Enhancing humic acid removal using surface modified granular media

Enhancing Resin/Dentin bond durability: The Effect of Chitosan/Riboflavin Modification By Umer Daood Thesis Submitted In Partial Fulfillment of the Requirements For the Degree of MASTER OF SCIENCE (RSH-FoD) At Discipline of Oral Sciences, Faculty of Dentistry National University of Singapore 2013 Supervisors: Assistant Professor Amr Fawzy (Main supervisor) Associate Professor Cao Tong ! "! (Co- supervisor) Declaration I hereby declare that the thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any University previously. _________________ Dr Umer Daood 20th November 2013 ! #! Acknowledgement My deepest appreciation to Almighty Allah for letting me through this academic journey. I would like to thank my supervisor and the scientific committee members on providing me full support for my work here at Faculty of Dentistry, National University of Singapore. My special thanks to Dr Amr Fawzy for his thoughtful and patient guidance throughout the course. I would also acknowledge my co-supervisor Associate Professor Cao Tong who had also helped me get through with this academic task. I am indebted for all the efforts that they have put in and helping me achieve this milestone. I would also want to thank Dr. Sudhiranjan Tripathy and Mr. Surani Dolmanan at Institute of Materials Research Engineering (IMRE, Singapore) for their technical assistance in micro-Raman analysis of the specimens. My special acknowledgements to my wife, son and family whose support had helped me throughout the entire course. Finally I would like to thank the members of the lab and the friends in NUS who took me from strength to strength. ! $! Table of Contents Acknowledgement ------------------------------------------------------------------- 2 Table of Contents -------------------------------------------------------------------- 3 !"#$%&'%(")*+,#!----------------------------------------------------------------------- 6 List of Tables ------------------------------------------------------------------------ 42 List of Abbreviations --------------------------------------------------------------- 48 List of Deliverables ------------------------------------------------------------------ 50 Abstract ------------------------------------------------------------------------------- 51 1. Introduction to Dentin Structure ------------------------------------------- 54 1.1. Intertubular dentin -------------------------------------------------------------- 55 1.2. Peritubular dentin --------------------------------------------------------------- 57 1.3. Tertiary dentin ------------------------------------------------------------------- 58 2. Riboflavin – Chemistry and Biological function ------------------------- 59 3. Collagen Structure ------------------------------------------------------------- 63 3.1. Distribution and Biosynthesis -------------------------------------------------- 64 3.2. Functions -------------------------------------------------------------------------- 66 3.3. Degradation ----------------------------------------------------------------------- 68 3.4. Bonding Hydrolysis -------------------------------------------------------------- 69 4. Chitosan Structure -------------------------------------------------------------- 70 4.1. pH ---------------------------------------------------------------------------------- 71 4.2. Applications ---------------------------------------------------------------------- 72 4.3. The Collagen Chitosan Relationship ------------------------------------------ 73 5. Singlet Oxygen Radical Theory ---------------------------------------------- 75 5.1.Riboflavin singlet oxygen -------------------------------------------------------- 77 5.2. Generation of singlet oxygen by light in the presence of ! %! Endogenous and exogenous sensitizers ---------------------------------------------- 78 5.3. Singlet oxygen and protein breakdown ----------------------------------------- 78 6. Collagen Crosslinking ----------------------------------------------------------- 80 7. Raman Spectroscopy ------------------------------------------------------------ 84 7.1. Raman Mapping and Imaging Instrumentation ------------------------------ 84 7.2. Sampling for Raman Spectroscopy; Applications --------------------------- 85 7.3. Protein Spectra ------------------------------------------------------------------- 86 7.4. Raman of the Hybrid Layer ----------------------------------------------------- 87 7.5. Raman spectroscopy is an effective technique for --------------------------the analysis of monomers and polymers 89 8. Resin bonding to dentin --------------------------------------------------------- 91 8.1. Adhesive systems ---------------------------------------------------------------- 94 8.2.Two-Step-etch-and-rinse -------------------------------------------------------- 95 8.3. Self-etch adhesive system – a drive for simplification---------------------- 97 8.4. Hybrid layer formation and degradation ------------------------------------ 98 8.5. Crosslinking and reinforcement of dentin collagen ------------------------ 103 9. Hypotheses and Aim!