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On the bulk biomechanical behavior of densely cross-linked dentin matrix: The role of induced-glycation, regional dentin sites and chemical inhibitor
Journal Pre-proof On the bulk biomechanical behavior of densely cross-linked dentin matrix: The role of induced-glycation, regional dentin sites and chemical inhibitor Yvette Alania, Livia T. Trevelin, Mohammad Hussain, Camila A. Zamperini, Gresa Mustafa, Ana K. Bedran-Russo PII:
S1751-6161(19)31032-X
DOI:
https://doi.org/10.1016/j.jmbbm.2019.103589
Reference:
JMBBM 103589
To appear in:
Journal of the Mechanical Behavior of Biomedical Materials
Received Date: 22 July 2019 Revised Date:
17 October 2019
Accepted Date: 7 December 2019
Please cite this article as: Alania, Y., Trevelin, L.T., Hussain, M., Zamperini, C.A., Mustafa, G., BedranRusso, A.K., On the bulk biomechanical behavior of densely cross-linked dentin matrix: The role of induced-glycation, regional dentin sites and chemical inhibitor, Journal of the Mechanical Behavior of Biomedical Materials (2020), doi: https://doi.org/10.1016/j.jmbbm.2019.103589. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
On the bulk biomechanical behavior of densely cross-linked dentin matrix: the role of induced-glycation, regional dentin sites and chemical inhibitor Graphical abstract
On the bulk biomechanical behavior of densely cross-linked dentin matrix: the role of induced-glycation, regional dentin sites and chemical inhibitor
Authors: Yvette Alaniaa, Livia T. Trevelina,1, Mohammad Hussaina, Camila A. Zamperinia,2, Gresa Mustafaa, Ana K. Bedran-Russo1
a
Department of Restorative Dentistry, College of Dentistry, University of Illinois at Chicago,
801 South Paulina St, Chicago, IL 60612, USA 1
Department of Restorative Dentistry, School of Dentistry, University of São Caetano do Sul,
Rua Santo Antônio 50, São Caetano do Sul, São Paulo, Brazil, 09521-160 2
Department of Operative Dentistry, College of Dentistry, University of Iowa, 801 Newton
Rd, Iowa city, IA, 52242, USA
Corresponding author: Ana K. Bedran-Russo Department of Restorative Dentistry, College of Dentistry, University of Illinois at Chicago, 801 South Paulina St, Room 531, Chicago, IL 60612, USA [email protected]
Abstract Collagen glycation takes place under physiological conditions during chronological aging, leading to the formation of advanced glycation end-products (AGEs). AGEs accumulation induces non-enzymatic collagen cross-links increasing tissue stiffness and impairing function. Here, we focused on determining the cumulative effect of induced glycation on the mechanical behavior of highly collagen cross-linked dentin matrices and assess the topical inhibition potential of aminoguanidine. Bulk mechanical characterization suggests that early glycation cross-links significantly increase the tensile strength and stiffness of the dentin matrix and promote a brittle failure response. Histologically, glycation yielded a more mature type I collagen in a densely packed collagen matrix. The time-dependent effect of glycation indicates cumulative damage of dentin matrices that is partially inhibited by aminoguanidine. The regional dentin sites were differently affected by induced-glycation, revealing the crown dentin to be mechanically more affected by the glycation protocol. These findings in human dentin set the foundation for the proposed in vitro ribose-induced glycation model, which produces an early matrix stiffening mechanism by reducing tissue viscoelasticity and can be partially inhibited by topical aminoguanidine.
Keywords: glycation, advanced glycation end-products, aminoguanidine, ribose, tensile strength, dentin
1. Introduction Glycation is a non-enzymatic physiological process in human aging resulting in the accumulation of advanced glycation end-products (AGEs) in proteins. The accumulation of AGEs aggravates existing diseases and increases the risk for the development of other pathologies [1], such as complications found in diabetes [2]. AGEs are the result of a molecular process involving multiple and complex reactions beginning with the formation of a non-stable Schiff base through Maillard reaction between a carbohydrate (typically glucose or ribose) [3] and an amino group (lysine or arginine) from a long-lived protein such as collagen [4,5]. Through rearrangements, the unstable Schiff base becomes a stable ketoamine Amadori product. Schiff bases and Amadori products, early glycation products, are reversible. However, with time, cross-links and irreversible protein adducts are formed [5,6]. Carboxymethylysine (CML), methylglyoxal (MGO), pentosidine, furosine and glucosepane, are some of the AGEs previously identified and commonly found in glycated tissues [1,2,7,8]. AGEs diminish functional aspects of tissues by accumulation in major proteins of the extracellular matrix (ECM) [2]. Glycated collagen shows non-enzymatic intermolecular cross-linking of adjacent fibrils/fibers leading to increased tissue stiffness and decreased flexibility [5,9–14]. Also, changes in the molecular charge profile and the addition of AGEs in the side chain of collagen hinder molecular recognition by modifying specific binding sites reducing cell interactions that can ultimately affect self-repair [9,15]. Dentin, a mineralized tissue with high collagen content, does not turnover, and thus accumulates ECM modifications over a lifetime. AGEs can accumulate in dentin [8,16], with few reports pointing to direct effect on the mechanical properties such as higher hardness [8] and lower flexural strength and toughness [17]. AGEs accumulation appears higher in older teeth [8,16,17]. Moreover, AGEs accumulation is predominant in the peritubular area of root
dentin, likely influenced by the dentin metabolism receiving nourishment through the dentinal tubules [8,16,18]. Synthetic and natural compounds can inhibit the formation of AGEs or cleavage the already formed AGEs’ cross-links [2,6,19]. However, clinical effectiveness of systemic usage is inconsistent [6]. An example is aminoguanidine chloride (AMG), a nucleophilic hydrazine, identified as an inhibitor of the formation of early glycation products [2,6,13,20,21], and herein topical usage investigated as proof-of-concept. Given the increased life expectancy and retention of the natural dentition [22], the aim of this study was two-fold: (1) to determine an experimental model of accumulation of early glycation to assess the mechanical properties of root and crown dentin matrices and (2) to assess the in vitro inhibitory potential of aminoguanidine during the accumulation of early glycation of dentin. The null hypothesis tested was that ribose-induced glycation would not affect the bulk mechanical properties of dentin matrix, regardless of the cumulative exposure time and regional dentin site. 2. Materials and methods 2.1 Experimental Design and Dentin Specimens Preparation This in vitro experiment included three (3) study factors: dentin site (2 levels: crown and root); conditions (3 levels: induced glycation, inhibition of glycation with aminoguanidine, and control); cumulative time (3 levels: 7, 14, and 21 days). The outcome variables were ultimate tensile strength, energy to fracture and apparent modulus of elasticity (n = 15 per group). Histological imaging of collagen with picrosirius red staining assessed structural and conformation changes to dentin matrix. Dentin sections (0.5 mm thickness and 6mm length, Fig. 1) from crown and root sites were obtained from extracted human third molars stored dry at -20°C. The use of extracted human
molars was approved by Institutional Review Board (protocol # 2011-0312). The edges of each specimen were covered with nail polish [23]. Sections were further trimmed (#835 diamond bur, Brasseler, Savannah, GA, USA) to produce hour-glass shaped specimens with a neck of 0.5 ± 0.1 mm2 in mid-dentin (Fig. 1). Specimens were demineralized with 0.5 M EDTA (pH 8) for 14 days at 4°C, and the specimens from each tooth were randomly assigned into experimental groups according to dentin conditions (Gly, AMG and control).
