Injectable, Shear-Thinning, Self-Healing, and Self-Cross-Linkable Benzaldehyde-Conjugated Chitosan Hydrogels as a Tissue Adhesive.

Benzaldehyde-conjugated chitosan (CH-CBA) was synthesized by a coupling reaction between chitosan (CH) and carboxybenzaldehyde (CBA). The pH-sensitive self-cross-linking can be achieved through the Schiff base reaction. The degree of substitution (DS) of CH-CBA was controlled at 1.4-12.7% by optimizing the pH and reagent stoichiometry. The dynamic Schiff base linkages conferred strong shear-thinning and self-healing properties to the hydrogels. The viscosity of the 2 wt/v % CH-CBA hydrogel decreased from 5.3 × 107 mPa·s at a shear rate of 10-2 s-1 to 2.0 × 103 mPa·s at 102 s-1 at pH 7.4. The CH-CBA hydrogel exhibited excellent biocompatibility in vitro and in vivo. Moreover, the hydrogel adhered strongly to porcine small intestine, colon, and cecum samples, comparable to commercial fibrin glue, and exhibited effective in vivo tissue sealing in a mouse cecal ligation and puncture model, highlighting its potential as a biomaterial for application in tissue adhesives, tissue engineering scaffolds, etc.

Injectable Extracellular Matrix-Inspired Hemostatic Hydrogel Composed of Hyaluronan and Gelatin with Shear-Thinning and Self-Healing.

Injectable ECM-inspired hydrogels composed of hyaluronic acid and gelatin are biocompatible and potentially useful for various medical applications. We developed injectable hydrogels composed of monoaldehyde-modified hyaluronic acid (HA-mCHO) and carbohydrazide-modified gelatin (GL-CDH), "HA/GL gel", whose ratios of HA-mCHO to GL-CDH were different. The hydrogels exhibited gelation times shorter than 3 s. In addition, the hydrogels showed strong shear-thinning and self-healing properties, mainly because of the dynamic covalent bonding of Schiff bases between HA-mCHO and GL-CDH. This hydrogel degraded in the mice's peritoneum for a week and showed excellent biocompatibility. Moreover, the hydrogel showed a higher breaking strength than fibrin glue in the lap shear test of porcine skin. Finally, the hydrogels decreased bleeding to as low as fibrin glue without using thrombin and fibrinogen in a mouse liver bleeding model in both single- and double-barreled syringe administrations. HA/GL gels have the potential for excellent biocompatibility and hemostasis in clinical settings.

pH responsive cationic guar gum-borate self-healing hydrogels for muco-adhesion.

We developed a new muco-adhesive hydrogel composed of cationic guar gum (CGG) and boric acid (BA). The CGG-BA precursor solution of 0.5-2% w/v concentration exhibited fluidity at low pH (3-5), while gelation occurred within 1 min at physiological pH (7-8) conditions. Scanning electron microscopy and Fourier-transform infrared spectroscopy results confirmed the change in physical and chemical behavior, respectively, with change in pH. The pH-responsive self-healing ability was analyzed through microscopy and rheology. CGG-BA hydrogels showed good self-healing property at pH 7.4. The in vitro biocompatibility test of the hydrogel studied using NIH3T3 and NHEK cells showed that it was non-toxic at concentrations of CGG-BA below 2% w/v. Ex vivo mucoadhesive tests confirmed the hydrogel's potential for use as a muco-adhesive. Burst pressure tests were conducted using pig esophageal mucosa and the results showed that at pH 7.4, 1% w/v CGG-BA self-healable hydrogel resisted about 8 ± 2 kPa pressure, comparable to that of Fibrin glue. This was higher than that at solution (pH 5) and brittle gel (pH 10) conditions. To confirm the good adhesive strength of the self-healable hydrogels, lap shear tests conducted, resulted in adhesive strengths measured in the range of 1.0 ± 0.5-2.0 ± 0.6 kPa, which was also comparable to fibrin glue control 1.8 ± 0.6 kPa. Hydrogel weight measurements showed that 40-80% gel lasted under physiological conditions for 10 h. The results suggest that CGG-BA hydrogel has potential as a pH responsive mucosal protectant biomaterial.

Developing a Glyoxal-Crosslinked Chitosan/Gelatin Hydrogel for Sustained Release of Human Platelet Lysate to Promote Tissue Regeneration.

