Biomaterial-based combinatorial approach of aescin-comprised zein-coated gelatin nanoparticles alleviates synovial inflammation in experimental inflammatory arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease that mostly affects joints. Although RA therapy has made significant progress, difficulties including extensive medication metabolism and its quick clearance result in its inadequate bioavailability. The anti-inflammatory effect of zein was reported with other medications, but it has certain limitations. There are reports on the anti-oxidant and anti-inflammatory effect of aescin, which exhibits low bioavailability for the treatment of rheumatoid arthritis. Also, the combinatorial effect of zein with other effective drug delivery systems is still under investigation for the treatment of experimental collagen-induced rheumatoid arthritis. The focus of this study was to formulate and define the characteristics of zein-coated gelatin nanoparticles encapsulated with aescin (Ze@Aes-GNPs) and to assess and contrast the therapeutic effectiveness of Ze@Aes-GNPs towards collagen-induced RA in Wistar rats. Nanoprecipitation and the layer-by-layer coating process were used to fabricate Ze@Aes-GNPs and their hydrodynamic diameter was determined to be 182 nm. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to further validate the size, shape, and surface morphology of Ze@Aes-GNPs. When tested against foreskin fibroblasts (BJ), these nanoparticles demonstrated significantly high cytocompatibility. Both Aes and Ze@Aes-GNPs were effective in treating arthritis, as shown by the decreased edoema, erythema, and swelling of the joints, between which Ze@Aes-GNPs were more effective. Further, it was demonstrated that Aes and Ze@Aes-GNPs reduced the levels of oxidative stress (articular elastase, lipid peroxidation, catalase, superoxide dismutase and nitric oxide) and inflammatory indicators (TNF-α, IL-1ß and myeloperoxidase). The histopathology findings further demonstrated that Ze@Aes-GNPs considerably reduced the infiltration of inflammatory cells at the ankle joint cartilage compared to Aes. Additionally, immunohistochemistry examination showed that treatment with Ze@Aes-GNPs suppressed the expression of pro-inflammatory markers (COX-2 and IL-6) while increasing the expression of SOD1. In summary, the experiments indicated that Aes and Ze@Aes-GNPs lowered the severity of arthritis, and critically, Ze@Aes-GNPs showed better effectiveness in comparison to Aes. This suppression of oxidative stress and inflammation was likely driven by Aes and Ze@Aes-GNPs.

Enzymatically produced acylglycerol and glycerin monostearate additives improved the characteristics of gelatin-stabilized omega-3 emulsions and microcapsules.

The impacts of enzymatically produced acylglycerol and glycerin monostearate on the characteristics of gelatin-stabilized omega-3 emulsions and microcapsules were investigated. Tuna oil was enzymatically produced and the resulting acylglycerol was mixed with tuna oil at 12.5% (w/w) to prepare a novel oil phase. This oil phase was stabilized by gelatin to prepare oil-in-water emulsions and subsequent microcapsules via complex coacervation. The tuna oil with glycerin monostearate (GMS) at 1 and 2% (w/w) were used as controls. Results showed that both acylglycerol and GMS significantly reduced the emulsion droplet size and zeta potential, while increasing the viscoelasticity and stability. The diacylglycerol/monoacylglycerol were involved in the oil/water interfacial layer formation by lowering interfacial tension and increasing droplet surface hydrophobicity. Overall, the changed emulsion properties promoted the complex coacervation and contributed to the formation of microcapsules with improved oxidative stability. Therefore, enzymatically produced acylglycerol can develop high-quality stable omega-3 microencapsulated novel food ingredients.

[Effects of gelatin methacrylate anhydride hydrogel loaded with small extracellular vesicles derived from human umbilical cord mesenchymal stem cells in the treatment of full-thickness skin defect wounds in mice].

