Biomimetic remineralization of artificial caries dentin lesion using Ca/P-PILP

ObjectiveTo assess the ultrastructural change of demineralized dentin collagen during calcium phosphate polymer-induced liquid precursor (Ca/P-PILP) mediated remineralization process and to evaluate the biomimetic remineralization potential of high concentration Ca/P-PILP at demineralized artificial caries dentin lesion, additionally to investigate the bond interfacial integrity as well as the bonding strength of the biomimetic remineralized artificial caries dentin lesion.MethodsDemineralized dentin collagen of 5 μm thick was biomimetically remineralized with low, medium concentration Ca/P-PILP for 10 days and high concentration Ca/P-PILP for 10, 15, 20 days. Artificial caries dentin lesion at a thickness of 150 ± 50 μm was biomimetically remineralized with high concentration Ca/P-PILP for 20 days. The biomimetic remineralization of demineralized dentin collagen was observed by scanning electron microscopy (SEM). The biomimetic remineralization intensity and depth of artificial caries dentin lesion was assessed by Electron Probe Micro Analyzer (EPMA). The bonding interfacial integrity between remineralized artificial caries dentin and composite resin was observed by Swept-source optical coherence tomography (SS-OCT) and the bonding strength of remineralized artificial caries dentin was evaluated by micro-tensile bond strength analysis (μTBS).ResultsSolely PAA-PASP solution and solely saturated Ca/P solution can’t achieve dentin collagen remineralization. Increased concentration of Ca/P-PILP and prolonged remineralization time can enhance the biomimetic remineralization intensity of demineralized dentin collagen. After treating with high concentration Ca/P-PILP, a 150 ± 50 μm thick layer of demineralized artificial caries dentin lesion was not fully remineralized, and the biomimetic remineralization intensity reached up to 88.0%. Furthermore, a better bonding interfacial integrity with less microgap and increased bond strength at both baseline level and aging level were observed when artificial caries dentin lesion was biomimetically remineralized with high concentration Ca/P-PILP.SignificanceBiomimetic remineralization of demineralized caries dentin lesion promotes its clinical properties for resin composited adhesive restoration.     

牙本质的仿生再矿化

近年来,在修复由龋病等原因引起的牙本质脱矿的研究中,牙本质仿生再矿化技术以其精确控制无定形矿物质前体在脱矿牙本质胶原纤维内有序沉积、形成的磷灰石晶体与天然矿化牙本质相似、自下而上的再矿化方式不依赖于籽晶存在等优点,逐渐成为这一领域的研究热点。本文回顾了脱矿牙本质再矿化的理念和实践的进展,并着重对牙本质仿生再矿化策略的相关研究进行综述,文献复习结果表明,传统的牙本质再矿化方法通常是脱矿牙本质与矿物质晶体的无序混合,这样矿化后的牙本质在形态特征和机械性能上均无法与天然矿化牙本质相媲美;而近年逐渐兴起的牙本质仿生再矿化技术则复现了天然矿化牙本质中矿物质在牙本质胶原纤维内迭序排列的结构特点,其微观结构、理化性能均得到极大提高,有望在树脂⁃牙本质粘结混合层和龋坏牙本质脱矿层的再矿化研究领域实现新的突破。目前牙本质仿生再矿化在临床应用上需要克服的技术障碍在于如何在再矿化过程中持续补充矿化所需的各种有效成份,并在缓慢释放各成份的同时保持母体材料的机械性能不变,研究者们已相继提出了三步法输送仿生再矿化原材料,以及预先制备聚合物稳定的矿化前体、再使用介孔硅纳米材料作为输送矿化成份的系统的构想,为牙本质仿生再矿化策略向临床应用的转化提供了初步体外实验基础。     

Biomimetic remineralization of dentin

Objectives

Remineralization of demineralized dentin is important for improving dentin bonding stability and controlling primary and secondary caries. Nevertheless, conventional dentin remineralization strategy is not suitable for remineralizing completely demineralized dentin within hybrid layers created by etch-and-rinse and moderately aggressive self-etch adhesive systems, or the superficial part of a caries-affected dentin lesion left behind after minimally invasive caries removal. Biomimetic remineralization represents a different approach to this problem by attempting to backfill the demineralized dentin collagen with liquid-like amorphous calcium phosphate nanoprecursor particles that are stabilized by biomimetic analogs of noncollagenous proteins.

