Standardized human adipose-derived stem cell spheroids can be harvested abundantly and the differentiation capability of cell spheroids performed well in the enzyme-crosslinked gelatin hydrogel.

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.

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.

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-β1 (TGF-β1) 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.

Surface coating with sulfobetaine methacrylate (SBMA) containing polymers is a simple method for reducing non-specific protein adsorption and cell adhesion to biomaterials. It has been shown that copolymers of zwitterionic monomers and butyl methacrylate (BMA) could be adsorbed onto hydrophobic substrates in order to provide anti-fouling properties. However, the copolymers of BMA/SBMA dissolved in organic solvents such as DMSO and THF, which is harmful to the host and environment, but not in environmentally friendly solvents, such as methanol and ethanol. Hydroxyethyl methacrylate (HEMA) was introduced in the copolymers in order to enhance the solubility of BMA/SBMA copolymers in methanol and ethanol. The solubility of the copolymers in methanol, ethanol and DMSO were examined. BMA/SBMA/HEMA copolymers in different solvents were coated on polystyrene (PS) plates, and the surface hydrophilicity and anti-fouling capacity were investigated. On the other hand, many studies have pointed out that spheroid formations of stem cells have the abilities to boost functionality and enhance their therapeutic potential. Therefore, the spheroid formation of human adipose-derived stem cells (hASCs) was studied on a plate coated with copolymers. The substrates coated with the copolymers prevented the adhesion of human adipose-derived stem cells (hASCs); moreover, hASCs formed spheroids after 24 hours of culture. In conclusion, the addition of HEMA in the BMA/SBMA copolymers made the copolymers soluble in methanol and ethanol. With the coating of copolymers, cell adhesion was inhibited and the stem cell spheroids were formed on the plate. The copolymers showed a potential for surface modification under an environmentally friendly condition for anti-fouling and stem cell spheroid application.

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.