Objective: To investigate the effects of gelatin methacrylate anhydride (GelMA) hydrogel loaded with small extracellular vesicles derived from human umbilical cord mesenchymal stem cells (hUCMSCs-sEVs) in the treatment of full-thickness skin defect wounds in mice. Methods: This study was an experimental study. hUCMSCs-sEVs were extracted by ultracentrifugation, their morphology was observed through transmission electron microscope, and the expression of CD9, CD63, tumor susceptibility gene 101 (TSG101), and calnexin was detected by Western blotting. The human umbilical vein endothelial cells (HUVECs), the 3rd and 4th passages of human epidermal keratinocytes (HEKs) and human dermal fibroblasts (HDFs) were all divided into blank control group (routinely cultured) and hUCMSC-sEV group (cultured with the cell supernatant containing hUCMSCs-sEVs). The cell scratch test was performed and the cell migration rates at 6, 12, and 24 h after scratching were calculated, the cell Transwell assay was performed and the number of migration cells at 12 h after culture was calculated, and the proportion of proliferating cells was detected by 5-acetylidene-2'-deoxyuridine and Hoechst staining at 24 h after culture, with sample numbers being all 3. The simple GelMA hydrogel and the GelMA hydrogel loaded with hUCMSCs-sEVs (hereinafter referred to as hUCMSC-sEV/GelMA hydrogel) were prepared. Then the micromorphology of 2 kinds of hydrogels was observed under scanning electron microscope, the distribution of hUCMSCs-sEVs was observed by laser scanning confocal microscope, and the cumulative release rates of hUCMSCs-sEVs at 0 (immediately), 2, 4, 6, 8, 10, and 12 d after soaking hUCMSC-sEV/GelMA hydrogel in phosphate buffer solution (PBS) were measured and calculated by protein colorimetric quantification (n=3). Twenty-four 6-week-old male C57BL/6J mice were divided into PBS group, hUCMSC-sEV alone group, GelMA hydrogel alone group, and hUCMSC-sEV/GelMA hydrogel group according to the random number table, with 6 mice in each group, and after the full-thickness skin defect wounds on the back of mice in each group were produced, the wounds were performed with PBS injection, hUCMSC-sEV suspenson injection, simple GelMA coverage, and hUCMSC-sEV/GelMA hydrogel coverage, respectively. Wound healing was observed on post injury day (PID) 0 (immediately), 4, 8, and 12, and the wound healing rates on PID 4, 8, and 12 were calculated, and the wound tissue was collected on PID 12 for hematoxylin-eosin staining to observe the structure of new tissue, with sample numbers being both 6. Results: The extracted hUCMSCs-sEVs showed a cup-shaped structure and expressed CD9, CD63, and TSG101, but barely expressed calnexin. At 6, 12, and 24 h after scratching, the migration rates of HEKs (with t values of 25.94, 20.98, and 20.04, respectively), HDFs (with t values of 3.18, 5.68, and 4.28, respectively), and HUVECs (with t values of 4.32, 19.33, and 4.00, respectively) in hUCMSC-sEV group were significantly higher than those in blank control group (P<0.05). At 12 h after culture, the numbers of migrated HEKs, HDFs, and HUVECs in hUCMSC-sEV group were 550±23, 235±9, and 856±35, respectively, which were significantly higher than 188±14, 97±6, and 370±32 in blank control group (with t values of 22.95, 23.13, and 17.84, respectively, P<0.05). At 24 h after culture, the proportions of proliferating cells of HEKs, HDFs, and HUVECs in hUCMSC-sEV group were significantly higher than those in blank control group (with t values of 22.00, 13.82, and 32.32, respectively, P<0.05). The inside of simple GelMA hydrogel showed a loose and porous sponge-like structure, and hUCMSCs-sEVs was not observed in it. The hUCMSC-sEV/GelMA hydrogel had the same sponge-like structure, and hUCMSCs-sEVs were uniformly distributed in clumps. The cumulative release rate curve of hUCMSCs-sEVs from hUCMSC-sEV/GelMA hydrogel tended to plateau at 2 d after soaking, and the cumulative release rate of hUCMSCs-sEVs was (59.2±1.8)% at 12 d after soaking. From PID 0 to 12, the wound areas of mice in the 4 groups gradually decreased. On PID 4, 8, and 12, the wound healing rates of mice in hUCMSC-sEV/GelMA hydrogel group were significantly higher than those in the other 3 groups (P<0.05); the wound healing rates of mice in GelMA hydrogel alone group and hUCMSC-sEV alone group were significantly higher than those in PBS group (P<0.05). On PID 8 and 12, the wound healing rates of mice in hUCMSC-sEV alone group were significantly higher than those in GelMA hydrogel alone group (P<0.05). On PID 12, the wounds of mice in hUCMSC-sEV/GelMA hydrogel group showed the best wound epithelization, loose and orderly arrangement of dermal collagen, and the least number of inflammatory cells, while the dense arrangement of dermal collagen and varying degrees of inflammatory cell infiltration were observed in the wounds of mice in the other 3 groups. Conclusions: hUCMSCs-sEVs can promote the migration and proliferation of HEKs, HDFs, and HUVECs which are related to skin wound healing, and slowly release in GelMA hydrogel. The hUCMSC-sEV/GelMA hydrogel as a wound dressing can significantly improve the healing speed of full-thickness skin defect wounds in mice.

