In vitro remineralization of hybrid layers using biomimetic analogs

In vitro remineralization of hybrid layers using biomimetic analogs 牙本质粘结混合层仿生再矿化的体外研究 1 first-author 1 corresp ‡Corresponding Authors ‡Corresponding Authors †E-mail:junlin2@126.comcmehl@proth.uni-kiel.de †E-mail:junlin2@126.comcmehl@proth.uni-kiel.de junlin2@126.com 1 1 2 corresp ‡Corresponding Authors ‡Corresponding Authors †E-mail:junlin2@126.comcmehl@proth.uni-kiel.de †E-mail:junlin2@126.comcmehl@proth.uni-kiel.de junlin2@126.com Department of Stomatology, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China Department of Stomatology, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China HarderMehl Dental Clinic, Volkartstraße 5, 80634 Munich, Germany HarderMehl Dental Clinic, Volkartstraße 5, 80634 Munich, Germany Resin-dentin bond degradation is a major cause of restoration failures. The major aim of the current study was to evaluate the impact of a remineralization medium on collagen matrices of hybrid layers of three different adhesive resins using nanotechnology methods. Coronal dentin surfaces were prepared from freshly extracted premolars and bonded to composite resin using three adhesive resins (FluoroBond II, Xeno-III-Bond, and iBond). From each tooth, two central slabs were selected for the study. The slabs used as controls were immersed in a simulated body fluid (SBF). The experimental slabs were immersed in a Portland cement-based remineralization medium that contained two biomimetic analogs (biomineralization medium (BRM)). Eight slabs per group were retrieved after 1, 2, 3, and 4 months, respectively and immersed in Rhodamine B for 24 h. Confocal laser scanning microscopy was used to evaluate the permeability of hybrid layers to Rhodamine B. Data were analyzed by analysis of variance (ANOVA) and Tukey’s honest significant difference (HSD) tests. After four months, all BRM specimens exhibited a significantly smaller fluorescent area than SBF specimens, indicating a remineralization of the hybrid layer ( P ≤0.05). A clinically applicable biomimetic remineralization delivery system could potentially slow down bond degradation. 目的 2 探讨再矿化介质聚乙烯磷酸和聚丙烯酸对3种粘结剂（FluoroBond II、Xeno-III和iBond）和牙本质所形成的树脂-牙本质界面再矿化程度的影响。 创新点 2 （1）多数仿生再矿化研究集中于通过透射电子显微镜观察牙本质粘结剂界面的再矿化情况，鲜有采用激光共聚焦显微镜（CLSM）作为观察手段的。虽有研究应用CLSM，但也仅是从定性的角度比较再矿化效应，没有定量分析比较，难以令人信服。而本项研究从定性和定量两方面分析对比。（2）本研究从当前牙本质粘结剂系统（共七代）常用的后四代中选择典型代表产品为研究对象，便于横向比较各类粘结剂。（3）本研究的定量结果除了比较同一时间实验组和对照组的矿化情况，还增加了纵向分析思路，进一步比较矿化程度随时间的变化以及不同牙本质粘结剂粘结界面的矿化情况。 方法 2 将96颗健康前磨牙按照FluoroBond II、Xeno-III和iBond粘结剂随机分为3组，每颗牙均暴露表层牙本质。3种粘结剂分别严格按照各自产品说明书处理牙本质表面，牙合面堆制5 mm厚的树脂。每颗牙沿牙合-龈向切成0.9 mm厚的薄片，获取中间两片样本用于矿化组和模拟组。矿化组采用含聚乙烯磷酸和聚丙烯酸的再矿化液浸泡；模拟组采用不含聚乙烯磷酸和聚丙烯酸的模拟体液浸泡。各组标本在储存1、2、3和4个月后，各取出8片，经苏丹明B荧光染料染色24 h，冲洗，吹干，置CLSM下观察渗入混合层及粘结层的荧光情况，测量荧光面积、平均荧光量及总荧光量。所有数据统计方法采用方差分析（ANOVA）和Tukey’s HSD检验分析。 结论 2 本研究中聚丙烯酸和聚乙烯磷酸双仿生类似物分子对FluoroBond II、Xeno-III及iBond粘结剂均显示不同程度的再矿化效应，其中对iBond粘结剂再矿化效应最明显，FluoroBond II粘结剂次之，Xeno-III粘结剂再矿化效应较差，但能够起到抵制基底矿物继续丢失或阻止胶原降解的作用。CLSM结合应用苏丹明B是量化混合层再矿化的一项有效手段。因此，上述双仿生类似物分子应用于口腔粘结剂修复材料促使混合层再矿化，具有良好的应用前景，值得进一步深入研究。 Remineralization Dentin Adhesive resin Biomimetic analog Altered collagen Confocal laser scanning microscopy (CLSM) Fluorescence 再矿化 牙本质 粘结树脂 仿生类似物 激光共聚焦显微镜 荧光 Introduction 1 The stability of the hybrid layer is inadvertently compromised during the bonding of resin and dentin. Firstly, etching and rinsing can degenerate the denuded collagen fibrils within the hybrid layer (Carrilho et al., 2007 2007 ). Secondly, demineralized collagen fibrils may collapse via inter-peptide hydrogen bonding when they are air-dried during bonding, restricting the infiltration of adhesive resin monomers (Pashley et al., 2007 2007 ; Mai et al., 2009 2009 ). Thirdly, due to fluid movement within the dentinal tubules during resin infiltration, often aggravated by the