In this study, a macro-scale bone-tooth organ from a human was discretized to millimeter sized blocks to evaluate the site-specific properties of a narrow 5-50 μm bone-PDL-cementum complex. The site-specific adaptation was identified by mapping changes in physicochemical properties at the PDL-bone and PDL-cementum entheses and respective tissues. The properties were correlated to plausible root association with the bony socket as a result of functional loads. Ultimately, overall functional adaptation resulting in function efficiency of an organ can be understood by identifying changes in range of motion of the joint [32
] and in this case the tooth within the bony socket.
The functional efficiency of the bone-tooth fibrous joint lies not only in the tissues of the tooth or the binding interfaces (dentin-enamel junction, cementum-dentin junction, bone-PDL or cementum-PDL), but also in the continuum with which all the tooth-related tissues interface with the vascularized bone and the vascularized/innervated PDL. As a result, functional efficiency of an organ can be impaired if any of the above mentioned sites or tissues adapt significantly. Parameters indicative of a loss in functional efficiency include a significant change in range of joint motion, and/or altered tissues mechanics i.e. tissue “quality”. Based on results from this study, another parameter would be a “compromised interface”, which is defined as a significant deviation from the original graded characteristics of a FGI. Naturally graded interface due to functional demands can deviate toward a discontinuous interface, eventually decreasing the functional efficiency of a dynamic joint. It is this characteristic feature that will be discussed in this study.
In musculoskeletal and dental orthopedics, macro-scale form-function behavior/adaptation at the organ level provides an insight to local behavior at the tissue level and resulting cellular responses and subsequent genetic and molecular expressions [33
]. These hierarchical length-scale events provide a continuum-like effect to address functional homeostasis. During function, at an organ level, joints facilitate relative motions between members through a combination of soft and hard tissues and their interfaces. Tissues continue to adapt locally over time via principles of mechanobiology and mechanotransduction [34
] under normal functional loads. However, significant deviation from functional loads in the form of magnitude and/or frequency, eccentric loads, which individually or combined can impair range of joint motion, i.e. a loss in overall functional efficiency of a joint.
A normal bone-tooth functional joint has a PDL-space ranging from 150-380 μm [20
]. The lower value of 150 μm corresponds to a uniform PDL-space with a decrease in functional space as age increases [20
]. However, apparently normally functioning teeth can also have localized regions of narrower PDL-space (). In this study such narrowed spaces were identified and their structure and properties were evaluated. Such specimens with narrow PDL-spaces of 5-50 μm had narrowing induced in two ways: 1) apposition of long stratified layers of bone with rich basophilic lines, 2) bony protrusions (). It is conceivable that as the bone grows more into the PDL-space (), tooth mobility in the bony socket is minimized because of the physical hindrance, thus compromising range of motion and reducing joint efficiency. These initial observations using a fundamental technique of light microscopy coupled with conventional histology raised subsequent questions. Is PDL attachment at the bone and cementum entheses similar to normal conditions i.e. 150-380 μm PDL-space? What is the chemical composition at the constricted sites? What is the elastic modulus of respective tissues and entheses at the constriction site? What are the biomechanical reasons that generated the observed constriction sites in certain regions of a bone-tooth fibrous joint extracted from a healthy human? In order to address these questions we furthered our investigation of the narrow PDL-complex using immunohistochemistry, site-specific higher resolution imaging for structural and chemical analyses, and wet nanoindentation techniques.
Based on the seminal and fundamental concept by Julius Wolff, the long-term effect of functional aberrations at a macro-scale could result in pathological deformations to maintain function [37
]. Over time pathological deformations of mineralized tissues change the internal architecture, but will continue to function [37
]. These spatiotemporal changes in physical and chemical properties of soft and mineralized tissues including their interfaces result in an overall change in form-function relationship, commonly termed as functional or biomechanical adaptation [9
]. In the bone-tooth fibrous joint, eccentric loads such as those imposed by parafunctional habits with varying magnitudes and frequencies [40
] can cause functional adaptation. Other significant load-induced perturbations include traumatic and therapeutic loads from orthodontic braces [24
] all of which can change the form-function relationship and decreased functional efficiency of the bone-tooth fibrous joint. Clinically, the narrowed PDL-space can be considered asymptomatic, but when superimposed with other clinical interventions can cause failure of the bone-tooth complex.
Analogous interfaces within the musculoskeletal system include bone-ligament, muscle-tendon, bone-cartilage. Interestingly in all these systems, adaptation in the form of movement of mineralized fronts into softer tissues due to cyclic loads at higher than normal functional loads have been observed. Repeated loading of the interfaces caused loss of joint mobility [7
]. On the other hand, lack of loading (disuse) caused loss in skeletal bone density [18
], or in case of cranial bone, premature fusion due to bone growth into softer tissue when the sutures (another representative fibrous joint) were not exercised [43
]. From these long established arguments, it is conceivable that every joint has its own functional limits and when exercised above or below its functional threshold limits for a prolonged time results in adaptive changes leading to decreased functional efficiency. Based on the results from this study, we propose that “higher strains at the soft-hard tissue interfaces act as sites of altered mechanotransduction due to activation of various mechanoreceptors between matrix to cells, and cell to cell. The bone-PDL and cementum-PDL entheses are “regulators
” of cellular and extracellular temporal events; earlier events result in strain-induced local cell gene expression and later events that result in matrix biochemistry and physico-chemical properties”. This fundamental mechanism could explain tissue, interface development, regeneration, and/or adaptation in various biomechanically active organs.
