Bone is currently the second most transplanted tissue, second only to blood. However, significant hurdles including graft supply and implant failure continue to plague researchers and clinicians. Currently, standard clinical procedures include autologous and allogeneic grafting. Autologous grafts may achieve functional repair; yet, they are available in limited supply and are associated with donor site morbidity. Allogeneic grafts are available in greater supply, but have a higher risk of infection. To overcome the disadvantages of current grafts, tissue engineering has become a major focus for the regeneration of bone. The goal of tissue engineering is to use a multidisciplinary approach to create biomimetic constructs that stimulate osteogenic regeneration to heal bone defects and restore tissue function. Biodegradable scaffolds are used in tissue engineering strategies as an interim template for tissue regeneration. The scaffold architecture provides mechanical support for cell attachment and tissue regeneration. Biocompatible poly(lactic-co-glycolic acid) (PLGA) has been processed through a number of techniques to create porous 3D architectures. Hydroxyapatite (HAP) and tricalcium phosphate have been used in conjunction with polymer scaffolds due to their osteoconductivity and biocompatibility, but they often lack osteoinductivity and are resistant to biodegradation. Conversely, amorphous calcium phosphate (ACP) is a mineral that solubilizes under aqueous conditions, releasing calcium and phosphate ions, which have been postulated to enhance osteoblast differentiation and mineralization. Controlled dissolution can be achieved by stabilizing ACP with divalent cations such as zinc or copper. Furthermore, incorporation of such osteogenic ACPs within a biodegradable PLGA scaffold could enhance the osteoconductivity of the scaffold while providing calcium and phosphate ions to differentiating osteoprogenitor cells, thereby stimulating osteogenesis when implanted in vivo. In this research, the effect of zinc on the differentiation of osteoprogenitor cells was investigated. Zinc supplementation of the culture media had no stimulatory effect on cell proliferation or differentiation. ACPs were synthesized using zirconium (ZrACP) and zinc (ZnACP) as stabilizers to achieve sustained ion release. Elevated concentrations suggested sustained ion release over the course of 96 hours and enhanced solubility of ZrACP and ZnACP. X-ray diffraction analysis showed a conversion of ZrACP to a semi-crystalline material after 96 hours, but ZnACP showed no conversion after 96 hours. Composite scaffolds were fabricated by incorporating HAP, zirconium-stabilized ACP (ZrACP), or zinc-stabilized ACP (ZnACP) into a sintered PLGA microsphere matrix and then characterized to determine the effect of the minerals on the in vitro differentiation of MC3T3-E1 cells. Scanning electron microscopy revealed a porous microsphere matrix with calcium phosphate powders distributed on the surface of the microspheres. Measurements of mechanical properties indicated that incorporation of 0.5 wt% calcium phosphates resulted in a 30% decrease in compressive modulus. When cells were cultured in the scaffolds, composite ACP scaffolds stimulated proliferation and ALP activity, while HAP scaffolds stimulated osteoblast gene expression. Overall, the results of this work indicate the addition of calcium phosphate minerals to PLGA scaffolds supported cell growth and stimulated osteogenic differentiation, making the scaffolds a promising alternative for bone tissue regeneration. / Ph. D.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24836号 / 医博第5004号 / 新制||医||1068(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 安達, 泰治, 教授 森本, 尚樹, 教授 上杉, 志成 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Objectives: To determine whether treatment of demineralized enamel white spot lesions with CPP-ACP paste, CPP-ACFP paste or microabrasion technique decreases lesion depth in vitro. Additionally, to determine whether treatment of demineralized enamel white spot lesions with microabrasion technique in combination with CPP-ACP paste or CPP-ACFP paste decreases lesion depth greater than any of the three techniques alone. Background: White spot lesions (WSLs) after the removal of orthodontic appliances remain a problem for clinicians and patients. Previous studies suggest that