| Pulpitis and periapical periodontitis are two common diseases in dental clinic. Microorganisms are major etiological agents in pulpal and periapical disease. Root canal therapy is a method of treating pulp necrosis and periapical diseases with the help of periapical tissues which are characterized by abundant blood flow and strong ability of repair and regeneration. The main goal of endodontic therapy is the elimination of microorganism from the root canal system and the prevention of subsequent reinfection, so as to cure the inflammation and conserve the sick tooth. But the persistence or reinfection of bacteria in the complicated root canal system often leads to failure of root canal treatment. Although most of the bacteria, necrotized pulp and infected dentin are removed from the root canals after chemical and mechanical preparation, there still have etiological agents such as residual bacteria, endotoxin in collateral pulp canals, deep part of dentinal tubules and periapex. Therefore, root canal disinfection is not a single procedure can be omitted. There are many different endodontic sterilizing medicines after longtime clinic and research work. Therefore, to thoroughly kill and avoid again infection of these microorganisms, endodontic sterilizing medicine should ideally have a powerful and broad-spectrum antibacterial capability.Humanβ-defensin-3 (hBD3) are a novel human epithelial-derived endogenous cationic antimicrobial peptides, which exhibits a strong, quickly and broad spectrum bactericidal action, in addition, with low susceptibility to acquired bacterial resistance. Consequently, the antibacterial properties of hBD3 have attracted the attention of researchers in the field of dentistry. However, there is nothing in the literature regarding the antibacterial efficacy of hBD3 against bacteria in the root canals. The aim of this study was to obtain recombinant hBD3 by means of gene engineering technique, moreover, evaluate and compare the antimicrobial effectiveness of hBD3 with other regular intracanal medicaments, so as to develop a new antimicrobial agent that suitable to treat pulp and periodontal inflammation, especially, severe infections. Our research and outcomes:1. Cloning of hBD3 cDNA from human gingival epithelium Two primers were designed based on hBD3 nucleotide sequence data registered in GenBank and used to clone hBD3-specific sequence from the mRNA template, which obtained from normal human gingival epithelium tissue's total RNA. After the RT-PCR, the product of expected size was inserted into cloning vector pGEM-T. Recombinant pGEM-T / HBD-3 vector was transduced into competent cell DH5αfor amplification, followed by sequencing. The result display that hBD3 gene was successfully cloned from human gingival tissue. Agarose gel electrophoresis showed an expected single strap about 250 bp and sequence of the product was coincident with the cDNA of hBD3 nucleotide order (GenBank TM accession nos.AJ237673).2. Expression of hBD3 in E. coli, purification and antimicrobial bioactivity assayhBD-3 gene was ligated to vector pET-32a(+). The expression vector was constructed, called pET32a/ HBD-3, which was finally transformed into E. coli BL21 (DE3) following the standard procedure. The foreign protein expression was induced by adding IPTG to transformed monoclone. According to SDS-PAGE analysis, the target protein was successfully expressed. A molecular weight of about 24 kd fusion protein was obtained, and was further confirmed by western blotting analysis. Expression optimization: the maximum was obtained when the expression condition is 35℃,0.6mmol/L IPTG and 6 hours induction.Cytorrhyctes, products of the massive expression, were dissolved by guanidine hydrochloride. After metal-affinity chromatography, rhBD3, with the 6Histagged fusion protein, was renatured by means of dilution for protein refolding. Then, rhBD3 was further purified by cationic exchange chromatography and desalted by gel chromatography. The SDS-PAGE assay showed the results. The mature peptide was obtained from fusion protein after cut by enterokinase, and about 5.1kd, similar with the reference.The antimicrobial bioactivity assay of recombinant rhBD3 display that both the fusion protein and the mature peptide exhibit antimicrobial activity against S. aureus and E. coli.3. In vitro study of the antibacterial activity of recombinant hBD3 against pathogenic bacterium from the infected root canalsSome distinct pathogenic bacterium (including anaerobe and facultative anaerobe) in endodontic infections: F. nucleatum, B. melanogenicus, P. anaerobius, S. mutans, A. naeslundii, E. faecalis, C. albicans, S. aureus and E. coli were performed in the antimicrobial activity assay. Assess the ability of peptides to kill bacteria, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values were obtained using a conventional microdilution method. Results presented that the recombination protein exhibits a broad spectrum antimicrobial activity. As the colony of bacteria remaining illustrated that the growth of bacteria was dramatically suppressed by rhBD3. And MIC and MBC of each bacterium were different respectively. Analyses of the in vitro antimicrobial properties of rhBD3 revealed antimicrobial activity against common facultative anaerobes: S. aureus and E. coli as well as three potential pathogenic anaerobes in endodontic infections: F. nucleatum, B. melanogenicus, and P. anaerobius. Furthermore, rhBD3 kills A. naeslundii, C. albicans and vancomycin-resistant E. faecium at similar low concentrations. But it has not so effective antimicrobial activity against S. mutans.Next, the study of the antibacterial activities of rhBD3 on pathogenic bacterium from the infected root canals in vitro showed that all experimental medicaments were effective against microorganism after sealed in the infected root canals. A statistically significant difference was found between the experimental groups and negative control groups (P<0.005). Among all of the medicament groups, FC Group, iodoform Group and rhBD3 Group were found to be more effective than Ca(OH)2 Group against anaerobes (P<0.05), however, no significant difference was found between FC Group, iodoform Group and rhBD3 Group (P>0.05). Significantly, the final product——rhBD3 can markedly kill and inhibit the multiplication of bacteria, especially anaerobe, in root canals.4. In vivo study of rhBD3 used as an intracanal medicament on infected dog's root canalsThe animal model of infected root canal was made to evaluate the antibacterial activities on pathogenic strains in the root canals. X-ray picture confirm the existence of periapical periodontitis in dog's incisors. The results showed that comparing with control group, the detection rate of bacteria, no matter anaerobes or aerobes, in experiment groups (Fc, Ca(OH)2 paste, rhBD3) decrease obviously. X2 analysis showed a statistically significant difference between the experimental groups and negative control groups (P<0.05). They all can reduce the amount of strains in root canals after sealing. Among all of the medicament groups, FC Group and rhBD3 Group were found to be more effective than Ca(OH)2 Group, especially against anaerobes (P<0.05).The HE slice was also made to evaluate the influence on the inflammation of periapical disease among four different groups. All the medicaments have influence on periapical inflammation. A statistically significant difference was found between the experimental groups and negative control groups (P<0.05).The level of inflammation of FC Group was found to be more serious than Ca(OH)2 Group (P<0.05). We conclude that it may due to the intense cytotoxicity. However, significant difference was found between rhBD3 Group and Ca(OH)2 Group (P<0.05). So, rhBD3 can also alleviate the inflammation of periapical disease to some extent, but not so good as Ca(OH)2 paste, which may be related to its indirect immune activation.In conclusion, hBD3, obtained by gene engineering technique, exhibits a broad spectrum antibacterial activity, at low micromolar concentrations, especially to many critical infected pathogenic microbes in root canals. The results indicate that rhBD3 has the potential to eliminate bacteria in the local microenvironment of the root canal system, thus lead to the success of endodontic treatment. The prospect of the application of rhBD3 as an intracanal medicament is good, and more deep research should be done in the future. |