A
B n = 15
1 mm 0.5 mm
Fig. 1. Schematic of specimen preparation for tensile strength test. (A) Crown and root dentin were sectioned axially into 0.5 mm-thick slabs. Sections were trimmed into an hour-glass shape to have the tubule orientation perpendicular to the tensile crosshead direction. (B) Demineralized hour-glass shaped specimen before and after tensile fracture. 2.2 In vitro glycation and chemical inhibition of glycation protocols In vitro glycation (Gly) of dentin matrices was induced by 0.6 M D-Ribose (Sigma Aldrich, St. Louis, MO, USA) in 10 mM phosphate-buffered saline (PBS) containing protease inhibitor (Pierce™ Protease Inhibitor Mini tablets, EDTA-free, Thermo Scientific, Rockford, IL, USA) and 0.01% NaN3 at 37°C. The inhibition of glycation (AMG) was carried out by the simultaneous incubation of dentin with 0.6 M D-ribose and 0.05 M aminoguanidine hydrochloride (Sigma Aldrich, St. Louis, MO, USA) [20]. Control groups were incubated in
PBS buffer solution only. Incubation solutions were replaced every 2 days per week up to 21 days. 2.3 Mechanical Properties of Dentin 2.3.1 Ultimate tensile strength (UTS) Mechanical measurements of dentin were determined in a universal testing machine (EZ Graph, Shimadzu, Kyoto, Japan) with a 200 N load cell. The edges of the specimens were attached onto a Ciucchi’s tensile testing jig. Tensile forces were applied at a crosshead speed of 1 mm/min. All specimens were kept hydrated in distilled water prior and during the tensile test. The peak load force was determined and the ultimate tensile strength individually calculated using the cross-sectional area at the neck region of each specimen. The UTS was expressed in MPa. 2.3.2 Energy to fracture The energy to fracture of dentin matrices was estimated from the area under the stress-strain curves generated during the tensile testing. The stress-strain data were extracted from the Trapezium software of the EZ Graph and analyzed using the trapezoidal rule (TRAPZ command) in the software MatLab (MathWorks Inc., Natick, MA, USA) [24]. Results were expressed in MPa.m1/2. 2.3.3 Apparent modulus of elasticity The apparent modulus of elasticity (E) was estimated from the stress-strain curve plots by selecting the longest section of the linear region closest to the beginning of the slope of the stress-strain curve (elastic slope). The beginning and end-points of the section were then entered into the trapezoidal equation coded in MatLab to calculate the apparent E modulus, defined as E=∆σ/∆ε, where ∆σ is the difference in stress and ∆ε is the difference in strain of
the slope in the linear segment corresponding to the elastic region. Results were expressed in MPa. 2.3.4 Data analysis Intragroup variability was assessed using Levene’s test and found to not meet the assumption of homogenous distribution (UTS: p = 0.001, energy to fracture: p < 0.001 and apparent modulus of elasticity: p < 0.001). Thus, datasets of UTS, energy to fracture and apparent modulus of elasticity were statistically analyzed by ANOVA and Games-Howell post hoc tests for multiple comparisons (α = 0.05). 2.4 Qualitative and Quantitative Histological Analysis of Dentin Matrix Structural and conformational modifications to type I collagen were histologically evaluated using Picrosirius red staining by enhancing their natural birefringence under polarized light [25,26,27]. Briefly, crown dentin specimens were treated in the same manner described above up to 21 days incubation (n = 5). Specimens were then cryo-sectioned (CM 3050 S, Leica Biosystems, Nussloch, Germany) into 8 µm-thick slices. Sections were stained with 0.1% picrosirius red (Electron Microscopy Sciences) in saturated picric acid for 90 min followed by a 0.01 N HCl rinse (1 min twice). Sections were dehydrated, sealed in xylene and mounted for observation under polarized light microscope (Axioscope 40, Carl Zeiss, Goettingen, Germany). Images were captured at 40x magnification using a digital camera (AxioCam HRc, Zeiss). Quantitative assessment was done by converting the images to grayscale and inverting bright and dark regions (ImageJ software, NIH, USA). Threshold tool settings were determined to quantify the counts of staining and expressed in area percentage [25]. Data were analyzed using two-way ANOVA and Tukey post hoc test for comparisons among groups (α = 0.05). 2.5 Spectroscopic analysis of the dentin matrix
The collagen quality/maturity [28,29,30], the post-translational modifications of the extracellular matrix [28,30] and the presence of pentosidine [28,31,32] were determined in crown and root dentin following mechanical measurements after 21-day incubation using Raman spectral range from 820 to 1900 cm-1, recorded with a 633 nm excitation diode laser (inVia Raman, Renishaw, Wooton Under Edge, UK). Spectral acquisitions were done (accumulations: 60; time for each spectrum: 1 s) at a laser power of 100%. Six spectra for each specimen were obtained with a resolution of ± 4 cm-1. Each spectrum was baseline corrected and normalized using WiRE 4.3 software (Renishaw). The collagen maturity was estimated by the ratio of non-reducible trivalent (1656 cm-1)/reducible divalent collagen crosslinks (1684 cm-1) peak areas. The extracellular matrix modifications were evaluated by the ratio of hydroxyproline (873 cm-1)/proline (917 cm-1) band area. Pentosidine accumulation was measured by the peak area ratio of pentosidine band (1495 cm-1 and 1363cm-1)/CH2 wag (1450 cm-1). Data were analyzed using two-way ANOVA and post hoc tests for comparisons among groups (α = 0.05).
3. Results 3.1 Mechanical properties of dentin matrix The ultimate tensile strength (UTS) results (Fig. 2) showed no significant interactions between the studied factors (dentin site vs. condition vs. cumulative time; p > 0.05), except between dentin site vs. cumulative time (p = 0.007). However, statistical differences were observed within study factors (dentin, condition and cumulative time, all at p = 0.001). Ribose-induced glycation (Gly) resulted in statistically higher UTS when compared to control (p < 0.001). Intermediate UTS values from AMG group, which were not statistically different from control and Gly groups (p > 0.05), indicated partial inhibitory role of aminoguanidine.
The induced glycation was time-dependent as values were significantly reduced after 21 days incubation (p < 0.028), suggesting cumulative damage. Overall, root dentin exhibited statistically higher UTS than crown dentin (p = 0.001).
A
DENTIN
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Fig. 2. Ultimate tensile strength. (A) Results of UTS (mean and standard deviation) of the dentin matrix for all variables (pooled means): condition, cumulative time and dentin site. Interactions among study factors were only significant for dentin site vs. time (p = 0.007). Statistical differences were observed within all study factors (p = 0.001). Different symbols represent statistically significant differences (p < 0.05) among groups in each graph. (B) Graphs describe UTS results from crown and root dentin ECM as a function of time. Different symbols represent statistically significant differences (p < 0.05) from all conditions among time points. Gly: ribose-induced glycation, AMG: aminoguanidine-mediated inhibition of glycation.
The energy to fracture values of the dentin matrix (Fig. 3) were only significant for dentin site vs cumulative time (p = 0.004). In general, the energy to fracture significantly decreased with time (p < 0.003). Interestingly, only the energy to fracture from the root dentin exhibited a significant increase after 14 days followed by a decrease after 21 days, regardless of any
condition (p < 0.003). UTS results from root dentin also showed this behavior. Statistical differences were observed within dentin site (p < 0.001) and time (p < 0.001), but not condition (p = 0.443), indicating that neither the induced glycation nor the glycation inhibitor affected the energy to fracture. However, the reduction of the energy to fracture shown at 21 days by crown and root dentin from Gly group was of 45% and 30%, respectively, whereas the reduction in the control group was less than 25%. Overall, root dentin required higher energy to fracture when compared to crown dentin (p < 0.001).