application of casein phosphopeptide amorphous calcium phosphate (CPP-ACP) and casein phosphopeptide amorphous calcium fluoride phosphate (CPP-ACFP) may promote enamel remineralization. Recently, microabrasion of enamel was proposed as another treatment modality of white spot lesions. A review of literature showed that there was no comprehensive in vitro study that combined microabrasion, casein phosphopeptide amorphous calcium phosphate and casein phosphopeptide amorphous calcium fluoride phosphate for treatment of WSLs. Methods: A total of one hundred and twelve bovine incisor teeth were randomly assigned to seven treatment groups: 1. Control 1 (demineralization control), 2. Control 2 (remineralization solution control), 3. CPP-ACP paste, 4. CPP-ACFP paste, 5. Microabrasion, 6. Microabrasion with CPP- ACP paste, and 7. Microabrasion with CPP-ACFP paste. Teeth in all groups were placed in demineralizing solution for 96 hours to produce artificial caries-like lesions. At the end of the 96 hr period, teeth in Control 1 group were sectioned to establish adequate amount of demineralization. The rest of samples were treated with assigned regimen once a day for 10 days and stored in remineralization solution. At the end of ten days, teeth were sectioned with a hard tissue microtome and observed under polarized microscopy to analyze enamel lesion depth. One-way ANOVA at α=0.05 was performed to assess difference in lesion depth between groups followed with post hoc Tukey's test. Results: Statistical analysis showed a significant difference between groups (pConclusions: Based on the results from this study, we can conclude that: (1) treatment of WSLs with CPP-ACP paste, CPP-ACFP paste or microabrasion decreases lesion depth in vitro; (2) microabrasion in combination with CPP-ACP paste or CPP-ACFP paste did not decrease lesion depth greater than that observed with either paste technique alone; (3) both CPP-ACP and CPP-ACFP pastes in combination with microabrasion treatments showed greater decrease in lesion depth than microabrasion alone.

Apesar do caráter cada vez mais preventivo, a Odontologia ainda lida com a realidade terapêutica de reabilitação de uma população cujo mais grave problema de saúde bucal é a ausência dental. A implantodontia apareceu como uma opção para essas reabilitações, porém, não raro, se vê diante de reabsorções ósseas que inviabilizam a instalação direta de implantes. A utilização de enxertos ósseos é o grande recurso para essas áreas. Os enxertos autógenos são uma excelente opção, mas vêm acompanhados de alguns inconvenientes como maior morbidade e risco de infecção, maior tempo de recuperação e maior custo. Esses dificultadores têm contribuído para o desenvolvimento de materiais alternativos. Um desses materiais é o fosfato de cálcio amorfo (FCA), cuja forma cristalina é a hidroxiapatita (HA). Nesse estudo utilizou-se de uma inédita apresentação do mesmo, em forma filamentosa, para avaliar a neoformação óssea na reparação alveolar. A hidroxiapatita em grânulos foi utilizada como referência comparativa. Foram utilizados 44 animais da espécie Rattus Novergicus albinus, linhagem Wistar, machos e adultos. Estes foram submetidos à exodontia do incisivo superior cuja loja alveolar foi imediatamente preenchida com um biomaterial, a saber: FCA, HA e FCAHA (mistura de 50%-50% de cada um deles). Ao término de 28 dias foram sacrificados e seus alvéolos analisados em microscopia de luz convencional quantificando a formação de coágulo, a neoformação óssea, a deposição de tecido osteóide e a presença de vasos no tecido de reparação. As células típicas do tecido ósseo se mostraram presentes. Osteoblastos e osteoclastos na superfície das trabéculas e profusão de fibroblastos no tecido ósseo imaturo, ocupando os espaços intertrabeculares. A deposição de tecido osteóide e de tecido ósseo neoformado apresentou-se em todos os grupos, porém com diferenças em relação à sua distribuição. O grupo controle apresentou reparação óssea compatível com o clássico descrito em literatura, ou seja, de maneira centrípeta. Também foi evidenciada a presença de um coágulo central ou paracentral em relação ao alvéolo. No grupo FCA a quantidade de tecido ósseo neoformado era mais evidente do que nos demais. Já no grupo HA a presença dos cristais sugeriu uma concorrência espacial com a formação tecidual. No grupo FCAHA a deposição tecidual