We have developed a novel method to induce spontaneous hyaline cartilage regeneration in vivo for a large osteochondral defect by implanting a plug made from a double-network hydrogel composed of poly(2-acrylamido-2-methylpropanesulfonic acid) and poly(N,N'-dimethylacrylamide) at the bottom of the defect, leaving the cavity vacant. In cells regenerated in the treated defect, type-2 collagen, Aggrican, and SOX9 mRNAs were highly expressed and the regenerated matrix was rich in proteoglycan and type-2 collagen at 4 weeks. This fact gave a significant modification to the commonly established concept that hyaline cartilage tissue cannot regenerate in vivo. This study prompted an innovative strategy in the field of joint surgery to repair an osteochondral defect using an advanced, high-function hydrogel.

Abstract:We investigated the behavior of chondrogenic ATDC5 cells on synthetic polymer gels with various charge densities: negative charged poly (2-acrylamido-2-methyl-1-propanesulfonic acid) gel, neutral poly (dimethylacrylamide) gel, and copolymer gels of 2-acrylamido-2-methyl-1-propanesulfonic acid and dimethylacrylamide with different compositions (molar fraction of 2-acrylamido-2-methyl-1-propanesulfonic acid, F=0.25, 0.5, 0.75). In insulin-free maintenance medium, the ATDC5 cells cultured on the highly negative charged gels;poly (2-acrylamido-2-methyl-1-propanesulfonic acid) gel and the copolymer gels of 2-acrylamido-2-methyl-1-propanesulfonic acid and dimethylacrylamide (F=0.75), spread and became confluent at day 7, and interestingly formed nodules at day 14, expressing type II collagen and proteoglycan. This result demonstrates that the highly negative charged gels can induce chondrogenic differentiation of ATDC5 cells even in insulin-free maintenance medium, in which the ATDC5 cells cultured on the standard polystyrene dish cannot differentiate into chondrocytes. In insulin-supplemented differentiation medium, ATDC5 cells cultured on the poly (dimethylacrylamide) gel made focal adhesions, rapidly aggregated, and formed large nodules within 7 days, expressing significantly greater levels of type II collagen and proteoglycan than cells cultured on the polystyrene dish and the negative charged gels. These results showed that the neutral gel accelerated chondrogenic differentiation of ATDC5 cells cultured in the differentiation medium. We suggest that the highly negative charged poly (2-acrylamido-2-methyl-1-propanesulfonic acid) gel and the neutral poly (dimethylacrylamide) gel are interesting biomaterials for cartilage tissue engineering as a scaffold with the potential to induce chondrogenic differentiation.3 Keywords: ATDC5; Chondrogenic differentiation; Poly (2-acrylamido-2-methyl-1-propanesulfonic acid) (PAMPS); Poly (dimethylacrylamide) (PDMAAm); Copolymer gels of 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) and dimethylacrylamide (DMAAm) (P(AMPS-co-DMAAm) gels); Type II collagen; Aggrecan.4

Electrical stimulation (ES) is known to guide the development and regeneration of many tissues. However, although preclinical and clinical studies have demonstrated superior effects of ES on cartilage repair, the effects of ES on chondrogenesis remain elusive. Since mesenchyme stem cells (MSCs) have high therapeutic potential for cartilage regeneration, we investigated the actions of ES during chondrogenesis of MSCs. Herein, we demonstrate for the first time that ES enhances expression levels of chondrogenic markers, such as type II collagen, aggrecan, and Sox9, and decreases type I collagen levels, thereby inducing differentiation of MSCs into hyaline chondrogenic cells without the addition of exogenous growth factors. ES also induced MSC condensation and subsequent chondrogenesis by driving Ca2+/ATP oscillations, which are known to be essential for prechondrogenic condensation. In subsequent experiments, the effects of ES on ATP oscillations and chondrogenesis were dependent on extracellular ATP signaling via P2X4 receptors, and ES induced significant increases in TGF-β1 and BMP2 expression. However, the inhibition of TGF-β signaling blocked ES-driven condensation, whereas the inhibition of BMP signaling did not, indicating that TGF-β signaling but not BMP signaling mediates ES-driven condensation. These findings may contribute to the development of electrotherapeutic strategies for cartilage repair using MSCs.

ABSTRACT:Polyelectrolyte gels are charged polymer networks with macro-ions fixed on polymer chains. This present paper introduces fundamental aspects, properties and application of negatively charged polyelectrolyte gels, focusing on the electrical properties of polyelectrolyte gels, diffusion of proteins in polyelectrolyte gels, interactions between polyelectrolyte gels and oppositely charged molecules, and mechanical strength of polyelectrolyte based gels. These characteristic properties of polyelectrolyte gels have considerable potential for applications, such as soft and wet scaffolds of cells, soft actuators and replacement of biological tissues.

The skeletal elements of embryonic limb are prefigured by prechondrogenic condensation in which secreted molecules such as adhesion molecules and extracellular matrix have crucial roles. However, how the secreted molecules are controlled to organize the condensation remains unclear. In this study, we examined metabolic regulation of secretion in prechondrogenic condensation, using bioluminescent monitoring systems. We here report on ATP oscillations in the early step of chondrogenesis. The ATP oscillations depended on both glycolysis and mitochondrial respiration, and their synchronization among cells were achieved via gap junctions. In addition, the ATP oscillations were driven by Ca2+ oscillations and led to oscillatory secretion in chondrogenesis. Blockade of the ATP oscillations prevented cellular condensation. Furthermore, the degree of cellular condensation increased with the frequency of ATP oscillations. We conclude that ATP oscillations have a critical role in prechondrogenic condensation by inducing oscillatory secretion.