presence of a positive pulpal pressure, monomers only inadequately infiltrate the dentin (Wang and Spencer, 2005 2005 ; Hashimoto, 2010 2010 ; Lin et al., 2010 2010 ). These procedural obstacles accelerate in vivo degradation of hybrid layers (Kim J. et al., 2010 2010 ). Degradation occurs as early as six months after initial intraoral function (Kim J. et al., 2010 2010 ). It is triggered by hydrolysis of suboptimally polymerized hydrophilic resin components and degradation of water-rich, resin-sparse collagen matrices by matrix metalloproteinases and cysteine cathepsins (Tjaderhane et al., 1998 1998 ; Pashley et al., 2004 2004 ; Liu et al., 2011 2011 ). Subsequently, the compromised hybrid layer causes leakage in the nano-space of the collagen fibers, leading to restoration failure (Li et al., 2000 2000 ). Several strategies have been developed to stabilize the bond between composite resin and dentin and thus prevent restoration failure (Liu et al., 2011 2011 ). Biomimetic remineralization is a recent approach using analogs of matrix proteins to progressively replace water with intrafibrillar and extrafibrillar apatites in order to exclude exogenous collagenolytic enzymes and fossilize endogenous collagenolytic enzymes (Zhang, 2003 2003 ; Tay et al., 2007 2007 ; Tay and Pashley, 2008 2008 ). Most biomimetic remineralization studies have used transmission electron microscopy to evaluate the interface between adhesive and resin and the degree of remineralization (Kinney et al., 2003 2003 ; Carrilho et al., 2005 2005 ; Trebacz and Wojtowicz, 2005 2005 ; Tay and Pashley, 2009 2009 ; Gu et al., 2010 2010 ; Kim Y.K. et al., 2010b 2010b ). Confocal laser scanning microscopy (CLSM) using Rhodamine B uptake by demineralized or incompletely resin-infiltrated collagen fibers seems to be a promising new method to evaluate remineralization effects (Kim J. et al., 2010 2010 ). Hence, the current study aimed to use CLSM to evaluate three commercial representative bonding adhesive resins based on three different working mechanisms. The null-hypotheses with regard to Rhodamine B uptake of the hybrid layers to be tested were: (1) there is no difference between a two-step etch-and-rinse adhesive resin, a self-etching one-step two-bottle adhesive resin, and a self-etching one-bottle adhesive resin; (2) the placement of the experimental specimens in a remineralization medium has no effect in comparison to a control group; and, (3) the duration of immersion has no effect. Materials and methods 1 Tooth preparation 2 Ninety-six intact human premolars were extracted and stored in 0.5% (5 g/L) thymol-saturated isotonic saline solution at 4 °C. The teeth were collected after the patients’ informed consents were obtained under a protocol approved by the Ethics Committee of the Faculty of Dentistry at the Zhejiang University, China. All teeth were used within three months after extraction. A flow chart of the experimental process can be found in Fig. 1 1 . FL: FluoroBond II; BRM: biomineralization medium; SBF: simulated body fluid; CLSM: confocal laser scanning microscopy The occlusal enamel was removed perpendicular to the longitudinal axis of each tooth using a slow-speed diamond saw (Isomet, Buehler, Lake Bluff, IL, USA) under water cooling (Lin et al., 2014 2014 ). The resulting flat dentin surface was checked with a stereomicroscope (Wild Makroskop M420, Heerbrugg, Switzerland) at 10× magnification for the absence of enamel (Lin et al., 2014 2014 ). The dentin bonding surfaces were polished with a 600-grit silicon carbide paper (Buehler) to create a standardized smear layer in mid-coronal dentin (MP-260E, Weiyi Instrument Test Equipment Manufacturing, Laizhou City, China) (Yang et al., 2006 2006 ). For all specimens, the dentin was kept wet during all preparations by storage in