Genipin Crosslinking Research Articles

Fabrication and Properties of Hydrogel Dressings Based on Genipin Crosslinked Chondroitin Sulfate and Chitosan.

Multifunctional hydrogel dressings remain highly sought after for the promotion of skin wound regeneration. In the present study, multifunctional CHS-DA/HACC (CH) hydrogels with an interpenetrated network were constructed using hydroxypropyl trimethyl ammonium chloride modified chitosan (HACC) and dopamine-modified chondroitin sulfate (CHS-DA), using genipin as crosslinker. The synthesis of HACC and CHS-DA was effectively confirmed using Fourier transform infrared (FT-IR) analysis and 1H nuclear magnetic resonance (1H NMR) spectroscopy. The prepared CH hydrogels exhibited a network of interconnected pores within the microstructure. Furthermore, rheological testing demonstrated that CH hydrogels exhibited strong mechanical properties, stability, and injectability. Further characterization investigations showed that the CH hydrogels showed favorable self-healing and self-adhesion properties. It was also shown that increasing HACC concentration ratio was positively correlated with the antibacterial activity of CH hydrogels, as evidenced by their resistance to Escherichia coli and Staphylococcus aureus. Additionally, Cell Counting Kit-8 (CCK-8) tests, fluorescent images, and a cell scratch assay demonstrated that CH hydrogels had good biocompatibility and cell migration ability. The multifunctional interpenetrated network hydrogels were shown to have good antibacterial properties, antioxidant properties, stable storage modulus and loss modulus, injectable properties, self-healing properties, and biocompatibility, highlighting their potential as wound dressings in wound healing applications.

Fabrication and characterization of electrospun core-shell nanofibers of bilayer zein/pullulan emulsions crosslinked by genipin

In this study, the electrospun core-shell nanofibers of zein/pullulan stabilized bilayer emulsions before and after genipin crosslinking were fabricated. The experimental results indicated that the addition of pullulan increased the apparent viscosity and elastic modulus of the bilayer emulsions, which was further increased after the chemical crosslinking of genipin. The nanofiber diameter increased from 102.9 nm to 169.9 nm with the increasing ratio of pullulan, but increased significantly to a range of 405.6–708.0 nm after genipin crosslinking. The electrospun nanofiber films of crosslinked emulsions had higher thermal stability and stronger water stability. The FTIR result proved the existence of hydrogen bond interaction between the zein, pullulan, and genipin molecules. In addition, before and after crosslinking, the encapsulation efficiency of electrospun fiber films for camellia oil was >77.68 %, and the maximum encapsulation efficiency could reach 87.94 %, and there was no significant change during the 7-day storage period, showing good stability. These research results can provide a theoretical basis for the encapsulation of hydrophobic active substances in zein-based emulsion electrospun core-shell nanofibers.

Effects of Genipin Crosslinking of Porcine Perilimbal Sclera on Mechanical Properties and Intraocular Pressure.

The mechanical properties of sclera play an important role in ocular functions, protection, and disease. Modulating the sclera's properties by exogenous crosslinking offers a way to expand the tissue's range of properties for study of the possible influences on the eye's behavior and diseases such as glaucoma and myopia. The focus of this work was to evaluate the effects of genipin crosslinking targeting the porcine perilimbal sclera (PLS) since the stiffness of this tissue was previously found in a number of studies to influence the eye's intraocular pressure (IOP). The work includes experiments on tensile test specimens and whole globes. The specimen tests showed decreased strain-rate dependence and increased relaxation stress due to the cross-linker. Whole globe perfusion experiments demonstrated that eyes treated with genipin in the perilimbal region had increased IOPs compared to the control globes. Migration of the cross-linker from the target tissue to other tissues is a confounding factor in whole globe, biomechanical measurements, with crosslinking. A novel quantitative genipin assay of the trabecular meshwork (TM) was developed to exclude globes where the TM was inadvertently crosslinked. The perfusion study, therefore, suggests that elevated stiffness of the PLS can significantly increase IOP apart from effects of the TM in the porcine eye. These results demonstrate the importance of PLS biomechanics in aqueous outflow regulation and support additional investigations into the distal outflow pathways as a key source of outflow resistance.

