Mathematical modelling of genipin-bovine serum albumin interaction using fluorescence intensity measurements.

The interaction between genipin and a model protein bovine serum albumin (BSA), with and without the addition of acetic acid, has been studied experimentally and by modelling. The number of amino groups available to react was determined to be 5.6 % of the total number of amino acid building blocks on BSA. Fluorescence intensity was used to record the progress of the reaction over the 24 h, while the modelling study focused on capturing the kinetic profiles of the reaction. The experiments revealed a slow start to the BSA and genipin interaction, that subsequently accelerated in an S-shaped curve which the modelling study linked with the existence of the feedback cycle for both reactive amino groups and genipin. At BSA concentrations ≥30 mg/mL the reaction was accelerated in the presence of acid, while below 30 mg/mL the acidified conditions delayed the onset of the reaction. Contrary to the reaction mechanisms previously proposed, a degree of breakdown of the fluorescent links in the products formed was denoted both experimentally and in a modelling study. This indicated the reversibility of the processes forming fluorescent product/s and suggested feasibility of the successful release of the protein following prospective encapsulation within the genipin-crosslinked hydrogel structure.

New Findings Regarding the Effects of Selected Blue Food Colorants (Genipin, Patent Blue V, and Brilliant Blue FCF) on the Hemostatic Properties of Blood Components In Vitro.

Natural and synthetic colorants present in food can modulate hemostasis, which includes the coagulation process and blood platelet activation. Some colorants have cardioprotective activity as well. However, the effect of genipin (a natural blue colorant) and synthetic blue colorants (including patent blue V and brilliant blue FCF) on hemostasis is not clear. In this study, we aimed to investigate the effects of three blue colorants-genipin, patent blue V, and brilliant blue FCF-on selected parameters of hemostasis in vitro. The anti- or pro-coagulant potential was assessed in human plasma by measuring the following coagulation times: thrombin time (TT), prothrombin time (PT), and activated partial thromboplastin time (APTT). Moreover, we used the Total Thrombus formation Analysis System (T-TAS, PL-chip) to evaluate the anti-platelet potential of the colorants in whole blood. We also measured their effect on the adhesion of washed blood platelets to fibrinogen and collagen. Lastly, the cytotoxicity of the colorants against blood platelets was assessed based on the activity of extracellular lactate dehydrogenase (LDH). We observed that genipin (at all concentrations (1-200 µM)) did not have a significant effect on the coagulation times (PT, APTT, and TT). However, genipin at the highest concentration (200 µM) and patent blue V at the concentrations of 1 and 10 µM significantly prolonged the time of occlusion measured using the T-TAS, which demonstrated their anti-platelet activity. We also observed that genipin decreased the adhesion of platelets to fibrinogen and collagen. Only patent blue V and brilliant blue FCF significantly shortened the APTT (at the concentration of 10 µM) and TT (at concentrations of 1 and 10 µM), demonstrating pro-coagulant activity. These synthetic blue colorants also modulated the process of human blood platelet adhesion, stimulating the adhesion to fibrinogen and inhibiting the adhesion to collagen. The results demonstrate that genipin is not toxic. In addition, because of its ability to reduce blood platelet activation, genipin holds promise as a novel and valuable agent that improves the health of the cardiovascular system and reduces the risk of cardiovascular diseases. However, the mechanism of its anti-platelet activity remains unclear and requires further studies. Its in vivo activity and interaction with various anti-coagulant and anti-thrombotic drugs, including aspirin and its derivatives, should be examined as well.

Maintained particulate integrity of soy protein nanoparticles during gastrointestinal digestion via genipin crosslinking enhancing stability and bioavailability of curcumin.