Physiological remodeling and load-mediated modeling resulting adapted alveolar bone were identified as necessary anabolic and catabolic events [18
] to address functional demands on a tooth-bone fibrous joint. For example, resorption of alveolar bone occurs when left unloaded [46
]. Results of this study indicate adaptive form of bone and PDL with no detectable physical effects in cementum. Adaptive form of bone interfacing with PDL is termed bundle bone. Bundle bone is identified by dominant radial fibers with a woven fabric-like structure compared to the commonly known lamellar bone. Bundle bone, a term normally used in oral histology refers to 1-2 μm wide (mineralized) radial inserts of collagen fiber bundles from the PDL that forms a strong interface with lamellar bone (). Interestingly, natural mesial drift of teeth throughout the life span of a human mediated through functional loads can also cause bone modeling, resulting in bundle bone, but an uniform PDL-space [48
]. Hence, the observed bundle bone could be due to abnormal loads (nonphysiological loads) resulting in a non uniform PDL-space. This observation in the bone-tooth complex parallels a distinctive pattern of bone formation causing enthesophytes in osteotendinous and osteoligamentous regions, a pathological condition in the diarthroidal joints of the human musculoskeletal system [39
]. Pathology in these studies was described as non-physiological mineralization and advancement of mineral fronts into softer tissue impeding range of joint motion [39
]. Other, identifiable load-mediated changes within tissues include altered concentration of polyanionic molecules i.e. proteoglycan (PG) content which in turn could regulate mineral formation. In diarthroidal joints the relatively large range of motion is also impeded due to cartilage mineralization as a result of significant changes in PG concentration [50
PG concentration controls many aspects of extracellular matrix properties including tissue structure, mineralization, and mechanical integrity resulting in load resisting and dampening properties of tissues. Under healthy conditions PGs along with collagen are promoters of functional mineralization which maintain the overall load bearing characteristics of joints. Changes in load can cause changes in glycosaminoglycans and respective tissue hydration characteristics resulting in altered load bearing conditions. Although several small and higher molecular weight PGs have been implicated toward tissue quality and joint efficiency, asporin was not correlated with loads. However, its existence in several load bearing tissues, such as cartilage and PDL was reported [50
]. More importantly and quite relevant to this study is that this PDL-specific SLRP prevents non-physiological mineralization [50
] and allows maintenance of the PDL-space, was also observed in the narrowed PDL-space (). Patchy localization of asporin compared to an uniform distribution in normal 150-380 μm wide PDL-complex [16
] was observed with no evidence of a mineralized PDL or mineral nodules within the PDL. Moreover, the narrowed PDL retained its organic phenotype by consistent demonstration of characteristic collagen periodicity ( and ). Though not shown, we also observed several other SLRPs within the PDL-complex including biglycan, decorin and fibromodulin. These consistently observed proteoglycans could be responsible for the hygroscopic nature of the PDL-inserts within bone and cementum () and maintenance of the PDL-space. This is complemented by the lower X-ray attenuating PDL-inserts demonstrating lack of mineral as seen at a higher magnification using Micro XCT™ (). It should also be noted, that the narrowed PDL between the two mineralized tissues demonstrates minimal hygroscopicity owing to higher packing density of the collagen fibers and not necessarily higher mineral content (). Along with the aforementioned proteoglycans [53
], tenascin and fibronectin have been found by others [54
] at the entheses, and it was suggested that they accommodate mechanical loads at the soft-hard tissue interfaces. We propose that the combination of local tensile strain along with polyanionic constituents could promote crystal growth and precipitation within or between collagenous structures of the PDL, more commonly known as intra- or extra-fibrillar mineral related to collagen fibrils [55
]. That is, the mineral growth process is controlled by organic cues that could change in molecular weight, molecular confirmation, and concentration with time so that the inorganic phase over prolonged time is directed along the mechanically strained fibers of the PDL. Supporting this argument is the presence of biglycan at the entheses and within PDL which continues to be reported as a PG with an osteogenic potential [57
]. The presence of biglycan predominantly at the entheses along with the patchy appearance of asporin within the PDL-space could be the necessary chemical cues to make the locally stretched fibers more osteogenic in nature promoting a controlled and time-dependent vectorial bone growth [58
]. The microscopic area over which the tensile loads occur at the bone-PDL tethered ends seen as scallops (white arrow heads, ) could either promote bone growth 1) over a macroscopic surface resulting in higher ordered structures seen as stratified bone with basophilic lines (black arrows, ), or 2) if load acted over a shorter surface area could promote bone growth seen as protrusion (star burst , ). This argument parallels the proposed theory in our previous work [16
] that states that pulling forces exist at the tethered ends of the PDL that can induce biological activity promoting biomineralization. These mechanically prompted biomineralization events could cause increased levels of Ca and P in narrowed PDL-complex, compared to similar amounts in a normal 150-380 μm PDL-complex (). However, several studies related to type of mineral, the stochiometry of the developed crystal, crystal size and texture within the strained organic matrix should be identified to better elucidate events regulating site-specific biomineralization. The combined effects of physically narrowed 5-50 μm PDL and increased Ca and P elements indicate reduced mechanical damping of PDL to functional loads. It is only conceivable that the observed physical and chemical changes in PDL and bundle bone can significantly alter the matrix stiffness from gradual varying to an abrupt change i.e. a discontinuity as seen in this study (). Despite the presence of heterogeneous adaptive bone attached to commonly known lamellar bone (), and the subsequent changes in the stiffness gradients, the question that still remains to be answered is, what are the biomechanical reasons that generated the observed constriction sites in certain regions of a bone-tooth fibrous joint extracted from a healthy human ()?