Non-carrier immobilization of yeast cells by genipin crosslinking for the synthesis of prebiotic galactooligosaccharides from plant-derived galactose

Galactooligosaccharides (GOS), as mimics of human milk oligosaccharides, are important prebiotics for modulating the ecological balance of intestinal microbiota. A novel carrier-free cell immobilization method was established using genipin to cross-link Kluyveromyces lactis CGMCC 2.1494, which produced β-galactosidase, an enzyme essential for GOS synthesis. The resulting immobilized cells were characterized as stable by thermogravimetric analysis and confirmed to be crosslinked through scanning electron microscopy analysis (SEM) and Fourier transform infrared spectroscopy (FTIR). The Km and Vmax values of β-galactosidase in immobilized cells towards o-nitrophenyl β-D-galactoside were determined to be 3.446 mM and 2210 μmol min−1 g−1, respectively. The enzyme in the immobilized showed higher thermal and organic solvent tolerance compared to that in free cells. The immobilized cells were subsequently employed for GOS synthesis using plant-derived galactose as the substrate. The synthetic reaction conditions were optimized through both single-factor experiments and response surface methodology, resulting in a high yield of 49.1 %. Moreover, the immobilized cells showed good reusability and could be reused for at least 20 batches of GOS synthesis, with the enzyme activity remaining above 70 % at 35 °C.

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.

Multi-form antibacterial dressings based on a deep eutectic supramolecular polymer promote healing of burn wounds through a weakly acidic microenvironment

Burn damage causes loss of the barrier function of the skin against bacteria, but providing a weakly acidic environment is beneficial for preventing bacterial invasion and promoting wound healing. The utilization of the natural antibacterial properties of chitosan is ideal for treating wounds. In this work, a deep eutectic supramolecular polymer (DESP) prepared with a cyclodextrin and an organic acid effectively improved the antibacterial properties of chitosan through its cavity structure and provided a weakly acidic environment for wound healing. Through genipin crosslinking, the mechanical strength and swelling rate of the prepared dressing reached 9.79 MPa and 1100 %, respectively. As a wound dressing, this new material shows good flexibility, breathability and stability. The cell survival rate when using this new wound dressing was greater than 90.0 %, and the hemolysis rate was less than 5.0 %. It had high biocompatibility and cell compatibility and facilitated cell proliferation. The dressing showed excellent antibacterial effects on E. coli and S. aureus, and the size of the inhibition rings was up to 11 mm and 14 mm. In in vivo wound healing experiments, new capillaries and collagen fibers appeared earlier, and the dressing in multiple forms could reduce inflammation, promote angiogenesis, and accelerate wound healing. Therefore, the use of a DESP with a cavity structure provides an effective strategy for the preparation of future medical materials.

Modulation of Decellularized Lacrimal Gland Hydrogel Biodegradation by Genipin Crosslinking.

Hydrogels derived from decellularized tissues are promising biomaterials in tissue engineering, but their rapid biodegradation can hinder in vitro cultivation. This study aimed to retard biodegradation of a hydrogel derived from porcine decellularized lacrimal glands (dLG-HG) by crosslinking with genipin to increase the mechanical stability without affecting the function and viability of lacrimal gland (LG)-associated cells. The effect of different genipin concentrations on dLG-HG stiffness was measured rheologically. Cell-dependent biodegradation was quantified over 10days, and the impact on matrix metalloproteinase (MMP) activity was quantified by gelatin and collagen zymography. The viability of LG epithelial cells (EpCs), mesenchymal stem cells (MSCs), and endothelial cells (ECs) cultured on genipin-crosslinked dLG-HG was assessed after 10days, and EpC secretory activity was analyzed by β-hexosaminidase assay. The 0.5-mM genipin increased the stiffness of dLG-HG by about 46%, and concentrations > 0.25mM caused delayed cell-dependent biodegradation and reduced MMP activity. The viability of EpCs, MSCs, and ECs was not affected by genipin concentrations of up to 0.5mM after 10days. Moreover, up to 0.5-mM genipin did not negatively affect EpC secretory activity compared to control groups. A concentration of 0.5-mM genipin increased dLG-HG stiffness, and 0.25-mM genipin was sufficient to prevent MMP-dependent degradation. Importantly, concentrations of up to 0.5-mM genipin did not compromise the viability of LG-associated cells or the secretory activity of EpCs. Thus, crosslinking with genipin improves the properties of dLG-HG for use as a substrate in LG tissue engineering.

