Scanning mode nanoDMA was used to evaluate the dynamic mechanical properties of intertubular and peritubular dentin.
The intertubular dentin from the old donor teeth exhibited higher storage and complex moduli than those of young donor teeth.
The root canal treated teeth exhibited higher storage.
Complex moduli and lower capacity for viscous deformation than that of the vital matched pairs.
Dentin undergoes irreversible changes in microstructure with aging that involve gradual filling of the tubule lumens with mineral. Known as dental sclerosis, this process begins at the root apex, progresses coronally, and is associated with a degradation in the resistance to fracture of dentin.
To determine i) age-related changes of intertubular dentin with aging, particularly within the root, and ii) the differences in age-related degradation between vital and pulpless (i.e. non-vital) teeth.
We performed nanoscopic dynamic mechanical analysis (nanoDMA) in scanning mode on the intertubular and peritubular dentin of teeth from young and old adults. The complex, loss and storage moduli, as well as the tan delta parameter were evaluated for teeth with no restorations and teeth with root canal treatment (non-vital).
There were significant changes in the dynamic moduli of intertubular dentin with age, which were most substantial in the apical third of the root. The storage modulus of the intertubular dentin, which quantifies the purely elastic resistance to deformation, was significantly (p < 0.0005) larger for both the old vital and non-vital teeth than that of the young teeth, over the entire root length. However, the tan delta parameter, which quantifies the relative capacity for viscous deformation, was significantly lower in these two groups (p < 0.005).
Radicular dentin undergoes an embrittlement with aging, involving reduced capacity for viscous deformation. The extent of degradation is largest in the apical third. Removal of the pulp appears to accelerate the aging process or compound the extent of degradation.
An increase in life expectancy coupled with rapid advancements in oral healthcare have led to an increase in the population of partially and fully dentate seniors over past decades [ ]. As a consequence, there will be growth in the population of seniors seeking dental care. The increase in dentate seniors has brought new challenges to the profession. One such concern is cracked teeth and other forms of mechanical degradation [ , ]. While tooth fractures are routinely encountered in dental practice irrespective of patient age [ ], vertical root fractures most commonly occur in patients 40 to 60 years old [ ]. These types of failures cannot often be repaired successfully by root canal treatment (RCT) and necessitate tooth extraction [ ].
Tooth fractures that initiate within the root, or that extend from the line angles within the crown typically involve dentin. Dentin is highly mineralized tissue consisting of approximately 45 % mineral by volume [ ] and serves as a foundation for the enamel. It is traversed by a network of microscopic channels (i.e. tubules) that extend radially from the pulp towards the dentin-enamel junction (DEJ) and cementum. The tubule lumens are occupied by dentin fluid and odontoblastic extensions. They are bounded by a highly mineralized cuff of peritubular dentin that consists primarily of apatite crystals. The region between the tubules is occupied by the intertubular dentin, which consists of a mesh of collagen fibrils that is reinforced by inter- and extra-fibrillar apatite crystallites.
The microstructure of dentin in adults is dynamic. It undergoes a gradual transition in translucency with increasing age that results from filling of the dentin tubules with mineral, a process regarded as sclerosis [ ]. Dentin sclerosis occurs first and is most severe in tissue near the root apex [ ], which could be attributed to the tubule morphology in the root. The dentin tubules have a smaller lumen diameter and density in the root in relation to the crown [ ].
Although the mechanisms of dentin sclerosis are unclear [ , ], the consequences are far more well known. In particular, the age-related changes in microstructure of dentin reduce the damage tolerance of the tooth [ ]. Previous studies that addressed aging and the mechanical behavior of dentin have shown that the crown [ ] and root [ ] undergo reductions in strength. Furthermore, the fatigue strength [ , ], fracture toughness [ , ] and fatigue crack growth resistance [ , ] also undergo a significant reduction with age. However, there are two key limitations of prior studies on this topic, namely: i) most work has concentrated on the coronal dentin, and ii) investigations of the root have not considered spatial variations. Furthermore, the degradation in damage tolerance of dentin with aging has largely been attributed to what is most noticeable, i.e. the mineral that fills the dentin tubules. In comparison, far less effort has focused on the changes to the intertubular dentin.