Poor stability during gastrointestinal digestion is a major challenge for the applications of protein-based nanoparticles as oral delivery systems. In this work, genipin was used to crosslink the partially enzymatic hydrolyzed soy protein nanoparticles, aiming to improve their performance in gastrointestinal tract as delivery carrier. Results showed that the obtained genipin-crosslinked soy protein nanoparticles (GSPNPs) were still spherically monodisperse with a diameter around 60 nm. Encapsulation with GSPNPs significantly improved the solubility of curcumin (Cur) and its stability against UV light as well as long-term storage. Compared to those un-crosslinked nanoparticles, particles crosslinked by genipin had a more compact structure less sensitive to ionic effect and digestive enzymes, showing enhanced digestion stability. The well-maintained nanoparticulate structure of GSPNPs further contributed to the enhanced bioaccessibility and facilitated absorption by epithelial cells. Furthermore, in vivo experiment on rats showed that Cur encapsulated in GSPNPs exhibited a slowed down and sustained absorption manner with an 8.11-fold improvement in its bioavailability. These suggested that GSPNPs could be a promising nanocarrier to enhance the bioavailability of functional factors.

Genipin's potential as an anti-cancer agent: from phytochemical origins to clinical prospects.

This comprehensive review delves into the multifaceted aspects of genipin, a bioactive compound derived from medicinal plants, focusing on its anti-cancer potential. The review begins by detailing the sources and phytochemical properties of genipin, underscoring its significance in traditional medicine and its transition into contemporary cancer research. It then explores the intricate relationship between genipin's chemical structure and its observed anti-cancer activity, highlighting the molecular underpinnings contributing to its therapeutic potential. This is complemented by a thorough analysis of preclinical studies, which investigates genipin's efficacy against various cancer cell lines and its mechanisms of action at the cellular level. A crucial component of the review is the examination of genipin's bioavailability and pharmacokinetics, providing insights into how the compound is absorbed, distributed, metabolized, and excreted in the body. Then, this review offers a general and updated overview of the anti-cancer studies of genipin and its derivatives based on its basic molecular mechanisms, induction of apoptosis, inhibition of cell proliferation, and disruption of cancer cell signaling pathways. We include information that complements the genipin study, such as toxicity data, and we differentiate this review by including commercial status, disposition, and regulation. Also, this review of genipin stands out for incorporating information on proposals for a technological approach through its load in nanotechnology to improve its bioavailability. The culmination of this information positions genipin as a promising candidate for developing novel anti-cancer drugs capable of supplementing or enhancing current cancer therapies.

Near-infrared-II responsive ovalbumin functionalized gold-genipin nanosystem cascading photo-immunotherapy of cancer.

Synergistic cancer therapies have attracted wide attention owing to their multi-mode tumor inhibition properties. Especially, photo-responsive photoimmunotherapy demonstrates an emerging cancer treatment paradigm that significantly improved treatment efficiency. Herein, near-infrared-II responsive ovalbumin functionalized Gold-Genipin nanosystem (Au-G-OVA NRs) was designed for immunotherapy and deep photothermal therapy of breast cancer. A facile synthesis method was employed to prepare the homogeneous Au nanorods (Au NRs) with good dispersion. The nanovaccine was developed further by the chemical cross-linking of Au-NRs, genipin and ovalbumin. The Au-G-OVA NRs outstanding aqueous solubility, and biocompatibility against normal and cancer cells. The designed NRs possessed enhanced localized surface plasmon resonance (LSPR) effect, which extended the NIR absorption in the second window, enabling promising photothermal properties. Moreover, genipin coating provided complimentary red fluorescent and prepared Au-G-OVA NRs showed significant intracellular encapsulation for efficient photoimmunotherapy outcomes. The designed nanosystem possessed deep photothermal therapy of breast cancer and 90% 4T1 cells were ablated by Au-G-OVA NRs (80µg ml-1concentration) after 1064 nm laser irradiation. In addition, Au-G-OVA NRs demonstrated outstanding vaccination phenomena by facilitating OVA delivery, antigen uptake, maturation of bone marrow dendritic cells, and cytokine IFN-γsecretion for tumor immunosurveillance. The aforementioned advantages permit the utilization of fluorescence imaging-guided photo-immunotherapy for cancers, demonstrating a straightforward approach for developing nanovaccines tailored to precise tumor treatment.

Biofunctionalized hydrogel composed of genipin-crosslinked gelatin/hyaluronic acid incorporated with lyophilized platelet-rich fibrin for segmental bone defect repair.

