In vitro evaluation of novel Zeolite-hydroxyapatite blended scaffold for dental tissue engineering

Main purpose of tissue engineering is creating appropriate conditions for the regeneration of tissues. Dental pulp-derived stem cells due to differentiation capacity and angiogenic properties have potential to regenerate dental pulp tissue. In the current experimental study poly caprolactone and poly L-lactic acid were synthesized by ring-opening polymerization method. The nano-hydroxyapatite and Zeolite were obtained by hydrothermal method. Morphological features and crystals properties of nHA and Zeolite were studied by X-ray diffraction. Nanofibers were fabricated using electrospinning method and investigated by FT-IR spectroscopy. DPSCs obtained from human source and proliferation and viability of them on electrospun scaffolds were evaluated by MTT assay. Also, the adhesion and proliferation of hDPSCs were investigated by SEM. The results showed that hDPSCs have the most viability and proliferation on the 1 st , 7 th , 14 th days on PCL-PLA/Zeolite scaffolds and maximum on the 3 rd day on PCL-PLA/nHA scaffolds. On the days of 7 th and 14 th , cell growth on scaffolds containing both nHA and Zeolite is better than sample that nHA is used alone with PCL-PLA. Briefly, by these results can be understand that Zeolite is a good agent in bone and tooth tissue engineering applications. More studies requires to investigate Zeolite effect on scaffold properties.

difference of human DPSCs on 4 types of designed scaffolds.

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.

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, roundbottom 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 PCL/Zeolite and PLA-PCL/nHA/Zeolite scaffolds in 4 groups to check the effects of using Zeolite on viability and proliferation of DPSCs.

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.   The electrospinning method for fabrication of nanofibers The electrospinning method for fabrication of nanofibers Micrograph of static contact angle Micrograph of static contact angle Figure 10 Contact angles of nanofibers