deionized water or placement on a wet dish. Tooth conditioning and bonding procedures 2 The composition of the main materials used in this study can be found in Table 1 1 . The three adhesive resins were chosen as representatives of commercially available bonding adhesive resins with different bonding methodologies. A two-step etch-and-rinse adhesive resin (FluoroBond II, Shofu, Japan), and two one-step adhesive resins (Xeno-III-Bond, Dentsply, York, PA, USA and iBond, Heraeus, Hanau, Germany) were used according to the manufacturers’ instructions. Following the adhesive procedures, a composite resin filling material was built up incrementally to a height of 5 mm (Valux Plus Restorative, 3M Espe, St. Paul, MN, USA). Each layer was light-cured separately for 20 s (output intensity 1500 mW/cm 2 ; Satelec, Bordeaux, France). Each tooth was sectioned occluso-gingivally. Only the two central 0.9 mm thick slabs were used. Overall, 24 experimental groups with 8 specimens were tested (3 adhesive resins, storage for 1, 2, 3, or 4 months and control group/experimental group; Fig. 1 1 ). Before experimentation, all specimens were stored in deionized water at room temperature and used within 24 h. Name Etching/steps Company & batch No. Principal component FluoroBond II Two-step Shofu, Kyoto, Japan; 0609 Primer: ethanol, carboxylic acid monomer, phosphoric acid monomer and initiator, water; Bonding agent: S-PRG filler on fluoroboroaluminosilicate glass, UDMA, TEGDMA, 2-HEMA, initiator Xeno-III-Bond One-step (mixed) Dentsply, York, PA, USA;  0909001714 Liquid A: HEMA, ethanol, BHT, 2,6-di- tert -butyl- p -hydroxytoluol, water, nanofillers; Liquid B: pyro-EMA, PEM-F, BHT, camphorquinone, p -dimethylamino-ethyl-benzoat iBond One-step (not mixed) Heraeus, Hanau, Germany;  010102 An acetone/water-based formulation of light-activated methylacrylate resins 4 4 S-PRG: surface pre-reacted glass-ionomer; UDMA: urethane dimethacrylate; TEGDMA: triethylene-glycol-dimethacrylate; HEMA: hydroxyethyl-methacrylate; BHT: butylated hydroxy toluidine; Pyro-EMA: tetramethacryloxyethyl pyrophosphate; PEM-F: pentamethacryloxyethyl cyclophosphazen mono fluoride Biomimetic remineralization procedures and control group preparation 2 For the control group, simulated body fluid (SBF) solution was prepared by dissolving 136.8 mmol/L NaCl, 4.2 mmol/L NaHCO 3 , 3.0 mmol/L KCl, 1.0 mmol/L K 2 HPO 4 ·3H 2 O, 1.5 mmol/L MgCl 2 ·6H 2 O, 2.5 mmol/L CaCl 2 , and 0.5 mmol/L Na 2 SO 4 in deionized water (Kokubo et al., 1990 1990 ), and adding 3.08 mmol/L sodium azide to stop bacteria from growing. Tris base at 0.1 mol/L and HCl at 0.1 mol/L were used to buffer the SBF to a pH value of 7.4 and the resulting solution was filtered (Kim J. et al., 2010 2010 ). For the experimental groups, initially a Portland cement block was prepared. For that purpose, deionized water was mixed with Type I white Portland cement (Lehigh Cement Company, Allentown, PA, USA) in a water-to-powder ratio of 0.35:1 (w/w). Before it was used for this study, the resulting solution was filtered and stored at 100% relative humidity for one week in silicone molds (Tay et al., 2007 2007 ; Mai et al., 2009 2009 ; Sauro et al., 2015 2015 ). The experimental slabs were then placed on the established blocks of Portland cement (ca. 1 g) inside a glass cylinder (Mai et al., 2009 2009 ; Kim J. et al., 2010 2010 ). The biomineralization medium (BRM) was produced by adding 500 µg/ml of polyacrylic acid (PAA; molecular weight (MW) 1800; Sigma-Aldrich, St. Louis, MO, USA) and 200 µg/ml of polyvinylphosphonic acid (PVPA; MW 10177; Sigma-Aldrich) to the SBF. The solution was buffered again to a pH of 7.4 (Mai et al., 2009 2009 ; Kim J. et al., 2010 2010 ). The glass cylinders containing the slabs were either filled with 15 ml of the SBF (control group) or BRM (experimental group), and stored at 37 °C (Mai et al., 2009 2009 ; Kim J. et al., 2010 2010 ). The remineralization medium was changed every month (Kim J. et al., 2010 2010 ). To ensure that apatite was formed instead of octacalcium phosphate (Meyer and Weatherall, 1982 1982 ; Eanes, 2001 2001 ), the pH (after placing on the Portland cement blocks) was monitored weekly and adjusted if necessary to maintain it above 9.25 (Mai et al., 2009 2009 ; Kim J. et al., 2010 2010 ). After 1, 2, 3, and 4 months, respectively, eight slabs for each adhesive resin were retrieved and immersed in a 0.1 mmol/L Rhodamine B solution (CAS81-88-9, MW 479, Shanghai Chemicals, China). After 24 h, the dye-infiltrated slabs were rinsed with deionized water for 30 s and examined using a CLSM that was coupled with a helium neon gas laser (Laser Scanning Confocal Microscope TCS-SP2, Leica, Wetzlar, Germany; 50% of 543 nm excitation, 383 V, and magnification of 40×4). Images were captured starting from 10 µm beneath the surface to avoid superficial specimen preparation artifacts. The total fluorescent area, average fluorescence (AF), and total fluorescence (TF) were measured using Image-Pro-Plus 6.0 software in the region of interest (ROI). The ROI was defined as the region between the composite and the dentin, including the hybrid layer and adhesive resin layer. The fluorescent area was the area of mineral deficiency in the hybrid layers (Sidhu and Watson, 1998 1998 ; Watson et al., 2008 2008 ); the AF represented the fluorescence intensity of the defect area in the hybrid layers; and the TF represented the sum of all fluorescent pixels in the mineral defect area. Statistical analysis 2 The data were analyzed using a statistical software package (SPSS 18.0, Chicago, IL, USA). Since data were distributed normally (Shapiro-Wilks and Kolmogorov-Smirnov tests), parametric tests were employed (three-way analysis of variance (ANOVA) (least significant difference (LSD)) and Tukey’s honest significant difference (HSD) tests). All testing was performed at a confidence level of 95%. Results 1 Three-way ANOVA results are presented in Table 2 2 . For each adhesive resin, the fluorescent area, AF, and TF are presented in Tables 3 3 – 5 5 . The choice of adhesive resin, the use of a remineralizing medium (SBF vs. BRM), and the duration of immersion had a significant impact on the fluorescent area and TF ( P ≤0.05). For the AF, only the duration of immersion was significantly influential ( P ≤0.05). 2 Source 2 Area 2 AF 2 TF F value P value F value P value F value P value  Adhesive resins 212.490 0.000 0.620 0.539 192.587 0.000   FluoroBond II   Xeno-III-Bond   iBond  Remineralizing medium 20.510 0.000 0.077 0.782 13.591 0.000   SBF   BRM  Immersed time 2.674 0.049 11.819 0.000 3.443 0.018   First month   Second month   Third month   Forth month All data are expressed as mean (SD), n =8. The same superscript lowercase letters indicate no statistical significance between the different immersion times of the adhesive resins for each experimental group ( P > 0.05). The same subscript uppercase letters indicate no statistical significance between SBF and BRM for each immersion time ( P > 0.05) 2 Immersed time (month) 2 Area (μm 2 ) 2 AF (×10 −3 ) 2 TF (μm 2 ) SBF BRM SBF BRM SBF BRM 1 2232.2 (501.2) a A 2060.4 (226.2) a A 113.2 (5.5) a A 109.6 (7.5) a A 302.5 (71.6) a A 281.0 (42.7) a A 2 1914.7 (252.6) a A 1974.8 (582.2) a,b A 100.8 (12.3) b A 98.9 (9.5) b A 232.1 (27.8) b A 236.7 (78.4) a,b A 3 2240.0 (384.4) a A 1953.2 (497.6) a,b A 108.8 (8.5) b A 110.7 (6.0) a,b A 284.6 (52.9) a,b A 263.7 (70.7) a,b A 4 2162.4 (564.2) a A 1573.3 (435.4) b B 108.1 (12.9) b A 110.8 (9.1) a A 268.2 (67.7) a,b A 218.1 (67.3) b A All data are expressed as mean (SD), n =8. The same superscript lowercase letters indicate no