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UC San Francisco Previously Published Works
Title The plastic nature of the human bone-periodontal ligament-tooth fibrous joint.
Permalink https://escholarship.org/uc/item/3sf0s7mk
Journal Bone, 57(2)
ISSN 8756-3282
Authors Ho, Sunita P Kurylo, Michael P Grandfield, Kathryn et al.
Publication Date 2013-12-01
DOI 10.1016/j.bone.2013.09.007
Peer reviewed

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Bone. Author manuscript; available in PMC 2014 December 01. Published in final edited form as:
Bone. 2013 December ; 57(2): 455–467. doi:10.1016/j.bone.2013.09.007.
The Plastic Nature of the Human Bone-Periodontal LigamentTooth Fibrous Joint
Sunita P. Ho*, Michael P. Kurylo#, Kathryn Grandfield#, Jonathan Hurng, Ralf-Peter Herber1, Mark I. Ryder2, Virginia Altoe3, Shaul Aloni3, Jian Q. (Jerry) Feng4, Samuel Webb5, Grayson W. Marshall, Donald Curtis, Joy C. Andrews5, and Piero Pianetta5 Division of Biomaterials and Bioengineering, Department of Preventive and Restorative Dental Sciences, University of California San Francisco, San Francisco, CA
1Divisions of Orthodontics, Department of Orofacial Sciences, University of California San Francisco, San Francisco, CA
2Division of Periodontics, Department of Orofacial Sciences, University of California San Francisco, San Francisco, CA
3Materials Science Division, The Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, CA
4Biomedical Sciences, Baylor College of Dentistry, Texas A&M, Dallas, TX
5Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Menlo Park, CA
Abstract
This study investigates bony protrusions within a narrowed periodontal ligament space (PDLspace) of a human bone-PDL-tooth fibrous joint by mapping structural, biochemical, and mechanical heterogeneity. Higher resolution structural characterization was achieved via complementary atomic force microscopy (AFM), nano transmission X-ray microscopy (nanoTXM), and micro tomography (Micro XCT™). Structural heterogeneity was correlated to biochemical and elemental composition, illustrated via histochemistry and microprobe X-ray fluorescence analysis (μ-XRF), and mechanical heterogeneity evaluated by AFM-based nanoindentation. Results demonstrated that the narrowed PDL-space was due to invasion of bundle bone (BB) into PDL-space. Protruded BB had a wider range with higher elastic modulus values (2-8 GPa) compared to lamellar bone (0.8-6 GPa), and increased quantities of Ca, P and Zn as revealed by μ-XRF. Interestingly, the hygroscopic 10-30 μm interface between protruded BB and lamellar bone exhibited higher X-ray attenuation similar to cement lines and lamellae within
© 2013 The Authors. Published by Elsevier Inc. All rights reserved. *To whom correspondence should be addressed: Sunita P. Ho, Ph.D., Division of Biomaterials and Bioengineering, Department of Preventive and Restorative Dental Sciences, 707 Parnassus Avenue, University of California San Francisco, CA 94143, [email protected], Phone: 415-514-2818. #both authors contributed equally. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. 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.

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bone. Localization of the small leucine rich proteoglycan biglycan (BGN) responsible for mineralization was observed at the PDL-bone interface and around the osteocyte lacunae. Based on these results, it can be argued that the LB-BB interface was the original site of PDL attachment, and that the genesis of protruded BB identified as protrusions occurred as a result of shift in strain. We emphasize the importance of bony protrusions within the context of organ function and that additional study is warranted.