------------------------------------------------------------ 107 10. Materials and Methods ------------------------------------------------------- 109 10.1. Phase I ------------------------------------------------------------------------- 109 10.2. Phase II ------------------------------------------------------------------------ 116 10.3. Phase III ----------------------------------------------------------------------- 122 11. Results --------------------------------------------------------------------------- 127 11.1. Phase I ------------------------------------------------------------------------- 127 11.2. Phase II ------------------------------------------------------------------------ 130 11.3. Phase III ----------------------------------------------------------------------- 133 ! &! 12. Discussion!----------------------------------------------------------------------- 137 12.1. Introduction ------------------------------------------------------------------- 137 12.2. Phase I Discussion ----------------------------------------------------------- 142 12.3. Phase II Discussion ---------------------------------------------------------- 148 12.4. Phase III Discussion --------------------------------------------------------- 153 13. Summary, conclusions and Future Work ------------------------------ 158 14. Bibliography ------------------------------------------------------------------ 162 ! '! List of Figures Fig 1. SEM of 35% phosphoric acid etched dentin showing open dentinal tubules lined with peritubular dentin as indicated by arrow (adapted from SEM evaluation of the interaction pattern between dentin and resin after cavity preparation using ER:YAG laser; Journal of Dentistry (2003) 31, 127–135). ! (! Fig 2. Schematic representation of the peritubular and mineralized intertubular dentin. ! )! Fig 3. Tertiary dentin also known as Reactive Dentin is seen clearly in this tooth model produced as a reaction to the caries. (http://www.dentalcaries.com/page.asp?pid=605). ! *! Fig 4. Schematic presentation of the chemical structure of riboflavin indicating the CH2OH positioning by the transfer of electrons in alloxan, and oxylene. ! "+! Fig 5. Type I collagen shown as a molecular structure of fibrillar collagen with various subdomains with cleavage sites for N- and C-procollagenases (adapted and redrawn from ‘Collagens—structure, function, and biosynthesis’; Advanced Drug Delivery Reviews 55 (2003) 1531– 1546) ! ""! Fig 6. The lysyl mediated mature crosslinks formed within the collagen Type I fiber; lysyl pyridinoline and hyroxylysyl-pyridinoline. ! "#! Fig 7. The collagen triple helix. (a) The crystal structure of collagen molecule; (b) view down the axis of triple helix with three strands with space filling, ball stick and ribbon presentation; (c) ball and stick profile of collagen triple helix; (d) stagger for three strands (Proteins: Three Dimensional Structure; Section 6-1. Secondary Structure). ! "$! Fig 8. The degree of N-acetylation in the physiochemical nature of chitin and chitosan (adapted from Biomedical Activity of Chitin/Chitosan Based Materials—Influence of Physicochemical Properties Apart from Molecular Weight and Degree of NAcetylation; Polymers 2011, 3(4), 1875-1901; doi:10.3390/polym3041875 Review). ! "%! Fig 9. Patterns of cross-linking collagens. Collagen types I (2), III (4), and IV (6) show a banding pattern distinct from the other two shown. The riboflavin sensitization with UVA causes the collagen Type I to almost disappear (3) [Effects of UltravioletA and riboflavin on the Interaction of Collagen and Proteoglycans during Corneal Cross-linking; Published, JBC Papers in Press, February 18, 2011, DOI 10.1074/jbc.M110.169813; Yuntao Zhang1, Abigail H. Conrad, and Gary W. Conrad. ! "&! Fig 10. The Type I mechanism leading to electron transfer as a result of hydrogen atom abstraction, thus yielding free radicals, which in turn can react with the available oxygen species to form the superoxide ion. The Type II mechanism results in collision of the excited sensitizer and the triplet excited oxygen that also results in an energy