statistical significance between the different immersion times of the adhesive resins for each experimental group ( P > 0.05). The same subscript uppercase letters indicate no statistical significance between SBF and BRM for each immersion time ( P > 0.05) 2 Immersed time (month) 2 Area (μm 2 ) 2 AF (×10 –3 ) 2 TF (μm 2 ) SBF BRM SBF BRM SBF BRM 1 555.7 (101.3) a A 630.9 (132.4) a A 102.1 (11.7) a A 118.5 (14.5) a B 66.4 (11.5) a A 82.6 (17.8) a B 2 924.0 (225.6) b A 664.0 (233.0) a,b B 104.4 (11.0) a A 100.2 (5.5) b A 118.1 (30.8) b A 80.2 (29.9) a,b B 3 889.6 (84.0) b A 479.2 (69.7) b B 106.1 (13.7) a A 108.8 (11.8) a,b A 113.4 (37.9) b A 59.2 (11.7) b B 4 989.6 (141.8) b A 517.7 (114.2) a,b B 109.7 (8.1) a A 117.6 (11.4) a A 121.6 (17.7) b A 69.7 (17.4) a B All data are expressed as mean (SD), n =8. The same superscript lowercase letters indicate no statistical significance between the different immersion times of the adhesive resins for each experimental group ( P > 0.05). The same subscript uppercase letters indicate no statistical significance between SBF and BRM for each immersion time ( P > 0.05) 2 Immersed time (month) 2 Area (μm 2 ) 2 AF (×10 −3 ) 2 TF (μm 2 ) SBF BRM SBF BRM SBF BRM 1 2640.1 (876.5) a A 2321.7 (819.3) a A 105.5 (3.8) a A 102.3 (10.3) a A 355.0 (126.3) a,b A 294.9 (111.9) a A 2 2308.9 (234.3) a A 1921.3 (267.9) a,b B 97.9 (9.2) b A 101.0 (8.5) a A 283.9 (43.0) a,b A 240.9 (32.5) a B 3 1980.9 (578.1) a A 1571.1 (465.4) b A 116.7 (10.6) c A 110.5 (8.5) a A 257.4 (71.4) a A 212.7 (67.0) a A 4 2549.6 (466.5) a A 1984.4 (553.1) a,b B 114.3 (11.9) a,c A 107.3 (4.7) a A 333.0 (78.1) b A 268.6 (72.0) a A iBond specimens and FluoroBond II specimens showed the largest fluorescent area ( P > 0.05), followed by Xeno-III-Bond with a significantly lower fluorescent area ( P ≤0.05). With the exception of Xeno-III-Bond specimens after one month and FluoroBond II specimens after two months, all BRM groups showed a lower Rhodamine B uptake than the SBF groups (fluorescent area, Fig. 2 2 ). For FluoroBond II and iBond, no statistical differences could be found for SBF groups among the different storage times ( P > 0.05). In contrast, using Xeno-III-Bond, the fluorescent area was significantly larger after 2, 3, and 4 months than after 1 month ( P =0.002). Most importantly, a statistical significant difference could be found for all adhesive resins when comparing the SBF and BRM groups at the 4th month ( P ≤0.05). This finding was supported by CLSM images (Figs. 3 3 , 4 4 , and 5 5 ), in which Rhodamine B uptake was visually equal for all SBF specimens, while BRM specimens at the 4th month showed less Rhodamine B infiltration and stabilization of the dentinal collagen fiber network. All data are expressed as mean±SD, with n =8 C: composite; D: dentin. (a) The CLSM image of a specimen immersed in SBF for one month shows that most of the hybrid layer (between short arrows, the same below) and the superficial dentinal tubules were permeable to Rhodamine B, but not the mineralized peritubular dentin; (b) The CLSM image of a specimen immersed in BRM for one month shows that the hybrid layer was permeable to Rhodamine B, with no significant difference from that of (a); (c) The CLSM image of a specimen immersed in SBF for two months shows uniform Rhodamine B uptake throughout the hybrid layer and the dentinal tubules; d) The CLSM image of a specimen immersed in BRM for two months shows the same fluorescence stripe as in (c), just slightly wider; (e) Three-month CLSM image of a specimen immersed in SBF shows a 25-μm thick hybrid layer infiltrated with Rhodamine B; (f) Three-month CLSM image of a specimen immersed in BRM exhibits a 25-μm thick red fluorescent stripe, with no significant difference from that of (e); (g) Four-month CLSM image of a specimen immersed