Zn-containing polymer nanogels promote cervical dentin remineralization

1  Thismanuscripthasbeenpublishedonlinein:Clinical Oral Investigations 2019March https://doi.org/10.1007/s00784-018-2548-1  2  Authors: Manuel Toledano1, Inmaculada Cabello1, Estrella Osorio1, Fátima S. Aguilera1, Antonio Luis Medina-Castillo2, Manuel Toledano-Osorio1*, Raquel Osorio1. Title: Zn-containing polymer nanogels promote cervical dentin remineralization. Institution: 1University of Granada, Faculty of Dentistry, Dental Materials Section. Colegio Máximo de Cartuja s/n 18071 – Granada - Spain. 2University of Granada, NanoMyP. Spin-Off Enterprise. Edificio BIC-Granada. Av. Innovación 1. 18016 - Armilla, Granada, Spain. *Corresponding author: Mr. Manuel Toledano-Osorio. University of Granada, Faculty of Dentistry Dental Materials Section Colegio Máximo de Cartuja s/n 18071 – Granada - Spain. Tel.: +34-958243788 Fax: +34-958240809 Email: [email protected] Acknowledgements This work was supported by grants MINECO/FEDER MAT2014-52036-P and MINECO/FEDER MAT2017-85999-P. Authors do not have a financial relationship with the organization that sponsored the research. 3  Abstract Objective: Nanogels designing for effective treatment of eroded cervical dentin lesions. Materials and Methods: Polymethylmetacrylate-based nanoparticles (NPs) were doxycycline (D), calcium or zinc loaded. They were applied on eroded cervical dentin. Treated surfaces were characterized morphologically by atomic force and scanning electron microscopy, mechanically probed by a nanoindenter to test nanohardness and Young modulus, and chemically analyzed by Raman spectroscopy at 24 h and 7 d of storage. Data were submitted to ANOVA and Student-Newman-Keuls multiple comparisons tests. Results: Dentin treated with Zn-NPs attained the highest nanomechanical properties, mineralization and crystallinity among groups. Nanoroughness was lower in Zn-treated surfaces in comparison to dentin treated with undoped gels. Dentin treated with Ca-NPs created the minimal calcification at the surface and showed the lowest Young modulus at peritubular dentin. Intertubular dentin appeared remineralized. Dentinal tubules were empty in samples treated with D-NPs, partially occluded in cervical dentin treated with undoped NPs and Ca-NPs, and mineral covered when specimens were treated with Zn-NPs. Conclusions: Zn-loaded NPs permit functional remineralization of eroded cervical dentin. Based on the tested nanomechanical and chemical properties, Zn-based nanogels are suitable for dentin remineralization. Clinical Relevance: The ability of zinc-loaded nanogels to promote dentin mineralization may offer new strategies for regeneration of eroded cervical dentin and effective treatment of dentin hypersensitivity. Keywords: remineralization, calcium, zinc, nanoparticles, cervical dentin. 4  Introduction Non-carious cervical lesions are pathological processes characterized by loss of dental hard tissues near the cementoenamel junction, in absence of caries [1]. They are significant predisposing factors for dentin hypersensitivity (DH) [2], which is generated by an extracellular matrix demineralization producing dentinal tubules exposure. Dentin is a highly mineralized tissue whose mechanical properties play an indispensable role in maintaining the stress/strain tooth behavior. Dentin extracellular matrix is composed by carbonate rich and calcium deficient hydroxyapatite (HAp) crystallites [3], disposed at extrafibrillar and intrafibrillar compartments of the collagen fibers. Dentinal tubules are surrounded by intertubular dentin (ID), which consists of a collagen matrix reinforced by apatite crystals similar to those of peritubular dentin (PD) [4]. Erosion and chemical degradation represent the etiology of DH [5]. Erosion is a complex process where the initial dissolution of the mineral exposes the organic matrix leaving a layer of fully demineralized organic matrix [6]. Degradation of the dentin matrix occurs after it has become accessible by the removal of mineral, i.e. the dentin matrix cannot be degraded unless it is demineralized [7]. Tubules occlusion and dentin remineralization are considered two of the main objectives of DH treatment [8, 9]. New biomaterials should facilitate dentin remineralization [10, 11] focusing on promoting regenerative processes of the extracellular matrix through interactions with the host tissue to release dentin bioactive