transfer. [Reproduced from (1995) Royal Chemical Society]. ! "'! Fig 11. The allysine crosslinking pathway with lysine residues for intermolecular crosslinking. The skin collagen involves histidine forming mature crosslinks (Adapted from Collagen Cross-Links; Top Curr Chem (2005) 247: 207–229). ! "(! Fig 12. Hydroxylation of crosslinking lysine residues showing bone tissue specific crosslinking (Adapted from Collagen Cross-Links; Top Curr Chem (2005) 247: 207– 229). ! ")! Fig 13. Raman and Rayleigh scattering compared in the Jablonski diagram in which a molecule getting excited to a virtual state in lower energy with photon losing its energy in Stokes Raman shift and gains a photon in Anti-Stokes Raman shift. ! "*! Fig 14. Raman spectra obtained from procine cartilage explants with minimal 960 cm1 peak [Adapted from Early detection of biomolecular changes in disrupted porcine cartilage using polarized Raman spectroscopy; J. Biomed. Opt. 2011;16(1):017003017003-10. doi:10.1117/1.3528006]. ! #+! Fig 15. Schematics (redrawn) showing acid etching of mineralized dentin removing the smear layer leading to demineralization and exposing the collagen fibrils. (Redrawn from Pashely DH, Ciucchi B, and Sano H. Dentin as a bonding substrate. Dtsch Zahn Z 1994;49:760-63) ! #"! Fig 16. Scanning electron micrograph of the resin-dentin interface bonded with 3M Single Bond ESPE (1500x). (a) dental composite; (b) adhesive bond; (c) hybrid layer; (d) resin tags. ! ##! Fig 17. Scanning electron microscopic image of the resin-dentin interface bonded with 3M Single Bond ESPE. A uniform hybrid layer (HL) formation can be seen with visible resin tag (RT) formation. ! #$! Fig 18. Collagenolysis in the presence of collagenase and unwinding of the triple helix in the alpha chain (Nagase H, Fushimi K. Elucidating the function of noncatalytic domains of collagenases and aggrecanases. Connective Tissue Research. 2008;16:9:74). ! #%! Fig 19. Obtained Raman spectrum of the (a) control demineralized specimen; (b) Adper TM Single Bond; (c) and chitosan in the region of 700-1700 cm-1 (Daood et al; Effect of chitosan/riboflavin modification on resin/dentin interface: Spectroscopic and microscopic investigations). ! #&! Fig 20. Schematic representation of the specimen preparation for the micro-tensile bond strength (!TBS) test and SEM analysis. (A) Removal of occlusal surface to expose the flat dentin surface; (B) preparation of the dentin surface to receive adhesive with or without RF; (C) cutting of resin-dentin beams from the center of bonded specimen; (D) attachment of the single resin-dentin beam for immediate !TBS testing; (E) storage of resin-dentin beams in artificial saliva for 9-months storage for !TBS testing and SEM analysis. ! #'! Fig 21. Bonded specimen glued to custom-made metallic jig mounted to Universal testing machine with cyanoacrylate adhesive. ! #(! Fig 22. Representative Raman spectra of (A) Adper TM Single bond adhesive, (B) demineralized dentin specimen, and (C) resin impregnated dentin recorded in the region between 800-1800 cm-1. The P-O bond at 960 cm-1of the mineral component is well represented for demineralized and resin impregnated dentin. The peaks at 1667 cm-1 and 1246 cm-1 are associated with organic components for dentin collagen. ! #)! Fig 23. Representative SEM micrographs of the etched dentin resulting from different bio-modification procedures. Images showing an illustrative area of the dentin surface of (A) control; bio-modified with (B) 0.1%RF; (C) Ch/RF 1:4 and (D) Ch/RF 1:1 specimens. The 0.1%RF and Ch/RF 1:4 specimens show open dentinal tubules with intact collagen fibers whereas the Ch/RF 1:1 specimens exhibit a discontinuous structure. The dentin of all specimens were conditioned for 15 s with 37% phosphoric acid (Daood et al; Effect of chitosan/riboflavin modification on resin/dentin interface: Spectroscopic and microscopic investigations). ! #*! Fig 24. SEM images of the control, 0.1% RF and Ch/RF (1:4 and 1:1) crosslinked resin/dentin interfaces surfaces treated with AdperTM Single bond 2; 3M ESPE. The hybrid layer (HL) and many resin tags (RT) were found at the adhesive interface between resin cement and dentin; (A) control, (B) A uniform hybrid layer with long resin tags can be observed in the specimen interface treated with 0.1%RF crosslinking prior to dentin bonding agent application. The resin cement penetrated deeply and many long resin tags were observed at the demineralized interface. (C) A funnelshaped configuration of the resin tags also seen at the base of Ch/RF 1:4-treated specimens. The resin tags exhibited a slightly rough texture. (D) The