mostrou-se nitidamente irregular. Nos grupos onde foi utilizado o FCA a deposição de tecido ósseo neoformado foi superior àqueles onde esse biomaterial não estava presente. Nosso estudo sugere que o FCA tem uma característica osteoindutora mais evidente do que a HA. Justifica-se por, dentro do período estudado, verificar-se o maior depósito de tecido neoformado e a menor quantidade de tecido osteóide quando os comparamos com os outros grupos. Concluímos que o FCA apresentado na forma de filamentos comportou-se in vivo com características comparáveis a de outros materiais biocompatíveis, mostrando boa resposta osteoindutora, e uma reparação e neoformação mais rápida que a da HA / Despite it increasingly preventive character, dentistry also deals with the reality of therapeutic rehabilitation of a population whose most serious oral health problem is dental absence. Dental implant appeared as an option for these rehabilitations, however, often, is faced with bone resorption that prevent direct implant placement. The use of bone grafts is a great resource for those areas. Autogenous grafts are an excellent option, but comes accompanied by some drawbacks as higher morbidity and risk of infection, longer recovery time and higher cost. These have contributed to hindering the development of alternative materials. One such material is amorphous calcium phosphate (ACP), which is the crystalline form hydroxyapatite (HA). In this study, we used an unprecedented presentation of the same in filamentous form, to evaluate new bone formation in the alveolar repair. The hydroxyapatite granules was used as a comparative reference. 44 animals were used species Rattus Novergicus Albinus, Wistar, adult males. These were subjected to extraction of the upper incisor whose cellular store was immediately filled with a biomaterial, namely: ACP, and ACPHA (mixture of 50% -50% of each). At the end of 28 days they were sacrificed and their alveoli analyzed by conventional light microscopy quantifying clot formation, bone formation, deposition of osteoid tissue and the presence of vessels in tissue repair. The cells typical of bone tissue showed present. Osteoblasts and osteoclasts on the surface of trabecular and profusion of fibroblasts in the immature bone tissue, occupying the intertrabecular spaces. The deposition of osteoid tissue and newly formed bone appeared in all groups, but with differences regarding their distribution. The control group exhibited bone repair compatible with the classic described in the literature, centripetal manner. In addition, it revealed the presence of a central clot or para in relation to the alveolus. ACP group in the amount of newly formed bone was more evident than in others. Already in the HA group the presence of crystals suggested a space competition with tissue formation. The group ACPHA tissue deposition was shown to be clearly irregular. In groups which was used ACP deposition of newly formed bone was higher than those where this biomaterial were not present. Our study suggests that the ACP has clearer osteoinductive characteristic of the HA. Justified by, within the study period, check it the largest deposit of newly formed tissue and the least amount of osteoid tissue when compared with the other groups. We conclude that the ACP presented in the form of filaments behaved in vivo comparable characteristics of other biocompatible materials, showing good osteoinductive response, a faster repair and neogenesis the HA

O presente trabalho avaliou o efeito in situ de uma goma de mascar comercialmente disponível contendo caseína fosfopeptídea - fosfato de cálcio amorfo (CPP-ACP) na erosão dentária. Para a primeira etapa do estudo (capacidade remineralizadora) utilizaram-se 72 blocos de esmalte humano selecionados pela dureza de superfície (SHi) e erodidos in vitro pela imersão em Coca Cola®, pH 2,4 por 3 min (avaliação da dureza - SHd). Os blocos foram randomizados entre os grupos: GI Trident Fresh® (sem CPP-ACP), GII controle (sem chiclete) e GIII Trident Total® (com CPPACP). Doze voluntários utilizaram dispositivos intrabucais palatinos por 24 h em 3 fases cruzadas. Nas fases de GI e GIII os voluntários mascaram um chiclete (30 min) e em todas as fases após 2h, a dureza foi avaliada (SHf1). Os blocos foram reposicionados e os dispositivos usados por mais 22 h (+ 3 ciclos de mastigação de chiclete - GI e