molecules [12]. Some resins, varnishes and remineralizing agents [9] have been proposed for DH treatment, but most of them do not promote functional remineralization, do not demonstrate enough compatibility [13], and their effects are often transitory [14]. In dentin treatment, demineralized dentin infiltration with polymeric nanoparticles (NPs) as calcium and phosphate sequestering materials has been proposed. Anionic carboxylate sequences (COO-) are on the NPs surfaces, allowing their complexation to cationic ions or molecules [11]. Metalloproteinases (MMPs) are activated when pH is lowered, as it occurs during caries and erosion processes [6]. Zinc inhibits MMPs-mediated collagen degradation and favors dentin remineralization [15]. Biological apatite is calcium deficient, and contains substantial amounts of carbonate. Carbonated apatite is a precursor of HAp, but when it is precipitated in the presence of zinc, there is an in vitro exchange between Zn2+ and Ca2+, forming a substituted apatite compound [16]. Furthermore, zinc incorporation into NPs raises the potential for intrafibrillar remineralization at partially demineralized collagen matrices [15]. Doxycycline potentiates hard tissues regeneration in periodontal defects when locally administrated due to its anticollagenolytic effect [17]. 5  The degree and the quality of the mineralization will affect the mechanical properties of dentin. Indeed, the extrafibrilar minerals act as a granular material that can withstand load, but in the absence of intrafibrilar mineralization. Intrafibrilar mineralization is the key factor for ensuring that collagen fibrils have the same high modulus of elasticity as occurs in natural biomineralized dentin [18]. Atomic force microscopy (AFM) assisted nano-indentation does represent a specific applied mean of testing mechanical properties of some materials or substrates [19]. Raman spectroscopy is an analytical technique able to measure the molecular composition of dentin containing information regarding chemical changes within the samples [20, 21]. The combination of biochemical data with atomic force microscopy (AFM) techniques appears to be a valuable tool for applying in dentin remineralization studies [22]. The aim of this study was to investigate the efficacy of different nanogels to remineralize eroded cervical dentin. Surface roughness, mechanical and chemical changes occurring after treating cervical dentin surfaces with four different NPs solutions were analyzed. The null hypothesis that was established is that no changes in surface profilometry, mechanical and chemical properties were produced at in vitro eroded cervical dentin surfaces after different NPs application. Material and Methods Nanoparticles production PolymP-n Active nanoparticles (NPs) (NanoMyP, Granada, Spain) were fabricated trough polymerization precipitation [23]. NPs are composed by 2-hydroxyethyl methacrylate (backbone monomer), ethylene glycol dimethacrylate (cross-linker) and methacrylic acid (functional monomer). Calcium-doped NPs (Ca-NPs) and Zinc-doped NPs (Zn-NPs) were produced. For zinc and calcium complexation 30 mg of NPs were immersed at room temperature, during 3 days under continuous shaking in 15 ml aqueous solutions of ZnCl2 or CaCl2 (containing zinc or calcium at 40 ppm at pH 6.5), in order to reach the adsorption equilibrium of metal ions. Then, the suspensions were centrifuged and the particles were separated from the supernatant. Attained ion complexation values are 0.96 ± 0.04 µg Ca/mg NPs and 2.15 ± 0.05 µg Zn/mg NPs [24]. A third group of NPs doped with doxycycline was introduced in the study. 30 mg of NPs were immersed in 18 ml of 40 mg/ml aqueous solution of doxycycline hyclate (Sigma Aldrich, ChemieGmbh, Riedstr, Germany), during 4 hours, under continuous shaking. Then, the suspensions were centrifuged and the particles were separated from the supernatant. Following this procedure, loading efficacy is 70%, incorporated doxycycline to NPs is 900 µg/mL (per mg of NPs). Four 6  different nanogels were tested: 1) NPs (NPs), 2) NPs doped with Ca (Ca-NPs), 3) NPs doped with Zn (ZnNPs), and 4) NPs doped with doxycycline hyclate (D-NPs). Specimen preparation 15 sound, single-rooted teeth were obtained with informed consent from donors (18 to 25 yr of age), under a protocol approved by the Institution review board (405/CEIH/2017). Two dentin blocks from the buccal surface of each