DL-lactide and ɛ-caprolactone were purchased from Sigma–Aldrich (Co., Steinem, Germany) and recrystallized twice from ethyl acetate, and dried under high vacuum at room temperature before usage. Stannous 2-ethyl hexanoate (stannous octoate, Sn(Oct)2) was purchased from Sigma-Aldrich (USA). Glutaraldehyde (25% aqueous solution) and all the solvents purchased from Merck Inc. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) was purchased from Sigma-Aldrich. Dulbecco Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS) and trypsin– EDTA were purchased from Gibco.
2.2. Synthesis of PLA
PLA was synthesized by ring-opening polymerization method. For this purpose, a certain amount of DL-lactide monomer was heated at 150 °C for 1 hour inside the balloon (With continuous flow of nitrogen atmosphere). Then, 0.1gr of Sn(Oct)2 (as catalyst) was added to lactide while being stirred. After that, the temperature of reaction was decreased to 120 °C and kept constant at this temperature for 6 hour. In order to remove excess catalyst and unreacted monomers, the product was dissolved in dichloromethane and precipitated in cold diethyl ether.
2.3. Synthesis of PCL
PCL was synthesized by ring-opening polymerization of ɛ-caprolactone in the presence of stannous octoate as catalyst. A certain amount of Ԑ-caprolactone was heated at 150 °C in a two-necked, round-bottom flask (With continuous flow of nitrogen atmosphere). After that, 0.1gr of Sn(Oct)2 was added to reaction. Then, the temperature of reaction decreased to 120 °C and kept constant at this temperature for 6 hours. Excess catalyst and unreacted monomers removed with dissolving in dichloromethane and transferred in a cold diethyl ether.
Both PCL and PLA were placed in to the avon vacuum for polymerization process completion.
nHydroxyapatite (nHA) and Zeolite powders were synthesized by hydrothermal method separately .
2.4. Designing and Fabricating of Nanofibrous Scaffolds by Electrospinning Method
Fibers were designed in 4 category: PCL-PLA, PCL-PLA/nHA, PCL-PLA/Zeolite and PCL-PLA/nHA/Zeolite. For all the samples, uniform solution of them was transferred in to the 5 mL plastic syringe with a metal needle tip (gauge 18). The process of the electrospinning for fabrication of nanofibers was shown in Figure 1. Electrospinning device settings (Fanavaran Nano-Meghyas, Iran) explained as follow: Drum speed: 130 rpm; Potential difference (Voltage): 20-25Kv; Temperature: 25 °C; Distance of collector and needle tip: 15 cm; Injection rate: 2ml/h.
2.4.1. Fabrication of PCL-PLA Nanofibers:
PCL-PLA with a molar ratio of 4:1 was dissolved in DCM: Methanol (4:1 v/v ratio) to prepare 15 wt% solution. The prepared solution was put in to container and be stirred for 24 hours at 25˚C temperature. Then, the electrospinning method was used for fabrication of PCL-PLA nanofibers.
2.4.2. Fabrication of PCL-PLA/nHA Nanofibers:
PCL-PLA with a molar ratio of 4:1 was dissolved in DCM: Methanol (4:1 v/v ratio) to prepare 15 wt% solution. Then, 0.1 gr HA nanoparticles were added to the PCL-PLA solution. The prepared mixture was stirred for 24 hours at 25˚C temperature. The electrospinning method was used for fabrication of PCL-PLA/nHA nanofibers.
2.4.3. Fabrication of PCL-PLA/Zeolite Nanofibers:
PCL-PLA with a molar ratio of 4:1 was dissolved in DCM: Methanol (4:1 v/v ratio) to prepare 15 wt% solution. After that, 0.1 gr Zeolite nanoparticles were added to co-polymers solution. The mixture of PCL-PLA solution and Zeolite was put in to container and be stirred for 24 hours at 25˚C temperature. The electrospinning method was used for fabrication of PCL-PLA/Zeolite nanofibers.
2.4.4. Fabrication of PCL-PLA/nHA/Zeolite Nanofibers:
PCL-PLA with a molar ratio of 4:1 was dissolved in DCM: Methanol (4:1 v/v ratio) to prepare 15 wt% solution. Then, 0.05 gr Zeolite nanoparticles and 0.05 gr HA nanoparticles were added to PCL-PLA solution and stirred for 24 hours at 25˚C temperature. The electrospinning method was used for fabrication of PCL-PLA/nHA/Zeolite nanofibers.
All the fabricated nanofibers dried for 3 days before cell culturing on them.
