Glow discharge argon plasma etching

The apatite-coated PLGA scaffold was synthesized through the process of solvent casting and particulate leaching methodology as previously established^{15 (link)}. In general, PLGA/Chloroform mixed solution was blended with 200–300 µm diameter of sucrose to form a porosity of approximate 92% (volume fraction), compressed into a Teflon mold and freeze-dried overnight at −110 °C and 100 mTorr (SP Industries, Inc., PA). Subsequently, the scaffolds were plunged in double-distilled (dd) H₂O to remove sucrose and then sterilized in 70% ethanol up to 30 mins, followed by several rinses in sterile ddH₂O. And then the dried scaffold sheets were sliced into small plates at the size of 5 × 5 × 2 mm. Finally, the PLGA scaffold plate was coated an apatite layer through incubating it in simulated body fluid (SBF): The scaffold was first subjected to glow discharge argon plasma etching (Harrick Scientific, Pleasantville, NY); The etched scaffold was then incubated in SBF1 formulated with the mixture of CaCl₂, MgCl₂· 6H₂O, NaHCO₃, K₂HPO₄· 3H₂O, Na₂SO₄, KCl and NaCl for 24 hours and subsequently in SBF2 with the mixture of CaCl₂, K₂HPO₄· 3H₂O, KCl and NaCl for another 24 hours at 37 °C.

Apatite-coated poly(lactic-coglycolic acid) (Ap-PLGA) scaffolds were prepared through solvent casting and particulate leaching processes following an established protocol as previously described.^{65 (link),66 (link)} Briefly, PLGA/CHCl₃ solution was mixed with sucrose (200–300 µm in diameter) to achieve a porosity of 92% (v/v), and the slurry was compressed into Teflon molds. After being lyophilized overnight, the scaffolds were immersed in Milli-Q water to leach out sucrose. The scaffolds were then sterilized in 70% EtOH for 30 min and rinsed with sterile water. Last, the scaffold sheets were shaped into round discs (3 mm in diameter and 0.5 mm in height) with a biopsy punch. The PLGA scaffolds were further coated with apatite layers by incubating scaffolds in simulated body fluid (SBF). The scaffold discs were subjected to glow discharge argon plasma etching (Harrick Scientific). The etched scaffolds were incubated in SBF1 composed of CaCl₂, MgCl₂•6H₂O, NaHCO₃, K₂HPO₄•3H₂O, Na₂SO₄, KCl and NaCl for 24 h and then further incubated in SBF2 composed of CaCl₂, K₂HPO₄•3H₂O, KCl and NaCl for another 24 h at 37 °C. The Ap-PLGA scaffolds were loaded with phosphate-buffered saline (PBS) or E2 (1, 5, 25 μmol·L^-1) and lyophilized for further usage.