| Objective: Periodontal disease, a chronic progressive devastating disease, can cause periodontal tissue defects , often leading to tooth loss. For a long time, researchers have been trying to find methods to effectively increase new attachment formation on teeth or reconstruct periodontal supporting tissues in order to treat periodontal disease, but the available approaches still need improvement. In recent years, the emergence of periodontal tissue engineering technology has opened up a new approach to the clinical treatment of periodontal diseases. Periodontal tissue engineering is regenerating new form and function with the original periodontal tissue cooperated different fields together between material science and biology, in order to repair or reconstruct periodontal tissue. Tissue engineering involves three key factors: porous scaffold, growth factors, seeding cell.Scaffold material affects cell bio-action and foster efficiency, and decides cell's adaptation and regeneration after implantation. Porous scaffold is a key factor restricted tissue engineering clinical application. It is very important to manufacture porous scaffold with properties, such as high porosity, interconnection and good biocompatability, to meet the requirements of tissue engineering. Because zein has a unique solubility, heat resistance, film-formation and natural biodegradable properties, it meets the general requirements of biomedical materials. So, this study tries to fabricate porous scaffold with zein. The properties of the scaffold are evaluated by determining its pore structure, porosity, degradation and biocompatibility, in order to explore the suitability of zein for periodontal tissue engineering.Methods:1 Preparation of zein scaffold: Zein was dissolved in 65% alcohol. NaCl was added to the solution as porogen, and the resulting mixture was heated with stirring until NaCl was evenly dispersed into the gel system. The gel was cast into a vessel. Subsequently, the gel was placed in deionized water at 37℃. Finally, the gel was dried in a biochemical incubator, and porous zein scaffold was obtained.2 Porosity: Under the same size of NaCl particle, three different mass concentrations of NaCl (70%, 75%, and 80%) were prepared for zein scaffolds to measure scaffold porosity, as described in the pycnometer method.3 Pore size trend observed: under 80% mass concentration of NaCl, three different sizes of NaCl particles (180~250μm, 250~300μm, 300~425μm) were used for the zein scaffold in order to observe the relationship between NaCl particles and scaffold pore size with the scanning electron microscopy.4 Degradation: The samples weighing about W-0 were placed in 37℃collagenase, removed at the time 1, 2, 3, 4, 5, 6, 7d, then washed by deionized water 3 times, drying to constant weight W_d. Degradation rate (η) = (W-0-W_d)/W-0×100%.5 Cytotoxicity testing5.1 Preparation of scaffold extract: Based on a certain equivalent, scaffolds were transferred into DMEM medium, obtaining 100% scaffold extract. The 100% scaffold extract was followed by serial dilutions using DMEM to a final concentration of 10%, 50%, and 100%.5.2 Liquid extraction experiment: Human periodontal ligament cells were cultured by tissue method. The third passage cells were seeded in 96-well plates incubating for 24h. Culture medium was removed and cells were randomly divided into three groups: experimental group (containing 10%, 50%, and 100% of scaffold extract), positive control group (containing DMEM with 0.64% phenol), and negative control group (containing DMEM alone). All samples were cultured in the corresponding medium for 24, 48, 72h. Cell viability was measured by MTT assay. Then we could calculate the relative growth rate, evaluate the toxicity of the scaffold.6 Direct contact assay: The zein scaffold was treated with ultraviolet, and soaked in DMEM solution overnight. The cells in good condition were inoculated into the scaffold to observe the cell morphology.Results:1 The zein scaffold displayed a high porosity with a sponge-like structure. The pores on the zein scaffold were distributed evenly and the pore sizes were mostly similar.2 With the same size of NaCl, when the mass concentrations of NaCl was 70%, 75%, and 80%, zein scaffold porosity was 64.1%,70.5%,78.0%.3 Under the same amount of porogen, not the same size, zein scaffolds showed the porous network structure. Also, as the porogen particle diameter increased, the size of the pores also increased.4 The degradation of zein scaffold was gradually increasing with the increase of immersing time in collagenase. At the seventh day, the degradation was 8.19%.5 The toxicity of zein scaffolds was rated as the 0~1 grade.6 These cells showed a tight attachment to zein scaffolds, fully stretched along the edge of the pore.Conclusions:1 We could fabricate porous zein scaffold using the solvent casting/particulate leaching method, with different porosity and pore size by adjusting the percentage and particle size of porogens.2 Zein scaffold was a biodegradable material.3 Zein scaffold had interconnected pores and affluent microns pores, which attributed to its good biocompatibility. Also, it could provide places for cells host growth, differentiation and proliferation. |
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