects.

Development of a Stretchable and Water-Resistant Hydrogel with Antibacterial and Antioxidant Dual Functions for Wound Healing in Movable Parts.

The treatment of wounds that develop on moving parts of the body, such as joints, is considered a challenge due to poor mechanical matching and secondary injury caused by continuous motion and inflammation. Herein, a stretchable, multifunctional hydrogel dressing utilizing the dual cross-linking of chitosan (CS) and acrylic acid (AA) and modified with caffeic acid (CA) and aloin (Alo) was developed. Mechanical testing demonstrated that the hydrogel possessed excellent stretching capability (of approximately 869%) combined with outstanding adhesion (about 56 kPa), contributing to its compatibility with moving parts and allowing complete coverage of wound sites without limiting joint and organ motion. Bioinformatics analysis confirmed that use of the hydrogel resulted in upregulated expression of multiple genes related to angiogenesis and cell proliferation. Furthermore, antibacterial testing indicated that the dressing suppressed the growth of Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA), providing a better microenvironment for wound healing. An in vivo wound defect model on movable skin verified that the wound healing observed with the hydrogel dressing was superior to that observed with a commercially available dressing. Taken together, the results suggest that a stretchable multifunctional hydrogel dressing represents a promising alternative wound dressing with therapeutic potential for superior healing, especially for moving parts of the body.

Inhibition of TWEAK/Tnfrsf12a axis protects against acute liver failure by suppressing RIPK1-dependent apoptosis.

Acute liver failure (ALF) is a severe clinical syndrome characterized by massive death of hepatocytes in a short time, resulting in coagulopathy and hepatic encephalopathy, with a high mortality in patients without pre-existing liver disease. Effective treatment of ALF is currently limited to liver transplantation, highlighting the need for new target therapies. Here, we found that expression of hepatic tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor tumor necrosis factor receptor superfamily member 12A (Tnfrsf12a) were significantly increased during ALF induced by thioacetamide (TAA) or acetaminophen (APAP). Inhibition of TWEAK/Tnfrsf12a axis markedly attenuated TAA or APAP-induced ALF. Moreover, our results demonstrated that TWEAK/Tnfrsf12a axis induced receptor-interacting protein kinase 1 (RIPK1)-dependent apoptosis of hepatocytes, instead of necroptosis or pyroptosis. Notably, hepatic TNFRSF12A and TWEAK levels were also significantly increased in liver biopsies from ALF patients. In summary, our results demonstrate that during ALF, TWEAK/Tnfrsf12a axis activates RIPK1 in hepatocytes, leading to RIPK1-dependent apoptosis and subsequent liver injury. Therefore, inhibition of either TWEAK/Tnfrsf12a axis or RIPK1-dependent apoptosis attenuates liver injury, providing a new potential therapeutic target for the treatment of ALF.

Low-Swelling Adhesive Hydrogel with Rapid Hemostasis and Potent Anti-Inflammatory Capability for Full-Thickness Oral Mucosal Defect Repair.

Full-thickness oral mucosal defects are accompanied by significant blood loss and frequent infections. Instead of conventional therapies that separate hemostasis and anti-inflammation in steps, emerging hydrogels can integrate multiple functions for the successive process after defect including hemostasis/inflammatory phase, proliferative phase, and remodeling phase. However, these functions can be easily compromised by rapid swelling and degradation of hydrogels in wet oral environment. Herein, a low-swelling adhesive hydrogel with rapid hemostasis and potent anti-inflammatory capability was developed using a dual cross-linking strategy as well as a safe and facile fabrication method. It was double cross-linked hydrogel consisting of gelatin methacrylate (GelMA), nanoclay, and tannic acid (TA) (referred to as GNT). GNT hydrogel exhibited low-swelling (one-eighth of that of GelMA), excellent stretchability (211.86%), and good adhesive properties (5 times the adhesive strength of GelMA). Physicochemical characterization illuminated the close interactions among the three components. A systematic investigation of the therapeutic effects of GNT hydrogels was performed. In vitro and in vivo experimental results demonstrated the potent hemostatic property and excellent antibacterial and anti-inflammatory effects of GNT hydrogels. The RNA sequencing analysis results for rat full-thickness oral mucosal samples showed that GNT reduced inflammation levels by down-regulating the expression of multiple inflammation-related pathways, including TNF and IL-17 pathways. It also enhanced the expression levels of tissue regeneration-related genes and thus accelerated defective mucosal repair. More importantly, the therapeutic effects of GNT were superior to those of a commercial oral tissue repair membrane when applied for full-thickness oral mucosal defect repair in rabbits. In summary, the prepared low-swelling adhesive GNT hydrogel with rapid hemostasis and potent anti-inflammatory is a promising therapy for full-thickness mucosal defect in the moist and dynamic oral environment.