Methods

This paper reviewed the changing concepts in calcium phosphate mineralization of fibrillar collagen, including the recently discovered, non-classical particle-based crystallization concept, formation of polymer-induced liquid-precursors (PILP), experimental collagen models for mineralization, and the need for using phosphate-containing biomimetic analogs for biomimetic mineralization of collagen. Published work on the remineralization of resin–dentin bonds and artificial caries-like lesions by various research groups was then reviewed. Finally, the problems and progress associated with the translation of a scientifically sound concept into a clinically applicable approach are discussed.

Results and significance

The particle-based biomimetic remineralization strategy based on the PILP process demonstrates great potential in remineralizing faulty hybrid layers or caries-like dentin. Based on this concept, research in the development of more clinically feasible dentin remineralization strategy, such as incorporating poly(anionic) acid-stabilized amorphous calcium phosphate nanoprecursor-containing mesoporous silica nanofillers in dentin adhesives, may provide a promising strategy for increasing of the durability of resin–dentin bonding and remineralizing caries-affected dentin.     

氯化钠溶液对人牙根面牙本质早期龋再矿化作用的影响

目的：探讨单纯用氯化钠（NaCl)溶液处理人牙根面牙本质早期龋后，提高根面早期龋再矿化作用的可行性，方法：在人牙根面上形成早期人工龋，将标本分组，分别用0．5mol/L,NaCl溶液和0．5mol/L EDTA二钠盐溶液浸泡后，用再矿化液处理，扫描电镜下观察比较早期龋矿化前牙根表面形态及矿化后牙根表面矿物盐沉积情况，显微X线照像及其图像分析比较矿化后矿物含量的不同。结果：人牙根面早期龋表面用NaCl溶液浸泡前后无明显改变，早期龋用EDTA二钠盐溶液浸泡后表面见大量牙本质小管开放，再矿化后，前者比后者表层明显增厚，表层阻射度明显增强。结论：用NaCl溶液处理人牙根面早期龋以后，再矿化效果明显增强，NaCl溶液对早期龋表面无不良影响。     

牙本质中可溶性磷蛋白对再矿化的抑制作用

目的 研究去除牙本质中可溶性磷蛋白后对进一步再矿化的影响，了解磷蛋白在牙本质矿化中的作用机制。方法 用EDTA脱矿法去除牙本质中的固有可溶性磷蛋白，获得脱矿的牙本质籽晶，进行再矿化实验，并与以正常牙本质和乙酸脱矿法（可保留可溶性磷蛋白）获得的牙本质再矿化过程对照。实验应用等组分晶体生长体系，记录再矿化过程添加液何种随时间的增加量，以计算再矿化速率。结果 经EDTA脱矿0.5h及2h的牙本质籽晶的再矿化速率比正常牙本质粉明显增快，在200min时观察到的速率增加可超过100%，而脱矿5h和10h以及所有乙酸脱矿牙本质籽晶的再矿化速率均较正常未脱矿籽晶慢。结论 牙本质固有的可溶性磷蛋白具有抑制脱矿牙本质再矿化的作用。     