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Keywords bone-PDL-tooth fibrous joint; periodontal ligament; alveolar bone; bundle bone; bone functional adaptation; discontinuities
1. INTRODUCTION
Operating under Wolff’s principles of organ use and disuse, the bone-periodontal ligament (PDL)-tooth fibrous joint and its respective tissue components react to occlusal inputs, that is, physiological and/or non-physiological loads. As such, the tissues adapt to functional demands, a cycle which may continue in perpetuity under prolonged loading. It is proposed that optimal function of the system is preserved by a characteristic functional PDL-space of 150–380 μm [1], maintained via balance of anabolic and catabolic activity of bone, and equivalent turnover of the PDL and lamellar cementum deposition [2] in response to the stress and strain fields generated during function.
Ideally, the occlusal loads placed on the tooth should result in the external architecture of the tooth to form a geometric conformity with bone and vice versa, under loaded conditions. The tooth is subjected to a variety of loads and when the majority of the compressive loads align with the anatomical axis of the tooth, the PDL could undergo shear, a mixed mode of tension and compression strain fields, supplemented by flexural moments at the tethered ends of the ligament, i.e. the radial PDL-inserts within bone and cementum [3]. As described by D’Arcy Thompson, force fields within organs and tissues promote the internal architecture [4]. In addition, Wolff’s fundamental law of bone remodeling states that changes in the internal architecture of bones, when pathophysiologically altered (e.g. via extraneous loading or increased frequency of loading), can change the overall form of bone [5]. Bone cells are capable of adapting to local mechanical stresses via feedback loops, remodeling and modeling micro and macro-structural changes to remain “functional” bone, despite existing in a pathological state [5]. As a result, it seems inevitable that tissues and their interfaces adapt towards a pathological state to accommodate the significant shifts in loads and frequencies.
Previous studies have demonstrated, at a macroscale, heterogeneous composition in the form of physicochemical gradients within normal bone-PDL-tooth specimens [6]. It has been proposed that the graded interfaces within a joint consisting of uniform PDL-space can optimize distribution of functional loads, thus promoting functional life of the organ. Interestingly, in our recent study we demonstrated the presence of mechanical, structural, and chemical discontinuities (abrupt transitions between two dissimilar materials), localized at the alveolar bone-PDL and cementum-PDL interfaces within the complex [6]. Sharp
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gradients in the form of elastic discontinuities were observed within bone at the lamellar and protruded bone interface. These protruded sites manifesting as elastic discontinuities can be regions of high stress localization in a load-bearing complex. As such, the complex is conducive to amplified strains and stresses leading to perpetuation of the discontinuous interfaces while still maintaining function. Results in this study also raise the question that if organs are predisposed with discontinuities, should therapeutic loads be imposed? While functional load adaptation have been described in skeletal orthopedics [7-12], to our knowledge there exist no studies on bone-PDL-tooth fibrous joint adaptations within oral and craniofacial orthopedics.
The goal of this study is to illustrate characteristics of the narrowed PDL space within the bone-PDL-tooth complex. The elastic graded properties of the inter-lamellae, and lamellae of lamellar bone, bundle-lamellar bone interface and bundle bone of a fibrous joint will be reported within the context of organ function by using erupted and undiseased specimens extracted from humans. This is because; a combination of matrix structure, biochemical and elemental composition, and elastic modulus of various tissues could affect biomechanical function. Hence, detailed PDL-bone, PDL-cementum and protruded bone-lamellar bone integration at the 5-50 μm wide reduced complex will be discussed based on evaluations using various complementary higher resolution characterization techniques, such as atomic force microscopy (AFM), nano X-ray computed tomography (nano- TXM), microprobe Xray fluorescence imaging (micro-XRF) and AFM-based nanoindentation.

2. MATERIALS AND METHODS
Specimens for Micro XCT™ imaging and physicochemical characterization
Specimens (N = 10) were acquired from patients undergoing orthodontic treatment where teeth along with proximal bone were removed. Teeth were removed due to crowding of dentition. Inclusion criterion for a specimen was proximal bone in the coronal half of the extracted tooth. Exclusion criteria were specimens with caries, periodontal disease, and/or root resorption. The protocol was approved by the UCSF Committee on Human Research. Specimens were sterilized using 0.31 Mrad of γ-radiation [13] and each specimen was scanned using a micro-X-ray computed tomography unit (μ-XCT, Micro XCT-200, Pleasanton, CA) [3]. The mandibular-molar complex was then imaged using a μ-XCT [3] to identify tooth-alveolar bone association when compressed with a finite load. Five specimens were used for immunohistochemistry and histology. Another five specimens were used for AFM, which were also used for site-specific mechanical properties using AFM-based nanoindentation and X-ray microprobe techniques. Thin-sectioned specimens (1-5 μm thickness) were prepared out of the remaining specimens and mounted on 100 nm think silicon nitride (Si3N4) membranes for nano-TXM. It should be noted that specimens were taken from the root using the first two-thirds for acellular cementum and last one-third for secondary cementum as location indicators [14].