Fully synthetic, tunable poly(α-amino acids) as the base of bioinks curable by visible light.

Bioinks play a crucial role in tissue engineering, influencing mechanical and chemical properties of the printed scaffold as well as the behavior of encapsulated cells. Recently, there has been a shift from animal origin materials to their synthetic alternatives. In this context, we present here bioinks based on fully synthetic and biodegradable poly(α,L-amino acids) (PolyAA) as an alternative to animal-based gelatin methacrylate (Gel-Ma) bioinks. Additionally, we first reported the possibility of the visible light photoinitiated incorporation of the bifunctional cell adhesive RGD peptide into the PolyAA hydrogel matrix. The obtained hydrogels are shown to be cytocompatible, and their mechanical properties closely resemble those of gelatin methacrylate-based scaffolds. Moreover, combining the unique properties of PolyAA-based bioinks, the photocrosslinking strategy, and the use of droplet-based printing allows the printing of constructs with high shape fidelity and structural integrity from low-viscosity bioinks without using any sacrificial components. Overall, presented PolyAA-based materials are a promising and versatile toolbox that extends the range of bioinks for droplet bioprinting.

Embosphere microspheres size for bronchial artery embolization in patients with hemoptysis caused by bronchiectasis: a retrospective comparative analysis of 500-750 versus 700-900 µm microspheres.

BACKGROUND: Bronchial arterial embolization (BAE) has been accepted as an effective treatment for bronchiectasis-related hemoptysis. However, rare clinical trials compare different sizes of specific embolic agents. This study aims to evaluate whether different Embosphere microsphere sizes change the outcome of BAE. METHODS: A retrospective review was conducted on consecutive patients with bronchiectatic hemoptysis who were scheduled to undergo BAE treatment during a period from January 2018 to December 2022. The patients received BAE using microspheres of different sizes: group A patients were treated with 500-750 µm microspheres, and group B patients were treated with 700-900 µm microspheres. The cost of embolic microspheres (Chinese Yuan, CNY), duration of hospitalization, complications, and hemoptysis-free survival were compared between patients in group A and those in group B. A Cox proportional hazards regression model was used to identify predictors of recurrent hemoptysis. RESULTS: Median follow-up was 30.2 months (range, 20.3-56.5 months). The final analysis included a total of 112 patients (49-77 years of age; 45 men). The patients were divided into two groups: group A (N = 68), which received 500-750 µm Embosphere microspheres, and group B (N = 44), which received 700-900 µm Embosphere microspheres. Except for the cost of embolic microspheres(group A,5314.8 + 1301.5 CNY; group B, 3644.5 + 1192.3 CNY; p = 0.042), there were no statistically significant differences in duration of hospitalization (group A,7.2 + 1.4 days; group B, 8 + 2.4days; p = 0.550), hemoptysis-free survival (group A, 1-year, 2-year, 3-year, 85.9%, 75.8%, 62.9%; group B, 1-year, 2-year, 3-year, 88.4%, 81.2%,59.4%;P = 0.060), and complications(group A,26.5%; group B, 38.6%; p = 0.175) between the two groups. No major complications were observed. The multivariate analysis results revealed that the presence of cystic bronchiectasis (OR 1.61, 95% CI 1.12-2.83; P = 0.001) and systemic arterial-pulmonary shunts (SPSs) (OR 1.52, 95% CI 1.10-2.72; P = 0.028) were independent risk factors for recurrent bleeding. CONCLUSIONS: For the treatment of BAE in patients with bronchiectasis-related hemoptysis, 500-750 µm diameter Embosphere microspheres have a similar efficacy and safety profile compared to 700-900 µm diameter Embosphere microspheres, especially for those without SPSs or cystic bronchiectasis. Furthermore, the utilization of large-sized (700-900 µm) Embosphere microspheres is associated with the reduced cost of an embolic agent.