in SBF shows no difference from that of (a), (c), and (e); (h) Four-month CLSM image of a specimen immersed in BRM displays areas of remineralization in the hybrid layer (long thin arrows), which could indicate mineral re-deposition C: composite; D: dentin. (a) One-month CLSM image of specimens immersed in SBF shows that the entire hybrid layer (between short arrows, the same below) was permeated by Rhodamine B; (b) One-month CLSM image of specimens immersed in BRM exhibits no difference from that of (a); (c) Three-month CLSM image of specimens immersed in BRM exhibits a solidly permeated hybrid layer and tubular dentin (long thin arrows); (d) Three-month CLSM image of specimens immersed in SBF shows the entire hybrid layer infiltrated with Rhodamine B, but the tubular dentin mainly left unpermeated C: composite; D: dentin. (a) One-month CLSM image of a specimen immersed in SBF shows that the entire hybrid layer (between short arrows, the same below) of about 30 μm thickness was completely permeable to Rhodamine B; (b) One-month CLSM image of a specimen immersed in BRM exhibits no difference from that of (a); (c) Three-month CLSM image of a specimen immersed in SBF shows that the entire hybrid layer of about 20 μm thickness was infiltrated by Rhodamine B; (d) Three-month CLSM image of a specimen immersed in BRM shows zones of reduced fluorescence in irregularly shaped and varied sizes (long thin arrows) Discussion 1 The null hypotheses could not be accepted, as the factors adhesive resin, remineralization medium, and duration of immersion were each found to have significant effects on Rhodamine B uptake of the hybrid layer. Because collagen fibers are reinforced with minerals, healthy dentin can provide the tooth with very good strength and toughness (Kinney et al., 2003 2003 ; Mai et al., 2009 2009 ). To be incorporated into the gap zones of the collagen fibers, these minerals need to be sufficiently small and be arranged in a specific way (Xu et al., 2007 2007 ; Mai et al., 2009 2009 ). Collagen fibers are first formed during the mineralization of biological hard tissues, in which water from the intrafibrillar compartments is replaced by apatites (Glimcher, 2006 2006 ). This process is dependent on the modulation of type I collagen fibers for growth of crystals, in which apatite crystals, assembled by type I collagen fibers, and hydroxyapatite are arranged along the long axis within the mineralized collagen fibers (Glimcher, 2006 2006 ). The passage of molecules is dramatically reduced when demineralized dentin collagen is replaced by apatite minerals (Glimcher, 2006 2006 ). Water-soluble molecules below a molecular mass of 6 kDa can diffuse into the water compartments of a collagen fiber, while those greater than 40 kDa cannot (Toroian et al., 2007 2007 ). As the channels become narrower during mineralization, the speed and volume of water-soluble molecules diffusing through mineralized collagen matrix decrease (Toroian et al., 2007 2007 ). With a molecular mass of 0.479 kDa, Rhodamine B molecules easily diffuse into the demineralized or incompletely resin-infiltrated collagen fibers. After three months, the amount of Rhodamine B molecules penetrating the resin-sparse regions of the hybrid layer was reduced for all adhesive resins immersed in remineralizing solution, indicating mineral deposition in the collagen matrices. In contrast, Rhodamine B uptake from specimens immersed in SBF remained almost constant. Compared to transmission electron microscopy, CLSM requires no preparation of thin specimens and is therefore simple and fast (Fontana et al., 1996 1996 ; Pfarrer et al., 1996 1996 ; Zhong et al., 2015 2015 ). When using CLSM, the intensity of a fluorescent dye penetrating an artificial carious dentin lesion is proportional to the porosity or loss