3D-printed PCL framework assembling ECM-inspired multi-layer mineralized GO-Col-HAp microscaffold for in situ mandibular bone regeneration.

BACKGROUND: In recent years, natural bone extracellular matrix (ECM)-inspired materials have found widespread application as scaffolds for bone tissue engineering. However, the challenge of creating scaffolds that mimic natural bone ECM's mechanical strength and hierarchical nano-micro-macro structures remains. The purposes of this study were to introduce an innovative bone ECM-inspired scaffold that integrates a 3D-printed framework with hydroxyapatite (HAp) mineralized graphene oxide-collagen (GO-Col) microscaffolds and find its application in the repair of mandibular bone defects. METHODS: Initially, a 3D-printed polycaprolactone (PCL) scaffold was designed with cubic disks and square pores to mimic the macrostructure of bone ECM. Subsequently, we developed multi-layer mineralized GO-Col-HAp microscaffolds (MLM GCH) to simulate natural bone ECM's nano- and microstructural features. Systematic in vitro and in vivo experiments were introduced to evaluate the ECM-inspired structure of the scaffold and to explore its effect on cell proliferation and its ability to repair rat bone defects. RESULTS: The resultant MLM GCH/PCL composite scaffolds exhibited robust mechanical strength and ample assembly space. Moreover, the ECM-inspired MLM GCH microscaffolds displayed favorable attributes such as water absorption and retention and demonstrated promising cell adsorption, proliferation, and osteogenic differentiation in vitro. The MLM GCH/PCL composite scaffolds exhibited successful bone regeneration within mandibular bone defects in vivo. CONCLUSIONS: This study presents a well-conceived strategy for fabricating ECM-inspired scaffolds by integrating 3D-printed PCL frameworks with multilayer mineralized porous microscaffolds, enhancing cell proliferation, osteogenic differentiation, and bone regeneration. This construction approach holds the potential for extension to various other biomaterial types.

MAM7 from Vibrio parahaemolyticus: Expression, purification and effects on RAW264.7 cells.

V. parahaemolyticus is a Gram-negative bacterium that causes gastroenteritis. Within the realm of bacterial interactions with the gut, the outer membrane protein MAM7 plays a key role. However, the precise function of MAM7 in intestinal inflammation, particularly its interactions with macrophages, remains unclear. In this study, we successfully expressed and purified recombinant MAM7. After optimization of the MAM7 expression condition, it was found that the optimal concentration and temperature were 0.75 mM and 15 °C, respectively, resulting in a 27-fold increase in its yield. Furthermore, RAW264.7 cytotoxicity assay was conducted. The CCK-8 results revealed that MAM7 substantially stimulated the proliferation of RAW264.7 cells, with its optimal concentration determined to be 7.5 µg/mL. Following this, the NO concentration of MAM7 was tested, revealing a significant increase (p < 0.05) in NO levels. Additionally, the relative mRNA levels of IL-1ß, IL-6, and TNF-α in RAW264.7 cells were measured by qRT-PCR, showing a remarkable elevation (p < 0.05). Moreover, ELISA results demonstrated that MAM7 effectively stimulated the secretion of IL-6 and TNF-α by RAW264.7 cells. In summary, these findings strongly suggest that MAM7 serves as a proinflammatory adhesion factor with the capacity to modulate immune responses.

Identification of a potential antigen stimulating immune response against Vibrio parahaemolyticus infection in hybrid tilapia (Oreochromis aureus♂ × Oreochromis niloticus♀).

Vibrio parahaemolyticus (V. parahaemolyticus) is a major pathogen that causes substantial losses in the marine fishery. With the emergence of antibiotic resistance, vaccines have become the most effective approach against V. parahaemolyticus infection. Adhesion factors on the cell surface are pivotal in the colonization and pathogenesis of V. parahaemolyticus within the host, highlighting their potential as vaccine candidates. This study aims to assess the immunogenicity and potential of recombinant V. parahaemolyticus MAM7 (rMAM7) as a vaccine candidate. Initially, we cloned and purified the MAM7 protein of V. parahaemolyticus. Moreover, after 4 weeks of vaccination, the fish were challenged with V. parahaemolyticus. rMAM7 demonstrated a certain protective effect. Immunological analysis revealed that rMAM7 immunization-induced antibody production and significantly increased acid phosphatase (ACP) and alkaline phosphatase (AKP) activity in hybrid tilapia. Furthermore, serum bactericidal tests demonstrated a lower bacterial survival rate in the rMAM7 group compared to PBS and rTrxa. qRT-PCR results indicated that rMAM7 significantly upregulated CD4, CD8 and IgM gene expression, suggesting the induction of Th1 and Th2 responses in hybrid tilapia. Overall, these findings highlight the potential application of MAM7 from V. parahaemolyticus in the development of protein vaccines.