Segmental bone defects can arise from trauma, infection, metabolic bone disorders, or tumor removal. Hydrogels have gained attention in the field of bone regeneration due to their unique hydrophilic properties and the ability to customize their physical and chemical characteristics to serve as scaffolds and carriers for growth factors. However, the limited mechanical strength of hydrogels and the rapid release of active substances have hindered their clinical utility and therapeutic effectiveness. With ongoing advancements in material science, the development of injectable and biofunctionalized hydrogels holds great promise for addressing the challenges associated with segmental bone defects. In this study, we incorporated lyophilized platelet-rich fibrin (LPRF), which contains a multitude of growth factors, into a genipin-crosslinked gelatin/hyaluronic acid (GLT/HA-0.5 % GP) hydrogel to create an injectable and biofunctionalized composite material. Our findings demonstrate that this biofunctionalized hydrogel possesses optimal attributes for bone tissue engineering. Furthermore, results obtained from rabbit model with segmental tibial bone defects, indicate that the treatment with this biofunctionalized hydrogel resulted in increased new bone formation, as confirmed by imaging and histological analysis. From a translational perspective, this biofunctionalized hydrogel provides innovative and bioinspired capabilities that have the potential to enhance bone repair and regeneration in future clinical applications.

Enhanced fabrication of size-controllable chitosan-genipin nanoparticles using orifice-induced hydrodynamic cavitation: Process optimization and performance evaluation.

Chitosan nanoparticles (NPs) possess great potential in biomedical fields. Orifice-induced hydrodynamic cavitation (HC) has been used for the enhancement of fabrication of size-controllable genipin-crosslinked chitosan (chitosan-genipin) NPs based on the emulsion cross-linking (ECLK). Experiments have been performed using various plate geometries, chitosan molecular weight and under different operational parameters such as inlet pressure (1-3.5 bar), outlet pressure (0-1.5 bar) and cross-linking temperature (40-70 °C). Orifice plate geometry was a crucial factor affecting the properties of NPs, and the optimized geometry of orifice plate was with single hole of 3.0 mm diameter. The size of NPs with polydispersity index of 0.359 was 312.6 nm at an optimized inlet pressure of 3.0 bar, and the maximum production yield reached 84.82 %. Chitosan with too high or too low initial molecular weight (e.g., chitosan oligosaccharide) was not applicable for producing ultra-fine and narrow-distributed NPs. There existed a non-linear monotonically-increasing relationship between cavitation number (Cv) and chitosan NP size. Scanning electron microscopy (SEM) test indicated that the prepared NPs were discrete with spherical shape. The study demonstrated the superiority of HC in reducing particle size and size distribution of NPs, and the energy efficiency of orifice type HC-processed ECLK was two orders of magnitude than that of ultrasonic horn or high shear homogenization-processed ECLK. In vitro drug-release studies showed that the fabricated NPs had great potential as a drug delivery system. The observations of this study can offer strong support for HC to enhance the fabrication of size-controllable chitosan-genipin NPs.

Genipin improves obesity through promoting bile secretion and changing bile acids composition in diet-induced obese rats.

OBJECTIVES: Bile acids (BAs), as signaling molecules to regulate metabolism, have received considerable attention. Genipin is an iridoid compound extracted from Fructus Gradeniae, which has been shown to relieve adiposity and metabolic syndrome. Here, we investigated the mechanism of genipin counteracting obesity and its relationship with BAs signals in diet-induced obese (DIO) rats. METHODS: The DIO rats were received intraperitoneal injections of genipin for 10 days. The body weight, visceral fat, lipid metabolism in the liver, thermogenic genes expressions in brown fat, BAs metabolism and signals, and key enzymes for BAs synthesis were determined. KEY FINDINGS: Genipin inhibited fat synthesis and promoted lipolysis in the liver, and upregulated thermogenic gene expressions in brown adipose tissue of DIO rats. Genipin increased bile flow rate and upregulated the expressions of aquaporin 8 and the transporters of BAs in liver. Furthermore, genipin changed BAs composition by promoting alternative pathways and inhibiting classical pathways for BAs synthesis and upregulated the expressions of bile acid receptors synchronously. CONCLUSIONS: These results suggest that genipin ameliorate obesity through BAs-mediated signaling pathways.