Utilization of Lumpfish (Cyclopterus lumpus) Skin as a Source for Gelatine Extraction Using Acid Hydrolysis.

Lumpfish (Cyclopterus lumpus) is an underutilized marine resource that is currently only being exploited for roe. Lumpfish skin was pre-treated with alkali (0.1M NaOH) and acid (0.1M HCl) at a skin to chemical ratio of 1:10 for 24 h at 5 °C to remove non-collagenous proteins and minerals. The pre-treated skin was washed, and gelatine was extracted with 0.1M of acetic acid at three different ratios (1:5, 1:10, and 1:15), time (12,18, and 24 h), and temperature combinations (12, 28, and 24 °C). The highest total extraction yield (>40%) was obtained with combinations of extraction ratios of 1:15 and 1:10 with a longer time (24 h) and higher temperature (18-24 °C). The highest gelatine content was obtained with an extraction period of 24 h and ratio of 1:10 (>80%). SDS-PAGE analysis confirmed the presence of type-I collagen. A rheological evaluation indicated melting and gelling temperatures, gel strength, and viscosity properties comparable to existing cold-water gelatine sources.

Creation of Porous, Perfusable Microtubular Networks for Improved Cell Viability in Volumetric Hydrogels.

The creation of large, volumetric tissue-engineered constructs has long been hindered due to the lack of effective vascularization strategies. Recently, 3D printing has emerged as a viable approach to creating vascular structures; however, its application is limited. Here, we present a simple and controllable technique to produce porous, free-standing, perfusable tubular networks from sacrificial templates of polyelectrolyte complex and coatings of salt-containing citrate-based elastomer poly(1,8-octanediol-co-citrate) (POC). As demonstrated, fully perfusable and interconnected POC tubular networks with channel diameters ranging from 100 to 400 µm were created. Incorporating NaCl particulates into the POC coating enabled the formation of micropores (∼19 µm in diameter) in the tubular wall upon particulate leaching to increase the cross-wall fluid transport. Casting and cross-linking gelatin methacrylate (GelMA) suspended with human osteoblasts over the free-standing porous POC tubular networks led to the fabrication of 3D cell-encapsulated constructs. Compared to the constructs without POC tubular networks, those with either solid or porous wall tubular networks exhibited a significant increase in cell viability and proliferation along with healthy cell morphology, particularly those with porous networks. Taken together, the sacrificial template-assisted approach is effective to fabricate tubular networks with controllable channel diameter and patency, which can be easily incorporated into cell-encapsulated hydrogels or used as tissue-engineering scaffolds to improve cell viability.

Antibiofilm activity of polyethylene glycol-quercetin nanoparticles-loaded gelatin-N,O-carboxymethyl chitosan composite nanogels against Staphylococcus epidermidis.