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.

Directed Assembly of Elastic Fibers via Coacervate Droplet Deposition on Electrospun Templates.

Elastic fibers provide critical elasticity to the arteries, lungs, and other organs. Elastic fiber assembly is a process where soluble tropoelastin is coacervated into liquid droplets, cross-linked, and deposited onto and into microfibrils. While much progress has been made in understanding the biology of this process, questions remain regarding the timing of interactions during assembly. Furthermore, it is unclear to what extent fibrous templates are needed to guide coacervate droplets into the correct architecture. The organization and shaping of coacervate droplets onto a fiber template have never been previously modeled or employed as a strategy for shaping elastin fiber materials. Using an in vitro system consisting of elastin-like polypeptides (ELPs), genipin cross-linker, electrospun polylactic-co-glycolic acid (PLGA) fibers, and tannic acid surface coatings for fibers, we explored ELP coacervation, cross-linking, and deposition onto fiber templates. We demonstrate that integration of coacervate droplets into a fibrous template is primarily influenced by two factors: (1) the balance of coacervation and cross-linking and (2) the surface energy of the fiber templates. The success of this integration affects the mechanical properties of the final fiber network. Our resulting membrane materials exhibit highly tunable morphologies and a range of elastic moduli (0.8-1.6 MPa) comparable to native elastic fibers.

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.

Enhancing the angiogenesis and osteogenesis of low elastic modulus Ti-Nb-Zr-Sn alloy surface through grafting growth factors-containing gelatin nanoparticles

The study aimed to apply a multiple surface modification for low elastic modulus Ti-24 % Nb-4 % Zr-8 % Sn (Ti2448) alloy, to enhance its biological properties for bone implant applications. We employed a combined sandblasting/acid etching/alkaline etching process to modify the Ti2448 alloy surfaces with mixed nano/submicron/micron-scaled pits and super wettability. We prepared gelatin nanoparticles (GNPs) loaded with growth factors: vascular endothelial growth factor/platelet-derived growth factor (VEGF/PDGF) and/or bone morphogenetic protein-2 (BMP-2). The growth factors-containing GNPs were then sequentially grafted onto the surface-modified Ti2448 alloy in a layered (single layer: BMP-2 or VEGF/PDGF; double layer: inner BMP-2 and outer VEGF/PDGF) or mixed BMP-2/VEGF/PDGF manner. Surface characterizations, including morphology, roughness, surface energy, and cytotoxicity, of Ti2448 alloy were analyzed. For biological response, we investigated the tube formation ability of human umbilical vein endothelial cells (HUVECs) and the cell responses (adhesion, proliferation, and mineralization) of human bone marrow mesenchymal stem cells (hMSCs). The results demonstrated the successful preparation of GNPs with 200∼300 nm particle size and their successful loading with the growth factors using natural crosslinker genipin. The growth factor-containing GNPs were effectively grafted onto the surface-modified hydrophilic Ti2448 alloy without significantly reducing its wettability (water contact angle remained below 40°). The results of the study showed that the GNPs-grafted Ti2448 alloy surfaces, with the GNPs loaded with VEGF/PDGF and/or BMP-2, exhibited enhanced biological properties. The GNPs-grafted surfaces were found to be non-toxic and capable of promoting tube formation of HUVECs, particularly when single VEGF/PDGF layer or double (inner BMP-2/ outer VEGF/PDGF) layer was loaded on GNPs (enhancing the cumulative tube length approximately 1.4 times that of no GNPs-grafted surfaces). Moreover, the GNPs-grafted surfaces enhanced the adhesion-related proteins and extracellular matrix mineralization of hMSCs, particularly with the loading of single VEGF/PDGF or mixed VEGF/PDGF/BMP-2 on GNPs (enhancing αvβ3 integrin patch number > 3.5 times, vinculin patch number > 2 times, and mineralization approximately 1.4 times). Based on these findings, we concluded that the proposed Ti2448 alloy surfaces, when grafted with VEGF/PDGF- and/or BMP-2-containing GNPs, have the potential for bone implant applications. The surface modifications on the Ti2448 alloy may promote angiogenesis in the early stage of bone repair (through VEGF/PDGF) and osteogenesis in the late stage (through BMP-2), thereby accelerating the overall bone healing process.

