Establishment Of An Antimicrobial Model Of Peri-implantitis And The Potential Application Of An Antimicrobial Peptide GL13K In GBR Treatment

Dentition defects have been seriously affecting humans' normal oral function for many years,which greatly reduce the efficiency of chewing hence reduce the quality of lives.In modern dentistry,dental implant restoration has been widely used as an excellent method for the treatment of dentition defects which has shown the advantages of high chewing efficiency,no damage to adjacent teeth,and low peculiar sense.However,its long-term success rate is still affected by many factors.Since peri-implantitis has been one of the main causes of implant failure,the prevention and treatment strategies for peri-implantitis are particularly important.Oral hygiene routine has always been an important part of preventing peri-implantitis.Furthermore,the use of an oral mouthrinse to reduce the bacteria load around the implant to prevent peri-implantitis has been a crucial issue in oral hygiene routine.However,the research on the antibacterial effect of mouthwash against the plaque biofilm on implant surfaces is still lacking.On the other hand,patients with peri-implantitis who have been diagnosed with symptoms such as bone absorption should undergo surgical treatment at an early stage.Guided bone regeneration(GBR)is an effective surgical treatment for repairing bone defects by using bone substitute materials and membranes,which could extremely benefit the long-term survival rate of implants.Thus,membranes as a physical barrier,are playing an essential role in the treatment of GBR.However,most common membranes do not exhibit antibacterial activity or the ability to induce bone regeneration.We studied the prevention and treatment strategies of peri-implantitis through three parts.In Part I,the antibacterial effect of two common mouthwashes on plaque biofilm grown on titanium implant surfaces was evaluated.The results indicated the mouthwashes can effectively kill dental plaques at the early stage of biofilm growth on the titanium implant surfaces hence prevent peri-implantitis.In Part ?,we worked on the antibacterial mechanism of an antimicrobial peptide(AMPs)GL13K by evaluating the relationship between its secondary structure,self-assembly process and antibacterial effect.The conclusions provide the theory for the preparation of new antibacterial drugs or coatings.In Part?,we developed a new kind of biomaterial for GBR membranes by coating GL13 K on mineralized collagen gels to provide antibacterial activity and ability to induce bone regeneration.Part I Antimicrobial effects of mouthrinses on dental plaque biofilms growing on different titanium implant surfaces?Objective? The aim of this study was to evaluate the antimicrobial effects of mouthrinses on dental plaque biofilms regarding three types of titanium surfaces by using an open biofilm model.?Materials and Method? The average surface roughness(RA)of three types of titanium surfaces(Smooth,hydroxyapatite(HA),sandblast large grit,and acid-etching(SLA))were tested by atomic force microscope(AFM).Subgingival plaques were collected and cultured on titanium surfaces for 4 hours to 2 weeks.After treatment with mouthrinses(PBS,0.2%CHX,Colgate Plax and Listerine COOL MINT)for 3 or 10 min,the percentage of dead bacteria in biofilms was measured by confocal laser scanning microscopy(CLSM)and viability staining.Another group of samples were prepared for SEM test.?Results? It was revealed that the mouthrinses were effective on the killing of young dental plaque biofilms(4 h-7 d),while the more mature biofilm(14 d)exhibited a stronger resistance to mouthrinses used in this study.Different titanium surfaces also affect the adhesion and growth of dental plaque biofilm,thus affects the antibacterial effect of mouthrinses.?Conclusions? In conclusion,Colgate Plax and Listerine COOL MINT are effective mouthrinses to kill dental plaques on the titanium implant surfaces.However,the mature biofilm has higher resistance to the disinfectants,indicating that mouthrinse is more beneficial to be used at the early stage of dental plaque biofilm growth.Part ? Supramolecular Self-assembly and Antimicrobial Activity of L-and Denantiomers of GL13 K peptide: Effect of p H,Time,and Amino Acid Sequence?Objective? Here,we tested all L-and D-amino acid versions of GL13 K,an AMP derived from a human protein,to study structural links between the AMP secondary structure,supramolecular self-assembly dynamics,and antimicrobial activity.?Materials and Method? Three different sequences of GL13K(L-GL13 K,DGL13K,L-GL13K-R)were prepared into an aqueous solution with a concentration of 0.1 m M under different p H conditions.The secondary structure in GL13 K was observed with transmission electron microscopy followed by evaluating the content of secondary structure with circular dichroism analysis.The p Ka values of L-GL13 K,D-GL13 K and L-GL13K-R were subsequently determined using p H titration experiments.Finally,the antimicrobial activity of the three peptides against the Gram-positive bacteria S.gordonii M5 and the Gram-negative bacteria P.aeruginosa Xen41 was tested.?Results? p H dependence and the evolution of secondary structures were related to a self-assembly process with differences among these AMPs.The two GL13 K enantiomers formed analogous self-assembled twisted nanoribbon structures,but DGL13 K initiated self-assembly faster and had notably higher antimicrobial potency than L-GL13 K.A nonantimicrobial scrambled amino acid version of L-GL13 K assembled at a much higher p H to form distinctively different self-assembled structures than LGL13 K.?Conclusions? Our results support a functional relationship between the AMP selfassembly and their antimicrobial activity which benefits our understanding of its antibacterial mechanism,providing better application in biomedicine.Part ? Biomimetic Mineralized Collagen gels coated with GL13K?Objective? The aim of this study was to develop a kind of multi-functional absorbable membrane material with high mechanical strength by loading the antimicrobial peptide GL13 K onto mineralized collagen gels,which has both antibacterial activity and osteogenic induction.?Materials and Method? Collagen gels were prepared from type I collagen solution,followed by cross-linking and biomimetic mineralization(0,1,2,4,and 8 days).Physicochemical properties of mineralized collagen were detected by scanning electron microscopy,transmission electron microscopy,X-ray diffraction analysis,thermogravimetric analysis,and rheological test.The mineralized collagen gels were coated with GL13 K by dipping in a 1.4 mg / m L GL13 K solution,and its loading and release rate were evaluated.The antimicrobial activity of the mineralized collagen gel with GL13 K coating against Gram-positive bacteria S.gordonii and Gram-negative bacteria E.coli was evaluated with ATP assay and Live/Dead assay.?Results? The PILP biomimetic mineralization system used in this experiment was able to effectively control the minerals to be only intrafibrillar for up to 8-day mineralization.The mineralization degree increased over time and after 8 days,the collagen fiber was filled with dense hydroxyapatite which also exhibited much higher storage modulus.In addition,the mineralized collagen gels were more stably bound to GL13 K,which achieved a higher loading amount and a slower release rate than pristine collagen gels.Moreover,the mineralized collagen with GL13 K coating showed effective antimicrobial activity against Gram-positive bacteria S.gordonii and Gramnegative bacteria E.coli,and it was also shown that the GL13 K coating was more efficient in killing E.coli than S.gordonii.? Conclusions ? This experiment demonstrated that the cross-linking and mineralization method can effectively enhance the mechanical properties of collagen gels and also improve its ability to bind to the antimicrobial peptide GL13 K.Thus,the mineralized collagen gel coated with GL13 K has shown the potential of being a new type of multifunctional GBR membrane.