2.1. Deparaffinized sections for conventional histology and immunohistochemistry
Extracted molars (N = 5) containing PDL and alveolar bone were prepared for histology as stated in our previous work [6]. The sections were subsequently stained with hematoxylin

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and eosin (H&E) and picrosirius red (PSR). Stained tissues were characterized for structural orientation and integration of the PDL with bone and cementum, using a polarized light microscope (BX 51, Olympus America Inc., San Diego, CA) to enhance the birefringence of collagen in alveolar bone and cementum. Images were acquired using Image Pro Plus v6.0 software (Media Cybernetics Inc., Silver Springs, MD).

2.2. Antibody tagging and localization
Antibody tagging was performed using the procedures established and described in our previous works [3]. In brief, the small leucine rich proteins (SLRPs), biglycan (BGN) and fibromodulin (FMOD) within alveolar bone, periodontal ligament (PDL), bone-PDL and cementum-PDL entheses were identified following deparaffinization of mounted sections with xylene. Antigen retrieval was performed using trypsin digestion [3]. The following modifications to the manufacturer’s protocol were made: FMOD, BGN stains, and respective antibodies from polyclonal rabbit sera were acquired from Dr. Larry Fisher (NICDR/NIH, Bethesda, MD). Specimens were blocked at room temperature for 20 min in 1% bovine serum albumin (Sigma, St. Louis, MO), 1.5% mouse serum (Sigma, St. Louis, MO) in PBS. Antibody incubation was then performed overnight (18 h) at 4 °C, with the appropriate antibody diluted in blocking solution (1:50 for anti-BGN, 1:100 for antiFMOD). Slides were washed 3 times the following day in PBS for 5 min each. Secondary antibody incubation of mouse anti-rabbit-IgG conjugated to HRP (Sigma, St. Louis, MO), diluted 1:100 in blocking solution, was performed at room temperature for 30 min, and then washed 3 times in PBS 10 min each.
3,3′-Diaminobenzidine (DAB) Enhanced Liquid Substrate System (Sigma, St. Louis, MO) was used per manufacturer’s instructions with an incubation of 1 h to provide a brown coloration of epitope locations. The specimens were then counterstained with Gill’s III Hematoxylin (Sigma), dehydrated through serial solutions of 80% alcohol, 95% alcohol, 100% alcohol, and xylene, and mounted with Permount (Sigma). An Olympus BX51 light microscope was used for imaging with analyses using Image Pro software (Media Cybernetics Inc., Bethesda, MD).

2.3 AFM, AFM-based nanoindentation, and μ-XRF characterization
Light microscopy (BX 51, Olympus America Inc., San Diego, CA) was used to image the surface of ultrasectioned block specimens [15] (N = 5), to identify bundle and lamellar bone in alveolar bone, and cementum of the tooth. Block specimens were characterized using an AFM, AFM-based nanoindentation and microprobe for micro-XRF. Light micrograph image acquisition and analysis of lamellar bone (specifically interlamellae and lamellae regions), and bundle bone were conducted using Image Pro Plus v6.0 software (Media Cybernetics Inc., Silver Springs, MD).

2.3.1. AFM for structural analysis—Semi-qualitative data representative of bundlelamellar (LB-BB) bone interface, collagen periodicity, hygroscopicity of inter-lamellae, and PDL-inserts within bundle bone was performed using contact mode AFM (Nanoscope III, Multimode; DI-Veeco Instruments Inc., Santa Barbara, CA) under dry and hydrated

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conditions [16]. AFM micrographs were analyzed with Nanoscope III version 5.12r3 software (Nanoscope III, Multimode; DI-Veeco Instruments Inc., Santa Barbara, CA).

2.3.2. AFM-based nanoindentation for site-specific mechanical property evaluation—Nanoindentation was performed on the ultrasectioned block specimens using an AFM attached to a load displacement transducer (Triboscope, Hysitron Incorporated, Minneapolis, MN). A sharp diamond Berkovich indenter with a conventional radius of curvature less than 100 nm (Triboscope, Hysitron Incorporated, Minneapolis, MN) was fitted to the transducer. Site-specific measurements of reduced elastic modulus (Er) in cementum, PDL, bundle, and lamellar bone were made under wet conditions using a displacement control mode and a penetration depth of 500 nm, with a load, hold, and unload for 3s each. Fused silica was used to calibrate the transducer under dry and wet conditions [16-20].