Fish oil encapsulation by genipin-crosslinked complex coacervation between gelatin and different anionic polysaccharides

Herein, a combination approach of bovine bone gelatin (BBG)/anionic polysaccharide (AP) complex coacervation with genipin crosslinking was developed to successfully fabricate fish oil-loaded genipin-crosslinked BBG/AP (fish oil@BBG-genipin-AP) powders. The genipin crosslinking process promoted the formation of larger fish oil@BBG-genipin-AP particles from the BBG/AP particles and fish oil@BBG/AP particles. AP types (gum Arabic, GA; sodium alginate, SA; sodium carboxymethyl cellulose, CMC) and concentrations significantly affected the critical pH to form complex coacervated particles, the powder physicochemical properties, the powder oxidative stability, and the powder in vitro digestion. The fish oil@BBG-genipin-CMC powder at the CMC preparation concentration of 17.5 mg/mL showed the highest loading capacity value (44.3% ± 0.8%), almost the highest encapsulation efficiency value (90.9% ± 0.7%), and the highest encapsulation yield (91.3% ± 2.0%). Due to the environmental pH change, the powders changed to emulsion droplets in the gastric phase and then released free fatty acids in the small intestinal phase. The highest FFA release percentages after the gastrointestinal phase (2 h in the gastric phase and 2 h in the small intestinal phase) were dependent on both AP types and their concentrations (GA: 64.8 ± 2.9%, 62.5 mg/mL; SA: 55.0 ± 3.1%, 15 mg/mL; and CMC: 48.9 ± 1.5%, 12.5 mg/mL). GA at a concentration of 62.5 mg/mL might be the best system to prepare fish oil@gelatin-genipin-anionic polysaccharide powders for fish oil digestion. This work provided useful information to develop oil-encapsulation powders for functional foods and understand the influences of wall materials on the fabrication and properties of powder foods.

Chitosan-Based Hierarchical Scaffolds Crosslinked with Genipin

Osteochondral defects present significant challenges for effective tissue regeneration due to the complex composition of bone and cartilage. To address this challenge, this study presents the fabrication of hierarchical scaffolds combining chitosan/β-tricalcium phosphate (β-TCP) to simulate a bone-like layer, interconnected with a silk fibroin layer to mimic cartilage, thus replicating the cartilage-like layer to mimic the native osteochondral tissue architecture. The scaffolds were produced by freeze-drying and then crosslinking with genipin. They have a crosslinking degree of up to 24%, which promotes a structural rearrangement and improved connection between the different layers. Micro-CT analysis demonstrated that the structures have distinct porosity values on their top layer (up to 84%), interface (up to 65%), and bottom layer (up to 77%) and are dependent on the concentration of β-tricalcium phosphate used. Both layers were confirmed to be clearly defined by the distribution of the components throughout the constructs, showing adequate mechanical properties for biomedical use. The scaffolds exhibited lower weight loss (up to 7%, 15 days) after enzymatic degradation due to the combined effects of genipin crosslinking and β-TCP incorporation. In vitro studies showed that the constructs supported ATDC5 chondrocyte-like cells and MC3T3 osteoblast-like cells in duo culture conditions, providing a suitable environment for cell adhesion and proliferation for up to 14 days. Overall, the physicochemical properties and biological results of the developed chitosan/β-tricalcium phosphate/silk fibroin bilayered scaffolds suggest that they may be potential candidates for osteochondral tissue strategies.

A Sterile, Injectable, and Robust Sericin Hydrogel Prepared by Degraded Sericin.

