Preparation And Properties Of A Novel Root-End Filling Material

Various types of root-end filling materials have been used in endodontic surgery; however, each of them has its own disadvantages. An ideal root-end filling material should produce a complete apical seal, be non-toxic and non-carcinogenic, be dimensionally stable, be unaffected by moisture, be radiopaque, be easy to manipulate, have antibacterial activity and be biocompatible. However, currently there is no such root-end filling material which could achieve all above requirements. Therefore, it is very important to develop new ideal root-end filling materials.Hydroxyapatite is one of major inorganic components of human hard tissues, such as bones and teeth. Due to its excellent bioactivity and biocompatibility, hydroxyapatite has been widely used in dental materials. However, its relatively poor formability, mechanical properties and postoperative displacement have restricted its application in bone and dental materials. Calcium phosphate cement (CPC) is a self-curing non-ceramic hydroxyapatite bone repair material, with excellent biocompatibility and osteoconductivity. Under a physiological environment and body temperature, it forms a paste which self-hardens to form hydroxyapatite when mixed with water. However, its shortcomings, such as prolonged setting time, relatively low mechanical strength and dispersion upon early contact with blood or aqueous media, limit its clinical applications in endodontic surgery. To overcome the disadvantages of hydroxyapatite and calcium phosphate cement, a novel root-end filling material (NRFM), which is primarily based on natural hydroxyapatite, tetracalcium phosphate, solid polyacrylic acid, solid citric acid, sodium citrate and zirconium dioxide, was first developed. The NRFM is a promising ideal root-end filling material with good physicochemical properties and biocompatibility. This work consists of five parts.1. Synthesis, characterization and properties of the natural hydroxyapatite/tetracalcium phosphate/polyacrylic acid composites (NHA/TTCP/PAA)The NHA/TTCP/PAA was first prepared by blending a mixed powder with distilled water in the powder/liquid (P/L) weight ratio of 5:1, where the power contains 43.5%natural hydroxyapatite,43.5% tetracalcium phosphate,8.7% solid polyacrylic acid,2.15% solid citric acid and 2.15%sodium citrate. X-ray diffraction (XRD) patterns, Fourier transform infrared spectrophotometry (FTIR) spectra and scanning electron microscope (SEM) images revealed that NHA/TTCP/PAA was primarily composed of hydroxyapatite, calcium polyacrylate and calcium citrate. After mixing, dough-like NHA/TTCP/PAA was obtained, indicating its excellent operating performance. The mean and standard deviation setting time was 11.03± 0.78min. Its compressive strength was 25.58±2.68MPa and 38.21±5.72 MPa on 1 and 7 days respectively. Its storage solution showed an initial weak acidity and gradually increased to a weak alkalinity 24 hours later, with pH values ranging from 6.14 to 8.28. The porosity was 13.91% and solubility ranged from 1.28% to 1.33%. The NHA/TTCP/PAA paste balls did not disintegrate in phosphate-buffered solution, indicating that it had excellent anti-washout ability. The contact angle of water was 54.33°±8.99°, indicating excellent hydrophilicity. However, the radiopacity of NHA/TTCP/PAA was low, which was equivalent to 1.11±0.05mm of aluminum, and did not comply with the requirements of ISO 6876:2001. Its cytotoxicity was ranked from grade 0 to I, which complied with the requirements of GB/T16886.5-2003 standard. With good physical-chemical properties and cytocompatibility, NHA/TTCP/PAA presents a promising clinical application prospect.2. Development, characterization and properties of the novel root-end filling material (NRFM) based on NHA/TTCP/PAATo satisfy the acceptable level of radiopacity, three kinds of NHA/TTCP/PAA associated with different radiopacifying agents were developed by adding 20%bismuth oxide (NHA/TTCP/PAA-Bi), zirconium oxide (NHA/TTCP/PAA-Zr) and barium sulfate (NHA/TTCP/PAA-Ba) respectively. XRD patterns, FTIR spectra and SEM images revealed that they were primarily composed of hydroxyapatite, calcium polyacrylate, calcium citrate and the relative radiopacifier. These materials had good physicochemical properties which were similar to those of NHA/TTCP/PAA. In addition, the radiopacity of NHA/TTCP/PAA-Bi and NHA/TTCP/PAA-Zr were equivalent to 5.22±0.23mm and 3.63± 0.22mm of aluminum respectively, which complied with the requirements of ISO 6876:2001. Besides that, their compressive strengths were 53.28±2.78MPa and 62.19±8.87MPa after 7 days respectively, which were greater