Genipin Activates Autophagy and Promotes Myoblast Differentiation by Activating AMPK Pathway.

Cultured meat technology is expected to solve problems such as resource shortages and environmental pollution, but the muscle fiber differentiation efficiency of cultured meat is low. Genipin is the active compound derived from Gardenia jasminoides Ellis, which has a variety of activities. Additionally, genipin serves as a noncytotoxic agent for cross-linking, which is suitable as a foundational scaffold for in vitro tissue regeneration. However, the impact of genipin on myoblast differentiation remains to be studied. The research revealed that genipin was found to improve the differentiation efficiency of myoblasts. Genipin improved mitochondrial membrane potential by activating the AMPK signaling pathway of myoblasts, promoting mitochondrial biogenesis, and mitochondrial network remodeling. Genipin activated autophagy in myoblasts and maintained cellular homeostasis. Autophagy inhibitors blocked the pro-differentiation effect of genipin. These results showed that genipin improved the differentiation efficiency of myoblasts, which provided a theoretical basis for the development of cultured meat technology.

Pepsin immobilization on activated carbon and functionalized with glutaraldehyde and genipin for the synthesis of antioxidant peptides of goat casein.

In this article, the synthesis of antioxidant peptides in the enzymatic hydrolysis of caprine casein was analyzed at three different time points (60 min, 90 min, and 120 min) using immobilized pepsin on activated and modified carbon (AC, ACF, ACG 50, ACG 100). The immobilization assays revealed a reduction in the biocatalysts' activity compared to the free enzyme. Among the modified ones, ACG 50 exhibited greater activity and better efficiency for reuse cycles, with superior values after 60 min and 90 min. Peptide synthesis was observed under all studied conditions. Analyses (DPPH, ß-carotene/linoleic acid, FRAP) confirmed the antioxidant potential of the peptides generated by the immobilized enzyme. However, the immobilized enzyme in ACG 50 and ACG 100, combined with longer hydrolysis times, allowed the formation of peptides with an antioxidant capacity greater than or equivalent to those generated by the free enzyme, despite reduced enzymatic activity.

Bioselective and Radiopaque Zinc-Biopolymeric Complex-Based Porous Biomaterials Promote Mammalian Tissue Ingrowth In Vivo While Inhibiting Microbial Biofilm Gene Expression and Biofilm Formation.

Metal-organic complexes have shown astounding bioactive properties; however, they are rarely explored as biomaterials. Recent studies showed that carboxymethyl-chitosan (CMC) genipin-conjugated zinc biomimetic scaffolds have unique bioselective properties. The biomaterial was reported to be mammalian cell-friendly; at the same time, it was found to discourage microbial biofilm formation on its surface, which seemed to be a promising solution to addressing the problem of trauma-associated biofilm formation and development of antimicrobial resistance. However, the mechanically frail characteristics and zinc overload raise concerns and limit the potential of the said biomaterials. Hence, the present work is focused on improving the strength of the earlier scaffold formulations, testing its in vivo efficacy and reaffirming its action against biofilm-forming microbe Staphylococcus aureus. Scaling up of CMC proportion increased rigidity, and 8% CMC was found to be the ideal concentration for robust scaffold fabrication. Freeze-dried CMC scaffolds with or without genipin (GP) cross-linking were conjugated with zinc using 2 M zinc acetate solution. Characterization results indicated that the CMC-Zn scaffolds, without genipin, showed mechanical properties close to bone fillers, resist in vitro enzymatic degradation until 4 weeks, are porous in nature, and have radiopacity close to mandibular bones. Upon implantation in a subcutaneous pocket of Wistar rats, the scaffolds showed tissue in-growth with simultaneous degradation without any signs of toxicity past 28 days. Neither were there any signs of toxicity in any of the vital organs. Considering many superior properties among the other formulations, the CMC-Zn scaffolds were furthered for biofilm studies. CMC-Zn showed negligible S. aureus biofilm formation on its surface as revealed by an alamar blue-based study. RT-PCR analysis revealed that CMC-Zn downregulated the expression of pro-biofilm effector genes such as icaC and clfB. A protein docking study predicted the inhibitory mechanism of CMC-Zn. Although it binds strongly when alone, at high density, it may cause inactivation of the transmembrane upstream activators of the said genes, thereby preventing their dimerization and subsequent inactivation of the effector genes. In conclusion, zinc-conjugated carboxymethyl-chitosan scaffolds are mechanically robust, porous, yet biodegradable, harmless to the host in the long term, they are radiopaque and prevent biofilm gene expression in notorious microbes; hence, they could be a suitable candidate for bone filler applications.