The application of sericin hydrogels is limited mainly due to their poor mechanical strength, tendency to be brittle and inconvenient sterilization. To address these challenges, a sericin hydrogel exhibiting outstanding physical and chemical properties along with cytocompatibility was prepared through crosslinking genipin with degraded sericin extracted from fibroin deficient silkworm cocoons by the high temperature and pressure method. Our reported sericin hydrogels possess good elasticity, injectability, and robust behaviors. The 8% sericin hydrogel can smoothly pass through a 16 G needle. While the 12% sericin hydrogel remains intact until its compression ratio reaches 70%, accompanied by a compression strength of 674 kPa. 12% sericin hydrogel produce a maximum stretch of 740%, with breaking strength and tensile modulus of 375 kPa and 477 kPa respectively. Besides that, the hydrogel system demonstrated remarkable cell-adhesive capabilities, effectively promoting cell attachment and, proliferation. Moreover, the swelling and degradation behaviors of the hydrogels are pH responsiveness. Sericin hydrogel releases drugs in a sustained manner. Furthermore, this study addresses the challenge of sterilizing sericin hydrogels (sterilization will inevitably lead to the destruction of their structures). In addition, it challenges the prior notion that sericin extracted under high temperature and pressure is difficult to directly cross-linked into a stable hydrogel. This developed hydrogel system in this study holds promise to be a new multifunctional platform expanding the application area scope of sericin.

Biomimetic cryogel promotes the repair of osteoporotic bone defects through altering the ROS niche via down-regulating the ROMO1

Osteoporosis is a systemic bone disease that is prone to fractures due to decreased bone density and bone quality, and delayed union or nonunion often occurs in osteoporotic fractures. Therefore, it is particularly important to develop tissue engineering materials to promote osteoporotic fracture healing. In this study, a series of biomimetic cryogels prepared from the decellularized extracellular matrix (dECM), methacrylate gelatin (GelMA), and carboxymethyl chitosan (CMCS) via unidirectional freezing, photo- and genipin crosslinking were applied for the regeneration of osteoporotic fractures. Specifically, dECM extracted from normal or osteoporotic rats was applied for the preparation of the cryogels, named as GC-Normal dECM or GC-OVX dECM, respectively. It was verified that the GC-Normal dECM demonstrated superior performance in promoting the proliferation of BMSCs isolated from osteoporotic rats (OVX-BMSCs), and the differentiation of OVX-BMSCs into osteoblasts both in vitro and in vivo. RNA sequencing and further verifications confirmed that GC-Normal dECM cryogel could scavenge the intracellular reactive oxygen species (ROS) in OVX-BMSCs to accelerate the regeneration of osteoporotic fracture by down-regulating the reactive oxygen species modulator 1 (Romo1). The results indicated that by regulating the ROS niche of OVX-BMSCs, biomimetic the GC-Normal dECM cryogel was expected to be a clinical candidate for repairing osteoporotic bone defects.

Improved Physiochemical Properties of Chitosan@PCL Nerve Conduits by Natural Molecule Crosslinking.

Nerve conduits may represent a valuable alternative to autograft for the regeneration of long-gap damages. However, no NCs have currently reached market approval for the regeneration of limiting gap lesions, which still represents the very bottleneck of this technology. In recent years, a strong effort has been made to envision an engineered graft to tackle this issue. In our recent work, we presented a novel design of porous/3D-printed chitosan/poly-ε-caprolactone conduits, coupling freeze drying and additive manufacturing technologies to yield conduits with good structural properties. In this work, we studied genipin crosslinking as strategy to improve the physiochemical properties of our conduit. Genipin is a natural molecule with very low toxicity that has been used to crosslink chitosan porous matrix by binding the primary amino group of chitosan chains. Our characterization evidenced a stabilizing effect of genipin crosslinking towards the chitosan matrix, with reported modified porosity and ameliorated mechanical properties. Given the reported results, this method has the potential to improve the performance of our conduits for the regeneration of long-gap nerve injuries.

