Evaluation Of Cytotoxicity And Osteogenic Capacity Of A Novel Semaphorin 3A Loaded Chitosan Microspheres/ Silk Fibroin/α-TCP Composite.

Objective: The objective of this work is the fabrication of Semaphorin 3A loaded Chitosan Microspheres/Silk Fibroin/α-Tricalcium composite(Sema3A CMs/SF/α-TCP)and the evaluation of its cytotoxicity and osteogenic capacity in vitro.Methods: 1.The silk solution was prepared through four steps named: degumming, dissolving, dialyzing and enriching. The CMs were fabricated using emulsion-chemical cross-linking method, and absorbtion method was used to get Sema3 A loaded. Sema3 A CMs/SF/α-TCP was fabricated by mixing silk solution, Sema3 A CMs and α-TCP powder. 2. The Sema3 A absorption capacity of the CMs and the in vitro release of Sema3 A were measured by Sema3 A ELISA kit. 3. The extract of Sema3 A CMs/SF/α-TCP was prepared at concentrations of 100% and 50%, MC3T3-E1 cells were cultured in the extract of different concentrations, positive and negative controls were set, MTT assay and the activity of Lactate Dehydrogenase(LDH) were used to evaluate the cytotoxicity of the extract. 4. MC3T3-E1 cells were seeded on the surfaces of Sema3 A CMs/SF/α-TCP, CMs/SF/α-TCP and SF/α-TCP, Scanning electron microscopy was used to observe the cell morphology on Sema3 A CMs/SF/α-TCP. The supernatant’s ALP activity in different groups were tested using ALP activity kit, the relative expression of Runx2, Col-1, ALP genes were measured using RT-q PCR.Results: 1.The Sema3 A Loaded chitosan microspheres were in spherical shape and well distributed. The drug-loading capacity was 0.41μg/mg. The release of Sema3 A from Sema3 A CMs/SF/α-TCP turned out to be a biphasic kinetics with a burst initial phase and sustained release phase, 91.73% of Sema3 A was released after 30 days 2.The cytotoxicity grades of Sema3 A CMs/SF/α-TCP were zero or one. 3. The MC3T3-E1 cells seeded on the surface of Sema3 A CMs/SF/α-TCP were well-spread, presenting an elongated shape with a few contact points with the material with the help of pseudopodia. 4. A slightly higher level of ALP activity within the supernatant was observed in Sema3 A CMs/SF/α-TCP compared to others after 14 days of incubation, but no statistically significant differences were found(P>0.05). After 21 days of incubation the ALP activity of Sema3 A CMs/SF/α-TCP group was significantly higher than other groups(P<0.05). 5. The relative expression of osteogenic related genes of Sema3 A CMs/SF/α-TCP group were higher than other groups with statistically significant difference(P<0.05).Conclusion:Sema3A CMs/SF/α-TCP presents no obvious cytotoxicity and is able to support the adherence of MC3T3-E1 cells and contributes to elevated ALP activity and relative expression of osteogenic related genes.