牙本质磷蛋白对牙本质再矿化的影响

目的　探讨牙本质磷蛋白 (dentinphosphoprotein ,DPP)与脱矿牙本质再矿化的关系。方法　 (1 )用氯化钠提取脱矿牙本质中的易溶性DPP并鉴定。 (2 )用氯化钠去除和未去除易溶性DPP的脱矿人牙根牙本质磨片 ,经再矿化后通过原子吸收光谱、扫描电镜和显微放射照相的观测比较两组再矿化程度。结果　 (1 )证明了 1mol/L氯化钠可以提取脱矿牙本质中的易溶性DPP ;(2 )去除和未去除易溶性DPP的磨片组相比再矿化液的钙离子浓度显著减少 (P <0 0 1 ) ;磨片上生成较多量的矿物质沉积 ;显微射线照片平均吸光度值显著降低 (P <0 0 1 )。结论　易溶性DPP对脱矿牙本质再矿化有明显的抑制作用 ,在根面龋的再矿化中去除易溶性DPP能提高其再矿化的潜能。     

Effect of fluoride-releasing liner on demineralized dentin.

OBJECTIVE: The aim of this study was to evaluate the remineralization of residual demineralized dentin underneath restorations by fluoride-releasing liners in vitro. METHOD AND MATERIALS: Two glass-ionomer cement liners, Lining Cement (GC) and RK-141A (GC), and two light-cured resin liners with and without 5 wt% NaF were used in this study. The resin liners were made by mixing TEGDMA and bis-GMA. Class 5 cavities were prepared on extracted human premolars. Demineralized dentin was promoted by using a bacterial caries induction system at the cavity floor. Cavities were restored with a resin composite following application of each liner. Specimens were then stored for 4 weeks at 37 degrees C and 100% humidity. After storage, the specimens were cut through the restorations, and axial sections of about 80-microm thickness were prepared. Contact microradiographs were taken, and the radiopacity of the demineralized dentin layer underneath the liner was analyzed. RESULTS: The ratios of radiopacity of demineralized dentin under the fluoride-releasing liners were significantly higher than those under the resin liner without fluoride release. Although the amounts of fluoride release from the glass-ionomer cement liners were lower than that from the resin liner with fluoride release, there were no significant differences in the ratios of radiopacity in demineralized dentin layers among the 3 liners with fluoride release. CONCLUSION: Although the fluoride released from liners can enhance remineralization of demineralized dentin, the amount of fluoride release does not affect the ratio of radiopacity in the remineralized dentin.     

Effect of ethylene oxide sterilization on enamel and dentin demineralization in vitro

For in situ studies into caries prevention, sterilization of tooth samples is essential. However, sterilization may influence the caries process itself. The aim of this study was to assess the effect of sterilising sound human enamel and dentin with ethylene oxide on lesion depth and mineral loss before and after in vitro demineralization. Lesion depth and mineral loss were measured using transversal microradiography (TMR). The experiment was carried out with 32 enamel and 32 dentin samples. We found a significant reduction of lesion depth due to sterilization in demineralized enamel (-9.8microm; 95% CI: -15.1 to -4.4microm). The small effect of sterilization on demineralized enamel is considered to be irrelevant for in situ studies of de- and remineralization.     

杨梅素对脱矿牙本质再矿化及树脂-牙本质粘接效果的影响

目的 评价杨梅素(myricetin,MYR)对脱矿牙本质基质的再矿化效果及其对牙本质粘接强度的影响。方法 切取中层牙本质,使用600目碳化硅砂纸湿抛光模拟玷污层,35%磷酸酸蚀后分别给予蒸馏水浸泡、直接再矿化处理2 d和MYR预处理30 min后再矿化处理2 d。采用扫描电镜(scanning electron microscope,SEM)观察样本表面形态,X射线衍射(X-ray diffraction,XRD)和衰减全反射傅里叶变换红外光谱(attenuated total reflection Fourier transform infrared spectroscopy,ATR-FTIR)分析表面物质,微拉伸强度(micro tensile strength,μTBS)和纳米渗漏实验观察粘接效果。结果 SEM发现再矿化处理的各组牙本质片均可以形成晶体,其中经MYR预处理后再矿化的样本形成的晶体最为明显,新形成的晶体经XRD和ATR-FTIR分析为羟基磷灰石。μTBS和纳米渗漏实验发现MYR预处理后的牙本质试件再矿化可明显增加粘接强度,减少纳米渗漏。结论 MYR预处理可以促进脱矿牙本质的再矿化过程并改善树脂-牙本质粘接强度。     