Mechanism study of cross presentation of exogenous antigen induced by cholera toxin-like chimeric protein.

Tumor subunit vaccines have great potential in personalized cancer immunotherapy. They are usually administered with adjuvant owing to their low immunogenicity. Cholera toxin (CT) is a biological adjuvant with diverse biological functions and a long history of use. Our earlier study revealed that a CT-like chimeric protein co-delivered with murine granulocyte-macrophage colony stimulating factor (mGM-CSF) and prostate cancer antigen epitope could co-stimulate dendritic cells (DCs) and enhance cross presentation of tumor epitope. To further study the molecular mechanism of CT-like chimeric protein in cross presentation, major histocompatibility complex class I (MHC I)-restricted epitope 257-264 of ovalbumin (OVAT) was used as a model antigen peptide in this study. Recombinant A subunit and pentameric B subunit of CT protein were respectively genetically constructed and purified. Then both assembled into AB5 chimeric protein in vitro. Three different chimeric biomacromolecules containing mGM-CSF and OVAT were constructed according to the different fusion sites and whether the endoplasmic reticulum (ER) retention sequence was included. It was found that A2 domain and B subunit of CT were both available for loading epitopes and retaining GM1 affinity. The binding activity of GM1 was positively correlated with antigen endocytosis. Once internalized, DCs became mature and cross-presented antigen. KDEL helped the whole molecule to be retained in the ER, and this improved the cross presentation of antigen on MHC I molecules. In conclusion, hexameric CT-like chimeric protein with dual effects of GM1 affinity and ER retention sequence were potential in improvement of cross presentation. The results laid a foundation for designing personalized tumor vaccine based on CT-like chimeric protein molecular structure.

Graphene Oxide/Black Phosphorus Functionalized Collagen Scaffolds with Enhanced Near-Infrared Controlled In Situ Biomineralization for Promoting Infectious Bone Defect Repair through PI3K/Akt Pathway.

Infectious bone defects resulting from surgery, infection, or trauma are a prevalent clinical issue. Current treatments commonly used include systemic antibiotics and autografts or allografts. Nevertheless, therapies come with various disadvantages, including multidrug-resistant bacteria, complications arising from the donor site, and immune rejection, which makes artificial implants desirable. However, artificial implants can fail due to bacterial infections and inadequate bone fusion after implantation. Thus, the development of multifunctional bone substitutes that are biocompatible, antibacterial, osteoconductive, and osteoinductive would be of great clinical importance. This study designs and prepares 2D graphene oxide (GO) and black phosphorus (BP) reinforced porous collagen (Col) scaffolds as a viable strategy for treating infectious bone defects. The fabricated Col-GO@BP scaffold exhibited an efficient photothermal antibacterial effect under near-infrared (NIR) irradiation. A further benefit of the NIR-controlled degradation of BP was to promote biomineralization by phosphorus-driven and calcium-extracted phosphorus in situ. The abundant functional groups in GO could synergistically capture the ions and enhance the in situ biomineralization. The Col-GO@BP scaffold facilitated osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSC) by leveraging its mild photothermal effect and biomineralization process, which upregulated heat shock proteins (HSPs) and activated PI3K/Akt pathways. Additionally, systematic in vivo experiments demonstrated that the Col-GO@BP scaffold obviously promotes infectious bone repair through admirable photothermal antibacterial performance and enhanced vascularization. As a result of this study, we provide new insights into the photothermal activity of GO@BP nanosheets, their degradation, and a new biological application for them.

Engineering Ag-Decorated Graphene Oxide Nano-Photothermal Platforms with Enhanced Antibacterial Properties for Promoting Infectious Wound Healing.