2.3.3. Nano-TXM for structural analysis—Five extracted bone-tooth specimens were sectioned into 3 × 3 × 3 mm cubes using a diamond wafering blade and a low-speed saw (Isomet, Buehler, Lake Bluff, IL) under wet conditions. Specimens were mounted on AFM steel stubs (Ted Pella, Inc., CA) using epoxy and were ultrasectioned with a diamond knife (MicroStar Technologies, Huntsville, TX) to isolate 1-5 μm thick specimens. Nano-scale structural properties of mineralized tissue in bone-PDL-tooth sections mounted on Si3N4 membranes were determined by transmission X-ray microscopy (TXM) (Xradia, Pleasanton, CA) images collected at the Stanford Synchrotron Radiation Lightsource (SSRL) beamline 6-2 [21]. Absorption images of specimens were performed at a X-ray energy of 5.4 keV using monochromatic X-ray radiation. Samples were mounted orthogonal to the incident Xray beam with transmitted x-rays imaged on a transmission detector system comprised of a scintillator optically coupled by a 10X objective to a 1024 × 1024 pixel Peltier-cooled CCD detector. Zernike phase contrast to enable imaging low-Z materials was achieved by the addition of a 3.0 μm thick nickel phase ring [21].

2.4. Micro-XRF mapping of ultrasectioned specimens
Elemental distribution and localization within bone-PDL-tooth block specimens were determined by X-ray fluorescence images collected at the SSRL beam line 2–3. Data were acquired with incident X-ray energy of 12 keV, which was set by using a Si (111) double crystal monochromator [6]. The fluorescence lines of calcium (Ca), phosphorus (P), and Zinc (Zn) within the alveolar bone and cementum were monitored using a silicon drift Vortex detector (SII NanoTechnology USA Inc.). The microfocused beam of 2 × 2 μm was provided by a Pt-coated Kirkpatrick-Baez mirror pair (Xradia Inc.) The incident and transmitted x-ray intensities were measured with nitrogen-filled ion chambers. Specimens were mounted at 45 degrees to the incident x-ray beam and were spatially rastered with the microbeam using a Newport VP-25XA-XYZ stage. Additionally, the entire fluorescence spectrum was also collected at each data point. Using a beam exposure of 100 ms per pixel high resolution maps were generated with scanning step size of 1 μm.

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2.5. Transmission Electron Microscopy (TEM) of ultramicrotomed sections
90 nm thick ultramicrotomed sections, corresponding to the surface of sections studied by TXM, were coated with a thin (~5 nm) layer of carbon and analyzed in a JEOL 2100F Field Emission TEM operated at 120 keV. Conventional bright-field TEM images and high-angle annular dark-field (HAADF) scanning TEM (STEM) images with compositional contrast [22] were recorded. High-resolution TEM (HRTEM) was utilized to visualize lattice fringes representative of crystalline structure. The crystallinity and texture of lamellae and interlamallae regions was analyzed with selected-area electron diffraction (SAED) patterns.

3. RESULTS
3.1 Qualitative assessment of non-conforming surfaces revealed with Micro XCT™ imaging
Micro XCT™ 2D images of intact bone-PDL-tooth fibrous joint specimens provided two types of tissue associations within the joints that were qualitatively assessed: 1) conforming (complementing) tooth root and alveolar bone surfaces and 2) non-conforming root and bone surfaces characterized by the presence of protrusions contiguous with alveolar bone, “encroaching” into the PDL-space. Despite the presence of bony protrusions (Fig. 1), in situ images of the periodontium under no load illustrated a seemingly uniform functional PDLspace. While under loaded conditions, a qualitative observation of nonconforming root and bone surfaces was made. Loaded conditions illustrated narrowed PDL-space, highlighted by focal contacts of non-conforming tooth root and alveolar bone surface sites (Figs. 1C & D). It should be noted that cementum on the tooth root surface also demonstrated protrusions into the PDL-space.

3.2 Structural and biochemical heterogeneities identified with various imaging modalities and immunohistochemistry
High magnification light microscopy images of ultrasectioned compromised bone-PDLtooth specimens illustrated distinct structural constructs within cementum, PDL, and alveolar bone at narrowed/constricted PDL sites (Figs. 2A & B). Picrosirius red staining coupled with polarized light microscopy of lamellar bone (LB) demonstrated differential collagen fiber orientation between lamellae and interlamellae regions of lamellar bone characterized by birefringence (Fig. 2D). Similarly, within secondary cementum of the tooth root, structural heterogeneity in the form of lamellar and non-lamellar structures was identified with non-lamellar secondary cementum interfacing with the PDL-space (Fig. 2C). Within alveolar bone two regions distinguished by differential collagen fiber orientation were identified using hematoxylin and eosin staining (H&E) coupled with polarized light microscopy (Figs. 2E & F). Bony protrusions adjacent to the PDL-space, which demonstrated primarily radial fibers, were identified as bundle bone (secondary) interfacing circumferential collagen fibers characteristic of (primary) lamellar bone. Interestingly, H&E staining demonstrated directional bone growth illustrated by bony protrusions expressed at the PDL- alveolar bone entheses, however this was not observed at the cementum-PDL entheses (Figs. 2G & H).