Figure 8 a) Schematic of a healthy bone-tooth complex illustrating a normal PDL-space. b-d) Schematics provide plausible scenarios that can be activated locally due to macroscale loads eventually narrowing PDL-space. Macroscale functional loads can cause the tooth (more ...)
It is known that functional loads on a tooth can cause tilting of the root in the bony socket [17
] and a lateral displacement of the root toward bone (). Virtual dissection of higher resolution Micro XCT™ illustrated reorganized fibrillar structures of the PDL (), complementing light microscopy (, ), AFM () and SEM () micrographs. However, the radial integration of the narrowed PDL with cementum and bone was similar to that observed in normal 150-380 μm wide bone-PDL-cementum complex [16
]. Although the narrowed space illustrated pure circumferential-PDL using AFM (), radial fibrils (out of plane) from the PDL-space were also identified using SEM micrographs (). In the absence of the adaptive form of bone, i.e. bundle bone, the SEM results could support the theory that the PDL was physically constricted by lateral displacement of the root into the PDL-space. Under such circumstances calcification of degenerating PDL was observed in in vivo
] and schematically shown as one of the plausible biomechanical reasons to cause narrowed PDL-space (). However, AFM and SEM results illustrated intact fibrils with characteristic periodicity ( and ) demonstrating no mineralization, thus maintaining its organic phenotype despite the constriction. Hence, the observations in this study can also be a result of lateral translation due to shearing of the root against bone, promoting a combination of tensile and compressive strains at the entheses and within the PDL.
Based on fundamental biomechanics, the tooth is subjected to a variety of loads and when superimposed with higher compressive loads, the PDL tissue can undergo increasing shear (), a combination of tension and compression, supplemented by flexural moments at the tethered ends of the ligament i.e. the radial PDL-inserts with bone and cementum (). At the soft-hard tissue attachment sites it is known that the presence of hydrophilic molecules including various types of proteoglycans, helps modulate cell migration and adhesion, tissue/interface biomineralization, and other biochemical processes responsible for continuous remodeling of the mechanically strained PDL-cementum and PDL-bone attachment sites [53
] making them into micro-scale dynamic sites i.e. “local regulators” () eventually causing waves of mineral fronts toward PDL-space (). Therefore, any significant variation in macro-scale biomechanical load can alter the local strain levels in the PDL and at the PDL-bone and PDL-cementum attachment sites. Altered strains could lead to an altered mechanotransduction through integrin based cell-matrix response or cell to cell response identified through cell gene expressions causing detrimental downstream effects [25
]. These cellular expressions at the strained attachment sites during function could manifest into local changes in biochemical composition permitting mineral nucleation subsequently resulting in higher concentrations of Ca, and P at the bone-PDL and cementum-PDL attachment sites (). These higher concentrations along with structural changes in the PDL-space are indicative of the steeper gradients in modulus observed at the bone-PDL and cementum-PDL attachment sites and can be used as indicators of attachment site/enthesis adaptation to macro-scale loading. Hence, it could be that the observed bone growth in this study is due to significantly altered mechanotransduction at the highly strained dynamic sites relative to adequately strained attachment sites within the same periodontal complex. These significant changes in PDL-space and shape of the outer surface of the bony socket can be identified as necessary modeling to address functional demands.
Following the primary event of bone ingrowth is the secondary event causing function impairment. With the narrowing of the PDL-space, the strain amplification at the PDL-bone and PDL-cementum could be minimized; however, a discontinuity in modulus profile between the tooth and bone now would permit prolonged compromised mechanotransduction. The compromised mechanotransduction will continue upon further loading, as the constriction sites will become the new “load bearing sites” that eventually cause direct local fusion of bone with cementum.