ABSTRACT
Hydrogels based scaffolds are very promising materials for a wide range of medical applications including tissue engineering and drug delivery. This study reports a covalently cross-linked composite hydrogel embedded with microspheres basing natural polysaccharides as a protein delivery system for soft tissue engineering. This biodegradable composite hydrogel derived from water-soluble chitosan and alginate derivatives upon mixing, without addition of chemical cross-linking agents. The gelation is attributed to the Schiff-base reaction between amino and aldehyde groups of N-succinyl chitosan (N-Chi) and oxidized alginate (OAlg), respectively. Meanwhile, gel-like microspheres were prepared with a diameter of 2-10⯵m by conjugating sodium alginate with Ca2+ in an aqueous emulsion via the emulsion cross-linking technique. Bull Serum Albumin (BSA) was encapsulated into alginate gel microspheres and subsequently incorporated into OAlg/N-Chi hydrogels to produce a composite scaffold. In the current work, gelation rate, morphology, mechanical properties, swelling ratio, in vitro degradation and BSA release of the composite scaffolds were examined. The results show that mechanical and stable properties of gel scaffolds can be significantly improved by embedding alginate microspheres. The alginate microspheres can serve as a filler to toughen the soft OAlg/N-Chi hydrogels. Compressive modulus of composite gel scaffolds containing 0.5â¯mL volume of microspheres was 57.3â¯KPa, which was higher than the control hydrogel without microspheres. Moreover, the controlled release of BSA encapsulated within this composite hydrogels showed significantly lower rate when compared with control hydrogel or microspheres alone. These characteristics provide a potential opportunity to use this injectable composite gel scaffold in protein delivery and soft tissue engineering applications.
Subject(s)
Alginates/chemistry , Chitosan/chemistry , Hydrogels/chemistry , Microspheres , Serum Albumin, Bovine/chemistry , Tissue Engineering , Animals , Cattle , Immobilized Proteins/chemistryABSTRACT
Enterovirus 71 (EV71) is a causative agent of an array of childhood diseases with severe neurological manifestations implicated. EV71 infection is known to induce caspase-dependent apoptosis in cell cultures and animal models. However, whether an alternative apoptotic pathway independent of caspase activation can be triggered by EV71 infection has not been explored. In this study, we showed that calcium (Ca²âº)-activated calpains are capable of mediating caspase-independent pathway activation during EV71-induced apoptosis in HeLa cells. Results from subcellular fractionation analysis and confocal imaging indicated that during EV71 infection, apoptosis-inducing factor (AIF), a primary mediator of the caspase-independent pathway, became truncated and translocated from the mitochondrion to nucleus. This was accompanied by the release of cytochrome c, and sharply decreased mitochondrial membrane potential. AIF knockdown data indicated significant protection against apoptotic cell death, with greater protection provided by the addition of a pan-caspase inhibitor. The Ca²âº-dependent, calpain isoforms 1 and 2, but not cathepsins, were proven crucial for the altered AIF behaviour as studied by the pharmacological inhibitor and the knockdown approaches. We then analysed Ca²âº dynamics in the infected cells and found elevated levels of mitochondrial Ca²âº. Treatment with ruthenium red, a mitochondrial Ca²âº influx inhibitor, significantly blocked calpain activations and AIF cleavage. Our conclusion was that calpain activation via Ca²âº flux plays an essential role in eliciting an AIF-mediated, caspase-independent apoptotic pathway in EV71-infected cells. These findings should be useful for understanding the virus-induced cytopathology and the impact of Ca²âº homeostasis on EV71 infection.