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Composite atomic force micrographs of the surface of sectioned protruded bone-PDL-tooth block specimens acquired under wet and dry conditions illustrated radial fibers insertions sites within mineralized protrusions of both bundle bone and cementum at respective tissue entheses (Fig. 3A). Two structurally distinct regions were again demonstrated within alveolar bone characterized by an abrupt structural transition from radially oriented Sharpey’s fibers to circumferential lamellar bone, with bundle bone exhibiting more hygroscopic nature than lamellar bone, suggestive of differential degrees mineral and organic content (Fig. 3B). Numerous osteocyte lacunae were also noted at the LB-BB interface, in conjunction with the insertion of Sharpey’s fibers across BB and into the LB region (Fig. 3C). Additionally, within lamellar bone, inter-lamellae regions demonstrated greater hygroscopic nature, with increased swelling as compared to the lamellae. Interestingly, macroscale AFM scans in wet conditions illustrated a third structurally unique region characterized by hygroscopic network of transitioning radial-circumferential fibers within the lamellar bone-bundle bone interface (LB-BB) interface (Fig. 3).
Immunohistochemistry labeling for fibromodulin (FMOD) demonstrated localization of FMOD in Sharpey’s fibers of bundle bone (BB), cementum, PDL, vascular tissue, and within lamellar bone (LB) localizing as striations bordering alveolar bone vasculature spaces (Figs. 4A & B). Tooth-PDL-bone histology sections labeled for biglycan (BGN) illustrated localization at the alveolar bone-PDL tissue interface, commonly known as entheses, regardless of the presence or absence of protruded bundle bone (Fig. 4C). BGN localization at the cementum-PDL entheses was also observed (Fig. 4D).
X-ray absorption maps of thin bone-PDL-tooth specimens (1-5 μm) collected via nanotransmission X-ray Tomography (Nano-TXM) illustrated various attenuating regions within protruded alveolar bone (Fig. 5). Bony protrusions adjacent to the PDL-space were identified as bundle bone (BB) by the presence of hygroscopic organic inserts (Sharpey’s fibers) confirmed by high-resolution atomic force microscopy (AFM) under wet conditions. AFM also demonstrated hygroscopic radial PDL fibers in BB changed orientation into circumferential in LB at the interface between lamellar bone and bundle bone. Interestingly, this region of fiber orientation transition, or interface, corresponded with a 10-30 μm wide junction between BB and LB which exhibited higher X-ray attenuation. Additionally, alternating bands of higher and lower attenuating regions corresponding with lamellae and inter-lamellae regions of lamellar bone were also identified.
Further investigation of these regions with Transmission Electron Microscopy (TEM) in both conventional bright-field (Figs. 6A & B) and HAADF STEM compositional contrast images (Fig. 6D) demonstrated the alternating lamellae and inter-lamellae spaces. The periodic 67 nm collagen banding pattern, characteristic of Type I collagen, is evident in both lamellae and interlamellae regions (Fig. 6B). High-resolution micrographs (Fig. 6C) exhibited lattice fringes characteristic of a crystalline mineral phase, while selected area electron diffraction patterns (Fig. 6E) confirm both the crystalline nature of the lamellae and interlamellae regions and their respective preferential orientation (texture), as indicated by the 002 arcs corresponding to the alignment of the crystal’s c-axis.

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3.3. Elemental composition by mapping X-ray fluorescence of Ca, P, and Zn using microprobe
High resolution microprobe X-ray fluorescence spectroscopy of elemental Calcium (Ca), Phosphorus (P), and Zinc (Zn) (Fig. 7) demonstrated higher X-ray fluorescence counts of Ca and P in bony protrusions identified as bundle bone (BB) relative to the interfacing lamellar bone (LB). Line maps illustrated steep chemical gradients in Ca and P from LB to BB, originating at the LB-BB interface. Interestingly, a distinct localization of Zn, an element theorized as a potential marker for function- and disease-induced biomineralization, was noted in BB.

