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2.7. Pore size distribution of PLGA/PHBV scaffold The pore size distribution of the scaffold was determined by a Quantachrome (USA)PoreMaster 33 mercury intrusion porosimetry (MIP). The pore diameter (D) was calculated according to the Wasburn equation [34]: in which p, θ and γ are the adopted pressure, contact angle and hydrargyric surface tension of mercury on solid surface, respectively. The tests were conducted under low pressure condition with the minimum starting pressure. 2.9. Cell culture Human mesenchymal stem cells (hMSCs) were a kind gift donated by the Second Affiliated Hospital of Sun Yat-Sen University and were propagated in Dulbecco’s Modified Eagle’s Medium (DMEM) with supplements of 1.5 mg$ml-1 sodium bicarbonate,4.5 mg ml-1 glucose, 10% (v/v) fetal bovine serum (FBS), 3 mg ml-1 4-(2- hydroxyerhyl)piperazine-1-erhanesulfonic acid (HEPES). The cells were kept in a humidified incubator at 37℃ Cand5%CO2, and themediumwas changed every 3 days. 2.10. Cell seeding on the scaffolds PLGA/PHBV scaffolds were cut into disks of 6mmin diameter and 2mmin height and sterilized by exposing to gamma radiation (15 kGy). The pure PLGA scaffolds of the same size as that of PLGA/PHBV were immersed in 75% (v/v) ethanol aqueous solution for 2 h and followed by ultraviolet radiation for 30 min to sterilize. All the scaffolds were prewet in the DMEM solution for 24 h. Fifteen microlitres of passage 6 cells in suspension (7.5×104 cells/well) were seeded on each scaffold. 2 h later, 700 μl of culture medium was added into each well. The cells/scaffold constructs were incubated at 37℃ in a humidified incubator of 5% CO2 for pre-set days. |
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