A Energy to Fracture (MPa.m1/2 )
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CUMULATIVE TIME 10
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Fig. 3. Energy to fracture. (A) Results of the energy to fracture of the dentin matrix presented by studied variables (pooled means). Interactions among factors were only significant for dentin site vs. time (p = 0.004). Statistical differences were observed for study factors: dentin site (p < 0.001) and cumulative time (p < 0.001), but not condition (p = 0.443). Different symbols represent statistically significant differences (p < 0.05) among groups in each graph. (B) Graphs describe energy to fracture results of crown and root dentin ECM as a function of time. Different symbols represent statistically significant differences (p < 0.05) from all conditions among time points. Gly: ribose-induced glycation, AMG: aminoguanidine-mediated inhibition of glycation.
The results of apparent modulus of elasticity - E (Fig. 4) showed no significant interactions among study factors (p = 0.326), however, differences were observed within condition (p < 0.001) and cumulative time (p = 0.016). Ribose-induced glycation significantly increased the E of dentin matrices (p < 0.001). After 21 days ribose treatment, crown and root dentin showed a 1.9- and 1.3-fold stiffness increase, respectively, when compared to the control group Aminoguanidine partially prevented the increase of stiffness in the dentin matrix (p = 0.002), which was more evident on the root. Regardless of the condition, the E of dentin
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Apparent modulus of elasticity (MPa)
matrices increased after 21-day incubation (p = 0.023).
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Fig. 4. Apparent modulus of elasticity. (A) Results of the apparent modulus of elasticity of the dentin matrix presented by study factors (pooled means). No interactions were found between studied factors (p = 0.326). Differences were observed within condition (p < 0.001) and cumulative time (p = 0.016). Different symbols represent statistically significant differences (p < 0.05) among groups in each graph. (B) Graphs describe the apparent modulus of elasticity of crown and root dentin ECM as a function of their experimental conditions. Different symbols represent statistically significant differences (p < 0.05) among all conditions. Gly: ribose-induced glycation, AMG: aminoguanidine-mediated inhibition of glycation.
Representative stress-strain curves (Fig. 5) from mechanical test at 7, 14 and 21 days showed distinct dentin mechanical behavior under tensile stress among experimental condition and dentin site. Stress-strain curves from Gly and AMG groups showed lack of post-yield deformation before failure, indicating a brittle type of fracture. Regardless of the condition and time, root dentin exhibited a more pronounced flattening of the curve followed by a rise and final failure, indicating higher toughness.
A
Control Gly AMG
Control Gly AMG
B
Control Gly AMG
Control Gly AMG
C
Control Gly AMG
Control Gly AMG
Fig. 5. Representative tensile stress-strain curves from experimental groups (plots used force and known cross-section area to calculate the stress over the displacement deformation of the specimen) after 7 (A), 14 (B) and 21 (C) days from crown and root dentin. Different from control group, the stress-strain slope of the ribose-induced glycation group showed little to no plastic deformation upon fracture, revealing a brittle failure with more pronounced changes after 21 days. After 14 days, curves from root dentin ECM showed a characteristic initial plateau before a rise in the stress until fracture, likely due to the alignment of the fibrils towards the force direction. Gly: ribose-induced glycation, AMG: aminoguanidine-mediated inhibition of glycation.
3.2 Qualitative and Quantitative Histological Analysis of Dentin Matrix Representative images of experimental groups are depicted in Fig. 6. Dentin sections from Gly and AMG group (Fig 6B and 6C) showed stronger red birefringence consistent with mature type I collagen in a thicker and densely packed collagen matrix. Quantitative analysis of staining (Fig. 7) shows no significant interaction between factors (condition vs. time; p = 0.359). However, at all incubation periods, red birefringence (mature collagen) was statistically higher for the Gly and AMG group when compared to the control group (p < 0.001), and no statistical differences were found between Gly and AMG (p = 0.938).
7 days
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14 days
21 days
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50 µm
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GLY
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AMG
Fig. 6. Representative images of the picrosirius red staining of crown dentin extracellular matrix from control (A), Gly (B) and AMG (C) groups at different time points. Dentin matrix from Gly group showed more densely packed mature collagen as it increased 1.5 to 2 times the red birefringence when compared to control. Scale bar corresponds to 50 µm. Gly: ribose-induced glycation, AMG: aminoguanidine-mediated inhibition of glycation.
7 days
14 days
21 days
Picrosirius Red Birefringence (%)
100 90 80 70 60 50 40 30 20 10 0 Control
Gly
AMG
Fig. 7. Results of percentage of mature collagen detected under red birefringence polarized microscopy of crown dentin sections stained with picrosirius red under experimental conditions and different time points. No interaction was found between conditions and cumulative times (p = 0.359). Statistical differences were observed within conditions (p < 0.001). Bar represents lack of statistical significant differences (p > 0.05). Gly: riboseinduced glycation, AMG: aminoguanidine-mediated inhibition of glycation.
3.3 Spectroscopic analysis of the dentin matrix The chemical characterization of the collagen maturity, post-translations modifications and pentosidine content are depicted in Fig. 8. Collagen maturity was similar among groups except for the AMG group, which was significantly lower in the root dentin ECM (p = 0.003). Glycation induction or inhibition did not affect the post-translational modifications of the dentin ECM (p = 0.330). Induced glycation increased the pentosidine content 1.44 to 3.5 times in crown and root dentin, respectively. However, the increase was only significant for the root ECM (p < 0.001). Aminoguanidine significantly decreased the pentosidine content regardless of the type of dentin (p < 0.001). The pentosidine content was significantly lower in root than in the crown dentin (p < 0.001), irrespective of the condition.
COLLAGEN MATURITY
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MODIFICATION 1.4
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* * Crown
* Root
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Fig. 8. Raman spectroscopy analysis of the dentin matrix after 21-day incubation. Ratios were calculated with the integrated peak of designated Raman bands. Graphs exhibit ratios (mean and standard error) of collagen maturity (A), post-translational modifications of the extracellular matrix (B), and pentosidine content (C). The symbol (*) depicts statistically significant differences when compared to other groups (p < 0.05). Gly: ribose-induced glycation, AMG: aminoguanidine-mediated inhibition of glycation.