root, just below the cementodentinal junction were obtained by cutting with a diamond saw (Accutom-50 Struers, Copenhagen, Denmark) under copious water irrigation. The surfaces were polished through SiC abrasive papers from 800 up to 4000 grit followed by final polishing steps performed using diamond pastes through 1 µm down to 0.25 µm (Struers LaboPol-4; Struers GmbH, Hannover, Germany) (Fig. 1). Specimens were prescreened for tubule occlusion with AFM and those with occluded tubules were excluded [25]. Dentin samples were dipped into a citric acid solution (pH 3.8) for 1 min to ensure the patency of the dentinal tubules and remove the smear layer [25]. Dentin surfaces were washed and ultrasound treated for 10 min before NPs application. A phosphate buffered saline (PBS) suspension of NPs, Zn-NPs, CaNPs, D-NPs (10 mg/ml) or just a PBS solution were applied (30 s), in each of the five different experimental groups. Each block of the same treated teeth was stored in PBS at 37o C for 24 hours and 7 days. Nanoindentation An atomic force microscope (AFM-Nanoscope V, Digital Instruments, Veeco Metrology group, Santa Barbara, CA, USA) equipped with a Triboscope indentor system (Hysitron Inc., Minneapolis, MN, USA) and a Berkovich indenter (tip radius 20 nm) was employed in this study. For each subgroup, three slabs were tested. On each slab, five indentation lines were executed in five different mesio-distal positions along the dentin surface in a straight line. Indentations were performed with a load of 4000 nN and a time function of 10 s. The indenter was progressively (at a constant rate) pressed over the sample up to a peak load of 4000 µN. Specimens were scanned in a hydrated condition. To avoid dehydration a layer of ethylene glycol over the specimen surface was applied, preventing water evaporation during a typical 25-to-30-min scanning period [26]. The distance between each indentation was kept constant by adjusting the distance intervals in 5 (±1) µm steps [27]. Hardness (Hi) and modulus of elasticity (Ei) data were registered in GPa. The load, F, was obtained as a function of the penetration depth, h, of the indenter in the sample. From the slope of these load-vs.-depth curves the nanoindentation modulus (Young modulus) could be obtained by application of different theoretical models [28, 29]. One of these is the Oliver-Pharr method, 7  which is based on a continuum, isotropic, homogeneous elastic contact model to determine the reduced modulus, Er. In this model the slope, S, of the unloading portion of the load-vs.-depth data is used to obtain Er according to the following equation [29]: 2 r dF SEA dh   , (1) where A is the projected contact area of the hardness impression of the indenter. Then, Ei of the sample is obtained through the following expression:  22 11 1ind i rind i EE E    , (2) In this expression “i” subscript refers to the tested sample and “ind” subscript to the indenter.  and E are the Poisson’s ratio and the Young modulus, respectively. For hard materials, as dentin, usually, Eind>>Ei and, thus, the contribution of the indenter in equation (2) can be neglected. With regard to the nanohardness of the sample, H, it is defined as: max F H A , Where Fmax is the peak load. In this work, values of nanohardness and Young modulus were automatically calculated by using the software Triboscan Quasi version 8.4.2.0 (Hysitron, Inc). Data were analyzed by two-way ANOVA and Student-Newman-Keuls multiple comparisons tests (P < 0.05). AFM imaging and nanoroughness assessments An atomic force microscope (AFM Nanoscope V, Digital Instruments, Veeco Metrology group, Santa Barbara, CA, USA) was employed in this study for topography analysis. The imaging process was undertaken inside a wet cell in a fully hydrated state, using the tapping mode, with a calibrated verticalengaged piezo-scanner (Digital Instrument, Santa Barbara, CA, USA). A 10-nm-radius silicon nitride tip (Veeco) was attached to the end of an oscillating cantilever that came into intermittent contact with the surface at the lowest point of the oscillation. Changes in vertical position of the AFM tip at resonance frequencies near 330 kHz provided the height of the images registered as bright and dark regions. 