BACKGROUND: Biofilms, such as those from Staphylococcus epidermidis, are generally insensitive to traditional antimicrobial agents, making it difficult to inhibit their formation. Although quercetin has excellent antibiofilm effects, its clinical applications are limited by the lack of sustained and targeted release at the site of S. epidermidis infection. OBJECTIVES: Polyethylene glycol-quercetin nanoparticles (PQ-NPs)-loaded gelatin-N,O-carboxymethyl chitosan (N,O-CMCS) composite nanogels were prepared and assessed for the on-demand release potential for reducing S. epidermidis biofilm formation. METHODS: The formation mechanism, physicochemical characterization, and antibiofilm activity of PQ-nanogels against S. epidermidis were studied. RESULTS: Physicochemical characterization confirmed that PQ-nanogels had been prepared by the electrostatic interactions between gelatin and N,O-CMCS with sodium tripolyphosphate. The PQ-nanogels exhibited obvious pH and gelatinase-responsive to achieve on-demand release in the micro-environment (pH 5.5 and gelatinase) of S. epidermidis. In addition, PQ-nanogels had excellent antibiofilm activity, and the potential antibiofilm mechanism may enhance its antibiofilm activity by reducing its relative biofilm formation, surface hydrophobicity, exopolysaccharides production, and eDNA production. CONCLUSIONS: This study will guide the development of the dual responsiveness (pH and gelatinase) of nanogels to achieve on-demand release for reducing S. epidermidis biofilm formation.

Modulating peppermint oil flavor release properties of emulsion-filled protein gels: Impact of cross-linking method and matrix composition.

For some food applications, it is desirable to control the flavor release profiles of volatile flavor compounds. In this study, the effects of crosslinking method and protein composition on the flavor release properties of emulsion-filled protein hydrogels were explored, using peppermint essential oil as a model volatile compound. Emulsion-filled protein gels with different properties were prepared using different crosslinking methods and gelatin concentrations. Flavor release from the emulsion gels was then monitored using an electronic nose, gas chromatography-mass spectrometry (GC-MS), and sensory evaluation. Enzyme-crosslinked gels had greater hardness and storage modulus than heat-crosslinked ones. The hardness and storage modulus of the gels increased with increasing gelatin concentration. For similar gel compositions, flavor release and sensory perception were faster from the heat-crosslinked gels than the enzyme-crosslinked ones. For the same crosslinking method, flavor release and perception decreased with increasing gelatin concentration, which was attributed to retardation of flavor diffusion through the hydrogel matrix. Overall, this study shows that the release of hydrophobic aromatic substances can be modulated by controlling the composition and crosslinking of protein hydrogels, which may be useful for certain food applications.

Production of Antioxidant, Angiotensin-Converting Enzyme Inhibitory and Osteogenic Gelatin Hydrolysate from Labeo rohita Swim Bladder.

Optimization of antioxidants and angiotensin-converting enzyme (ACE) inhibitory potential gelatin hydrolysate production from Labeo rohita (rohu) swim bladder (SBGH) by alcalase using central composite design (CCD) of response surface methodology (RSM) was investigated. The maximum degree of hydrolysis (DH), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS), total antioxidants (TAO), and ACE inhibitory activity were achieved at 0.1:1.0 (w/w) enzyme to substrate ratio, 61 °C hydrolysis temperature, and 94-min hydrolysis time. The resulting SBGH obtained at 19.92% DH exhibited the DPPH (24.28 µM TE/mg protein), ABTS (34.47 µM TE/mg protein), TAO (12.01 µg AAE/mg protein), and ACE inhibitory (4.91 µg/mg protein) activity. Furthermore, SBGH at 100 µg/ml displayed osteogenic property without any toxic effects on MC3T3-E1 cells. Besides, the protein content of rohu swim bladder gelatin (SBG) and SBGH was 93.68% and 94.98%, respectively. Both SBG and SBGH were rich in glycine, proline, glutamic acid, alanine, arginine, and hydroxyproline amino acids. Therefore, SBGH could be an effective nutraceutical in functional food development.

3D printed cinnamon essential oil/banana peel carbon dots loaded corn starch/gelatin bilayer film with enhanced functionality for food packaging application.

Using 3D printing technology, a gelatin-polyvinyl alcohol­carbon dots (GPC) layer+corn starch-polyvinyl alcohol-cinnamon essential oil (CPC) layer active bilayer film with an external barrier function and an internal controlled-release effect was successfully produced for food preservation. The GPC film was provided with potent antioxidant and UV blocking properties by the banana peel carbon dots (CDs). The cinnamon essential oil (CEO) had the strongest interaction with the film matrix at 3% (w/w), causing the CPC film having the lowest surface wettability, good integrity, and lowest crystallinity. The CEO's stability and releasing effectiveness were greatly enhanced by the creation of a bilayer film. At 60% filling rate of the CPC layer, the bilayer film showed the highest CEO retention after drying and the best CEO release performance. Finally, the created active bilayer film was found to significantly improve the sensory quality stability of the spicy essential oil microcapsule powders. It also successfully extended the mangoes' shelf life by delaying browning and rot.