3
Discussion
This study investigated the effect of in vitro early glycation on the mechanical properties of crown and root dentin matrices; and also, the inhibitory effect of topical aminoguanidine. The findings revealed that ribose-induced glycation increased 23% the ultimate tensile strength and 31% the apparent modulus of the dentin ECM, indicating an early matrix stiffening mechanism. Therefore, the null hypothesis was rejected. The increase of densely packed collagen shown histologically, and the glycation products found in the ribose-glycated ECM through spectroscopy, pointed to non-enzymatic cross-linking of the collagen fibrils, supporting the efficacy of the proposed in vitro glycation model. Also, the expected browning of the dentin matrix, characteristic of the Maillard reaction [20], was observed on the glycated ECM. The low turnover of collagen and the enduring nature of non-enzymatic crosslinking lead to the progressive accumulation of AGEs in aged human tissues, contributing to its functional impairment [5,9–14,33]. Herein, glycated dentin ECM clearly showed a time-dependent
modification of the mechanical strength resulting in increased stiffness. Ribose-induced glycation forms both intra- and inter-fibrillar crosslinks along the collagen backbone, on the free ε-amino groups on the lysine side-chains or in the N-terminal amine group. Further reactions undergo to produce covalent intermolecular cross-links that ultimately constrain or suppress the fibril’s plasticity [10,33,34]. The increased stiffening revealed a key feature of glycated ECM expressed by the markedly low post-yielding plastic behavior, supporting findings that ribose-mediated crosslinking increase brittleness [5,17]. It was suggested that glycation takes effect at the surface of the fibril, based on the size of the reducing glycating sugars (< 1nm) [5,34]. Despite that, reports of AGEs-mediated changes at lower hierarchical levels (i.e. microfibrils and collagen molecules) contribute to the reduction in viscoelasticity [5,28,35]. Considering the hindering of intermolecular cross-links stretching, different mechanisms proposed involve the limitation in collagen fibril-fibril sliding [5] and the halted unfolding/unwinding of individual collagen molecules [11,33]. Thus, the capacity of deformation compensation from the collagen demonstrates its physiological function to increase toughness and reduce the risk of brittle fracture [11,33]. Although previous studies reported decreased toughness in glycated collagen [12,17,33], our results showed that the energy to fracture was not influenced by the ribose-induced glycation. One possible explanation is that the organization, density and distribution [36] of the collagen fibrils in the demineralized dentin, unlike tendon or bone, does not possess a preferred unidirectional orientation [11]. The lack of unidirectional orientation might grant dentin compensation for the glycation-induced stiffening by crack bridging [37,38]. Further, the amount of bound water in the matrix [39] and the absence of mineral phase might have outweighed the toughness of glycated ECM. Mostly ECM from the root exhibited higher tensile strength and energy to fracture when compared to the crown dentin matrix. In fact, root was differently affected by ribose than
crown, as energy and strength values after 21 days were similar to those at 7 days incubation. The density and cross-linking of the collagen, highly expressed in root dentin when compared to crown dentin [40–42], may partially explains variations here found between the crown and root dentin matrix. Interestingly, root dentin showed less pentosidine content than crown ECM (controls, Fig. 8), indicating a reverse relationship of pentosidine content and tensile strength of the untreated dentin ECM. While not investigated in this study, non-collagenous components such as proteoglycans could play a possible role as regulators of fibrillar spaces [21,24,25] and tissue mechanical viscous behavior [13,21]. Aminoguanidine, as an early glycation inhibitor, is known to trap reactive dicarbonyl groups from reducing sugars, avoiding their conversion to AGEs [2,43]. Thus, it would not have an effect on more advanced stages of glycation [2], as supported by the mechanical findings. Aminoguanidine exhibited a mild inhibitor effect (27.7 % decrease in UTS) within 7 days incubation, probably attributed to its short half-life [43]. Previous reports using aminoguanidine showed a partial reversal (from 19 to 55%) of the glycation effects of proteins at 0.01 M concentration [13,20,21,44]. Nonetheless, Raman spectroscopy showed that AMG significantly reduced pentosidine in both crown and root after 21 days. It also decreased the collagen maturity of root ECM, suggesting anti-aging characteristics [45]. The inhibitory properties of aminoguanidine may involve a specific complex mechanism in the collagen that does not reflect substantial mechanical improvement within the timeframe assessed in this study. Interestingly, picrosirius red (PSR) staining showed no difference in density and packing of collagen fibrils between Gly and AMG groups after 14 or 21 days, possibly due to the maturation of the Amadori products and glycation cross-links. Early phases of glycation consist of largely intrafibrillar cross-links along the helix of collagen molecules [46]. The fact that glycation occurs primarily on the fibril surface and thus at an
accessible location could have also important implication for therapeutic strategies based on enzymatic de-glycation [5]. More detailed knowledge of the complexities of AGE-mediated cross-links will aid in the understanding of the biomechanical functional consequences to the dentin and promote alternatives for glycation inhibition. Within the study limitations, an experimental model of early glycation of dentin matrices and testing method can prove valuable bulk mechanical properties. Since the effects of AGEs accumulation are time- and site-dependent, in vitro glycation should include time and dentin area as variables. The potential of topical inhibition of glycation using aminoguanidine was modest as it partially reduced the AGEs mechanical effect in the dentin matrix. However, it indicates that topical usage of chemical agents may be a strategy to prevent and reduce glycation of the dentin ECM. 4
Conclusions
The findings strongly suggest that in vitro ribose-induced early glycation of the dentin matrix reduced tissue viscoelasticity and increased tensile strength by 23%, revealing an abnormal stiffening mechanism. In addition, results were time-dependent, indicating the accumulation of glycation products in the dentin matrix. The regional dentin sites were differently affected by induced-glycation, where crown dentin matrix was mechanically more affected than root dentin. Glycation of dentin matrix can be partially inhibited by topical exposure to aminoguanidine.
Acknowledgements The authors gratefully acknowledge UIC Health Navigators Summer research program. We thank Yasmin Bakir for contributing to the image analysis of the histology experiments. We greatly appreciate Dr. Mariana Cavalcante dos Reis for the technical assistance with Raman
microscope. This work was supported by the National Institutes of Health/National Institute of Dental and Craniofacial Research [grant DE021040]. Author contributions Yvette Alania: Investigation, Methodology, Data curation, Visualization, Writing - Original draft preparation. Livia Trevelin: Investigation. Mohammad Hussain: Investigation. Camila A. Zamperini: Investigation, Visualization, Writing- Review & Editing. Gresa Mustafa: Investigation, Software, Visualization. Ana Bedran-Russo: Conceptualization, Methodology, Writing – Review & Editing, Supervision. Declarations of interest: none References [1] A. Ilea, A.M. Băbţan, B.A. Boşca, M. Crişan, N.B. Petrescu, M. Collino, R.M. Sainz, J.Q. Gerlach, R.S. Câmpian, Advanced glycation end products (AGEs) in oral pathology, Arch Oral Biol 93 (2018) 22–30. https://www.doi.org/10.1016/j.archoralbio.2018.05.013. [2] P. Gkogkolou, M. Böhm, Advanced glycation end products: Key players in skin aging?, Dermatoendocrinol 4 (2012) 259–270. https://www.doi.org/10.4161/derm.22028. [3] S.K. Grandhee, V.M. Monnier, Mechanism of formation of the Maillard protein crosslink pentosidine. Glucose, fructose, and ascorbate as pentosidine precursors, J Biol Chem 266 (1991) 11649–11653. [4] N. Verzijl, J. DeGroot, S.R. Thorpe, R.A. Bank, J.N. Shaw, T.J. Lyons, J.W. Bijlsma, F.P. Lafeber, J.W. Baynes, J.M. TeKoppele, Effect of collagen turnover on the accumulation of advanced glycation end products, J Biol Chem 275 (2000) 39027–39031. https://www.doi.org/10.1074/jbc.M006700200. [5] A. Gautieri, F.S. Passini, U. Silvan, M. Guizar-Sicairos, G. Carimati, P. Volpi, M. Moretti, H. Schoenhuber, A. Redaelli, M. Berli, J.G. Snedeker, Advanced glycation endproducts: Mechanics of aged collagen from molecule to tissue, Matrix Biol 59 (2017) 95– 108. https://www.doi.org/10.1016/j.matbio.2016.09.001. [6] N. Ahmed, Advanced glycation endproducts - role in pathology of diabetic complications, Diabetes Res Clin Pract 67 (2005) 3–21. https://www.doi.org/10.1016/j.diabres.2004.09.004. [7] F. Greis, A. Reckert, K. Fischer, S. Ritz-Timme, Analysis of advanced glycation end products (AGEs) in dentine: useful for age estimation?, Int J Legal Med 132 (2018) 799–805. https://www.doi.org/10.1007/s00414-017-1671-x. [8] J. Miura, K. Nishikawa, M. Kubo, S. Fukushima, M. Hashimoto, F. Takeshige, T. Araki, Accumulation of advanced glycation end-products in human dentine, Arch Oral Biol 59 (2014) 119–124. https://www.doi.org/10.1016/j.archoralbio.2013.10.012. [9] N.C. Avery, A.J. Bailey, The effects of the Maillard reaction on the physical properties and cell interactions of collagen, Pathol Biol (Paris) 54 (2006) 387–395. https://www.doi.org/10.1016/j.patbio.2006.07.005. [10] N. Verzijl, J. DeGroot, Z.C. Ben, O. Brau-Benjamin, A. Maroudas, R.A. Bank, J.