CPA-seq reveals small ncRNAs with methylated nucleosides and diverse termini.

High-throughput sequencing reveals the complex landscape of small noncoding RNAs (sRNAs). However, it is limited by requiring 5'-monophosphate and 3'-hydroxyl in RNAs for adapter ligation and hindered by methylated nucleosides that interfere with reverse transcription. Here we develop Cap-Clip acid pyrophosphatase (Cap-Clip), T4 polynucleotide kinase (PNK) and AlkB/AlkB(D135S)-facilitated small ncRNA sequencing (CPA-seq) to detect and quantify sRNAs with terminus multiplicities and nucleoside methylations. CPA-seq identified a large number of previously undetected sRNAs. Comparison of sRNAs with or without AlkB/AlkB(D135S) treatment reveals nucleoside methylations on sRNAs. Using CPA-seq, we profiled the sRNA transcriptomes (sRNomes) of nine mouse tissues and reported the extensive tissue-specific differences of sRNAs. We also observed the transition of sRNomes during hepatic reprogramming. Knockdown of mesenchymal stem cell-enriched U1-5' snsRNA promoted hepatic reprogramming. CPA-seq is a powerful tool with high sensitivity and specificity for profiling sRNAs with methylated nucleosides and diverse termini.

Enhanced osteogenesis and angiogenesis by PCL/chitosan/Sr-doped calcium phosphate electrospun nanocomposite membrane for guided bone regeneration.

Membranes play pivotal role in guided bone regeneration (GBR) technique for reconstruction alveolar bone. GBR membrane that is able to stimulate both osteogenic and angiogenic differentiation of cells may be more effective in clinic practice. Herein, we fabricated the Sr-doped calcium phosphate/polycaprolactone/chitosan (Sr-CaP/PCL/CS) nanohybrid fibrous membrane by incorporating 20 wt% bioactive Sr-CaP nanoparticles into PCL/CS matrix via one-step electrospinning method, in order to endow the membrane with stimulation of osteogenesis and angiogenesis. The physicochemical properties, mechanical properties, Sr2+ release behavior, and the membrane stimulate bone mesenchymal stem cell (BMSCs) differentiation were evaluated in comparison with PCL/CS and CaP/PCL/CS membranes. The SEM images revealed that the nanocomposite membrane mimicked the extracellular matrix structure. The release curve presented a 28-day long continuous release of Sr2+ and concentration which was certified in an optimal range for positive biological effects at each timepoint. The in vitro cell culture experiments certified that the Sr-CaP/PCL/CS membrane enjoyed excellent biocompatibility and remarkably promoted rat bone mesenchymal stem cell (BMSCs) adhesion and proliferation. In terms of osteogenic differentiation, BMSCs seeded on the Sr-CaP/PCL/CS membrane showed a higher ALP activity level and a better matrix mineralization. What's more, the synergism of the Sr2+ and CaP from the Sr-CaP/PCL/CS membrane enhanced BMSCs angiogenic differentiation, herein resulting in the largest VEGF secretion amount. Consequently, the Sr-CaP/PCL/CS nanohybrid electrospun membrane has promising applications in GBR.