GIII). A dureza foi reavaliada (SHf2) para cálculo do percentual de recuperação de dureza (%SHR) após 2 e 24h. Na segunda etapa do estudo (ciclagem erosiva) 48 blocos de esmalte humano hígidos foram aleatorizados entre os grupos (GI, GII e GIII) e 8 voluntários utilizaram dispositivos intrabucais palatinos em fases cruzadas de 7 dias cada (washout de 7 dias). O protocolo de ciclagem erosiva foi de 4 imersões diárias do dispositivo intrabucal em 150 ml de Coca Cola® durante 5 min. Nos grupos I e III após cada desafio erosivo e reinserção do dispositivo na cavidade bucal, os voluntários mascaram um chiclete durante 30 min. A alteração da superfície do esmalte foi mensurada por perfilometria (&#x3BC;m). Os dados foram submetidos à ANOVA (2 critérios - etapa 1; 1 critério - etapa 2) e teste Tukey (&#x3B1;=0,05). Os resultados da recuperação de dureza demonstraram haver diferença significativa entre os grupos e os tempos (p<0,05). O Trident Total® (2h = 50,0%; 24h = 95,9%) promoveu maior recuperação de dureza que o Trident Fresh® (2h= 30,0%; 24h= 71,1%) e o grupo controle (2h = 15,7%; 24h = 40,9%). No desafio erosivo prolongado, o Trident Total® (5,2± 2,8 &#x3BC;m) e o Trident Fresh® (3,8 ± 1,5 &#x3BC;m) reduziram significativamente o desgaste dentário em relação ao grupo controle (6,8 ± 3,5 &#x3BC;m), no entanto, não houve diferença significativa entre os chicletes (p>0,05). Conclui-se que o efeito remineralizador da saliva foi maior após utilização de goma de mascar, sendo significativamente aumentado em função do período de remineralização e pela presença de CPP-ACP na goma de mascar. Contudo, para o protocolo de desafio erosivo adotado, apesar da goma de mascar convencional apresentar efeito na inibição do desgaste erosivo, o comportamento da goma de mascar com incorporação de CPP-ACP foi similar. / This study evaluated the in situ effect of a commercial chewing gum containing casein phosphopeptide - amorphous calcium phosphate (CPP-ACP) on dental erosion. On the first stage (remineralizing effect) 72 human enamel blocks, which were selected by surface hardness (SHi) and eroded in vitro by immersion in cola drink, pH 2,4 for 3 minutes (hardness evaluation - SHd) were used. Blocks were randomized into 3 groups: GI Trident Fresh® (conventional gum, without CPP-ACP), GII control (no gum) and GIII Trident Total® (with CPP-ACP). Twelve volunteers wore intraoral palatal devices for 24 h in 3 crossover phases. In phases of GI and GIII volunteers chewed a gum (30 min) and in all phases after 2h, the surface hardness was evaluated (SHf1). The blocks were reinserted and the devices used for additional 22h (+ 3 cycles of chewing gum - GI e GIII). The surface hardness was reassessed (SHf2) to calculate the percentage of surface hardness recovery (%SHR) after 2 and 24h. In the second stage (erosive cycling) 48 healthy human enamel blocks were randomized between groups (GI, GII and GIII) and 8 volunteers wore intraoral palatal devices in crossover phases of 7 days each (washout of 7 days). The cycling protocol consisted of 4 daily immersions of the intraoral device into 150 ml of cola drink for 5 min. In groups I and III after each erosive challenge and oral device reinsertion into oral cavity, the volunteers chewed a gum for 30 min. The enamel surface alterations were measured by profilometry (&#x3BC;m). Data were analyzed by Anova (Two way - stage 1, One way - stage 2) and Turkeys test (&#x3B1;=0,05). The results of percentage of surface hardness recovery showed significant differences for factors groups and time (p<0.05). Trident Total® (2h = 50.0%; 24h = 95.9%) showed higher percentage of surface hardness recovery than the Trident Fresh® (2h = 30.0%; 24h = 71.1%) and control group (2h = 15.7% 24h = 40.9%). In the prolonged erosive challenge, Trident Total® (5.2 ± 2.8 &#x3BC;m) and Trident Fresh® (3.8 ± 1.5 µm) significantly reduced tooth wear compared to control group (6.8 ± 3, 5 µm). However, there was no significant difference between chewing gums. It is concluded that the saliva remineralizing effect increased after the use of conventional chewing gum and was enhanced by prolonged period of remineralization and by the presence of CPPACP in the chewing gum. However for the adopted erosive challenge, despite the conventional chewing gum being able to diminish erosive wear, the chewing gum containing CPP-ACP showed similar effect.