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Highlights -
Glycation of highly cross-linked dentin extracellular matrix results in an immediate brittle-like biomechanical behavior.
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Distinct regional effects indicates higher susceptibility to glycation of crown extracellular matrix when compared to root dentin.
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Damage to the biomechanics of dentin matrices is cumulative, and only partially inhibited by topical exposure to aminoguanidine.
S1751-6161(19)31032-X
DOI:
https://doi.org/10.1016/j.jmbbm.2019.103589
Reference:
JMBBM 103589
To appear in:
Journal of the Mechanical Behavior of Biomedical Materials
Received Date: 22 July 2019 Revised Date:
17 October 2019
Accepted Date: 7 December 2019
Please cite this article as: Alania, Y., Trevelin, L.T., Hussain, M., Zamperini, C.A., Mustafa, G., BedranRusso, A.K., On the bulk biomechanical behavior of densely cross-linked dentin matrix: The role of induced-glycation, regional dentin sites and chemical inhibitor, Journal of the Mechanical Behavior of Biomedical Materials (2020), doi: https://doi.org/10.1016/j.jmbbm.2019.103589. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
On the bulk biomechanical behavior of densely cross-linked dentin matrix: the role of induced-glycation, regional dentin sites and chemical inhibitor Graphical abstract
On the bulk biomechanical behavior of densely cross-linked dentin matrix: the role of induced-glycation, regional dentin sites and chemical inhibitor
Authors: Yvette Alaniaa, Livia T. Trevelina,1, Mohammad Hussaina, Camila A. Zamperinia,2, Gresa Mustafaa, Ana K. Bedran-Russo1
a
Department of Restorative Dentistry, College of Dentistry, University of Illinois at Chicago,
801 South Paulina St, Chicago, IL 60612, USA 1
Department of Restorative Dentistry, School of Dentistry, University of São Caetano do Sul,
Rua Santo Antônio 50, São Caetano do Sul, São Paulo, Brazil, 09521-160 2
Department of Operative Dentistry, College of Dentistry, University of Iowa, 801 Newton
Rd, Iowa city, IA, 52242, USA
Corresponding author: Ana K. Bedran-Russo Department of Restorative Dentistry, College of Dentistry, University of Illinois at Chicago, 801 South Paulina St, Room 531, Chicago, IL 60612, USA [email protected]
Abstract Collagen glycation takes place under physiological conditions during chronological aging, leading to the formation of advanced glycation end-products (AGEs). AGEs accumulation induces non-enzymatic collagen cross-links increasing tissue stiffness and impairing function. Here, we focused on determining the cumulative effect of induced glycation on the mechanical behavior of highly collagen cross-linked dentin matrices and assess the topical inhibition potential of aminoguanidine. Bulk mechanical characterization suggests that early glycation cross-links significantly increase the tensile strength and stiffness of the dentin matrix and promote a brittle failure response. Histologically, glycation yielded a more mature type I collagen in a densely packed collagen matrix. The time-dependent effect of glycation indicates cumulative damage of dentin matrices that is partially inhibited by aminoguanidine. The regional dentin sites were differently affected by induced-glycation, revealing the crown dentin to be mechanically more affected by the glycation protocol. These findings in human dentin set the foundation for the proposed in vitro ribose-induced glycation model, which produces an early matrix stiffening mechanism by reducing tissue viscoelasticity and can be partially inhibited by topical aminoguanidine.
Keywords: glycation, advanced glycation end-products, aminoguanidine, ribose, tensile strength, dentin
1. Introduction Glycation is a non-enzymatic physiological process in human aging resulting in the accumulation of advanced glycation end-products (AGEs) in proteins. The accumulation of AGEs aggravates existing diseases and increases the risk for the development of other pathologies [1], such as complications found in diabetes [2]. AGEs are the result of a molecular process involving multiple and complex reactions beginning with the formation of a non-stable Schiff base through Maillard reaction between a carbohydrate (typically glucose or ribose) [3] and an amino group (lysine or arginine) from a long-lived protein such as collagen [4,5]. Through rearrangements, the unstable Schiff base becomes a stable ketoamine Amadori product. Schiff bases and Amadori products, early glycation products, are reversible. However, with time, cross-links and irreversible protein adducts are formed [5,6]. Carboxymethylysine (CML), methylglyoxal (MGO), pentosidine, furosine and glucosepane, are some of the AGEs previously identified and commonly found in glycated tissues [1,2,7,8]. AGEs diminish functional aspects of tissues by accumulation in major proteins of the extracellular matrix (ECM) [2]. Glycated collagen shows non-enzymatic intermolecular cross-linking of adjacent fibrils/fibers leading to increased tissue stiffness and decreased flexibility [5,9–14]. Also, changes in the molecular charge profile and the addition of AGEs in the side chain of collagen hinder molecular recognition by modifying specific binding sites reducing cell interactions that can ultimately affect self-repair [9,15]. Dentin, a mineralized tissue with high collagen content, does not turnover, and thus accumulates ECM modifications over a lifetime. AGEs can accumulate in dentin [8,16], with few reports pointing to direct effect on the mechanical properties such as higher hardness [8] and lower flexural strength and toughness [17]. AGEs accumulation appears higher in older teeth [8,16,17]. Moreover, AGEs accumulation is predominant in the peritubular area of root
dentin, likely influenced by the dentin metabolism receiving nourishment through the dentinal tubules [8,16,18]. Synthetic and natural compounds can inhibit the formation of AGEs or cleavage the already formed AGEs’ cross-links [2,6,19]. However, clinical effectiveness of systemic usage is inconsistent [6]. An example is aminoguanidine chloride (AMG), a nucleophilic hydrazine, identified as an inhibitor of the formation of early glycation products [2,6,13,20,21], and herein topical usage investigated as proof-of-concept. Given the increased life expectancy and retention of the natural dentition [22], the aim of this study was two-fold: (1) to determine an experimental model of accumulation of early glycation to assess the mechanical properties of root and crown dentin matrices and (2) to assess the in vitro inhibitory potential of aminoguanidine during the accumulation of early glycation of dentin. The null hypothesis tested was that ribose-induced glycation would not affect the bulk mechanical properties of dentin matrix, regardless of the cumulative exposure time and regional dentin site. 2. Materials and methods 2.1 Experimental Design and Dentin Specimens Preparation This in vitro experiment included three (3) study factors: dentin site (2 levels: crown and root); conditions (3 levels: induced glycation, inhibition of glycation with aminoguanidine, and control); cumulative time (3 levels: 7, 14, and 21 days). The outcome variables were ultimate tensile strength, energy to fracture and apparent modulus of elasticity (n = 15 per group). Histological imaging of collagen with picrosirius red staining assessed structural and conformation changes to dentin matrix. Dentin sections (0.5 mm thickness and 6mm length, Fig. 1) from crown and root sites were obtained from extracted human third molars stored dry at -20°C. The use of extracted human
molars was approved by Institutional Review Board (protocol # 2011-0312). The edges of each specimen were covered with nail polish [23]. Sections were further trimmed (#835 diamond bur, Brasseler, Savannah, GA, USA) to produce hour-glass shaped specimens with a neck of 0.5 ± 0.1 mm2 in mid-dentin (Fig. 1). Specimens were demineralized with 0.5 M EDTA (pH 8) for 14 days at 4°C, and the specimens from each tooth were randomly assigned into experimental groups according to dentin conditions (Gly, AMG and control).
