Multiscale biomechanical responses of adapted bone-periodontal ligament-tooth fibrous joints

Abstract

Reduced functional loads cause adaptations in organs. In this study, temporal adaptations of bone-ligament-tooth fibrous joints to reduced functional loads were mapped using a holistic approach. Systematic studies were performed to evaluate organ-level and tissue-level adaptations in specimens harvested periodically from rats (N=60) given powder food for 6 months over 8,12,16,20, and 24 weeks. Bone-periodontal ligament (PDL)-tooth fibrous joint adaptation was evaluated by comparing changes in joint stiffness with changes in functional space between the tooth and alveolar bony socket. Adaptations in tissues included mapping changes in the PDL and bone architecture as observed from collagen birefringence, bone hardness and volume fraction in rats fed soft foods (soft diet, SD) compared to those fed hard pellets as a routine diet (hard diet, HD). In situ biomechanical testing on harvested fibrous joints revealed increased stiffness in SD groups (SD:239-605 N/mm) (p<0.05) at 8 and 12 weeks. Increased joint stiffness in early development phase was due to decreased functional space (at 8 weeks change in functional space was -33 μm, at 12 weeks change in functional space was -30 μm) and shifts in tissue quality as highlighted by birefringence, architecture and hardness. These physical changes were not observed in joints that were well into function, that is, in rodents older than 12 weeks of age. Significant adaptations in older groups were highlighted by shifts in bone growth (bone volume fraction 24 weeks: Δ-0.06) and bone hardness (8 weeks: Δ-0.04 GPa, 16 weeks: Δ-0.07 GPa, 24 weeks: Δ-0.06 GPa). The response rate (N/s) of joints to mechanical loads decreased in SD groups. Results from the study showed that joint adaptation depended on age. The initial form-related adaptation (observed change in functional space) can challenge strain-adaptive nature of tissues to meet functional demands with increasing age into adulthood. The coupled effect between functional space in the bone-PDL-tooth complex and strain-adaptive nature of tissues is necessary to accommodate functional demands, and is temporally sensitive despite joint malfunction. From an applied science perspective, we propose that adaptations are registered as functional history in tissues and joints.

Keywords: Biomechanics; Bone–PDL–tooth fibrous joint; Multiscale; Periodontal ligament; Rat; Stiffness.

Figure 1. In situ loading device coupled to a XRM system to image tooth motion relative to the alveolar socket

a) Hemimandibles were prepared with composite buildups for mechanical testing and were placed in the load cell as shown. b) To ensure a parallel surface between the anvil and the composite buildup, occlusal marking paper was used (not shown) to mark uniform area of contact. c) The output of load displacement curve was analyzed by comparing the change in load (Δ Load) vs. the change in displacement (Δ Disp). d) 2D virtual section relating tooth to the alveolar socket. Please note that 1d is a GIF file and should be viewed under slide show.

Figure 2. Stiffness values for fibrous joints obtained from those fed hard and soft diets respectively

a) The nonlinear plot illustrate joint stiffness values from all age groups when individually tested at different speeds (0.2, 0.5, 1.0, 1.5, 2.0 mm/min) and when loaded to 5 N (triangles – open and filled) and 15 N (squares – open and filled) respectively. Data for 6, 8, and 10 N are not shown but showed similar trends. The graph illustrates an increased range in displacement of the teeth from the HD group compared to displacement of teeth from SD group. 3D surface plots illustrate different spread of stiffness values with age (wks) and displacement (mm) when tested for peak loads of 5, 6, 8, 10, and 15 N in (b) HD groups and (c) SD groups. d) Statistical differences between stiffness values calculated from load-displacement relationships were analyzed using a mixed effects regression model and plotted as a function of age of the mammal. From the resulting 95% confidence intervals (blue – upper bound, green – lower bound, red – zero plane) the notable difference is that the joints from younger animals (8 and 12 week) in soft diet group were significantly stiffer than the joints from hard diet group under hard diet load-simulation conditions (higher load, lower displacement rate).

Figure 3. XRM tomography measurements of interradicular PDL-space

a) Representative tomogram illustrating a bone--PDL-tooth complex. Functional space was measured within the interradicular region (white dotted box). b) At younger groups, a narrower interradicular space was observed in soft diet groups compared with hard diet groups. However, with an increase in age, the difference between hard and soft diet groups became less significant.