than that of NHA/TTCP/PAA significantly (P<0.05). The cytotoxicity scores of both fresh specimens (set 10min) and aged specimens (set 24h) of them ranked from grade 0 to I, which complied with the requirements of GB/T16886.5-2003. Among all tested materials, the cytocompatibility of NHA/TTCP/PAA-Zr was the best. Due to its superior physical-chemical properties and cytocompatibility among the tested materials, NHA/TTCP/PAA-Zr was selected as the novel root-end filling material (NRFM).3. Comparison of the sealing ability of NRFM, MTA and GICSealing ability is one of key factors which affects success rate of endodontic surgery. Microleakage models were set up by using root canals of extracted human teeth and simulated root canals separately. The sealing ability of them was investigated by dying method. The order of mean dye microleakage values was MTA< NRFM< GIC and there were statistically significant differences among experimental groups (P<0.05). The marginal adaptation of three root-end filling materials and the dental wall of root canals of extracted human teeth was assessed by SEM. The results showed that MTA and NRFM had good marginal adaptation, but GIC did not.4. Comparison of biocompatibility of NRFM, MTA and GICThe biocompatibility of root-end filling materials is one of the most important requirements because the materials are in direct contact with periapical tissues. The general biocompatibility and cell biocompatibility of NRFM, MTA and GIC were assessed in the present research.1) The reactions of subcutaneous connective tissue to NRFM, MTA and GIC were evaluated by implanting these materials into the dorsal connective tissue of Sprague-Dawley rats. After 1 week, all tested materials induced an initial acute inflammatory response. With time prolonged, all tested materials and control specimens had significantly less severity of inflammation compared to the result of 1 week. No significant difference between NRFM and control specimens was observed in terms of extent and severity of inflammation. However, the extent and severity of inflammation of MTA and GIC was severer than that of NRFM and a few inflammatory cells were observed in them after 12 weeks (P<0.05). Thus, NRFM shows the best histocompatibility among all of tested materials.2) The proliferation and differentiation of NRFM, MTA and GIC on human osteoblast-like MG-63 cells was assessed by using a transwell cell culture system in vitro. Compared with the negative control group, NRFM did not inhibit the proliferation of MG-63 cells, but it significantly promoted the alkaline phosphatase (ALP) activity of them on day 5 and 7 (P<0.05). MTA also inhibited the proliferation and promoted the ALP activity of MG-63 cells on day 5 and 7 significantly (P<0.05). Different from MTA and NRFM, GIC inhibited the proliferation of MG-63 cells on day 5 and the ALP activity on day 3,5 and 7 significantly (P<0.05). Therefore, NRFM has minimum cytotoxicity on MG-63 cells and can promote cell osteogenic differentiation among all three materials.5. The molecular mechanism for effects of NRFM, MTA and GIC on MG-63 cells osteogenic differentiationGene expression profiles of MG-63 cells after co-cultured with NRFM, MTA and GIC for 5 days were obtained through gene expression microarray technology. After screening the data,2950,2273 and 404 differentially expressed genes were gained in NRFM, MTA and GIC group respectively (P<0.05). Gene ontology (GO) of all the differentially expressed genes were analyzed by DAVID database. In GO biological process classifications,6,5 and 1 classifications on osteogenic differentiation were involved in NRFM, MTA and GIC group respectively. There were also 2 classifications on odontogenesis differentiation involved in NRFM group. Biological pathway analysis of the differential expressed genes indicated that both PI3K-AKT and MAPK singaling pathways regulated MG-63 cells osteogenic differentiation in NRFM and MTA. And only PI3K-AKT singaling pathway regulated MG-63 cells osteogenic differentiation in GIC. The 6 genes related to osteogenic differentiation were chosen to be assessed by qRT-PCR. The results of qRT-PCR suggested that microarray experiments were reliable and reproducible.Compared with GIC and MTA, the novel root-end filling material (NRFM), whose main reaction products are hydroxyapatite, calcium polyacrylate, calcium citrate and zirconium oxide, has excellent biocompatibility and physicochemical properties in terms of setting time, washout resistance, pH and so on. It also can promote MG-63 cells osteogenic differentiation. Based on the results of the present study, the NRFM appears to be a promising ideal root-end filling material.