Discovery, optimization and biological activity evaluation of genipin derivatives as potential KRAS G12D inhibitors.

A series of genipin derivatives were designed and synthesized as potential inhibitors targeted KRAS G12D mutation. The majority of these compounds demonstrated potential antiproliferative effects against KRAS G12D mutant tumor cells (CT26 and A427). Notably, seven compounds exhibited the anticancer effects with IC50 values ranging from 7.06 to 9.21 µM in CT26 (KRASG12D) and A427 (KRASG12D) cells and effectively suppressed the colony formation of CT26 cells. One representative compound SK12 was selected for further investigation into biological activity and action mechanisms. SK12 markedly induced apoptosis in CT26 cells in a concentration-dependent manner. Moreover, SK12 elevated the levels of reactive oxygen species (ROS) in tumor cells and exhibited a modulatory effect on the KRAS signaling pathway, thereby inhibiting the activation of downstream phosphorylated proteins. The binding affinity of SK12 to KRAS G12D protein was further confirmed by the surface plasmon resonance (SPR) assay with a binding KD of 157 µM. SK12 also exhibited notable anticancer efficacy in a nude mice tumor model. The relative tumor proliferation rate (T/C) of the experimental group (50 mg/kg) was 31.04 % (P < 0.05), while maintaining a commendable safety profile.

An eco-friendly chitosan-genipin/SiO2 composite for reactive orange 16 dye removal: Insights into adsorption statistical modeling and mechanism.

The present work develops an effective bioadsorbent of cross-linked chitosan-genipin/SiO2 adsorbent (CHI-GNP/SiO2). The developed CHI-GNP/SiO2 was employed for the removal of organic dye (reactive orange 16, RO16) from simulated wastewater. The optimization of the fundamental adsorption variables (CHI-GNP/SiO2 dose, time, and pH) via the Box-Behnken design (BBD) was attained for achieving maximal adsorption capacity and high removal efficiency. The good agreement between the Freundlich isotherms and empirical data of RO16 adsorption by CHI-GNP/SiO2 indicates that the adsorption process follows a multilayer adsorption mechanism. The reasonable agreement between the pseudo-second-order model and the kinetic data of RO16 adsorption by CHI-GNP/SiO2 was obtained. The maximum RO16 adsorption capacity (qmax) of CHI-GNP/SiO2 was identified to be 57.1 mg/g. The adsorption capacity of CHI-GNP/SiO2 is attributed to its unique surface properties, including its highly porous structure and the presence of functional groups such as amino and hydroxyl groups. According to the results of this investigation, CHI-GNP/SiO2 has the potential to be an adsorbent for the removal of acidic dyes from wastewater.

Genipin crosslinked quaternary ammonium chitosan hydrogels for wound dressings.

Bacterial infection can lead to various complications, such as inflammations on surrounding tissues, which can prolong wound healing and thus represent a significant clinical and public healthcare problem. Herein, a report on the fabrication of a novel genipin/quaternized chitosan (CS) hydrogel for wound dressing is presented. The hydrogel was prepared by mixing quaternized CS and genipin under 35 °C bath. The hydrogels showed porous structure (250-500 µm) and mechanical properties (3000-6000 Pa). In addition, the hydrogels displayed self-healing ability and adhesion performance on different substrates. Genipin crosslinked quaternized CS hydrogels showed antibacterial activities againstE. coliandS. aureus. The CCK-8 and fluorescent images confirmed the cytocompatibility of hydrogels by seeding with NIH-3T3 cells. The present study showed that the prepared hydrogel has the potential to be used as wound dressing.