A
B n = 15
1 mm 0.5 mm
Fig. 1. Schematic of specimen preparation for tensile strength test. (A) Crown and root dentin were sectioned axially into 0.5 mm-thick slabs. Sections were trimmed into an hour-glass shape to have the tubule orientation perpendicular to the tensile crosshead direction. (B) Demineralized hour-glass shaped specimen before and after tensile fracture. 2.2 In vitro glycation and chemical inhibition of glycation protocols In vitro glycation (Gly) of dentin matrices was induced by 0.6 M D-Ribose (Sigma Aldrich, St. Louis, MO, USA) in 10 mM phosphate-buffered saline (PBS) containing protease inhibitor (Pierce™ Protease Inhibitor Mini tablets, EDTA-free, Thermo Scientific, Rockford, IL, USA) and 0.01% NaN3 at 37°C. The inhibition of glycation (AMG) was carried out by the simultaneous incubation of dentin with 0.6 M D-ribose and 0.05 M aminoguanidine hydrochloride (Sigma Aldrich, St. Louis, MO, USA) [20]. Control groups were incubated in
PBS buffer solution only. Incubation solutions were replaced every 2 days per week up to 21 days. 2.3 Mechanical Properties of Dentin 2.3.1 Ultimate tensile strength (UTS) Mechanical measurements of dentin were determined in a universal testing machine (EZ Graph, Shimadzu, Kyoto, Japan) with a 200 N load cell. The edges of the specimens were attached onto a Ciucchi’s tensile testing jig. Tensile forces were applied at a crosshead speed of 1 mm/min. All specimens were kept hydrated in distilled water prior and during the tensile test. The peak load force was determined and the ultimate tensile strength individually calculated using the cross-sectional area at the neck region of each specimen. The UTS was expressed in MPa. 2.3.2 Energy to fracture The energy to fracture of dentin matrices was estimated from the area under the stress-strain curves generated during the tensile testing. The stress-strain data were extracted from the Trapezium software of the EZ Graph and analyzed using the trapezoidal rule (TRAPZ command) in the software MatLab (MathWorks Inc., Natick, MA, USA) [24]. Results were expressed in MPa.m1/2. 2.3.3 Apparent modulus of elasticity The apparent modulus of elasticity (E) was estimated from the stress-strain curve plots by selecting the longest section of the linear region closest to the beginning of the slope of the stress-strain curve (elastic slope). The beginning and end-points of the section were then entered into the trapezoidal equation coded in MatLab to calculate the apparent E modulus, defined as E=∆σ/∆ε, where ∆σ is the difference in stress and ∆ε is the difference in strain of
the slope in the linear segment corresponding to the elastic region. Results were expressed in MPa. 2.3.4 Data analysis Intragroup variability was assessed using Levene’s test and found to not meet the assumption of homogenous distribution (UTS: p = 0.001, energy to fracture: p < 0.001 and apparent modulus of elasticity: p < 0.001). Thus, datasets of UTS, energy to fracture and apparent modulus of elasticity were statistically analyzed by ANOVA and Games-Howell post hoc tests for multiple comparisons (α = 0.05). 2.4 Qualitative and Quantitative Histological Analysis of Dentin Matrix Structural and conformational modifications to type I collagen were histologically evaluated using Picrosirius red staining by enhancing their natural birefringence under polarized light [25,26,27]. Briefly, crown dentin specimens were treated in the same manner described above up to 21 days incubation (n = 5). Specimens were then cryo-sectioned (CM 3050 S, Leica Biosystems, Nussloch, Germany) into 8 µm-thick slices. Sections were stained with 0.1% picrosirius red (Electron Microscopy Sciences) in saturated picric acid for 90 min followed by a 0.01 N HCl rinse (1 min twice). Sections were dehydrated, sealed in xylene and mounted for observation under polarized light microscope (Axioscope 40, Carl Zeiss, Goettingen, Germany). Images were captured at 40x magnification using a digital camera (AxioCam HRc, Zeiss). Quantitative assessment was done by converting the images to grayscale and inverting bright and dark regions (ImageJ software, NIH, USA). Threshold tool settings were determined to quantify the counts of staining and expressed in area percentage [25]. Data were analyzed using two-way ANOVA and Tukey post hoc test for comparisons among groups (α = 0.05). 2.5 Spectroscopic analysis of the dentin matrix
The collagen quality/maturity [28,29,30], the post-translational modifications of the extracellular matrix [28,30] and the presence of pentosidine [28,31,32] were determined in crown and root dentin following mechanical measurements after 21-day incubation using Raman spectral range from 820 to 1900 cm-1, recorded with a 633 nm excitation diode laser (inVia Raman, Renishaw, Wooton Under Edge, UK). Spectral acquisitions were done (accumulations: 60; time for each spectrum: 1 s) at a laser power of 100%. Six spectra for each specimen were obtained with a resolution of ± 4 cm-1. Each spectrum was baseline corrected and normalized using WiRE 4.3 software (Renishaw). The collagen maturity was estimated by the ratio of non-reducible trivalent (1656 cm-1)/reducible divalent collagen crosslinks (1684 cm-1) peak areas. The extracellular matrix modifications were evaluated by the ratio of hydroxyproline (873 cm-1)/proline (917 cm-1) band area. Pentosidine accumulation was measured by the peak area ratio of pentosidine band (1495 cm-1 and 1363cm-1)/CH2 wag (1450 cm-1). Data were analyzed using two-way ANOVA and post hoc tests for comparisons among groups (α = 0.05).
3. Results 3.1 Mechanical properties of dentin matrix The ultimate tensile strength (UTS) results (Fig. 2) showed no significant interactions between the studied factors (dentin site vs. condition vs. cumulative time; p > 0.05), except between dentin site vs. cumulative time (p = 0.007). However, statistical differences were observed within study factors (dentin, condition and cumulative time, all at p = 0.001). Ribose-induced glycation (Gly) resulted in statistically higher UTS when compared to control (p < 0.001). Intermediate UTS values from AMG group, which were not statistically different from control and Gly groups (p > 0.05), indicated partial inhibitory role of aminoguanidine.
The induced glycation was time-dependent as values were significantly reduced after 21 days incubation (p < 0.028), suggesting cumulative damage. Overall, root dentin exhibited statistically higher UTS than crown dentin (p = 0.001).
A
DENTIN
CONDITION
30
UTS (MPa)
25 20
30
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25
§
20
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∞§
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15
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10
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10
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5
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∞
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AMG
7 days
14 days
21 days
CUMULATIVE TIME
B 30 25 UTS (MPa)
TIME
30
30
CROWN ∞
20
∞
§
25
§ ∞
∞
20
15
15
10
10
5
5
ROOT
0
0 7 days
14 days
7 days
21 days
Control
Gly
14 days
21 days
AMG
Fig. 2. Ultimate tensile strength. (A) Results of UTS (mean and standard deviation) of the dentin matrix for all variables (pooled means): condition, cumulative time and dentin site. Interactions among study factors were only significant for dentin site vs. time (p = 0.007). Statistical differences were observed within all study factors (p = 0.001). Different symbols represent statistically significant differences (p < 0.05) among groups in each graph. (B) Graphs describe UTS results from crown and root dentin ECM as a function of time. Different symbols represent statistically significant differences (p < 0.05) from all conditions among time points. Gly: ribose-induced glycation, AMG: aminoguanidine-mediated inhibition of glycation.
