Preparation And Characterization Of Porous Microspheres Based Cell-loaded Scaffolds Sintered By Subcritical CO₂

Utilizing the “modularization” strategy to bond single microsphere together by plasticization, the microspheres based scaffolds play an important role in tissue engineering. CO₂, as a green sintering agent, has been applied in tissue engineering due to its unique advantages. In this study, the preparation of poly?lactic-co-glycolic acid??PLGA? porous microspheres by double emulsion was carried out at the first place, and then the porous microspheres based scaffolds sintered by subcritical CO₂ were investigated. The effects of operating parameters on the resulting microspheres and sintered scaffolds were studied, and the biological evaluations on the scaffolds were also investigated.Firstly, the porous microspheres were prepared by double emulsion using PLGA solution in dichloromethane?DCM? as the oil phase, the NH4HCO3 aqueous solution as the internal water phase, and the polyvinyl alcohol?PVA? solution as the external water phase. The effects of homogenization rate, water/oil ratio, oil phase concentration, foaming agent concentration, stirring speed and NH4HCO3 concentration on the particle size, pore size and water uptake ratio were investigated. The results indicate that porous microspheres with an average pore diameter larger than 20 ?m, particle size greater than 200 ?m and good water uptake ratio can be prepared with homogenizing rate of 10,000 rpm, water/oil ratio of 1:3.2, PLGA concentration of 6%, NH4HCO3 concentration of 10%, stirring speed of 200 rpm and PVA concentration of 0.5%. The DCM residue is far less than the limits of United States Pharmacopoeia USP467?600 ppm?.Secondly, the effects of sintering pressure, sintering time and particle size of microspheres on porosity and compressive strength of the resulting scaffolds were investigated. The results indicate that porous microspheres based scaffolds with pore size of more than 20 ?m, high compressive strength?>100 k Pa? and high porosity?>85%? can be prepared in the condition of particle size of microspheres ranges from 200³50 ?m, sintering pressure of 2 MPa and sintering time of 10 min. The results of Fourier transform infrared spectroscopy?FTIR?, X-ray diffraction?XRD? and differential scanning calorimetry?DSC? measurements show that subcritical process had no significant effect on the chemical structure, chemical group, crystallization and glass transition temperature of PLGA.Thirdly, the biocompatibility of the prepared scaffolds was evaluated. The cytotoxicity test shows that the scaffolds have no inhibitory effect on cell proliferation, the morphology of cell is normal and the relative growth rate is more than 95%, which belongs to level one. Acute toxicity test shows that the weight of mice increased significantly and the liver pathology detection which proves a non-toxic material. Hemolysis test shows that the hemolysis ratio of the materials was below the national standards of 5%, and had good blood compatibility. The results of effect of CO₂ on cell viability show that, the subcritical CO₂ had no significant influence on cell viability.Finally, the cell-loaded porous microsphere based scaffolds were prepared with two kinds of cells, C5.18 and L929, respectively. The cell viabilities on scaffolds were investigated. The results show that single step method improve the cell adhesion ratio on scaffolds.?C5.18: 60.00%±2.90%, L929: 80.13%±4.19%?. Pore of the porous microsphere not only provides sites for cell adhesion, but also induces the fibroblast proliferation in microspheres. Cells proliferated within 21 days, and the protein secretion results show that subcritical CO₂ did not affect the secreted protein of cells?C5.18: 1.04±0.065 mg/scaffold, L929: 0.86±0.050 mg/scaffold?.In conclusion, the cell-loaded porous microspheres based scaffolds were successfully developed in a single step. The strategy not only keeps cell viability in high-pressure environment, but also avoids the residue of organic solvents and inactivation of biological substances. It is worth to mention that the “modularization” strategy is in line with the structure of natural tissue, this provides a new way to tissue engineering.