10 x 10 µm digital images were recorded with a slow scan rate (0.1 Hz). For each image, 5 randomized boxes (2 x 2 µm) and (1 x 1 µm) were created to examine the ID and PD nanoroughness at 24 h and 7 d of storage. Nanoroughness (SRa, in nanometers) was measured with proprietary software (Nanoscope Software, 8  version V7). Data were submitted to ANOVA and Student-Newman-Keuls multiple comparisons tests (p<0.05). Raman spectroscopy The same dentin surfaces were, then, submitted to Raman analysis using a dispersive Raman spectrometer/microscope (Horiba Scientific Xplora, Villeneuve d´Ascq, France). A 785-nm diode laser through a X100/0.90 NA air objective was employed. Raman signal was acquired using a 600-lines/mm grafting centered between 400 and 1700 cm–1. Chemical mapping of the surfaces was performed. For each specimen two areas 12x12 m of the surfaces at different sites were mapped using 0.5 m spacing at X and Y axis (625 points per map). The output from a clustering algorithm was basically a statistical description of the cluster centroids with the number of components in each cluster. The biochemical content of each cluster was analyzed using the average cluster spectra. The natural groups of components (or data) based on some similarity and the centroids of a group of data sets were found by the clustering algorithm once calculated by the software and the Hierarchical Cluster Analysis (HCA). The observed spectra were described at 400-1700 cm-1 with 10 complete overlapping Gaussian lines, suggesting homogeneous data for further calculations [30]. At this point, the mineral component of dentin was assessed after the analysis of the relative presence of mineral, i.e, phosphate (960 cm-1) and carbonate (1070 cm-1) peaks and areas, relative mineral concentration of phosphate (PO43-) and carbonate (CO32-) referred to phenyl (RMCp and RMCc, respectively). Additional peaks were measured at 954 cm-1, 956 cm-1 and 963 cm-1 to analyze the calcification of the extracellular matrix, the additional substituted or amorphous-like apatite species and the stoichiometric hydroxyapatite (HAp), respectively [20,31,32]. Crystallinity was also assessed based on the full width at half maximum (FWHM) of the phosphate band at 960 cm-1, FWHMP [32]. The organic component of dentin was analyzed examining normalization at 1003 cm-1, crosslinking at 1030/1032.7 cm1 (Pyridinium ring vibration), and at 1550 cm-1 [AGEs, (advance glycation end products)-pentosidine], Nature of collagen at the amide III, CH2, amide I and proteoglycans peaks [20,32] Field Emission Scanning Electron Microscopy (FESEM) and energy dispersive (SEM/EDX) analyses Representative specimens of each group were fixed in a solution of 2.5% glutaraldehyde in 0.1 mol/L sodium cacodylate buffer for 24 h, rinsed three times in 0.1 mol/L sodium cacodylate buffer. Samples were placed in an apparatus for critical point drying (Leica EM CPD 300, Wien, Austria). They were, then, sputter-coated with carbon by means of a sputter-coating Nanotech Polaron-SEMPREP2 (Polaron 9  Equipment Ltd., Watford, UK) and observed with a field emission scanning electron microscope (FESEM Gemini, Carl Zeiss, Oberkochen, Germany) at an accelerating voltage of 3 kV. Energy-dispersive analysis was performed in selected points using an X-ray detector system (EDX Inca 300, Oxford Instruments, Oxford, UK) attached to the FESEM. Results The nanomechanical properties (Hi and Ei) of the cervical dentin surfaces at PD and ID were influenced by the type of NPs applied (P<0.05) and by the storage time (P<0.05). Interactions between factors were also significant (P<0.05). Mean and SD of Hi and Ei are represented in Figure 2. The highest Hi and Ei were achieved after 7 d of storage, at PD and ID, in specimens treated with Zn-NPs (Fig. 2). The lowest Hi values were reached at 7 d time point at ID in untreated dentin specimens. The lowest Ei after 7 d of storage was attained in dentin treated with Ca-NPs at both PD and ID. Intertubular and peritubular cervical dentin, untreated, decreased their Hi after 7 d. Zn-NPs application after 7 d contributed to increase Hi at PD. Ei decreased at ID in untreated cervical dentin surfaces after 7 d, and also at PD when surfaces were untreated or Ca-NPs treated. Dentin surfaces treated with Zn-NPs increased their Ei after 7 d of storage (Figs. 2a, 2b). AFM images of untreated and Zn-NPs treated dentin surfaces, after 7 d storage are shown in Figure 3. The strong remineralization pattern of the dentin surface, and the staggered periodicity banding of collagen fibrils are shown at cervical dentin after Zn-NPs treatment. Nanoroughness (SRa) of dentin surfaces were influenced by the type of NPs applied (P < 0.05) and by storage time (P < 0.05); interactions between factors were also significant (P < 0.05). Mean and SD of nanoroughness are presented in Figure 2C. At ID and PD after 7 d, the lowest SRa values were obtained when the dentin surface was treated with D-NPs. Roughness at PD could not be measured after treating with Zn-NPs as only some tubules orifices were partially visible (Figs. 2c, 3b). Results from Raman analysis are presented in Table 1 and Figure 5. Cervical dentin treated with Zn-NPs attained the highest degree of mineralization related to the phosphate (960 cm-1) and carbonate (1070 cm-1) at 7 d time point, as their peaks and areas showed the maximum values, among groups. The full width at half maximum (FWHMp) of the phosphate (PO43-) band at 961 cm-1, was the lowest when ZnNPs were applied, after 7 d of storage, showing the highest crystallinity among groups. This complied with the maximum height peak of the stoichiometric HAp band v1 (963 cm-1), 743 cm-1 (Table 1). The highest 16  [13] Niu LN, Zhang W, Pashley DH, Breschi L, Mao J, Chen JH, Tay FR (2014) Biomimetic remineralization of dentin. 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Tooth coronal section was discarded (1), a disc of cervical dentin was obtained (2), a longitudinal cut was also made to obtain two halves of the original specimen (3), two dentin blocks were prepared by cutting below the cementodentinal junction (4), surfaces were polished (5) to expose the cervical dentin Fig 2 a, Mean and standard deviation of nanohardness values (Hi) (GPa) at eroded cervical dentin surfaces of the different experimental groups. b, Mean and standard deviation of Young modulus values (Ei) (GPa) at eroded cervical dentin surfaces of the different experimental groups. c, Mean and standard deviation of average surface nanoroughness values (SRa) (nm) at eroded cervical dentin surfaces of the different experimental groups. Same letter (capital for 24 h and lowercase for 7 d) indicates no significant differences between treatment groups at the same dentin type. * indicates significant differences between the different storage periods in the same treatment group and dentin type. Abbreviations: NPs: unloaded nanoparticles; D-NPs: doxycycline doped nanoparticles; Ca-NPs: Ca doped nanoparticles; Zn-NPs: Zn doped nanoparticles Fig 3 a, (I) 10 x 10 μm top-view and surface plot image of untreated cervical dentin at 7 d of storage. Opened dentinal tubules are observed. Peritubular (PD) and intertubular (ID) dentin appear well differentiated. (II) AFM phase image (10 x 10 μm) of this partially demineralized dentin surface. b, (I) 10 x 10 μm top-view and surface plot image of cervical after applying Zn-NPs, at 7 d of storage. The dentin surface is totally remineralized. (II) AFM phase image (10 x 10 μm) showing the wider bandwidth of the collagen fibrils and the staggered pattern of collagen fibrils (faced arrows) Fig 4 a, Field emission scanning electron microscopy (FESEM) image of untreated eroded cervical dentin, shown after 7 d of storage (scale bar: 1 µm). A few crystals or mineral deposits were occasionally encountered on both intertubular and peritubular dentin (arrows). Tubules appeared mineral free with a clear ring of peritubular dentin (PD) (inset scale bar: 300 nm). b, Eroded cervical dentin treated with NPs is shown after 7 d of storage (scale bar: 1 µm). Peritubular (PD) and intertubular dentin (ID) were strongly mineralized. A robust and rougher PD was observed (pointers). Dentinal tubules appeared partially filled with mineral precipitates (asterisks) and mineralized NPs (doubled arrows) (inset scale bar: 100 nm). A dense network of fibrils completely mineralized may be observed covering the peritubular dentin (arrows). The prototypical D-periodicity banding of collagen fibrils was observed in multiple details (faced arrows). c, Eroded cervical dentin treated with D-NPs is shown after 7 d of storage (scale bar: 3 µm). Dentinal 21  tubules were empty. Intertubular (ID) and peritubular (PD) dentin were strongly mineralized (asterisks). At PD, mineral formed a lip around each tubule lumen detected. Mineralized collagen fibers and NPs formed the tubular wall (pointers). Crystal formations permit the observation of the subjacent collagen fibers which appeared clearly remineralized (arrows) (inset scale bar: 300 