The energy to fracture values of the dentin matrix (Fig. 3) were only significant for dentin site vs cumulative time (p = 0.004). In general, the energy to fracture significantly decreased with time (p < 0.003). Interestingly, only the energy to fracture from the root dentin exhibited a significant increase after 14 days followed by a decrease after 21 days, regardless of any
condition (p < 0.003). UTS results from root dentin also showed this behavior. Statistical differences were observed within dentin site (p < 0.001) and time (p < 0.001), but not condition (p = 0.443), indicating that neither the induced glycation nor the glycation inhibitor affected the energy to fracture. However, the reduction of the energy to fracture shown at 21 days by crown and root dentin from Gly group was of 45% and 30%, respectively, whereas the reduction in the control group was less than 25%. Overall, root dentin required higher energy to fracture when compared to crown dentin (p < 0.001).
A Energy to Fracture (MPa.m1/2 )
DENTIN
10
10
8 6
TIME
CONDITION
10
§
8
8
∞
6
∞
∞
∞
6
4
4
4
2
2
2
0
0 crown
∞§
∞ §
0
root
Control
B
Gly
AMG
7 days
14 days
21 days
Energy to Fracture (MPa.m1/2 )
CUMULATIVE TIME 10
10
§
CROWN 8
8
∞ ∞
6
∞ ∞
6
§
4 2
ROOT
4 2
0
0 7 days
14 days
21 days
Control
7 days
Gly
14 days
21 days
AMG
Fig. 3. Energy to fracture. (A) Results of the energy to fracture of the dentin matrix presented by studied variables (pooled means). Interactions among factors were only significant for dentin site vs. time (p = 0.004). Statistical differences were observed for study factors: dentin site (p < 0.001) and cumulative time (p < 0.001), but not condition (p = 0.443). Different symbols represent statistically significant differences (p < 0.05) among groups in each graph. (B) Graphs describe energy to fracture results of crown and root dentin ECM as a function of time. Different symbols represent statistically significant differences (p < 0.05) from all conditions among time points. Gly: ribose-induced glycation, AMG: aminoguanidine-mediated inhibition of glycation.
The results of apparent modulus of elasticity - E (Fig. 4) showed no significant interactions among study factors (p = 0.326), however, differences were observed within condition (p < 0.001) and cumulative time (p = 0.016). Ribose-induced glycation significantly increased the E of dentin matrices (p < 0.001). After 21 days ribose treatment, crown and root dentin showed a 1.9- and 1.3-fold stiffness increase, respectively, when compared to the control group Aminoguanidine partially prevented the increase of stiffness in the dentin matrix (p = 0.002), which was more evident on the root. Regardless of the condition, the E of dentin
B
DENTIN 60
50
50
40
∞
TIME
CONDITION
60
∞
60 50
§
40
∞
∞
30
30
20
20
20
10
10
10
0
0 Crown
§
40
∞§
∞
7 days
14 days
30
0
Root
Control
Gly
AMG
21 days
CONDITION Apparent modulus of elasticity (MPa)
A
Apparent modulus of elasticity (MPa)
matrices increased after 21-day incubation (p = 0.023).
60
CROWN
50 40
§
60
40
∞
∞§
30
∞§
∞ §
30
20
20
10
10
0
ROOT
50
0 Control
Gly
AMG
7 days
Control
14 days
Gly
AMG
21 days
Fig. 4. Apparent modulus of elasticity. (A) Results of the apparent modulus of elasticity of the dentin matrix presented by study factors (pooled means). No interactions were found between studied factors (p = 0.326). Differences were observed within condition (p < 0.001) and cumulative time (p = 0.016). Different symbols represent statistically significant differences (p < 0.05) among groups in each graph. (B) Graphs describe the apparent modulus of elasticity of crown and root dentin ECM as a function of their experimental conditions. Different symbols represent statistically significant differences (p < 0.05) among all conditions. Gly: ribose-induced glycation, AMG: aminoguanidine-mediated inhibition of glycation.
Representative stress-strain curves (Fig. 5) from mechanical test at 7, 14 and 21 days showed distinct dentin mechanical behavior under tensile stress among experimental condition and dentin site. Stress-strain curves from Gly and AMG groups showed lack of post-yield deformation before failure, indicating a brittle type of fracture. Regardless of the condition and time, root dentin exhibited a more pronounced flattening of the curve followed by a rise and final failure, indicating higher toughness.
A
Control Gly AMG
Control Gly AMG
B
Control Gly AMG
Control Gly AMG
C
Control Gly AMG
Control Gly AMG
Fig. 5. Representative tensile stress-strain curves from experimental groups (plots used force and known cross-section area to calculate the stress over the displacement deformation of the specimen) after 7 (A), 14 (B) and 21 (C) days from crown and root dentin. Different from control group, the stress-strain slope of the ribose-induced glycation group showed little to no plastic deformation upon fracture, revealing a brittle failure with more pronounced changes after 21 days. After 14 days, curves from root dentin ECM showed a characteristic initial plateau before a rise in the stress until fracture, likely due to the alignment of the fibrils towards the force direction. Gly: ribose-induced glycation, AMG: aminoguanidine-mediated inhibition of glycation.
3.2 Qualitative and Quantitative Histological Analysis of Dentin Matrix Representative images of experimental groups are depicted in Fig. 6. Dentin sections from Gly and AMG group (Fig 6B and 6C) showed stronger red birefringence consistent with mature type I collagen in a thicker and densely packed collagen matrix. Quantitative analysis of staining (Fig. 7) shows no significant interaction between factors (condition vs. time; p = 0.359). However, at all incubation periods, red birefringence (mature collagen) was statistically higher for the Gly and AMG group when compared to the control group (p < 0.001), and no statistical differences were found between Gly and AMG (p = 0.938).
7 days
A
14 days
21 days
CONTROL
50 µm
B
GLY
C
AMG
Fig. 6. Representative images of the picrosirius red staining of crown dentin extracellular matrix from control (A), Gly (B) and AMG (C) groups at different time points. Dentin matrix from Gly group showed more densely packed mature collagen as it increased 1.5 to 2 times the red birefringence when compared to control. Scale bar corresponds to 50 µm. Gly: ribose-induced glycation, AMG: aminoguanidine-mediated inhibition of glycation.
7 days
14 days
21 days
Picrosirius Red Birefringence (%)
100 90 80 70 60 50 40 30 20 10 0 Control
Gly
AMG
Fig. 7. Results of percentage of mature collagen detected under red birefringence polarized microscopy of crown dentin sections stained with picrosirius red under experimental conditions and different time points. No interaction was found between conditions and cumulative times (p = 0.359). Statistical differences were observed within conditions (p < 0.001). Bar represents lack of statistical significant differences (p > 0.05). Gly: riboseinduced glycation, AMG: aminoguanidine-mediated inhibition of glycation.
3.3 Spectroscopic analysis of the dentin matrix The chemical characterization of the collagen maturity, post-translations modifications and pentosidine content are depicted in Fig. 8. Collagen maturity was similar among groups except for the AMG group, which was significantly lower in the root dentin ECM (p = 0.003). Glycation induction or inhibition did not affect the post-translational modifications of the dentin ECM (p = 0.330). Induced glycation increased the pentosidine content 1.44 to 3.5 times in crown and root dentin, respectively. However, the increase was only significant for the root ECM (p < 0.001). Aminoguanidine significantly decreased the pentosidine content regardless of the type of dentin (p < 0.001). The pentosidine content was significantly lower in root than in the crown dentin (p < 0.001), irrespective of the condition.