O objetivo do presente estudo foi quantificar, in vitro, a permeabilidade da dentina tratada com dois agentes anti-hiperestésicos à base de oxalato e um à base de fosfato de cálcio, por meio de testes de condutividade hidráulica e de espectroscopia de impedância eletroquímica e, ainda, determinar se espectroscopia de impedância eletroquímica pode ser considerada um método alternativo para avaliar a capacidade obstrutiva de agentes antihiperestésicos. Para o teste de condutividade hidráulica foram utilizados vinte quatro discos de dentina, obtidos de terceiros molares humanos extraídos, que foram divididos em três grupos de oito espécimes, correspondentes aos três materiais testados: gel Experimental, Sensi Kill e BisBlock. Outros dezoito discos foram selecionados para o teste de espectroscopia de impedância eletroquímica, sendo divididos em três grupos de seis espécimes, correspondentes aos três agentes anti-hiperestésicos citados anteriormente. Para ambos os testes as medidas foram realizadas nas seguintes condições: na presença de smear layer; após condicionamento com ácido fosfórico a 37% e após a aplicação dos materiais na superfície oclusal dos discos de dentina. Com base na análise estatística dos resultados, verificou-se, por meio dos dois métodos utilizados, que o gel Experimental e o BisBlock apresentaram efeitos estatisticamente semelhantes na redução da permeabilidade e foram significantemente mais eficientes que o Sensi Kill. Pode-se concluir que os agentes anti-hiperestésicos Experimental e BisBlock mostraram-se efetivos na redução da permeabilidade dentinária, pelos dois métodos de avaliação, e que o método de espectroscopia de impedância eletroquímica pode ser considerado um método alternativo para a avaliação da capacidade obstrutiva de agentes anti-hiperestésicos. / The aim of the present study was to quantify in vitro the dentin permeability of dentin treated with two potassium oxalate dentin desensitizers and one calcium phosphate agent through hydraulic conductance (Lp) and eletrochemical impedance spectroscopy (EIS) tests and, yet, to determine whether EIS may be considered a suitable alternative method to evaluate the occlusive capacity of the dentin desensitizers. For the hydraulic conductance, twenty-four dentin discs were obtained from extracted human third molars, which were divided into three groups of eight specimens, corresponding to the materials to be tested: Experimental gel, Sensi Kill and BisBlock. Other eighteen dentin discs were selected for theEIS and divided into three groups of six specimens corresponding to the three dentin desensitizers mentioned before. For both tests, the following conditions were measured: in the presence of smear layer, after 37% phosphoric acid etching and after the application of the tested materials in the occlusal surfaces of dentin discs. Based upon the statistical analysis of the results, it was demonstrated by both methods that Experimental gel and BisBlock performed statistically similarly in reducing dentin permeability and were significantly more efficient that Sensi Kill. It can be concluded that the dentin desensitizers Experimental e BisBlock were demonstrated to be effective in the reduction of dentin permeability by both experimental methods and that EIS may be considered as an alternative method for the evaluation of the obstructive capacity of dentin desensitizers.