Natural physiological aging is not the only concern in root fractures. Teeth with non-vital pulp and those that have had the pulp removed during root canal treatment (RCT) are more susceptible to fracture than vital teeth [ ]. Vertical root fracture (VRF) occurs more frequently in teeth with prior RCT [ ]. According to the American Association of Endodontists, a VRF involves cracks that initiate in the apical region [ ] and extend coronally ( Fig. 1 A). There is some belief that VRFs initiate from internal dentin cracks [ ] that have resulted from the RCT. Indeed, dentin defects introduced during the endodontic procedures could be a contributing factor [ ]. However, previous studies have also been reported that no defects could be identified within the dentin of teeth subjected to RCT and/or their contribution to VRF is unlikely [ , ]. Clearly the cause of VRF and how RCT contributes to the process of failure is controversial and remains unclear [ ].
Removal of the pulp during RCT is associated with irreversible change to the dentin structure [ ]. In a comparison of the strength of root dentin between teeth with age- and donor-matched controls, those with prior RCT exhibited 30% lower strength [ ]. Yet, the progression of aging in non-vital teeth, specifically those that have received RCT followed by post-treatment function, and the spatial variations in degradation of the root are not understood. Root fractures in the teeth of seniors with RCT could be attributed to an acute form of degradation in mechanical properties at the apex and could manifest in the intertubular or peritubular components of dentin. Understanding this phenomenon is the first step towards the development of treatment modalities, or the development of dental materials, that account for the unique property variations.
Indentation methods have been adopted to characterize the changes in mechanical behavior of dentin with aging and sclerosis [ , ]. Macroscopic indentation methods are not able to discern the properties of distinct aspects of the microstructure or interfaces [ ], which is highly relevant to an evaluation of dentin. As such, nanoindentation has become a standard method for measuring the mechanical behavior of biological hard tissues at the microscopic scale and also enables an assessment of the viscous behavior. Nanoindentation or atomic-force microscopy is required to measure discrete changes in the intertubular and peritubular dentin [ ], which is relevant to the present investigation.
Nanoscopic Dynamic Mechanical Analysis (NanoDMA) is a special form of nanoindentation-based structural analysis. Scanning nanoDMA, performed using scanning probe microscopy, maintains the indentation stress within the elastic range, which is preferred for highly sensitive analyses. Recent applications include evaluations of dentin bonding [ ] and aging [ ]. It has also been applied in investigations related to remineralization of dentin [ ] and to assess the effects of collagen crosslinking [ , ]. However, no investigation has been reported on the use of nanoDMA mapping within tooth roots to delineate the unique age-related changes of the intertubular and peritubular dentin or the differences between vital and non-vital teeth.
In the present study, scanning mode nanoDMA was used to characterize the dynamic mechanical behavior of dentin in the roots of teeth with regards to donor age, pulp vitality and histological location. Two null hypotheses were defined: i) there is no significant difference in the mechanical behavior of intertubular dentin along the length of the root with regards to donor age, and ii) there is no significant difference in the properties of intertubular dentin between vital teeth and those with prior RCT.
Materials and methods
Human single-rooted non-carious teeth were obtained from participating clinics with an exempt protocol approved by the Institutional Review Board of the University of Washington. Single-rooted premolars were chosen for their simple physiology in comparison to molars with multiple roots. The teeth were stored in Hank’s balanced salt solution (HBSS) with record of donor age and gender. Those teeth with visible caries or structural defects were discarded. A total of 12 teeth from 8 patients were selected and divided into young (n = 4, age <25), old (n = 4, age >60) and old non-vital (n = 4, age >60) groups. The four teeth in the old and old non-vital groups were matched pairs of teeth. Each pair consisted of teeth from mirrored locations of the arch from the same donor that included a vital tooth and non-vital tooth with prior RCT. Obtaining matched pairs of teeth in mirrored locations of the arch is very difficult, but very beneficial, since it reduces the variations in tooth properties across donors.