Transcriptional profiling of geniposide bioconversion into genipin during gardenia fructus extract fermentation by Lactobacillus (Lactiplantibacillus) plantarum SN13T.

Lactiplantibacillus plantarum SN13T is a probiotic plant-derived lactic acid bacterium that can grow in various medicinal plant extracts. In this study, we fermented an aqueous extract of gardenia fructus, the fruit of a medicinal plant, with SN13T, such that the bioactivity of the extract was potentiated after fermentation to suppress the release of inflammatory mediators, such as nitric oxide (NO), reactive oxygen species (ROS), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α), as well as downregulate inflammatory genes in lipopolysaccharides (LPS)-stimulated RAW 264.7 cells. This increased antioxidant and anti-inflammatory activity was mediated through bioconversion of the iridoid glycoside geniposide to its aglycone genipin via the supposed hydrolytic action of ß-glucosidases harbored by SN13T. In the complete genome of SN13T, ten putative genes encoding ß-glucosidases of glycosyl hydrolase (GH) family 1 organized among eight gene operons were identified. Transcriptional profiling revealed that two 6-phospho-ß-glucosidase genes, pbg9 and SN13T_1925, located adjacently in the gene operon SN13T_1923, were transcribed significantly more than the remaining genes during fermentation of the gardenia extract. This suggests the role of these ß-glucosidases in bioconversion of geniposide to genipin and the subsequent enhanced bioactivity of the gardenia fructus extract after fermentation with SN13T.

Genipin ameliorates diabetic retinopathy via the HIF-1α and AGEs-RAGE pathways.

BACKGROUND: Traditional Chinese medicine (TCM) is useful in disease treatment and prevention. Genipin is an active TCM compound used to treat diabetic retinopathy (DR). In this study, a network pharmacology (NP)-based approach was employed to investigate the therapeutic mechanisms underlying genipin administration in DR. METHODS: The potential targets of DR were identified using the gene expression omnibus (GEO) database. TCM database screening and NP were used to predict the potential active targets and pathways of genipin in DR. Cell viability was tested in vitro to determine the effects of different doses of glucose and genipin on Human Retinal Microvascular Endothelial Cells (hRMECs). CCK-8, CCK-F, colony formation, CellTiter-Lum, Annexin V-FITC, wound healing, Transwell, tube-forming, reactive oxygen species (ROS), and other assay kits were used to detect the effects of genipin on hRMECs during high levels of glucose. In vivo, a streptozotocin (STZ)-mouse intraocular genipin injection (IOI.) model was used to explore the effects of genipin on diabetes-induced retinal dysfunction. Western blotting was performed to identify the cytokines involved in proliferation, apoptosis, angiogenesis, ROS, and inflammation. The protein expression of the AKT/ PI3K/ HIF-1α and AGEs/ RAGE pathways was also examined. RESULTS: Approximately 14 types of TCM, and nearly 300 active ingredients, including genipin, were identified. The NP approach successfully identified the HIF-1α and AGEs-RAGE pathways, with the EGR1 and UCP2 genes, as key targets of genipin in DR. In the in vitro and in vivo models, we discovered that high glucose increased cell proliferation, apoptosis, angiogenesis, ROS, and inflammation. However, genipin application regulated cell proliferation and apoptosis, inhibited angiogenesis, and reduced ROS and inflammation in the HRMECs exposed to high glucose. Furthermore, the retinal thickness in the genipin-treated group was lower than that in the untreated group. AKT/ PI3K/ HIF-1α and AGEs/ RAGE signaling was increased by high glucose levels; however, genipin treatment decreased AKT/ PI3K and AGEs/ RAGE pathway expressions. Genipin also increased HIF-1α phosphorylation, oxidative phosphorylation of ATP synthesis, lipid peroxidation, and the upregulation of oxidoreductase. Genipin was found to protect HG-induced hRMECs and the retina of STZ-mice, based on; 1 the inhibition of UCP2 and Glut1 decreased intracellular glucose, and glycosylation; 2 the increased presence of HIF-1α, which increased oxidative phosphorylation and decreased substrate phosphorylation; 3 the increase in oxidative phosphorylation from ATP synthesis increased lipid peroxidation and oxidoreductase activity, and; 4 the parallel effect of phosphorylation and glycosylation on vascular endothelial growth factor (VEGF), MMP9, and Scg3. CONCLUSION: Based on NP, we demonstrated the potential targets and pathways of genipin in the treatment of DR and confirmed its effective molecular mechanism in vitro and in vivo. Genipin protects cells and tissues from high glucose levels by regulating phosphorylation and glycosylation. The activation of the HIF-1α pathway can also be used to treat DR. Our study provides new insights into the key genes and pathways associated with the prognosis and pathogenesis of DR.