O efeito protetor da caseína fosfopeptídea fosfato de cálcio amorfo (CPP-ACP) contra a erosão dentária é controverso. Este estudo in situ teve como objetivo investigar a capacidade de uma goma de mascar com CPP-ACP em prevenir uma única desmineralização erosiva. Blocos de esmalte bovino (120) selecionados pela dureza superficial inicial foram divididos aleatoriamente entre os grupos: GI - goma de mascar com CPP-ACP, GII - goma de mascar sem CPP-ACP e GIII - controle negativo para avaliação do efeito protetor sem estimulação salivar (sem goma de mascar). Dezenove voluntários participaram do estudo durante três fases cruzadas de 2 h cada. Nas fases de GI e GII os voluntários usaram dispositivos intrabucais palatinos contendo 2 blocos de esmalte, durante 120 minutos e mascaram uma unidade da goma de mascar correspondente ao grupo nos últimos 30 minutos. No grupo controle os voluntários usaram o dispositivo intrabucal por 2h, sem uso de goma de mascar. Em cada fase, imediatamente após a utilização, os dispositivos intrabucais foram imersos em refrigerante tipo cola durante 5 minutos para promover a desmineralização erosiva. A dureza superficial final foi mensurada e os valores foram utilizados para o cálculo do percentual de perda de dureza. Os dados foram analisados por ANOVA de medidas repetidas e teste Tukey (&#x3B1; = 5%). Menor perda de dureza do esmalte foi encontrada após a utilização de goma de mascar com (GI - 32,7%) e sem (GII - 33,5%) CPP-ACP relação ao efeito salivar sem estimulação (GIII - 39,8%) (p <0,05). Não houve diferença entre GI e GII (p> 0,05). Os resultados sugerem que a utilização de goma de mascar imediatamente antes de uma desmineralização erosiva é capaz de diminuir a perda de dureza do esmalte. No entanto, a presença de CPP-ACP na goma de mascar não foi capaz de melhorar este efeito. / The erosion-protective effect of CPP-ACP is controversial. This in situ study aimed to investigate the ability of CPP-ACP chewing gum to prevent a single event of erosive demineralization. Bovine enamel blocks (120), after selection (initial surface hardness) were randomly assigned to groups: GI-chewing gum with CPP-ACP, GIIchewing gum without CPP-ACP and Control group-salivary effect without stimulation (no gum). Nineteen volunteers participated on this study during 3 crossover phases of 2 h. On GI and GII, the volunteers wore intraoral palatal devices for 120 min and chewed a unit of the corresponding chewing gum on the last 30 min. On Control group the volunteer wore the appliance for 2 h, without chewing gum. On each phase immediately after the intraoral use, devices were extra orally immersed in cola drink for 5 minutes to promote erosive demineralization. The percentage of surface hardness loss was calculated. The data were analyzed by Repeated Measures ANOVA and Turkeys test. Less enamel hardness loss was found after the use of chewing gum with (GI-32.7 %) and without (GII-33.5%) CPP-ACP when compared to salivary effect without stimulation (control- 39.8%) (p<0.05). There was no difference between GI and GII (p>0.05). The results suggest that the use of chewing gum immediately before an erosive demineralization is able to diminish the enamel hardness loss. However, the presence of CPP-ACP in the chewing gum cannot enhance this protective effect.