Fluoride releasing materials: their anti-cariogenic properties tested in in vitro caries models

The anti-cariogenic properties of three fluoride-releasing materials on root surfaces were evaluated using two different caries models. Standardized cavities were prepared in dentin specimens and restored with either glass-ionomer, resin-modified glass-ionomer, polyacid-modified resin composite or resin composite material. Two groups of 56 specimens were demineralized using a microbial caries model for three days, while another 56 specimens were demineralized using a chemical demineralization model for three days. Lesions around the restorations were measured with a confocal laser scanning microscope. Glass ionomers demonstrated significant anti-cariogenic properties when exposed to the chemical model. However, no significant anti-cariogenic properties were observed with the microbial caries model. In conclusion, the fluoride releasing materials showed different anti-cariogenic properties in root surfaces under the different caries models, suggesting that caution should be exercised when trying to extrapolate the results of in vitro studies to the clinical situation.     

Micro‐computed tomography assessment of root dentin around fluoride‐releasing restorations after demineralization/remineralization

Using micro‐computed tomography (micro‐CT), this study assessed the inhibitory effect of three different types of restorations on root‐caries formation under a cariogenic challenge. Bovine‐root dentin blocks with a cylindrical cavity were divided into three restoration groups: a fluoride‐free self‐etch adhesive and a resin composite (SE‐ES); a self‐etch adhesive and a resin composite with multi‐ion release (FL‐BF); and a glass ionomer cement (Fuji‐VII). After the restorative procedures, the specimens were stored in artificial saliva for 1 d, then subjected to a demineralization solution for 4 d and a remineralization solution for 28 d. Mineral density and mean mineral loss of dentin around the restorations were measured using micro‐CT. The mean mineral loss values of dentin around the restorations were highest among SE‐ES restorations and lowest among Fuji‐VII restorations. Fuji‐VII showed the highest mineral density and the lowest mineral loss after 28 d of remineralization. The observations made by scanning electron microscopy demonstrated that Fuji‐VII created the smallest outer lesion followed by FL‐BF and SE‐ES. Fuji‐VII has a larger inhibitory effect on root caries around the restorations and enhances remineralization more effectively than either FL‐BF or SE‐ES. Multi‐ion and fluoride release from the restorative materials may be beneficial for inhibition of root‐dentin caries around the restorations.     

Does ART have a place in preservative dentistry?

The ART technique consists of hand excavating carious tissue and placing a highly viscous glass ionomer cement as a restoration material and as a sealant. Although the results of several studies are promising, the retention rates of these restorations for primary teeth are not impressive. Materials and methods that yield greater success rates are needed to improve long-term caries management outcomes. In principle, ART should yield outcomes similar to those associated with preservative dentistry, including the potential for minimal surgical intervention, conservation of sound tooth structure, avoidance of pain and need for local anesthetic injections, reduced risk for subsequent endodontics and tooth extraction, and increased survival time of the affected teeth. The ideal direct-filling ART material would be biocompatible and tooth colored; "forgiving" in its handling properties; insensitive to moisture or desiccation; hardenable without special equipment; able to form stable bonds to enamel and dentin; able to seal marginal gaps against bacteria; capable of releasing fluoride or remineralization and antibacterial agents when demineralization is most likely; and resistant to chemical attack. The highly viscous glass ionomer materials currently used for ART meet several of this criteria, though they may be deficient in their ability to seal marginal gaps against bacteria and in their sensitivity to desiccation. Furthermore, although they release fluoride over the lifetime of the restoration, this fluoride release alone may not prevent caries progression in all cases. It is necessary for cases of high caries risk to use chlorhexidine in conjunction with fluoride to achieve caries arrest and remineralization of adjacent areas of the affected teeth. Thus, while the ART technique offers some benefits in restoring function and reducing the rate of caries progression, it is unlikely that current materials will be able to arrest caries progression completely in high-risk persons.     