Figure 4. Collagen birefringence and PDL directionality across groups and age

a) Histology sections were stained with picrosirius red. b) Imaging was primarily focused around the furcation and root-PDL (R-PDL) surrounding the distal root of the 2^nd molar. c) Birefringence signal was measured in the following regions: interradicular bone (IR-B), interradicular periodontal ligament (IR-PDL), and R-PDL. IR-PDL revealed a sponge-like configuration while R-PDL illustrated an increased collagen fiber orientation. Under polarized light, collagen orientation was quantified within IR-PDL, IR-B, and R-PDL. White bars are 100 µms. Trends show increased birefringence as a function of age within PDL regions and a decrease in birefringence as a function of age within interradicular bone. Statistical significance in birefringence between SD and HD is indicated with an (*) (p<0.05) and was only observed in the PDL of 8 week old. Graphical representation of averages and standard deviations show no age-related trends for both hard and soft diet groups. (d) Images using polarized light microscopy show regions of distal (yellow box) and mesial (red box) regions used to calculate fiber orientation. Representative histogram distributions of fiber orientation in (e) hard diet and (f) soft diet are shown for 8, 16, and 24 weeks with total group average and standard deviations are shown. g) As a function of age trends included a decreasing difference in distal- and mesial-PDL orientation within and across hard and soft diet groups with age.

Figure 5. Microindentation and interradicular functional space of bone-PDL-tooth complexes

Microindentation was performed on cementum, interdental bone and interradicular bone. In general, interradicular bone was always harder than interdental bone which was always harder than cementum. While age-related trends were not found, hard diet groups illustrated an increased interradicular bone hardness compared to bone from rats on a softer diet. Additionally, interradicular functional space was measured by taking standard virtual sections between the distal and mesial buccal roots. Results show that interradicular space decreased in younger rats fed softer diet. *indicates statistical significant difference (p<0.05).

Figure 6. Comparison of bone volume fraction (BVF) of interradicular alveolar bone by using XRM tomography

(a) Digitally reconstructed structure of tooth in the alveolar socket illustrate the volume of interest in alveolar bone. (b) Bone volume fraction is illustrated as the ratio between bone volume to total volume (bone volume + endosteal volume (c)). (c) Endosteal volume decreased with age in both groups. However, greater endosteal volume in interradicular bone from those fed softer diet were observed. (d) BVF of HD was significantly higher than that of SD at 24 weeks.

Figure 7. Comparison of reactionary responses of joints to loads

a) Higher reactionary response to load for HD older group was observed. b) Although similar trends were seen to a certain extent, the obvious trend was that the younger group fed SD demonstrated a higher reactionary response compared to its HD counterpart. Similar trend was not observed with an increase in age.

Figure 8. Postulated load-mediated adaptation of the dentoalveolar complex

a) Experimental workflow showing different food consistencies given to control (HD) and SD groups and changes in observed biological responses. (b) The fibrous joint compensates through decreased PDL-space through ingrowth of cementum and decreased orientation and organization of the collagen fibers. The shift in input signal prompts a shift in adaptive properties of tissues as indicated by site-specific measurements. However, if normal functional loads are placed on adapted tissues belonging to the SD diet group, they would likely be seen as traumatic loads prompting a damage response (yellow start bursts). (c) Shifts in outcomes due to load-mediated adaptations can be divided into two phases; early ages focused around the tissues within the PDL space, and shifts seen at later ages primarily as changes in bone mineral content and internal structure. (d) Normally chewing loads are accommodated by the dentoalveolar complex through compression of supporting tissues (PDL, bone, cementum) causing physiological strain due to tissue deformation (mixed-mode) and fluid flow from interstitial fluid movement. Mechano-responsiveness of localized cells in the regions of higher fluid flux and mechanical strain regulate a shift in tissue turnover in bone and the PDL morphology and material properties accordingly. A reduction of functional loads net a decrease in strain and is felt by the supporting tissue away from the physiological strain. Within bone, atrophy of tissue is usually seen, however, the opposite effect (growth) has been reported within cementum tissues.