Cyanophycin modifications for applications in tissue scaffolding.

Cyanophycin (CGP) is a polypeptide consisting of amino acids-aspartic acid in the backbone and arginine in the side chain. Owing to its resemblance to cell adhesive motifs in the body, it can be considered suitable for use in biomedical applications as a novel component to facilitate cell attachment and tissue regeneration. Although it has vast potential applications, starting with nutrition, through drug delivery and tissue engineering to the production of value-added chemicals and biomaterials, CGP has not been brought to the industry yet. To develop scaffolds using CGP powder produced by bacteria, its properties (e.g., biocompatibility, morphology, biodegradability, and mechanical strength) should be tailored in terms of the requirements of the targeted tissue. Crosslinking commonly stands for a primary modification method for renovating biomaterial features to these extents. Herein, we aimed to crosslink CGP for the first time and present a comparative study of different methods of CGP crosslinking including chemical, physical, and enzymatic methods by utilizing glutaraldehyde (GTA), UV exposure, genipin, 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS), and monoamine oxidase (MAO). Crosslinking efficacy varied among the samples crosslinked via the different crosslinking methods. All crosslinked CGP were non-cytotoxic to L929 cells, except for the groups with higher GTA concentrations. We conclude that CGP is a promising candidate for scaffolding purposes to be used as part of a composite with other biomaterials to maintain the integrity of scaffolds. The initiative study demonstrated the unknown characteristics of crosslinked CGP, even though its feasibility for biomedical applications should be confirmed by further examinations. KEY POINTS: • Cyanophycin was crosslinked by 5 different methods • Crosslinked cyanophycin is non-cytotoxic to L929 cells • Crosslinked cyanophycin is a promising new material for scaffolding purposes.

Genipin crosslinking promotes biomechanical reinforcement and pro-regenerative macrophage polarization in bioartificial tubular substitutes.

Traumatic nerve injuries are nowadays a significant clinical challenge and new substitutes with adequate biological and mechanical properties are in need. In this context, fibrin-agarose hydrogels (FA) have shown the possibility to generate tubular scaffolds with promising results for nerve repair. However, to be clinically viable, these scaffolds need to possess enhanced mechanical properties. In this line, genipin (GP) crosslinking has demonstrated to improve biomechanical properties with good biological properties compared to other crosslinkers. In this study, we evaluated the impact of different GP concentrations (0.05, 0.1 and 0.2% (m/v)) and reaction times (6, 12, 24, 72 h) on bioartificial nerve substitutes (BNS) consisting of nanostructured FA scaffolds. First, crosslinked BNS were studied histologically, ultrastructurally and biomechanically and then, its biocompatibility and immunomodulatory effects were ex vivo assessed with a macrophage cell line. Results showed that GP was able to improve the biomechanical resistance of BNS, which were dependent on both the GP treatment time and concentration without altering the structure. Moreover, biocompatibility analyses on macrophages confirmed high cell viability and a minimal reduction of their metabolic activity by WST-1. In addition, GP-crosslinked BNS effectively directed macrophage polarization from a pro-inflammatory (M1) towards a pro-regenerative (M2) phenotype, which was in line with the cytokines release profile. In conclusion, this study considers time and dose-dependent effects of GP in FA substitutes which exhibited increased biomechanical properties while reducing immunogenicity and promoting pro-regenerative macrophage shift. These tubular substitutes could be useful for nerve application or even other tissue engineering applications such as urethra.