Tensile strength of mineralized/demineralized human normal and carious dentin

The bond strengths of resins to caries-affected dentin are low. This could be due to weakened organic matrix. The purpose of this work was to determine if the ultimate tensile strength (UTS) of excavated carious dentin is weaker than that of normal dentin. Soft caries was excavated from extracted human molars, and the tooth was vertically sectioned into slabs. Each slab was trimmed to an hourglass shape, parallel or perpendicular to the tubule direction. Half of the specimens were mineralized, while the other half were completely demineralized in EDTA. ANOVA on ranks showed that the three-factor interactions (mineralization, caries, tubule direction) were all significant (p < 0.0001), indicating that mineralization and tubule direction gave different UTS results in normal and caries-affected dentin. No significant differences were seen between the UTS of normal and and that of caries-affected demineralized dentin in the parallel or perpendicular group. The matrix of demineralized caries-affected dentin was as strong as that of normal demineralized dentin when tested in the same direction.     

Dental Caries under Glass Ionomer Restorations

The Atraumatic Restorative Treatment (ART) technique has two potential limitations. Because only hand instruments are used, dental caries can be left during cavity preparation. Beyond this limitation, the glass ionomer filling material currently used has some unfavorable mechanical properties with respect to its strength, wear resistance, and shrinkage during setting. The purpose of this paper is to provide an overview of changes that can be expected in carious dentin when a glass ionomer restoration is placed. Restriction of the substrate supply and the release of different components from the glass ionomer have an inhibiting effect on cariogenic microorganisms. Whether this influence is sufficient to completely arrest the caries process is doubtful. The influence of glass ionomers on the remineralization of carious dentin needs further research.     

Effects of phosphoprotein moieties on the remineralization of human root caries.

The noncollagenous proteins, especially phosphoprotein, have been shown to modulate biomineralization. The objective of this study was to investigate the remineralization potential of human tooth root organic matrices which did or did not contain soluble non-collagenous proteins including phosphoprotein. Human tooth roots were completely demineralized using conditions that either removed or did not remove soluble phosphoprotein and were then subjected to remineralization conditions. Removal of soluble phosphoprotein resulted in remineralization while no remineralization occurred in tooth roots that still contained soluble phosphoprotein. Transmission electron microscopy and microradiography demonstrated that demineralized cementum did not remineralize under any of the conditions used in this study. Collagenase digestion of demineralized and salt-reextracted tooth root organic matrices revealed that a nonsoluble phosphoprotein was present in the matrices. Amino acid analysis and SDS-PAGE showed that this nonsoluble phosphoprotein was similar in composition to the soluble phosphoprotein. This work suggests that the removal of soluble, noncollagenous proteins, especially phosphoprotein from root caries lesions, may enhance their remineralization potential.     

Remineralization of caries lesions extending into dentin.

Remineralization is one aspect of the overall process of tooth decay. However, it is primarily studied in shallow lesions. The aim of this study was to explore whether caries lesions in enamel and extending into the dentin can be remineralized. A single-section model was developed for the longitudinal and non-destructive monitoring of changes in enamel and dentin. Lesions at least 200 microm into dentin were formed in undersaturated acetate buffers. Next, the lesions were divided into groups (three treatment and one control) and remineralized. The treatments were: weekly immersion in 1,000 ppm fluoride, single treatment with methanehydroxybisphosphonate, and a constant level of 1 ppm fluoride. De- and remineralization was assessed by transverse microradiography. Remineralization was observed in enamel, but also in dentin, indicating that, deep into dentin, the pores become supersaturated to apatite formation. Treatments affected remineralization only in the outer part of enamel. Both findings are explained by a relatively fast diffusion of mineral ions, with precipitation being rate-limiting. The results suggest that dentin remineralization, underneath enamel, can be achieved and could possibly be used in clinical treatment strategies.     