Exosome-coated oxygen nanobubble-laden hydrogel augments intracellular delivery of exosomes for enhanced wound healing.

Wound healing is an obvious clinical concern that can be hindered by inadequate angiogenesis, inflammation, and chronic hypoxia. While exosomes derived from adipose tissue-derived stem cells have shown promise in accelerating healing by carrying therapeutic growth factors and microRNAs, intracellular cargo delivery is compromised in hypoxic tissues due to activated hypoxia-induced endocytic recycling. To address this challenge, we have developed a strategy to coat oxygen nanobubbles with exosomes and incorporate them into a polyvinyl alcohol/gelatin hybrid hydrogel. This approach not only alleviates wound hypoxia but also offers an efficient means of delivering exosome-coated nanoparticles in hypoxic conditions. The self-healing properties of the hydrogel, along with its component, gelatin, aids in hemostasis, while its crosslinking bonds facilitate hydrogen peroxide decomposition, to ameliorate wound inflammation. Here, we show the potential of this multifunctional hydrogel for enhanced healing, promoting angiogenesis, facilitating exosome delivery, mitigating hypoxia, and inhibiting inflammation in a male rat full-thickness wound model.

Injectable Nanoengineered Adhesive Hydrogel for Treating Enterocutaneous Fistulas.

Enterocutaneous fistula (ECF) is a severe medical condition where an abnormal connection forms between the gastrointestinal tract and skin. ECFs are, in most cases, a result of surgical complications such as missed enterotomies or anastomotic leaks. The constant leakage of enteric and fecal contents from the fistula site leads to skin breakdown and increases the risk of infection. Despite advances in surgical techniques and postoperative management, ECF accounts for significant mortality rates, estimated between 15-20%, and causes debilitating morbidity. Therefore, there is a critical need for a simple and effective method to seal and heal ECF. Injectable hydrogels with combined properties of robust mechanical properties and cell infiltration/proliferation have the potential to block and heal ECF. Herein, we report the development of an injectable nanoengineered adhesive hydrogel (INAH) composed of a synthetic nanosilicate (Laponite®) and a gelatin-dopamine conjugate for treating ECF. The hydrogel undergoes fast cross-linking using a co-injection method, resulting in a matrix with improved mechanical and adhesive properties. INAH demonstrates appreciable blood clotting abilities and is cytocompatible with fibroblasts. The adhesive properties of the hydrogel are demonstrated in ex vivo adhesion models with skin and arteries, where the volume stability in the hydrated internal environment facilitates maintaining strong adhesion. In vivo assessments reveal that the INAH is biocompatible, supporting cell infiltration and extracellular matrix deposition while not forming fibrotic tissue. These findings suggest that this INAH holds promising translational potential for sealing and healing ECF.

Belugas (Delphinapterus leucas) create facial displays during social interactions by changing the shape of their melons.

Beluga whales are considered unique among odontocetes in their ability to visibly alter the appearance of their head by changing the shape of the melon, but only anecdotal observations are available to evaluate the use or potential function of these melon shapes. This study of belugas in professionally managed care aimed to establish an ethogram for the repertoire of categorizable melon shapes and then evaluate their potential function as intentional communication signals by determining if they were produced and elaborated during social interactions of varying behavioral contexts while in the line of sight of a recipient. Five different melon shapes were reliably identified in video observations of the primary study population (n = 4) and externally validated in a second aquarium population (n = 51). Among the 2570 melon shapes observed from the primary study subjects, melon shapes occurred 34 × more frequently during social interactions (1.72 per minute) than outside of social interactions (0.05 per minute). Melon shapes occurring during social interactions were performed within the line of sight of a recipient 93.6% of the time. The frequency of occurrence of the different melon shapes varied across behavioral contexts. Elaboration of melon shapes through extended duration and the occurrence of concurrent open mouth displays varied by shape type and across behavioral contexts. Melon shapes seem to function as visual displays, with some characteristics of intentional communication. This ability could yield adaptive benefits to belugas, given their complex social structure and hypothesized mating system that emphasizes pre-copulatory female mate choice.