2.5. Fourier Transforms Infrared (FT-IR) Spectroscopy
The chemical structure of the prepared electrospun nanofibers was studied by FTIR spectrometer to identified functional groups of scaffolds (Equinox 55 LS 101, Bruker, Germany). The sample was mixed with potassium bromide and pressed to form a disk. The IR spectra of scaffolds were obtained in the range of 400 to 4000 cm-1.
2.6. X-ray Diffraction (XRD) Analysis
XRD is an old and widely used technique for investigation of the crystalline structure. This technique was used to study the properties of crystal structure such as: network geometry, determination of crystalline phases, lattice defects, determining the crystal size and etc.
For this purpose, synthesized HA and Zeolite were studied by XRD technique to ensure the formation of respective phases and check size of synthesized particles. XRD analysis of nHA and Zeolite was done by Bruker Discover 8 X-ray diffractometer (Germany) operated at 40 mA and 40 kV which was measured with Cu-Kα radiation in a 2θ range from 5° to 70° at 0.05°/s. The d-spacing (d) corresponding to the XRD peak was determined from Bragg’s equation, nλ = 2dsinθ, where θ is the diffraction position, λ is the wavelength (which is 1.54 Å for a Cu target), and n is an integer.
2.7. Preparation of Scaffolds for Cell Culture Studies
The scaffolds with different combinations of materials including 4 groups: PLA- PCL, PLA- PCL/nHA, PLA- PCL/Zeolite and PLA- PCL/nHA/Zeolite was prepared for further cell compatibility studies. About 100 mg of each scaffolds placed in 12-well cell culture plates and dipped three times in a 70% ethanol solution for 20-30 minutes. Then, the ethanol was evaporated in the air and the scaffolds were rinsed three times with sterile PBS solution to remove the residual ethanol. In the next step, the scaffolds were incubated in a routine culture medium containing DMEM/10% FBS at 37 ºC for 48 h. Then, the scaffolds were seeded by DPSCs at a density of 5×105 cells/well using routine medium. The media refreshed every 2 days.
2.8. Dental Pulp Stem Cells (DPSCs) Preparation for Transforming on Scaffolds
In a previous study, the protocol of isolation and characterization of DPSCs has addressed by the authors . In the current study, we investigated DPSCs behavior on PLA- PCL, PLA- PCL/nHA, PLA- PCL/Zeolite and PLA- PCL/nHA/Zeolite scaffolds in 4 groups to check the effects of using Zeolite on viability and proliferation of DPSCs.
2.9. Cytotoxicity Analysis by MTT Assay
MTT assay is a method for investigation of cell viability and proliferation. Scaffolds were studied in two cases: cellular and cell free to remove the background absorbance. For this purpose, DPSCs were seeded on electrospun nanofibers; cell viability and proliferation were investigated in 1st, 3rd, 7th and 14th day. In brief: at the determined times, plates was taken out of incubator, the old medium was removed and 500 μL of MTT solution (Sigma) (10mg of MTT powder dissolve in 5ml PBS) and 1500 μL of sample medium was added to each wells and incubated for 4 hours at 37 ºC. Then, medium of each well was removed and 500 μl of dimethyl sulfoxide (DMSO) (Sigma) was added. The MTT was reduced by the mitochondrial dehydrogenase of living cells and DMSO dissolved the purple formazan crystals that can be readable by Elisa Reader. The optical density (OD) of each well measured at a certain wavelength (absorbance at 570 nm) by Elisa Reader machine (Awareness Technologies Stat Fax 2100 Microplate Reader). The viability was calculated using the formula: V = (ODsample - ODblank/ODcontrol - ODblank)×100. Where the blank is cell free scaffolds measured OD.
2.10. Cell Adhesion by Scanning Electron Microscopy (SEM) investigation
For cell attachment investigation, PCL-PLA, PCL-PLA/nHA, PCL-PLA/Zeolite, PCL-PLA /nHA/Zeolite scaffolds were seeded with DPSCs. The culturing period for cell attachment on nanofibers and morphological observation was 14 days. Preparation of nanofibers for SEM studies includes the following steps: At the determined time, scaffolds were rinsed twice with PBS, then cells on scaffolds fixed in 2.5% glutaraldehyde that diluted with PBS buffer (fixative solution), rinsed in PBS at 4 ºC for about 24 h, dehydrated in a series of ethanol, rinsed twice with PBS and finally dried at room temperature. In the next step scaffolds containing cells coated with nanometer-thick gold and observed by SEM (ChamScan MV2300).