Use of a self-assembling on the biomodification of caries-affected dentin: chemical and biomechanical analyses

Author(s)

Jossaria Pereira de Sousa

• Document type: Doctoral Thesis
• Press: Piracicaba, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Faculdade de Odontologia de Piracicaba
• Defense date: 2018-08-03

Examining board members

Regina Maria Puppin Rontani; Kamila Rosamilia Kantovitz; Fabiola Galbiatti de Carvalho; Alan Roger Santos Silva; Sérgio Luiz Pinheiro

• Advisor: Regina Maria Puppin Rontani

Abstract

Recently, a rationally designed self-assembling peptide P11-4 has been developed to enhance remineralization on initial caries lesions; yet, its applicability on dentin tissue remains unclear. Thus, the present study investigated the interaction of P11-4 with the inorganic and organic dentin compounds. Furthermore, the effect of the self-assembling peptide on the bonding interface to artificial caries-affected dentin (CaD) was studied. Experiments were divided in three main arms: 1- nucleation of hydroxyapatite, 2- interaction with collagen type-I, and 3- influence on the resin/CaD bonding interface. Fluorescence changes in the P11-4 tryptophan was measured to verify the affinity of P11-4 to calcium ions. Dynamical light scattering (DLS) and pH monitoring were used to evaluate the rate of HAP formation in the presence of P11-4, while Atomic Force Microscopy (AFM) screened the size of HAP particles formed. Surface Plasmon Resonance (SPR) and AFM were used to measure the interaction of the peptide P11-4 with collagen fibers. Fluorometric and SDS-PAGE assays were performed to evaluate collagen degradation inhibition by P11-4. Finally, the influence of P11-4 on bonding interface to CaD was verified. Eighty-four extracted human third molars were randomized in six different groups (n=11), according to dentin substrate (SD:sound dentin, CaD:caries-affected dentin, and CaD + P11-4:CaD treated with P11-4), and storage-time (24 h and 6 months). Adhesion to substrates were done with the two-step etch-and-rinse adhesive system AdperTM Single Bond 2, and a 4-mm-thick resin composite block was built. Samples were stored in deionized water at 37°C for 24 h. Afterwards, samples of each group were allocated into to three different analysis: microtensile bond strength -µTBS (n=8), nanoleakage (n=3) and in situ zymography (n=3). Kolmogorov Sminorv test was applied to evaluate data normality, and appropriate tests were chosen to verify difference between groups, p< 0.05. P11-4 had its intrinsic fluorescence decayed in function of the Ca2+ concentration (Kd= 0.63±0.07 mM). Dynamical light scattering and pH monitoring showed P11-4 increased significantly the rate of conversion of amorphous calcium phosphate (ACP) into octacalcium phosphate (OCP), while the velocity of conversion of OCP into HAP phase was decreased. AFM analysis demonstrated P11-4 standardized the HAP particle growth by reducing the particle size variability. SPR and AFM explorations showed P11-4 bind to immobilized collagen I fibers, increasing its diameter from 214±4 nm to 308±5 nm (p<0.0001). Complementary, SDS-PAGE demonstrated the increase of P11-4 concentrations impaired the collagen degradation from bacterial collagenase. Regarding bonding analysis, the immediate treatment of CaD with P11-4 enhanced the mechanical performance of bonding interface (p<0.0001), by increasing the µTBs values and to decrease the nanoleakage; additionally to impair the proteolytic activity of hybrid layer (p<0.05). Those effects were reduced overtime but still higher compared to CaD group. In conclusion, the present study observed that P11-4 induces the nucleation of hydroxyapatite and protects Collagen Type I against bacterial collagenase degradation. In addition, P11-4 improves the stability of the hybrid layer formed by artificial caries-affected dentin (AU)

• FAPESP's process: 14/22899-8 - Effect of remineralizing agents on proteolytic activity of caries-affected dentin and on and resin/dentin bond
• Grantee: Jossaria Pereira de Sousa
• Support Opportunities: Scholarships in Brazil - Doctorate