Exogenous Collagen Crosslinking is Highly Detrimental to Articular Cartilage Lubrication.

Healthy articular cartilage is a remarkable bearing material optimized for near-frictionless joint articulation. Because its limited self-repair capacity renders it susceptible to osteoarthritis (OA), approaches to reinforce or rebuild degenerative cartilage are of significant interest. While exogenous collagen crosslinking (CXL) treatments improve cartilage's mechanical properties and increase its resistance to enzymatic degradation, their effects on cartilage lubrication remain less clear. Here, we examined how the collagen crosslinking agents genipin (GP) and glutaraldehyde (GTA) impact cartilage lubrication using the convergent stationary contact area (cSCA) configuration. Unlike classical configurations, the cSCA sustains biofidelic kinetic friction coefficients (µk) via superposition of interstitial and hydrodynamic pressurization (i.e., tribological rehydration). As expected, glutaraldehyde- and genipin-mediated CXL increased cartilage's tensile and compressive moduli. Although net tribological rehydration was retained after CXL, GP or GTA treatment drastically elevated µk. Both healthy and "OA-like" cartilage (generated via enzymatic digestion) sustained remarkably low µk in saline- (≤0.02) and synovial fluid-lubricated contacts (≤0.006). After CXL, µk increased up to 30-fold, reaching values associated with marked chondrocyte death in vitro. These results demonstrate that mechanical properties (i.e., stiffness) are necessary, but not sufficient, metrics of cartilage function. Furthermore, the marked impairment in lubrication suggests that CXL-mediated stiffening is ill-suited to cartilage preservation or joint resurfacing.

Effect of final irrigation protocols with chitosan nanoparticle and genipin on dentine against collagenase degradation: An ex-vivo study.

AIM: Endodontic irrigants may affect the mechanical and chemical properties of dentine. This study evaluated the effects of various final irrigation protocols including the use of chitosan nanoparticle (CSnp) and cross-linking with genipin on the (1) mechanical and (2) chemical properties of dentine against enzymatic degradation. METHODOLOGY: CSnp was synthesized and characterized considering physiochemical parameters and stability. The root canals of 90 single-rooted teeth were prepared and irrigated with NaOCl. Dentine discs were obtained and divided into groups according to the following irrigation protocols: Group NaOCl+EDTA, Group NaOCl+CSnp, Group NaOCl+EDTA+CSnp, Group NaOCl+CSnp+Genipin, Group NaOCl+EDTA+CSnp+Genipin and Group distilled water. (1) Mechanical changes were determined by microhardness analysis using Vickers-tester. (2) Chemical changes were determined by evaluating molecular and elemental compositions of dentine using Fourier transform infrared spectroscopy (FTIR) analysis and scanning electron microscope (SEM)/energy dispersive X-ray spectroscopy (EDS) analysis, respectively. All analyses were repeated after the discs were kept in collagenase for 24 h. Data were analysed with repeated measures analysis of variance and Bonferroni correction for microhardness analysis, and Kruskal-Wallis and Wilcoxon tests for FTIR and SEM/EDS analyses (p = .05). RESULTS: (1) Collagenase application did not have a negative effect on microhardness only in Group NaOCl+EDTA+CSnp+Genipin when compared with the post-irrigation values (p > .05). Post-collagenase microhardness of Group NaOCl+EDTA+CSnp and Group NaOCl+CSnp+Genipin was similar to the initial microhardness (p > .05). (2) After collagenase, Amide III/ PO 4 3 - ratio presented no change in Group NaOCl+EDTA+CSnp, Group NaOCl+CSnp+Genipin and Group NaOCl+EDTA+CSnp+Genipin (p > .05), while decreased in other groups (p < .05). Collagenase did not affect CO 3 2 - / PO 4 3 - ratio in the groups (p > .05). There were no changes in the groups in terms of elemental level before and after collagenase application (p > .05). CONCLUSIONS: CSnp and genipin positively affected the microhardness and molecular composition of dentine. This effect was more pronounced when CSnp was used after EDTA.