促牙本质再矿化的生物活性树脂研究进展

诱导混合层裸露胶原再矿化是提高树脂牙本质粘接耐久性的重要方法.然而粘接界面有限的矿化离子源限制了牙本质再矿化的临床应用.采用无机材料对粘接树脂改性,可制备出具有生物活性的树脂修复材料,这些树脂在溶液中可缓慢释放矿化离子(如Ca2+、pO43-、OH-等),具有促进脱矿牙体组织再矿化的潜能.用于树脂改性的无机材料包括硅酸盐、磷酸钙盐、生物活性玻璃等.研究证实生物活性树脂修复材料具有促进脱矿牙本质再矿化、抑制胶原纤维降解的能力,但在树脂中添加无机材料会影响树脂的力学性能,制备具有良好力学性能的生物活性树脂是研究的重点和难点.本文将分类阐述不同生物活性树脂修复材料在牙本质再矿化中的作用.     

胶原酶对人根面牙本质胶原蛋白降解作用的实验研究

目的：探讨胶原酶对脱矿后的人牙根面牙本质胶原蛋白的降解作用。方法：根面牙本质标本用pH5.0的乳酸37℃孵育脱矿。分别于第1、7、14、21和28天取孵育液，10000r/min，4℃离心，上清液作Ca^2 测定和羟脯胺酸分析。脱矿后的标本加入pH7.4的胶原酶溶液，37℃孵育7d。孵育液10000r/min、4℃离心，上清液作羟脯胺酸分析。结果：随着时间的延长，Ca^2 释放量逐渐增加，增加量有显著性差异（P＜0．05）；胶原蛋白的释放量随时间的延长而增加，但增加量无显著性差异。如胶原酶降解后，当Ca^2 少于80.01μmol/cm^2时，胶原蛋白释放量增加明显（P＜0．001）；以后虽然Ca^2 浓度增加，胶原蛋白的增加量无显著性差异。结论：胶原酶对根面牙本质胶原蛋白的降解作用与Ca^2 密切相关。保留龈损区的矿物质对于预防胶原纤维的降解、促进早期根面龋的再矿化是非常重要的。     

Distinct decalcification process of dentin by different cariogenic organic acids: Kinetics,ultrastructure and mechanical properties

ObjectivesWe studied artificial dentin lesions in human teeth generated by lactate and acetate buffers (pH 5.0), the two most abundant acids in caries. The objective of this study was to determine differences in mechanical properties, mineral density profiles and ultrastructural variations of two different artificial lesions with the same approximate depth.Methods0.05 M (pH 5.0) acetate or lactate buffer was used to create 1) 180 μm-deep lesions in non-carious human dentin blocks (acetate 130 h; lactate 14days); (2) demineralized, ∼180 μm-thick non-carious dentin discs (3 weeks). We performed nanoindentation to determine mechanical properties across the hydrated lesions, and micro X-ray computed tomography (MicroXCT) to determine mineral profiles. Ultrastructure in lesions was analyzed by TEM/selected area electron diffraction (SAED). Demineralized dentin discs were analyzed by small angle X-ray scattering (SAXS).ResultsDiffusion-dominated demineralization was shown based on the linearity between lesion depths versus the square root of exposure time in either solution, with faster kinetics in acetate buffer. Nanoindentation revealed lactate induced a significantly sharper transition in reduced elastic modulus across the lesions. MicroXCT showed lactate demineralized lesions had swelling and more disorganized matrix structure, whereas acetate lesions had abrupt X-ray absorption near the margin. At the ultrastructural level, TEM showed lactate was more effective in removing minerals from the collagenous matrix, which was confirmed by SAXS analysis.ConclusionsThese findings indicated the different acids yielded lesions with different characteristics that could influence lesion formation resulting in their distinct predominance in different caries activities, and these differences may impact strategies for dentin caries remineralization.     

纳米技术在龋病治疗中应用的研究进展

近年来,纳米技术在牙科材料中表现出了较大的潜在应用价值,本文将主要从纳米技术在龋病早期的再矿化、纳米防龋填料复合树脂以及釉质仿生合成等方面进行综述。