nm). d, Eroded cervical dentin surfaces treated with Ca-NPs are shown after 7 d of storage (scale bar: 1 µm). The dentin surface exhibited amorphous clumps of minerals scattered and grouped as dense network of buttons-like materials. Tubule entrances were visible. Peritubular (PD) and intertubular dentin (ID) appeared totally remineralized. Dentin collagen fibrils were coated with nucleated crystals (pointers). Dentinal tubules appeared partially filled with mineral precipitates (asterisks) and NPs (arrows) (inset scale bar: 300 nm). NPs remained totally adhered to the remineralized collagen fibers (faced arrows). e, Eroded dentin surfaces treated with Zn-NPs after 7 d of storage (scale bar: 2 µm). Tubules were not observed. Mineral precipitated throughout the dense network of multilayered crystals on the dentin surface were shown (arrows). Mineralized collagen fibrils were not noticeable below this coat of new crystals (inset scale bar: 300 nm). f, Spectra from energy dispersive analysis is showing elemental composition of phosphorous (P) and calcium (Ca), as main components in untreated (I), NPs (II), D-NPs (III), Ca-NPs (IV), and Zn-NPs (V) specimens of eroded cervical dentin Fig 5 a, 2D micro-Raman map of the phosphate peak (961 cm-1) intensities at the untreated surfaces, at 7 d of storage. b, 2D micro-Raman map of the phosphate peak (961 cm-1) intensities at dentin surfaces treated with Zn-NPs, at 7 d of storage. c, Color mapping from hierarchical cluster analysis (HCA) image corresponding to the untreated surfaces, at 7 d of storage. d, Color mapping from hierarchical cluster analysis (HCA) image corresponding to dentin surfaces treated with Zn-NPs, at 7 d of storage. Three levels of HCA clustering are shown. Areas of distinct colors have differences in Raman spectral distribution and chemical composition. Each cluster, corresponds to a different dentin location, which is assigned to a different color (red, green and blue). e, Raman spectra from hierarchical cluster analysis (HCA) results of untreated surfaces at 7 days of storage. f, Raman spectra from hierarchical cluster analysis (HCA) results of dentin surfaces treated with Zn-NPs at 7 d of storage. 22  Table 1. Raman intensities (in arbitrary units) of mineral and organic components at eroded cervical dentin surfaces. Mineral components Untreated dentin NPs D-NPs Ca-NPs Zn-NPs 24h 7d 24h 7d 24h 7d 24h 7d 24h 7d Relative Presence of Mineral Phosphate (961) Peak 465.73 486.72 575.49 622.66 753.32 685.64 503.20 551.64 726.57 804.68 Area 11600.8 12371.7 14597.9 15790.2 19098.4 17392.0 12760.7 13996.5 18425.2 20284.6 RMCP 23.55 30.01 25.58 24.18 38.14 34.70 30.37 33.66 35.35 32.75 Carbonate (1070) Peak 57.65 58.50 72.22 71.70 103.96 91.26 66.45 72.18 99.80 108.66 Area 1940.45 1778.2 2390.7 2168.8 3439.1 3575.9 2603.6 2389.2 3612.7 3890.9 RMCC 2.91 3.61 3.21 2.78 5.26 4.62 4.01 4.40 4.84 4.42 Crystallinity (FWHMp) 19.01 19.40 19.36 19.35 19.35 19.36 19.36 19.36 19.35 19.24 v1 (954) 370.88 371.00 430.36 465.13 544.30 490.94 415.69 428.61 509.50 548.62 v1 (956) 417.33 465.41 491.13 530.67 630.41 567.23 462.40 485.84 592.72 640.49 v1 (963) 429.51 420.31 555.51 554.08 665.49 614.94 457.92 519.22 654.36 743.21 Organic components Normalization Phenyl (1003) 19.78 16.22 22.50 25.75 19.75 19.76 16.57 16.39 20.64 24.57 Crosslinking Pyridinium (1032) 28.67 21.69 36.68 38.69 35.51 32.95 31.37 28.72 32.71 40.20 AGEs-Pentosidine (1550) 6.39 7.20 5.14 4.98 9.40 7.98 5.31 6.04 9.85 9.29 Nature and secondary structure of collagen A-III (1246-1270) 34.82 36.26 40.17 43.56 53.49 48.51 42.1 38.55 52.41 55.88 CH2 (1450) 22.48 22.86 25.26 27.06 31.77 28.58 27.11 24.42 31.61 33.97 A-I (1655-1667) 4.45 5.24 3.98 4.72 11.05 10.59 6.44 6.16 12.02 11.22 Proteoglycans (1062) 34.65 32.07 42.55 44.26 52.03 46.34 35.93 35.83 45.45 51.40 For the mineral components, the peaks values had been normalized to the basis intensity of the symmetric phosphate band, near 996 cm-1. For the organic components, the peaks values had been normalized to the basis intensity of the Amide II band near 1510 cm-1. Peaks positions are expressed in cm-1. NPs: unloaded nanoparticles; D-NPs: doxycycline doped nanoparticles; Ca-NPs: Ca doped nanoparticles; Zn: Zn doped nanoparticles; RMC: Relative mineral concentration between mineral/Phenyl (1003 cm-1); FWHM: Full-width half-maximum; A: amide; AGEs: advanced glycation end products. 23  Figure 1 24  Figure 2 25  Figure 3