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Stem-cell-derived organoid can resemble in vivo tissue counterpart and mimic at least one function of tissue or organ, possessing great potential for biomedical application. The present study develops a hydrogel with cell-responsive switch to guide spontaneous and sequential proliferation and aggregation of adipose-derived stem cells (ASCs) without inputting artificial stimulus for in vitro constructing cartilaginous microtissues with enhanced retention of cell-matrix and cell-cell interactions. Polylactic acid (PLA) rods are surface-aminolyzed by cystamine, followed by being involved in the amidation of poly(( l-glutamic acid) and adipic acid dihydrazide (ADH) to form a hydrogel. Along with tubular pore formation in hydrogel after dissolution of PLA rods, aminolyzed PLA molecules with disulfide bonds on rod surfaces are covalently transferred to the tubular pore surfaces of poly(l-glutamic acid)/ADH hydrogel. Because PLA attaches cells, while poly(l-glutamic acid)/ADH hydrogel repels cells, ASCs are found to adhere and proliferate on the tubular pore surfaces of hydrogel first and then cleave disulfide bonds by secreting molecules containing thiol, thus inducing desorption of PLA molecules and leading to their spontaneous detachment and aggregation. Associated with chondrogenic induction by TGF-β1 and IGF-1 in vitro for 28 days, the hydrogel as an all-in-one incubator produces well-engineered columnar cartilage microtissues from ASCs, with the glycosaminoglycans (GAGs) and collagen type II (COL II) deposition achieving 64 and 69% of those in chondrocytes pellet, respectively. The cartilage microtissues further matured in vivo for 8 weeks to exhibit extremely similar histological features and biomechanical performance to native hyaline cartilage. The GAGs and COL II content, as well as compressive modulus of the matured tissue show no significant difference with native cartilage. The designer hydrogel may hold a promise for long-term culture of other types of stem cells and organoids.

Adipose-derived stem cell (ASC) spheroids exhibit enhanced angiogenic efficacy toward ischemia treatment. Thus, it is necessary to develop an all-in-one platform that enables efficient spheroid production, collection, and injectable implantation in vivo. The present study fabricated a poly(l-glutamic acid) (PLGA)-based porous hydrogel that can not only produce ASC spheroids but also conveniently collect spheroids for in vivo implantation via minimally invasive injection to treat hind limb ischemia. PLGA was cross-linked with cystamine (Cys), which contains disulfide bonds, to form a porous hydrogel that could realize "gel-sol" transition by the reduction effect of glutathione (GSH). For one thing, it was found that the introduction of the disulfide bond in the PLGA hydrogel promoted cellular adhesion via combining fibronectin, preventing the formation of spheroids, while the introduction of polyethylene glycol monomethyl ether (mPEG) could disturb the effect of the disulfide bond on cellular adhesion, supporting spheroid formation inside the porous hydrogel. For another, the porous hydrogel transferred into a syringe could turn into liquid polymer solution within about 40 min for collection of the produced spheroids and in vivo injection. In addition, because of the lubrication of polymer solution, the spheroids were protected during the injection of the spheroids/polymer suspensoid through a 25G syringe needle, avoiding damages from shearing. After the in vivo injection, the enhanced paracrine secretion of ASC spheroids resulted in promoted angiogenesis and muscle regeneration, exhibiting obvious therapeutic effect on limb ischemia in mice after 21 days. At the same time, PLGA-based material exhibited well-performed biocompatibility in vivo.

Multicellular mesenchymal stem cell (MSC) spheroids possess enhanced chondrogenesis ability and limited fibrosis, exhibiting advantage toward hyaline-like cartilage regeneration. However, because of the limited cell surfaces in spheroid exposed to DNA/vector, it is difficult to realize efficient gene transfection, most of which highly rely on cell-substrate interaction. Here, we report a poly(l-glutamic acid)-based porous scaffold with tunable inner surfaces that can sequentially realize cell-scaffold attachment and detachment, as well as the followed in situ spheroid formation. The attachment and detachment of cells from scaffold is achieved by the capture and release of fibronectin (Fn) via reversible imine linkage between aromatic aldehyde groups of scaffold and amino groups of Fn. Together with N, N, N-trimethyl chitosan chloride condensing plasmid DNA encoding transforming growth factor-β1 (pDNA-TGF-β1), cell attachment realizes efficient surface-mediated gene transfection. Conversion of scaffold stiffness can affect the adhesion shape of cells. Stiffer scaffold reinforces the adhesion, leading to the amplification of peripheral focal adhesions and the promotion of cell spreading, as well as the promotion of gene transfection efficiency. After cellular detachment from the scaffold via lysine treatment, the subsequent spheroid formation with extensive cell-cell interaction up-regulates the corresponding protein expression with a prolonged term. With the induction effect of the expressed TGF-β1, significantly enhanced chondrogenesis of MSCs in spheroids is achieved at 10 d in vitro. Well-regenerated cartilage at 8 weeks in vivo indicates that the present gene transfection system is a platform that can be potentially applied toward cartilage tissue engineering.