| Since the colonization by C. albicans of the silicone-based materials frequently used for maxillofacial prostheses is of importance in clinical terms, studies of fungal colonization on some commercial maxillofacial silicone prosthetic elastomers have been published.Clinical applications of elastomer focus on maxillofacial prosthetic rehabilitation after traumatic deficits and tumor resection. Thus, elastomer is in contact with wound surfaces and mucosa. In this way, elastomers tend to be contaminated with microorganisms from a patient's saliva and blood. The physical and microbiological properties related to the porosity of maxillofacial elastomer are partly disadvantageous in the use of these materials. The porosity allows for the colonization and infection of the silicone-based material surface by C. albicans and related Candida species, which has been reported to have serious consequences. Clinically, it has been pointed out that patients, particularly immunocompromised or medicated elderly hosts, would be at risk for unexpected infections from the continuous swallowing or aspiration of microorganisms from denture plaque. The maintenance of maxillofacial elastomer hygiene is important, yet, there are no sufficiently effective methods for doing this.Humanβ-defensin-3 (HBD3) is a novel human epithelial-derived endogenous cationic antimicrobial peptides, which exhibits a strong, quickly and broad spectrum bactericidal action, as well as some multidrug-resistant clinical nosocomial pathogens. Consequently, the antibacterial properties of HBD3 have attracted the attention of researchers in the field of dentistry. The purpose of this study was to evaluate the antimicrobial efficacy of rHBD3 on maxillofacial elastomer. This may provide a method that effectively and efficiently eliminates various kinds of bacteria in maxillofacial elastomers.Our research and outcomes:1. Expression of HBD3 in E. coli, purification and antimicrobial bioactivity assayThe expression vector was constructed, called pET32a/ HBD3, which was finally transformed into E. coli BL21 (DE3) following the standard procedure. The target protein fused with a His-tag was induced by isopropyl-1-thio-b-D-galactopyranoside (IPTG). Standard SDS-PAGE (15% gel) and western blotting analysis was used to check the expression of fusion protein. A fusion protein with a molecular weight of about 24 kDa was obtained and was further confirmed by western blotting. After cell disruption, rHBD3, along with the 6His-tagged fusion protein, was purified by means of metal-affinity chromatography and re-natured by means of dilution for protein refolding. The SDS-PAGE assay showed the results. HBD3, the recombination protein that was produced, exhibited antimicrobial activities against S. aureus and C. albicans in different temperature.Secondary structure of rHBD3 was predicted by circular dichroism. The ratio of composing componentnormal of secondary structure of rHBD3 which is similar to HBD3 of human gingival epithelium tissue.The isoelectric point by Isoelectric Focusing Electrophoresis was 8.6. Study on the Antibacterial Mechanism of rHBD3. Experimental studies on the cytotoxicity of rHBD3. Antimicrobial activities of recombinant HBD3 against S. aureus and C. albicans on minimum inhibitory concentration (MIC) would have no obvious influence on normal human dental pulp.2. Study on antibacterial properties and research for antibacterial mechanism of rHβD-3 to maxiliofacial silicone elastomer.Firstly, investigate the antibacterial activity of rHBD3 (25μg/m1) to Staphylococcus aureus and Candida albicans on the surface of maxiliofacial silicone elastomer. Results confirmed that an immersion time of 30min or higher could yield a good disinfection effect, both for the experiment group and the positive control. These results suggest that when rHBD3 is used in immersion disinfection in the clinic, the time of 30 min should be recommended.Secondly, confocal laser scanning microscopy of biofilm-associated C. albicans and biofilm-associated S. aureus were treated with rHBD3 in different times.The result show that a large number of pathogenic bacterium were killed cells stained red by SYTO9/PI after 30min.Thirdly, to develop an insight into the mechanisms by which C.albicans and S. aureusis possibly killed by HBD3, we examined the morphological changes of C.albicans and S. aureus exposed to HBD3 by transmission electron microscopy. The result shows signs of perforation of the peripheral cell wall, with explosion-like liberation of the plasma membrane within 30 min. After 1 h most cells undergo bacteriolysis with different degrees of cellular disintegration.3. Physical properties of a maxillofacial silicone elastomer immersed in human beta-defensin-3A study on the intrinsic or extrinsic colorant of specimens which be made of A-2186 silicone elastomer, were immersed in rHBD3 (25μg/ml). Color changes (ΔE*) were calculated with a spectrophotometer. Under D65 light source, color change was determined from the data obtained before storage (baseline) and after 6 months. The same groups of samples were used throughout the test, because they were able to serve as their own controls. In this study,ΔE* less than 2 was used as baseline and considered less effect on the color stability of the specimens tested.The another experimental procedures conformed to specifications for vulcanized rubber established by the American Society for Testing and Materials (ASTM). To study the mechanical function of silicone elastomer were immersed in PH5 or PH7.5 rHBD3, the tensile strength (MPa), the elongation at break (%), the tear strength (kN/m) and the Shore-A hardness were tested. Immersion in acidic rHBD3 gave a greater change in the mechanical properties compared with the alkaline solution.The tensile modulus ranged from 5.8 to 6.3 MPa; the tensile strength ranged from 13.08 to 13.5 MPa, and the elongation from 430% to 412%. Furthermore, the Shore A hardness varied from 36.3 to 41. Finally, the weight changes found ranged from 0.01% to 0.36% weight loss.Clearly, rHBD3 not only eliminated these microorganisms in the maxiliofacial silicone elastomer, but may also effective to stubborn biofilm- associated C. albicans and biofilm-associated S. aureus. The physical properties of the maxillofacial silicone elastomer, before and after immersion in acidic solutions, exhibited significant changes. These changes will not affect the application of rHBD3 in clinic.At the same time, rHBD3 has less effect on the color stability of the specimens te sted for some time. |
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