This body of research investigated the bonding efficiency of self-etching primer adhesives to enamel and dentine with various surface characteristics. A series of preliminary experiments was carried out to determine the effect of operator experience, dentine tubule orientation, bond strength test method and resin composite material used on bond strengths. The results of the preliminary tests concluded that it is essential to develop skills in material handling and the test methods used; 2-step self-etching primer adhesives exhibit higher but more variable microshear bond strengths (µSBS) than ‘all-in-one’ adhesives on dentine at different depths and tubule orientations; fracture toughness and bond strength test results suggest that the fracture toughness of a resin composite may not be of significant influence on microtensile and µSBS tests outcomes for nano-filled hybrid materials. / An investigation of the bonding ability of self-etching primer adhesives under various tooth preparation conditions was carried out. Enamel and dentine specimens were prepared from human teeth and finished with various rotary cutting instruments and the erbium, chromium:yttrium, scandium, gallium, garnet laser. Specimens were bonded with two 2-step self-etching primer adhesives and two ‘all-in-one’ adhesives with a resin composite. The results showed that one of the 2-step adhesives exhibited higher but more variable µSBS than the ‘all-in-one’ adhesives and a silorane-based self-etching primer adhesive system to enamel and dentine. / The relationship between enamel microhardness and µSBS was evaluated. Enamel specimens were prepared and finished with one half of the surface tested for hardness using the Vickers test. The other half of the enamel surface was bonded using either a 2-step self-etching primer adhesive or an ‘all-in-one’ adhesive and a hybrid resin composite. Mean Vickers hardness numbers and µSBS for each enamel surface were calculated. Analysis using Pearson’s parametric test for regression analysis evaluated the correlation between Vickers hardness and µSBS. The results revealed a weak negative insignificant correlation between VHN and µSBS for the 2-step adhesive and no correlation for the ‘all-in-one’ adhesive. / The effect of conditioning and casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) on bonding to bleached and unbleached enamel was investigated. Four groups of enamel specimens: untreated control; bleaching with 16% carbamide peroxide gel for 90 min daily x 14 days; treated with CPP-ACP paste (Tooth Mousse, GC Corp., Japan) for 60 min daily x 7 days and bleached and CPP-ACP-treated were used. The specimens were divided into a further two groups and bonded with a total-etch adhesive or a 2-step self-etching primer adhesive. Specimens bonded with the self-etching primer adhesive were sub-divided into four conditioning subgroups before bonding: no conditioning; 30 – 40% phosphoric; 15% EDTA; 20% polyacrylic acid. Specimens were tested in shear mode until failure and analysed by 2-way ANOVA, one-way ANOVA and Tukey’s post-hoc test. The µSBS of the total-etch adhesive was not affected by enamel treatment. Bleaching reduced the µSBS of the self-etching primer adhesive but preconditioning with phosphoric acid and polyacrylic acid improved bond strengths after CPP-ACP application. Bond failure analysis revealed a predominance of adhesive failures after bleaching, but prior conditioning reduced the proportion of adhesive failures. Scanning electron microscopy (SEM) revealed that the interfacial morphology produced by the 2-step self-etching primer adhesive was independent of enamel treatment except after bleaching. Phosphoric acid etching was not inhibited by CPP-ACP treatment. Resin tag formation was observed with prior phosphoric acid and polyacrylic acid conditioning. / The effect of conditioning and CPP-ACP application on dentine bonding was also investigated. Dentine specimens with and without the smear layer were prepared and divided into a further two groups, CPP-ACP paste applied to one group for 60 min daily x 7 days and the other group was untreated. The two groups were divided into three subgroups for conditioning: no conditioning; 30 - 40% phosphoric acid; 20% polyacrylic acid. The dentine was bonded using a 2-step self-etching primer adhesive and an ‘all-in-one’ adhesive, and tested as previously described. Statistical analysis was carried out using one-way ANOVA and Tukey’s post-hoc test at α = 0.05. The results showed that the µSBS of both adhesives were not significantly affected on smear-covered dentine but was affected on smearless dentine. Conditioning did not improve bond strengths. Bond failure analysis showed more adhesive failures for the ‘all-in-one’ adhesive, particularly on smearless dentine and with prior polyacrylic acid conditioning. SEM revealed a similar morphology of the bonded interface for the 2-step self-etching primer adhesive regardless of conditioning; and areas of bond failures for the ‘all-in-one’ adhesive. / The 2-step self-etching primer adhesives exhibited higher bond strength and more regular bond integrity than the ‘all-in-one’ adhesives, as shown on the SEM observations. However, the ‘all-in-one’ adhesives exhibited less variability in bond strengths to tooth surface characteristics.