resin tags in Ch/RF 1:1 specimens showed a regular cylindrical shape exhibiting a rough texture (arrow) on top of the resin tags and a relatively thicker and more textured hybrid layer; HL, hybrid layer; RT, resin tags (Daood et al; Effect of chitosan/riboflavin modification on resin/dentin interface: Spectroscopic and microscopic investigations). ! $+! Fig 25. Raman spectra of control, 0.1%RF and Ch/RF (1:4 and 1:1) crosslinked dentin specimens in the spectral range of 900–1700 cm-1. The peak assignments are represented in Table II. The C-H alkyl groups also appeared in the collagen spectrum in dentinal substrate with other peak areas of CAC bond, Amide I and Amide III. Schematic representation of CH3 inplane bending also shown. (a) control (b) 0.1%RF (c) Ch/RF 1:4 (d) Ch/RF 1:1. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com ; Daood et al; Effect of chitosan/riboflavin modification on resin/dentin interface: Spectroscopic and microscopic investigations]. ! $"! Fig 26. Raman line map (A) and images of the spectrum at the 4 !m (B) and 8 lm (C) crosslinked resin/dentin interfaces. Intensities at 960 cm and 1450 cm-1 for all other specimens are identified in the line map. The Raman images indicate the positions of spectra in the region of interest. The spectrum is characterized to (a) Ch/RF 1:1 (b) Ch/RF 1:4 (c) 0.1%RF (d) control. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com. Daood et al; Effect of chitosan/riboflavin modification on resin/dentin interface: Spectroscopic and microscopic investigations]. ! $#! Fig 27. SEM images of the resin-dentin interface after 24 h storage in artificial saliva. Well-defined uniform hybrid layer (white arrows) and with well-formed branched resin tags could be observed with control (A), 1%RF-modified adhesive (B), and 3%RF-modified adhesive (C) specimens. Relatively thick and textured hybrid layer with long well formed resin tags could be seen with the 5%RF-modified adhesive specimens (D). The 10%RF-modified adhesive specimens showed a very thin hybrid layer with lack of well-formed resin tags (E). C: resin composite; HL: hybrid layer; RT: resin tags. ! $$! Fig 28. SEM images showing the resin-dentin interface of the RF-modified adhesive system after 9-months aging period in artificial saliva. Relatively intact hybrid layer could be seen after 9-months storage for the control (A), 1% (B) and 3%RF-modified specimens (C) compared to the 5%RF-modified specimens (D). Hardly any hybrid layer could be observed in the 10%RF-modified specimens and only few short resin tags could be seen (E). ! $%! Figure 29: Distribution (%) of failure mode of control and RF-modified adhesive specimens after the micro-tensile strength testing of the immediate (A) and the 9months stored specimens (B) in artificial saliva. ! $&! Fig 30. Representative fracture surfaces of specimens bonded with control and RFmodified adhesives. Control group (A), without RF, showing a typical mixed fracture pattern at the outer rim, occurring mostly at the bottom of the hybrid layer (higher magnification shown in solid box); mixed failure in 1%RF-modified adhesive specimens with open dentinal tubules, and adhesive remnants (arrow) (B); 3%RFmodified adhesive specimens, with mixed failure pattern and with resin tags (C and D;); resin tags within the dentinal tubules of 3%RF-modified adhesive specimens (E; open arrows); 5%RF-modified adhesive specimens at lower magnification presenting dentin side of fracture with mixed failure pattern (solid box) and cohesive failure within the adhesive (dotted box) (F); adhesive failure within 10%RF-modified adhesive specimens (G); small cracks within 5%RF-modified adhesive specimens after 9-months of artificial saliva (H; arrows); Cohesive failure in 10%RF-modified adhesives (A=adhesive) after 9-months of storage (I). Horizontal fracture seen at the interfacial region in 10%RF-modified adhesive specimens after 9-months of storage (J). ! $'! Fig 31. The percentage of degree of conversion of control and different RF-modified adhesive specimens at different time intervals from the start of photoactivation (0 s) till 30 minutes. ! $(! Fig 32. Representative Raman spectrum for the five adhesives tested illustrating the principal functional groups. The groups can be identified at CH2CH3 1450 cm-1, C=O 1610 cm-1, C=O 1640 cm-1 and C=O 1720 cm-1. The arrows indicate shifts associated with C=O 1720 cm-1;(A) control=1717 cm-1, (B) 1%RF-modified adhesive=1711 cm1 , (C) 3%RF-modified adhesives=1710 cm-1, (D) 5%RF-modified adhesives =1713 cm-1 and (E) 10%RF-modified adhesives = 1727 cm-1. ! $)! Fig 33. Representative line map (scans) across