Introduction: Graphene oxide (GO) nanoparticles have emerged as a compelling photothermal agent (PHTA) in the realm of photothermal antibacterial therapy, owing to their cost-effectiveness, facile synthesis, and remarkable photostability. Nevertheless, the therapeutic efficacy of GO nanoparticles is commonly hindered by their inherent drawback of low photothermal conversion efficiency (PCE). Methods: Herein, we engineer the Ag/GO-GelMA platform by growing the Ag on the surface of GO and encapsulating the Ag/GO nanoparticles into the GelMA hydrogels. Results: The resulting Ag/GO-GelMA platform demonstrates a significantly enhanced PCE (47.6%), surpassing that of pure GO (11.8%) by more than fourfold. As expected, the Ag/GO-GelMA platform, which was designed to integrate the benefits of Ag/GO nanoparticles (high PCE) and hydrogel (slowly releasing Ag+ to exert an inherent antibacterial effect), has been shown to exhibit exceptional antibacterial efficacy. Furthermore, transcriptome analyses demonstrated that the Ag/GO-GelMA platform could significantly down-regulate pathways linked to inflammation (the MAPK and PI3K-Akt pathways) and had the ability to promote cell migration. Discussion: Taken together, this study presents the design of a potent photothermal antibacterial platform (Ag/GO-GelMA) aimed at enhancing the healing of infectious wounds. The platform utilizes a handy method to enhance the PCE of GO, thereby making notable progress in the utilization of GO nano-PHTAs.

Enhanced physiochemical, antibacterial, and hemostatic performance of collagen-quaternized chitosan-graphene oxide sponges for promoting infectious wound healing.

Bacteria-infected wound healing has attracted widespread attention in biomedical engineering. Wound dressing is a potential strategy for repairing infectious wounds. However, the development of wound dressing with appropriate physiochemical, antibacterial, and hemostatic properties, remains challenging. Hence, there is a motivation to develop new synthetic dressings to improve bacteria-infected wound healing. Here, we fabricate a biocompatible sponge through the covalent crosslinking of collagen (Col), quaternized chitosan (QCS), and graphene oxide (GO). The resulting Col-QCS-GO sponge shows an elastic modulus of 1.93-fold higher than Col sponge due to enhanced crosslinking degree by GO incorporation. Moreover, the fabricated Col-QCS-GO sponge shows favorable porosity (84.30 ± 3.12 %), water absorption / retention (2658.0 ± 113.4 % / 1114.0 ± 65.7 %), and hemostasis capacities (blood loss <50.0 mg). Furthermore, the antibacterial property of the Col-QCS-GO sponge under near-infrared (NIR) irradiation is significantly enhanced (the inhibition rates are 99.9 % for S. aureus and 99.9 % for E. coli) due to the inherent antibacterial properties of QCS and the photothermal antibacterial capabilities of GO. Finally, the Col-QCS-GO+NIR sponge exhibits the lowest percentage of wound area (9.05 ± 1.42 %) at day 14 compared to the control group (31.61 ± 1.76 %). This study provides new insights for developing innovative sponges for bacteria-infected wound healing.

Graphene Oxide Functionalized Gelatin Methacryloyl Microgel for Enhanced Biomimetic Mineralization and in situ Bone Repair.

Introduction: The formation of bone-like apatite (Ap) on natural polymers through biomimetic mineralization using simulated body fluid (SBF) can improve osteoconductivity and biocompatibility, while lowering immunological rejection. Nonetheless, the coating efficiency of the bone-like Ap layer on natural polymers requires improvement. Carbonyls (-COOH) and hydroxyls (-OH) are abundant in graphene oxide (GO), which may offer more active sites for biomimetic mineralization and promote the proliferation of rat bone marrow stromal cells (BMSCs). Methods: In this study, gelatin methacryloyl (GelMA) microgels were infused with GO (0, 0.5, 1, and 2 mg/mL) and embedded into microgels in SBF for 1, 7, and 14 days. Systematic in vitro and in vivo experiments were performed to evaluate the structure of the microgel and its effect on cell proliferation and ability to repair bone defects in rats. Results: The resulting GO-GelMA-Ap microgels displayed a porous, interconnected structure with uniformly coated surfaces in bone-like Ap, and the Ca/P ratio of the 1 mg/mL GO-GelMA-Ap group was comparable to that of natural bone tissue. Moreover, the 1 mg/mL GO-GelMA-Ap group exhibited a greater Ap abundance, enhanced proliferation of BMSCs in vitro and increased bone formation in vivo compared to the GelMA-Ap group. Discussion: Overall, this study offers a novel method for incorporating GO into microgels for bone tissue engineering to promote biomimetic mineralization.