Synthesis and Characterization of Amine and Aldehyde-Containing Copolymers for Enzymatic Crosslinking of Gelatine.

In tissue engineering (TE), the support structure (scaffold) plays a key role necessary for cell adhesion and proliferation. The protein constituents of the extracellular matrix (ECM), such as collagen, its derivative gelatine, and elastin, are the most attractive materials as possible scaffolds. To improve the modest mechanical properties of gelatine, a strategy consists of crosslinking it, as naturally occurs for collagen, which is stiffened by the oxidative action of lysyl oxidase (LO). Here, a novel protocol to crosslink gelatine has been developed, not using the commonly employed crosslinkers, but based on the formation of imine bonds or on aldolic condensation reactions occurring between gelatine and properly synthesized copolymers containing amine residues via LO-mediated oxidation. Particularly, we first synthesized and characterized an amino butyl styrene monomer (5), its copolymers with dimethylacrylamide (DMAA), and its terpolymer with DMAA and acrylic acid (AA). Three acryloyl amidoamine monomers (11a-c) and their copolymers with DMAA were then prepared. A methacrolein (MA)/DMAA copolymer already possessing the needed aldehyde groups was finally developed to investigate the relevance of LO in the crosslinking process. Oxidation tests of amine copolymers with LO were performed to identify the best substrates to be used in experiments of gelatine reticulation. Copolymers obtained with 5, 11b, and 11c were excellent substrates for LO and were employed with MA/DMAA copolymers in gelatine crosslinking tests in different conditions. Among the amine-containing copolymers, that obtained with 5 (CP5/DMMA-43.1) afforded a material (M21) with the highest crosslinking percentage (71%). Cytotoxicity experiments carried out on two cell lines (IMR-32 and SH SY5Y) with the analogous (P5) of the synthetic constituent of M21 (CP5/DMAA) had evidenced no significant reduction in cell viability, but proliferation promotion, thus establishing the biocompatibility of M21 and the possibility to develop it as a new scaffold for TE, upon further investigations.

Enhanced antibacterial activity of porous chitosan-based hydrogels crosslinked with gelatin and metal ions.

Addressing the increasing drug resistance in pathogenic microbes, a significant threat to public health, calls for the development of innovative antibacterial agents with versatile capabilities. To enhance the antimicrobial activity of non-toxic biomaterials in this regard, this study focuses on novel, cost-effective chitosan (CS)-based hydrogels, crosslinked using gelatin (GEL), formaldehyde, and metallic salts (Ag+, Cu2+, and Zn2+). These hydrogels are formed by mixing CS and GEL with formaldehyde, creating iminium ion crosslinks with metallic salts without hazardous crosslinkers. Characterization techniques like FTIR, XRD, FESEM, EDX, and rheological tests were employed. FTIR analysis showed metal ions binding to amino and hydroxyl groups on CS, enhancing hydrogelation. FESEM revealed that freeze-dried hydrogels possess a crosslinked, porous structure influenced by various metal ions. Antibacterial testing against gram-negative and gram-positive bacteria demonstrated significant bacterial growth inhibition. CS-based hydrogels containing metal ions showed reduced MIC and MBC values against Staphylococcus aureus (0.5, 8, 16 µg/mL) and Escherichia coli (1, 16, 8 µg/mL) for CS-g-GEL-Ag+, CS-g-GEL-Cu2+, and CS-g-GEL-Zn2+. MTT assay results confirmed high biocompatibility (84.27%, 85.24%, 84.96% viability at 10 µg/mL) for CS-based hydrogels towards HFF-1 cells over 48 h. Therefore, due to their non-toxic nature, these CS hydrogels are promising for antibacterial applications.

Evaluation of gelatin bloom strength on gelatin methacryloyl hydrogel properties.