resin-dentin interface of different control and riboflavin-modified adhesive specimens. The spectral contribution is recorded at 960 cm-1 (hydroxyapatite) and 1450 cm-1 (C-H Alkyl) intensities representing the penetration of different adhesives. ! $*! Fig 34. Comparison of Raman spectral data acquired in the region of 1030 cm-1 and 1670 cm-1 for the (A) Control, (B) 1%, (C) 3%, (D) 5%, and (E) 10%RF-modified adhesive specimens at 5!m levels. The principal bands identified are Amide bands (I and III) along with C-H alkyl groups in the resin-dentin specimens. The single arrow indicates the pyridinium ring group where accentuated intensity is observed in 3%RFmodified adhesive specimen. ! %+! Fig 35. Calculated ratios of 1610 cm-1 to 1640 cm-1 suggesting the ratio of aromatic to aliphatic groups. ! %"! Fig 36. (A) Confocal fluorescence images of the resin-dentin interfaces of (i) control, (ii) 1%, (iii) 3%, (iv) 5%, and (v) 10%RF-modified two-step etch-and-rinse adhesive specimens. The image shows variation in micro permeability within the interface. (B) 3D image of resin tags formed by 3%RF-modified adhesive specimens. ! %#! List of Tables % ,-./0!"1!Data representing the proportionate mixture of Chitosan and Riboflavin for crosslinking treatment.% -.%/&.%! 01"$/%2/3%4"5&'627"/%8&/8,/$+2$"&/%"/%$,+9#%&'%! ! !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!2345/!!!!!!!!!!!!!!!!!!!!!!!!!!!6745/!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!6-89:!:;!23467!590! "1! #1! &+! #+! &+! )+! "?"!! "?%! ! @//!A-5B/0A!C0>0!D>:AA/9EF0G!C983!+1"H!67!-EG!:>!23467!I"?%J!"?"K!;:>5=/-89:EA!-A!G0AD>9.0G!9E!830!L-80>9-/A!-EG! L083:GA! ! %$! Table 2. Typical band assignments of Raman spectrum of dentin collagen disc specimens in both innate state and after cross-linking treatment of dentin disc specimens. ! ! ! 0&662),/! :.;modified with different concentrations of riboflavin and study its effect on the bond strength and on the degree of conversion of the adhesive. In addition, investigation of the modification of commercially available two-step etchand-rinse adhesive with different concentrations of riboflavin was performed to study the effect of riboflavin modification on resin impregnation. Materials and Methods: In phase I of the study, dentin disc specimens were prepared from sound molars, acidetched with 35% phosphoric acid and modified with either 0.1% riboflavin or chitosan/riboflavin (Ch/RF ratios 1:4 or 1:1) and photo-activated by UVA. Dentin surfaces of sound molars were exposed, acid-etched, and modified as described before. Etch-and-rinse dentin adhesive was applied, light-cured, and layered with resin-restorative composite. The resin infiltration and resin/dentin interface were characterized by micro-Raman spectroscopy and SEM. Moreover, in phase II, an experimental adhesive-system was modified with different concentrations of riboflavin (0, 1, 3, 5 and 10 wt%). Similarly, dentin surfaces were etched with 37% phosphoric acid, bonded with respective adhesives, restored with restorative composite-resin, and sectioned into beams to be stored for 24 hour or 9-months in artificial saliva. Micro-tensile bond testing was performed along with scanning electron microscopy to analyze the failure distribution of debonded beams and to investigate resin-dentin interface morphology. The degree of conversion was evaluated by performing Fourier transform infrared spectroscopy at different timepoints from the start of irradiation. Data was analyzed with one-way and two-way ! &#! ANOVA followed by Tukey’s for pair-wise comparison. In phase III of the study, commercially available AdperTM Single Bond adhesive-system was modified with different concentrations of riboflavin (1, 3, 5 and 10 wt%) or left unmodified as a control. Dentin surfaces were again etched with 37% phosphoric acid, bonded with two coats of respective adhesives and restored with restorative composite-resin. The resin infiltration and resin-dentin interface were characterized by micro-Raman spectroscopy. Confocal microscopy was performed by adding Rhodamine B dye (0.01%) to the bonding resin. Data was analyzed with two-way ANOVA followed by Tukey’s for pair-wise comparison. Results: In Phase I of the study, an open-intact collagen network-structure, formation of uniform hybrid-layer and higher resin infiltration were found with 0.1%RF and Ch/RF 1:4 modifications. Raman analysis revealed chemical changes and shifts in Amide bands with the modification of dentin collagen-matrix. 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"*"! [...]