Hydrogen sulfide-releasing silk fibroin scaffold for bone tissue engineering.

Hydrogen sulfide (H2S)-based therapy is a promising therapeutic strategy for several biomedical applications. Following the observation that endogenous and exogenous H2S plays a prominent role as a bone anabolic agent, we recently developed a silk fibroin (SF) porous scaffold loaded with GYY4137 (GYY), an H2S donor, for applications in bone tissue engineering. Here, we assayed whether the combination of SF with H2S potentiates the osteoconductive properties of SF. Biocompatibility and osteoanabolic activity were assayed in vitro using human bone marrow mesenchymal stromal cells. Cell cultures were performed on a perfusion bioreactor to obtain results closer to the in vivo microenvironment. Cytotoxicity was excluded by lactate dehydrogenase and live/dead assays. Cell colonization and mineral apposition were evaluated by Haematoxylin & Eosin and Von Kossa/Alizarin Red-S stainings respectively. PCR array for human osteogenesis and immunohistochemical analyses were performed to identify pathways and targets involved. Our findings show that H2S-releasing SF scaffolds supported cell adhesion, proliferation and viability. Moreover, H2S activated genes and proteins involved in ossification, osteoblast differentiation, bone mineral metabolism and angiogenesis allowing a high and early mineralization. Based on these properties, we suggest the use of H2S-releasing SF scaffolds for bone healing and regeneration applications.

Silk Fibroin Porous Scaffolds Loaded with a Slow-Releasing Hydrogen Sulfide Agent (GYY4137) for Applications of Tissue Engineering.

Hydrogen sulfide (H2S) is a physiological gasotransmitter known to possess a regulatory role in several tissues, including bone. The exogenous administration by injection of solutions of H2S-releasing compounds (e.g., GYY4137) has been previously investigated as a novel therapeutic approach for the treatment of bone diseases. Here, GYY4137 was embedded into fibroin sponges, previously shown to be suitable as scaffolds for bone, thanks to their biocompatibility, scalable porous structure, and biodegradability rate. Fibroin porous scaffolds were produced by solvent casting and the particulate leaching method, and GYY4137 was successively incorporated by using dimethyl sulfoxide (DMSO) as vehicle. The process used to produce GYY4137-loaded scaffolds allowed the incorporation of different controlled amounts of GYY4137 into fibroin matrices. The loading process preserved the properties of the system components in the final products, as assessed by SEM, FT-IR, NMR, and different thermal analyses techniques. Release of H2S from GYY4137 incorporated into the scaffolds was monitored upon incubation in saline solution at physiological pH: H2S-release kinetic was found to be dependent on the amount of GYY4137. To ensure biocompatibility, mouse fibroblasts and human primary bone marrow stromal cells were seeded onto scaffolds, and short-term viability assays were performed. Results showed that the GYY4137-loaded scaffold did not induce cytotoxicity in any of the cell type tested. Our findings demonstrate that embedding an H2S-releasing donor in silk fibroin scaffold is a suitable strategy to achieve a long-lasting release of H2S that preserves cell viability and allows local delivery at sites of tissue injury.