Gelatin methacryloyl (GelMA) hydrogels are widely used for a variety of tissue engineering applications. The properties of gelatin can affect the mechanical properties of gelatin gels; however, the role of gelatin properties such as bloom strength on GelMA hydrogels has not yet been explored. Bloom strength is a food industry standard for describing the quality of gelatin, where higher bloom strength is associated with higher gelatin molecular weight. Here, we evaluate the role of bloom strength on GelMA hydrogel mechanical properties. We determined that both bloom strength of gelatin and weight percent of GelMA influenced both stiffness and viscoelastic ratio; however, only bloom strength affected diffusivity, permeability, and pore size. With this library of GelMA hydrogels of varying properties, we then encapsulated Swan71 trophoblast spheroids in these hydrogel variants to assess how bloom strength affects trophoblast spheroid morphology. Overall, we observed a decreasing trend of spheroid area and Feret diameter as bloom strength increased. In identifying clear relationships between bloom strength, hydrogel mechanical properties, and trophoblast spheroid morphology, we demonstrate that bloom strength should considered when designing tissue engineered constructs.

Silymarin enriched gelatin methacrylamide bioink imparts hepatoprotectivity to 3D bioprinted liver construct against carbon tetrachloride induced toxicity.

Three-dimensional liver bioprinting is an emerging technology in the field of regenerative medicine that aids in the creation of functional tissue constructs that can be used as transplantable organ substitutes. During transplantation, the bioprinted donor liver must be protected from the oxidative stress environment created by various factors during the transplantation procedure, as well as from drug-induced damage from medications taken as part of the post-surgery medication regimen following the procedure. In this study, Silymarin, a flavonoid with the hepatoprotective properties were introduced into the GelMA bioink formulation to protect the bioprinted liver against hepatotoxicity. The concentration of silymarin to be added in GelMA was optimised, bioink properties were evaluated, and HepG2 cells were used to bioprint liver tissue. Carbon tetrachloride (CCl4) was used to induce hepatotoxicity in bioprinted liver, and the effect of this chemical on the metabolic activities of HepG2 cells was studied. The results showed that Silymarin helps with albumin synthesis and shields liver tissue from the damaging effects of CCl4. According to gene expression analysis, CCl4 treatment increased TNF-α and the antioxidant enzyme SOD expression in HepG2 cells while the presence of silymarin protected the bioprinted construct from CCl4-induced damage. Thus, the outcomes demonstrate that the addition of silymarin in GelMA formulation protects liver function in toxic environments.

Adipose-derived stem cells loaded photocurable and bioprintable bioinks composed of GelMA, HAMA and PEGDA crosslinker to differentiate into smooth muscle phenotype.

Developing a polymer-based photocrosslinked 3D printable scaffolds comprised of gelatin methacryloyl (G) and hyaluronic acid methacryloyl (H) incorporated with two molecular weights of polyethylene glycol diacrylate (P) of various concentrations that enables rabbit adipose-derived stem cells (rADSCs) to survive, grow, and differentiate into smooth muscle cells (SMCs). Then, the chemical modification and physicochemical properties of the PGH bioinks were evaluated. The cell viability was assessed via MTT, CCK-8 assay and visualized employing Live/Dead assay. In addition, the morphology and nucleus count of differentiated SMCs were investigated by adopting TRAP (tartrate-resistant acid phosphatase) staining, and quantitative RT-PCR analysis was applied to detect gene expression using two different SMC-specific gene markers α-SMA and SM-MHC. The SMC-specific protein markers namely α-SMA and SM-MHC were applied to investigate SMC differentiation ability by implementing Immunocytofluorescence staining (ICC) and western blotting. Moreover, the disk, square, and tubular cellular models of PGH7 (GelMA/HAMA=2/1) + PEGDA-8000 Da, 3% w/v) hybrid bioink were printed using an extrusion bioprinting and cell viability of rADSCs was also analysed within 3D printed square construct practising Live/Dead assay. The results elicited the overall viability of SMCs, conserving its phenotype in biocompatible PGH7 hybrid bioink revealing its great potential to regenerate SMCs associated organs repair.