... for the micro-tensile bond strength (!TBS) test and SEM analysis (A) Removal of occlusal surface to expose the flat dentin surface; (B) preparation of the dentin surface to receive adhesive with or without RF; (C) cutting of resin-dentin beams from the center of bonded specimen; (D) attachment of the single resin-dentin beam for immediate !TBS testing; (E) storage of resin-dentin beams in artificial... (white arrows) and with well-formed branched resin tags could be observed with control (A), 1%RF -modified adhesive (B), and 3%RF -modified adhesive (C) specimens Relatively thick and textured hybrid layer with long well formed resin tags could be seen with the 5%RF -modified adhesive specimens (D) The 10%RF -modified adhesive specimens showed a very thin hybrid layer with lack of well-formed resin tags... images showing the resin-dentin interface of the RF -modified adhesive system after 9-months aging period in artificial saliva Relatively intact hybrid layer could be seen after 9-months storage for the control (A), 1% (B) and 3%RF -modified specimens (C) compared to the 5%RF -modified specimens (D) Hardly any hybrid layer could be observed in the 10%RF -modified specimens and only few short resin tags could... could be seen (E) ! $%! Figure 29: Distribution (%) of failure mode of control and RF -modified adhesive specimens after the micro-tensile strength testing of the immediate (A) and the 9months stored specimens (B) in artificial saliva ! $&! Fig 30 Representative fracture surfaces of specimens bonded with control and RFmodified adhesives Control group (A), without RF, showing a typical mixed fracture pattern... (higher magnification shown in solid box); mixed failure in 1%RF -modified adhesive specimens with open dentinal tubules, and adhesive remnants (arrow) (B); 3%RFmodified adhesive specimens, with mixed failure pattern and with resin tags (C and D;); resin tags within the dentinal tubules of 3%RF -modified adhesive specimens (E; open arrows); 5%RF -modified adhesive specimens at lower magnification presenting... the adhesive (dotted box) (F); adhesive failure within 10%RF -modified adhesive specimens (G); small cracks within 5%RF -modified adhesive specimens after 9-months of artificial saliva (H; arrows); Cohesive failure in 10%RF -modified adhesives (A=adhesive) after 9-months of storage (I) Horizontal fracture seen at the interfacial region in 10%RF -modified adhesive specimens after 9-months of storage (J) !... showing an illustrative area of the dentin surface of (A) control; bio -modified with (B) 0.1%RF; (C) Ch/RF 1:4 and (D) Ch/RF 1:1 specimens The 0.1%RF and Ch/RF 1:4 specimens show open dentinal tubules with intact collagen fibers whereas the Ch/RF 1:1 specimens exhibit a discontinuous structure The dentin of all specimens were conditioned for 15 s with 37% phosphoric acid (Daood et al; Effect of chitosan/riboflavin... cartilage explants with minimal 960 cm1 peak [Adapted from Early detection of biomolecular changes in disrupted porcine cartilage using polarized Raman spectroscopy; J Biomed Opt 2011;16(1):017003017003-10 doi:10.1117/1.3528006] ! #+! Fig 15 Schematics (redrawn) showing acid etching of mineralized dentin removing the smear layer leading to demineralization and exposing the collagen fibrils (Redrawn... chitosan/riboflavin modification on resin/dentin interface: Spectroscopic and microscopic investigations) ! #*! Fig 24 SEM images of the control, 0.1% RF and Ch/RF (1:4 and 1:1) crosslinked resin/dentin interfaces surfaces treated with AdperTM Single bond 2; 3M ESPE The hybrid layer (HL) and many resin tags (RT) were found at the adhesive interface between resin cement and dentin; (A) control, (B) A uniform hybrid... with cleavage sites for N- and C-procollagenases (adapted and redrawn from ‘Collagens—structure, function, and biosynthesis’; Advanced Drug Delivery Reviews 55 (2003) 1531– 1546) ! ""! Fig 6 The lysyl mediated mature crosslinks formed within the collagen Type I fiber; lysyl pyridinoline and hyroxylysyl-pyridinoline ! "#! Fig 7 The collagen triple helix (a) The crystal structure of collagen molecule; ... micro-tensile bond strength (!TBS) test and SEM analysis (A) Removal of occlusal surface to expose the flat dentin surface; (B) preparation of the dentin surface to receive adhesive with or without RF; (C)... cm-1;(A) control=1717 cm-1, (B) 1%RF -modified adhesive=1711 cm1 , (C) 3%RF -modified adhesives=1710 cm-1, (D) 5%RF -modified adhesives =1713 cm-1 and (E) 10%RF -modified adhesives = 1727 cm-1 ! $)!... RF -modified adhesive specimens after the micro-tensile strength testing of the immediate (A) and the 9months stored specimens (B) in artificial saliva ! $&! Fig 30 Representative fracture surfaces