COLLAGEN MATURITY
B
POST-TRANSLATIONAL
C
PENTOSIDINE
MODIFICATION 1.4
1.4
1.2
1.2
1.2
1.0
1.0
1.0
0.8
*
0.6
A1362 /A1450
1.4
A873 /A917
A1656 /A1684
A
0.8 0.6
0.8 0.6
0.4
0.4
0.4
0.2
0.2
0.2
0.0
0.0
0.0
Crown
Root
Crown
Control
Root
Gly
* * Crown
* Root
AMG
Fig. 8. Raman spectroscopy analysis of the dentin matrix after 21-day incubation. Ratios were calculated with the integrated peak of designated Raman bands. Graphs exhibit ratios (mean and standard error) of collagen maturity (A), post-translational modifications of the extracellular matrix (B), and pentosidine content (C). The symbol (*) depicts statistically significant differences when compared to other groups (p < 0.05). Gly: ribose-induced glycation, AMG: aminoguanidine-mediated inhibition of glycation.
3
Discussion
This study investigated the effect of in vitro early glycation on the mechanical properties of crown and root dentin matrices; and also, the inhibitory effect of topical aminoguanidine. The findings revealed that ribose-induced glycation increased 23% the ultimate tensile strength and 31% the apparent modulus of the dentin ECM, indicating an early matrix stiffening mechanism. Therefore, the null hypothesis was rejected. The increase of densely packed collagen shown histologically, and the glycation products found in the ribose-glycated ECM through spectroscopy, pointed to non-enzymatic cross-linking of the collagen fibrils, supporting the efficacy of the proposed in vitro glycation model. Also, the expected browning of the dentin matrix, characteristic of the Maillard reaction [20], was observed on the glycated ECM. The low turnover of collagen and the enduring nature of non-enzymatic crosslinking lead to the progressive accumulation of AGEs in aged human tissues, contributing to its functional impairment [5,9–14,33]. Herein, glycated dentin ECM clearly showed a time-dependent
modification of the mechanical strength resulting in increased stiffness. Ribose-induced glycation forms both intra- and inter-fibrillar crosslinks along the collagen backbone, on the free ε-amino groups on the lysine side-chains or in the N-terminal amine group. Further reactions undergo to produce covalent intermolecular cross-links that ultimately constrain or suppress the fibril’s plasticity [10,33,34]. The increased stiffening revealed a key feature of glycated ECM expressed by the markedly low post-yielding plastic behavior, supporting findings that ribose-mediated crosslinking increase brittleness [5,17]. It was suggested that glycation takes effect at the surface of the fibril, based on the size of the reducing glycating sugars (< 1nm) [5,34]. Despite that, reports of AGEs-mediated changes at lower hierarchical levels (i.e. microfibrils and collagen molecules) contribute to the reduction in viscoelasticity [5,28,35]. Considering the hindering of intermolecular cross-links stretching, different mechanisms proposed involve the limitation in collagen fibril-fibril sliding [5] and the halted unfolding/unwinding of individual collagen molecules [11,33]. Thus, the capacity of deformation compensation from the collagen demonstrates its physiological function to increase toughness and reduce the risk of brittle fracture [11,33]. Although previous studies reported decreased toughness in glycated collagen [12,17,33], our results showed that the energy to fracture was not influenced by the ribose-induced glycation. One possible explanation is that the organization, density and distribution [36] of the collagen fibrils in the demineralized dentin, unlike tendon or bone, does not possess a preferred unidirectional orientation [11]. The lack of unidirectional orientation might grant dentin compensation for the glycation-induced stiffening by crack bridging [37,38]. Further, the amount of bound water in the matrix [39] and the absence of mineral phase might have outweighed the toughness of glycated ECM. Mostly ECM from the root exhibited higher tensile strength and energy to fracture when compared to the crown dentin matrix. In fact, root was differently affected by ribose than
crown, as energy and strength values after 21 days were similar to those at 7 days incubation. The density and cross-linking of the collagen, highly expressed in root dentin when compared to crown dentin [40–42], may partially explains variations here found between the crown and root dentin matrix. Interestingly, root dentin showed less pentosidine content than crown ECM (controls, Fig. 8), indicating a reverse relationship of pentosidine content and tensile strength of the untreated dentin ECM. While not investigated in this study, non-collagenous components such as proteoglycans could play a possible role as regulators of fibrillar spaces [21,24,25] and tissue mechanical viscous behavior [13,21]. Aminoguanidine, as an early glycation inhibitor, is known to trap reactive dicarbonyl groups from reducing sugars, avoiding their conversion to AGEs [2,43]. Thus, it would not have an effect on more advanced stages of glycation [2], as supported by the mechanical findings. Aminoguanidine exhibited a mild inhibitor effect (27.7 % decrease in UTS) within 7 days incubation, probably attributed to its short half-life [43]. Previous reports using aminoguanidine showed a partial reversal (from 19 to 55%) of the glycation effects of proteins at 0.01 M concentration [13,20,21,44]. Nonetheless, Raman spectroscopy showed that AMG significantly reduced pentosidine in both crown and root after 21 days. It also decreased the collagen maturity of root ECM, suggesting anti-aging characteristics [45]. The inhibitory properties of aminoguanidine may involve a specific complex mechanism in the collagen that does not reflect substantial mechanical improvement within the timeframe assessed in this study. Interestingly, picrosirius red (PSR) staining showed no difference in density and packing of collagen fibrils between Gly and AMG groups after 14 or 21 days, possibly due to the maturation of the Amadori products and glycation cross-links. Early phases of glycation consist of largely intrafibrillar cross-links along the helix of collagen molecules [46]. The fact that glycation occurs primarily on the fibril surface and thus at an
accessible location could have also important implication for therapeutic strategies based on enzymatic de-glycation [5]. More detailed knowledge of the complexities of AGE-mediated cross-links will aid in the understanding of the biomechanical functional consequences to the dentin and promote alternatives for glycation inhibition. Within the study limitations, an experimental model of early glycation of dentin matrices and testing method can prove valuable bulk mechanical properties. Since the effects of AGEs accumulation are time- and site-dependent, in vitro glycation should include time and dentin area as variables. The potential of topical inhibition of glycation using aminoguanidine was modest as it partially reduced the AGEs mechanical effect in the dentin matrix. However, it indicates that topical usage of chemical agents may be a strategy to prevent and reduce glycation of the dentin ECM. 4
Conclusions
The findings strongly suggest that in vitro ribose-induced early glycation of the dentin matrix reduced tissue viscoelasticity and increased tensile strength by 23%, revealing an abnormal stiffening mechanism. In addition, results were time-dependent, indicating the accumulation of glycation products in the dentin matrix. The regional dentin sites were differently affected by induced-glycation, where crown dentin matrix was mechanically more affected than root dentin. Glycation of dentin matrix can be partially inhibited by topical exposure to aminoguanidine.
Acknowledgements The authors gratefully acknowledge UIC Health Navigators Summer research program. We thank Yasmin Bakir for contributing to the image analysis of the histology experiments. We greatly appreciate Dr. Mariana Cavalcante dos Reis for the technical assistance with Raman
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Highlights -
Glycation of highly cross-linked dentin extracellular matrix results in an immediate brittle-like biomechanical behavior.
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Distinct regional effects indicates higher susceptibility to glycation of crown extracellular matrix when compared to root dentin.
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Damage to the biomechanics of dentin matrices is cumulative, and only partially inhibited by topical exposure to aminoguanidine.