An increase in Wharton's jelly membrane osteocompatibility by a genipin-cross-link

Wharton's Jelly (WJ) has attracted significant interest in the field of tissue healing thanks to its biological properties, including antibacterial activity and immunomodulation. However, due to the fast degradation and poor mechanical behavior in biological environment, its application in bone regeneration is compromised. Here, we proposed to use genipin as an efficient cross-linking agent to significantly improve the elasticity and the enzymatical stability of the WJ matrix. The degree of cross-linking, linear elastic moduli, and collagenase resistance varied over a wide range depending on genipin concentration. Furthermore, our results highlighted that an increase in genipin concentration led to a decreased surface wettability, therefore impairing cell attachment and proliferation. The genipin cross-linking prevented rapid in vitro and in vivo degradation, but led to an adverse host reaction and calcification. When implanted in the parietal bone defect, a limited parietal bone regeneration to the dura was observed. We conclude that genipin-cross-linked WJ is a versatile medical device however, a careful selection is required with regards to the genipin concentration.

Optimization of chitosan adhesive properties by means of genipin crosslinking

ABSTRACT Chitosan is a polysaccharide resulting from chitin deacetylation. It has demonstrated interesting characteristics in the field of adhesion but loses most of its adhesive resistance in moist environment. A chemical crosslinking of chitosan with genipin has been implemented to improve its adhesive properties in the presence of water. The study of crosslinking kinetics using rheology revealed its dependency on crosslinking rate and chitosan concentration. The capacity of the chitosan-genipin systems for water absorption has been quantified as their Free Swelling Capacity. The cross-linkage of chitosan by genipin (1% w/w) decreased significantly its ability for water absorption. Finally, the adhesive strength of several chitosans be they supplemented or not with genipin and/or glycerol used as plasticizer have been performed on Thick Adherent Shear Test samples of beech wood according to standard EN 204:2016 and EN 205:2016. The chitosan adhesive formulation containing genipin and glycerol exhibits the better adhesive properties on wood and presents higher water resistance compared to the native chitosan formulation.

Reaction kinetics of chitosan nanogels crosslinked by genipin

Crosslinking of chitosan chains in dilute solution by natural crosslinker genipin leads to biocompatible nanogels. Here we investigated the reaction kinetics between chitosan and genipin in a 200 mM acetate buffer at 37 °C, and the structural and conformational evolutions of the nanogels during the crosslinking reaction by multi detection asymmetric flow field-flow fractionation (AF4). Upon crosslinking by genipin, the z-average hydrodynamic radius Rhz of the chitosan chains increased from 26 nm to 130 nm, while the weight average molar mass Mw increased from 2.0 × 105 g/mol to 1.8 × 107 g/mol. The crosslinking reaction appeared to be first-order and size-dependent. In particular, the intrachain crosslinking reaction was preferentially for nanogels having the larger size, leading to formation of branched chains/nanogels having a wide range of molar masses between 106 and 108 g/mol but a similar radius of gyration Rg ∼ 40 nm. For the largest nanogel fractions with M > 2.0 × 108 g/mol, both Rg and Rh showed a scaling relation with exponent 1/3 and a structure parameter Rg/Rh = 0.74, as expected for the hard sphere particle. The reaction was accompanied by a reduction of charge density and an increase in hydrophobicity of chitosan nanogels, which plays a key role in the formation of uniform size nanogels with chain density ρ(Rh) up to 0.45 g/cm3.

Curcumin-Laden Crosslinked Chitosan–PVA Films: The Synergistic Impact of Genipin and Curcumin on Accelerating Wound Closure

AbstractWound dressings play a critical role in healing by maintaining a moist environment and protecting against infection. Here, we fabricate crosslinked films of chitosan–polyvinyl alcohol–genipin–curcumin to investigate the synergistic wound-healing effect of genipin and curcumin. The chemical bonding, morphology, strength, water retention capacity, curcumin release characteristics, and cytotoxicity of the films were investigated. The results showed a 192% increase in tensile strength and good water retention, and the infrared analysis and scanning electron microscopy micrographs confirmed genipin crosslinking and the presence of curcumin in the films, whose morphology was uniform and continuous. The curcumin-loaded films were found to have insignificant cytotoxicity against 3T3 fibroblast cells, proving their biocompatibility. The curcumin dissolution tests determined the amount of curcumin released from the films with time, and assessed the release characteristics to be sustained at all pH levels. In vivo trials were carried out on rodents to evaluate the wound-healing effects of the films, and the results showed that the curcumin-incorporated crosslinked films accelerated wound healing compared to the uncrosslinked and curcumin-absent films, confirming their effectiveness for wound healing.