The teeth were cast in a polyester resin foundation within two weeks of receipt and sectioned axially in the mesial-distal (M–D) direction using a precision slicing/grinding machine according to established methods [ ]. The resulting halves were embedded in cold-cured epoxy resin (Epofix HQ Resin and Hardener, Struers) exposing the root canal and longitudinal section. The exposed dentin sections were polished using silicon carbide abrasive paper from #800 to #4000 mesh with water irrigation until halfway through the thickness of dentin and the dentin tubules became evident. Further polishing was performed with 3 μm diamond particle suspensions and 0.04 μm colloidal alumina suspension. The specimens were subjected to 20 minutes sonication to remove residual debris inside the dentin tubules. All of the aforementioned procedures were conducted with the tooth maintained fully hydrated in HBSS.
A dynamic mechanical analysis (DMA) of the polished sections of root dentin was performed using Scanning Probe Microscopy (SPM) on a commercial system for nanoindentation (Hysitron Inc., Model TI980 Triboindenter, Minneapolis, MN). The scanning-based evaluations were performed using a Berkovich diamond indenter with 90 nm nominal tip radius, which was measured in scanning mode with quartz standard according to [ ]. Prior to nanoDMA on teeth, a frequency sweep was performed on fused quartz from 100 Hz to 300 Hz to identify resonance components related to the machine operation. Based on the results of this process a scanning frequency of 200 Hz was used to maximize the signal to noise ratio in the evaluations of dentin. A 4 μN static indentation load and a dynamic sinusoidal load of 2 μN were applied following Ryou et al. [ ] The scanning mode nanoDMA was conducted using a window of evaluation of 20 μm × 20 μm, which constitutes an area involving 5–10 dentin tubules as shown in Fig. 2 A . The instrument performs the evaluation through 256 horizontal scans and with pixel density of 256 × 256. Properties of the intertubular and peritubular dentin were quantitatively evaluated within the apical, middle and coronal thirds ( Fig. 2 B). In each region, areas of interest within the window of evaluation were selected that corresponded to either the intertubular or peritubular dentin. Three independent nanoDMA scans were conducted in each region of each tooth to ensure that a statistical representative assessment was achieved. Then the average and standard deviation of the desired properties were determined from the scan data within that domain of relevance.
To minimize the effects of dehydration to the mechanical behavior [ ], the nanoDMA evaluations were performed in the hydrated condition with treatment of 99.4% ethylene glycol (EG) [ ]. Briefly, a thin layer of EG was smeared on the sample surface after removal from HBSS bath to prevent the evaporation of water during scanning. Previous results have shown that there is no influence of EG on the moduli estimated using scanning-based DMA [ ] and that this approach can maintain the hydration of the tissue for over an hour. Since the individual scans were completed with a period of less than 30 min, the process ensured that the properties were evaluated in the fully hydrated condition.
Within each region of evaluation, the storage ( E’ ) and loss ( E″ ) moduli were obtained from the nanoDMA scans, which represent the elastic behavior and dampening capacity of the material, respectively. These two parameters were used in estimating the complex modulus ( <SPAN role=presentation tabIndex=0 id=MathJax-Element-1-Frame class=MathJax style="POSITION: relative" data-mathml='E*’>?*E*
) according to <SPAN role=presentation tabIndex=0 id=MathJax-Element-2-Frame class=MathJax style="POSITION: relative" data-mathml="E*=(E’2+(E")2)1/2″>?*=((?‘)2+(?“)2)1/2E*=(E’2+(E”)2)1/2
E * = ( E ‘ 2 + ( E " ) 2 ) 1 / 2
, which represents a measure of the combined influence of the storage and loss behavior. The ratio of the loss and storage moduli were used to obtain the tan delta parameter, which provides a relative measure of the viscous response of the tissue that is independent of tip geometry. A statistical analysis of the parameters was performed using a two-way Analysis of Variance (ANOVA) test with significant differences identified at α = 0.05. The normality of the data was checked before performing the statistical analysis.
Representative property maps obtained for the apical dentin of a tooth root from the young group of donors are shown in Fig. 3 . Specifically, the surface topography and mechanical properties distributions representing the complex, storage and loss moduli, as well as the tan delta distribution are presented in this figure as denoted. These maps were obtained over a 20 μm × 20 μm window of evaluation. The dentin tubules and peritubular cuffs are clearly evident in the topography and mechanical property maps, which enabled precise identification of the regions corresponding to the intertubular and peritubular dentin. The center of the tubule lumens exhibit property values that are unrealistic due to boundary effects between the tip and open lumens. Such areas are artifacts of the indentation method and were excluded in the quantitative analysis.