The clinical application of human platelet lysate (HPL) holds promise for tissue regeneration, and the development of an efficient vehicle for its delivery is desired. Chitosan-based hydrogels are potential candidates, but they often exhibit weak mechanical properties. In this study, a chitosan/gelatin (CS-GE) hydrogel crosslinked by glyoxal was fabricated for sustained release of HPL. The influence of HPL on Hs68 fibroblast and human umbilical vein endothelial cell (HUVEC) culture was evaluated, and we found that supplementing 5% HPL in the medium could significantly improve cell proliferation relative to supplementing 10% fetal bovine serum (FBS). Moreover, HPL accelerated the in vitro wound closure of Hs68 cells and facilitated the tube formation of HUVECs. Subsequently, we fabricated CS-GE hydrogels crosslinked with different concentrations of glyoxal, and the release pattern of FITC-dextrans (4, 40 and 500 kDa) from the hydrogels was assessed. After an ideal glyoxal concentration was determined, we further characterized the crosslinked CS-GE hydrogels encapsulated with different amounts of HPL. The HPL-incorporated hydrogel was shown to significantly promote the proliferation of Hs68 cells and the migration of HUVECs. Moreover, the release pattern of transforming growth factor-ß1 (TGF-ß1) and platelet-derived growth factor-BB (PDGF-BB) from hydrogel was examined in vitro, demonstrating a sustained release profile of the growth factors. Finally, the chick chorioallantoic membrane assay revealed that HPL encapsulation in the hydrogel significantly stimulated angiogenesis in ovo. These results demonstrate the great potential of the crosslinked CS-GE hydrogel to serve as an effective delivery system for HPL to promote tissue regeneration.

Enzyme-Cross-linked Gelatin Hydrogel Enriched with an Articular Cartilage Extracellular Matrix and Human Adipose-Derived Stem Cells for Hyaline Cartilage Regeneration of Rabbits.

Hyaline cartilage regeneration remains clinically challenging. In this study, microbial transglutaminase was used to cross-link gelatin. The articular cartilage extracellular matrix (cECM), mainly comprising collagen type II and glycosaminoglycans (GAGs), which can support chondrogenesis, was enclosed in this enzyme-catalyzed hydrogel. After human adipose-derived stem cells (hASCs) were encapsulated in the hydrogel enriched with the cECM, the results demonstrated that the enzymatic cross-linking reaction is of low cytotoxicity. Moreover, the stem cells showed great proliferation and chondrogenic differentiation potential in the hydrogel. Most importantly, we assessed the therapeutic effects of applying a hydrogel enriched with the cECM and hASCs to repair a full-thickness osteochondral defect. At 8 weeks after surgery, the GCC group (hydrogel encapsulating cells and the cECM) exhibited a smooth articular surface with transparent new hyaline-like tissue macroscopically. According to histological analysis, inflammatory responses were hardly observed, and sound chondrocytes were aligned in the newly formed chondral layer. In addition, the GCC group exhibited significant improvement in the GAG content between weeks 4 and 8. In summary, the implantation of a gelatin hydrogel enriched with the cECM and hASCs could facilitate the hyaline cartilage regeneration significantly in rabbit knee joint models.

Enhancement of human adipose-derived stem cell spheroid differentiation in an in situ enzyme-crosslinked gelatin hydrogel.

Human adipose-derived stem cells (hASCs) can differentiate into multiple lineages and be harvested abundantly. However, the expression of pluripotency markers, which is important for the renewal and differentiation capabilities of hASCs, decreases during monolayer culture. Increasing evidence has proven that cells aggregated to form cell spheroids in 3D cell cultures better mimic the in vivo microenvironment and can enhance the expression of stemness markers. In this study, uniform hASC spheroids were formed by seeding cells in agarose microwell plates, and the size of the spheroids could be adjusted. Most importantly, the stemness expression of the spheroids increased significantly. Additionally, we utilized microbial transglutaminase (mTG), which is an enzyme that exhibits highly specific activity over a wide range of temperature and pH, to crosslink gelatin. The enzymatic crosslinking reaction is milder than physical and chemical methods, which may lead to cell death. The properties of the gelatin/mTG hydrogel were evaluated in detail. In addition, the spheroids were encapsulated in the 3D hydrogel successfully. The results showed that the hydrogel has low toxicity to the cells, which significantly proliferated in the 3D hydrogel. Moreover, the analysis of the differentiation potential indicated that the cell spheroids in the 3D hydrogel exhibited good activity, especially adipogenesis and chondrogenesis, compared to the cell suspension group. Furthermore, the in vivo data confirmed the excellent injectability and biocompatibility of the 3D hydrogel.

Modulation of Neural Differentiation through Submicron-Grooved Topography Surface with Modified Polydopamine.

Surface topography and bioactive molecules can generate physicochemical cues that control proliferation and differentiation of neural cells. In this study, polystyrene (PS) submicron-patterns with different widths (400 and 800 nm) and depths (100 and 400 nm) were prepared and subsequently modified with polydopamine (PDA) by a coating method. We examined neurites of PC12 cells and human adipose-derived stem cells (hADSCs) incubated in neuronal induction medium containing nerve growth factor (NGF) and basic fibroblast growth factor (bFGF), respectively. Then the differentiated cells on different grooved topographies were immunologically stained by Tuj-1 (a neuron marker) to compare the extent of neuronal differentiation. Our results showed that PC12 cells on grooved topography have predominantly bipolar neurite extension and align along the direction of the patterns, while flat surface has multipolar neurites. We demonstrated that the depths of topography have a strong impact on neurite outgrowth and alignment. In terms of the number of neurites, neurite length, and percentage of Tuj-1 positive cells, the 400/400 and 800/400 nm (widths/depths) PS grooves are appropriate for the cultivations of PC12 cells and hADSCs relative to those of other groups. In conclusion, the submicron-grooved topography and neurotrophic growth factors supported neurites outgrown and differentiated into neuron-like cells.