of mineral density within the lesion (van der Veen et al., 1996 1996 ; Jardine et al., 2016 2016 ). For this reason, re-or demineralization can be quantitatively measured by the fluorescence area, TF, and AF (Fontana et al., 1996 1996 ; Jardine et al., 2016 2016 ). The Portland cement-based remineralization protocol introduced in 2008 uses biomimetic analogs to guide apatite remineralization (Tay et al., 2007 2007 ; Tay and Pashley, 2008 2008 ). According to this approach, a PAA with a low MW is used to imitate the stabilizing function of dentin matrix protein 1 (DMP1) on amorphous calcium phosphate precursors (He et al., 2005 2005 ; Mai et al., 2009 2009 ; Zhong et al., 2015 2015 ). The interaction of the Portland cement with SBF reduces these precursors to a nanoscale (Olszta et al., 2003 2003 ; Mai et al., 2009 2009 ) and thus prevents their aggregation and precipitation (Cai and Tang, 2008 2008 ; Mai et al., 2009 2009 ; Zhong et al., 2015 2015 ). PVPA, the other biomimetic analog, is a polyanion that imitates the negative charges of phosphoproteins (e.g. DMP1), phosphophoryn, or bone sialoprotein and their roles in organizing parts of the mineralization process (Gajjeraman et al., 2007 2007 ; Baht et al., 2008 2008 ; Mai et al., 2009 2009 ). In our study, Figs. 3h 3h , 4d 4d , and 5d 5d and the quantitative analysis from specimens immersed in BRM show reduced fluorescence zones, suggesting mineral re-deposition, in agreement with the literature (Kim J. et al., 2010 2010 ; Kim Y.K. et al., 2010a 2010a ; Zhong et al., 2015 2015 ). Some fluoride-releasing adhesive resins may induce crystal growth within fluid-filled gaps of resin-dentin interfaces without biomimetic remineralization (Hashimoto et al., 2008 2008 ). However, note that in the current study apatite deposition occurred using non-fluoride-releasing adhesive resins in the absence of interfacial gaps. The remineralization effect of iBond was most obvious and had the fastest emergence time. Most of the remineralization with iBond occurred between one and three months and did not improve further after four months. This finding is similar to an apparent self-limitation of remineralization in primary dentin observed by Kim J. et al. ( 2010 2010 ) using Adper Prompt L-Pop, a one-step self-etch adhesive resin. The remineralization effect on specimens bonded with Xeno-III-Bond was not as obvious as for iBond. In FluoroBond II, one-month-old specimens, Rhodamine B-free areas could be found in the hybrid layer for specimens immersed in SBF and BRM alike (Figs. 3a and 3b 3a and 3b ). These areas were significantly reduced in two-and three-month-old specimens (Figs. 3c–3f 3c–3f ), while for the four-month-old specimens immersed in SBF the fluorescence of the hybrid layer remained unchanged. BRM specimens showed less Rhodamine B uptake, indicating a possible remineralization. Rhodamine B uptake was reduced for all adhesive resins tested in the current study when placed in the biomineralizing medium, which is a promising result. However, no solution-based systems can yet be used clinically and basic science may here only pave the way for the development of future usable techniques (Zhong et al., 2015 2015 ; Luo et al., 2016 2016 ). Conclusions 1 Hybrid layers of the adhesive resins used in this study could be remineralized by placement in a BRM. 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Informed consent was obtained from all patients for being included in the study. 10.1631/jzus.B1600151 Project supported by the National Natural Science Foundation of China (No. 81271955) and the Zhejiang Provincial Natural Science Foundation of China (No. Y2080338) 2016-04-05 2016-10-18 2016-06-23 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2016 2016 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 11 17