3.4. Mechanical gradients from AFM-based nanoindentation
Site specific nano-indentation of lamellar bone (LB) and adjacent bundle bone (BB) demonstrated significantly higher elastic moduli (Er) in bundle bone relative to lamellar bone, ranging from 2-8GPa, and 0.8-6GPa, respectively. It should be noted that due to the heterogeneous architecture and composition of the material, mechanical properties were reported as a range of values rather than an averages as illustrated via frequency plot (Fig. 8). Multiple line profiles starting from LB demonstrated a steep mechanical gradient from LB into BB (Fig. 8). Interestingly, from a mechanics perspective, the LB-BB interface is representative of a discontinuous interface, interfaces which are known to accumulate local strain amplification and subsequent mechanical failure.

4. DISCUSSION
In this investigation, the bone-PDL-tooth complex illustrated bony protrusions with physicochemical discontinuities. The structural phenotype of bundle bone is not typical of development, but is related to the latter stages of the organ when it comes into function (i.e. mastication) [14, 24-27] during which time mechanical loads are felt by the bone-ligamenttooth complex. Bone continuously adapts to accommodate mechanical loads identifying the “functional plasticity” of alveolar bone [14, 24-27]. Regardless, adaptation over time prompts a uniform periodontal ligament space of 150-380 μm [1, 3] which constitutes an optimum range of motion for the tooth inside the alveolar socket. However, bone growth of human dentition presented previously [6] and in this study hypothesizes a cause for bony protrusions from the lamellar bone that at the very least are unique sites that warrant investigation. Hence, the logical questions are: 1) What features are suggestive of functional adaptation of the fibrous organ? And of further importance: 2) Is there a critical point at which adaptation restricts or prevents organ function shifting it to a state of pathology? From a philosophical stand-point, where is the balance between adaptation and pathology and as adaptation occurs does that mean that the potential for adaptation decreases, or when the organ crosses a critical threshold does the next effort at adaptation lead to destruction?
Addressing the first question we made a qualitative observation of a change in tooth-alveolar bone association, i.e. the geometric conformation at no load that became nonconforming when loaded (Fig. 1). This observation highlighted the potential for nonconforming regions to experience strain amplification when under load. Hence it is plausible that the protrusion sites (stars in Fig. 1) act as potential culprits that shift the organ into malfunction. Sites that

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promote an irregular functional space of less than 100 μm are not to be confused with “hot spots” (strain amplified sites) when functional loads are well within physiological limits and bone-tooth geometric conformation is maintained [23]. The observations presented in this study raise specific questions; “How are these nonconforming regions or protrusions generated? What is the physicochemical makeup of these protrusions when compared to primary lamellar alveolar bone?” The first question can be systematically answered using animal models, but does not fall within the scope of this study. However, it is the second question that will be answered by discussing the measured graded physicochemical properties of the protruded bone-lamellar bone interface in a human bone-PDL-tooth fibrous joint.
The accepted theory on bone adaptation is driven by the reduction of resultant strain fields from dynamic mechanical loading [4]. According to Wolff “form follows function and that change in the internal architecture of bones deformed and stressed pathologically, entail secondary alterations in the external form of these bones, also following mathematical rules”. Additionally, the “development of an external shape which Roux later called “functional” occurs to meet functional demands; however, could have shifted the tissue and/or organ to a pathological state” [5]. Extending these concepts to alveolar bone, we speculate that the bony protrusions could occur due to nonphysiological functional demands at the tethered ends of the PDL-bone and PDL-cementum interfaces. The response to the shift in strain at the interfaces can be identified by tipping of the blastic and clastic events thus forming or resorbing mineral along the PDL-bone and PDL-cementum tethered ends, within bone per se, and that these local events strengthen the region to accommodate functional demands.
In this study, the pullout forces at the tethered ends are best evidenced by the hematoxylin and eosin histology sections (Fig. 2E & F). These sections demonstrated microscale protrusions, presence of basophilic groups at the PDL-bone and PDL-cementum attachment sites, and established a positive correlation with the presence of FMOD and BGN biomolecules at similar anatomical locations (Figs. 2, 4). It is proposed that BGN prompts mineralization at the soft-hard tissue attachment site [28] in the direction of the stretched PDL fibers. The direction of the PDL-inserts in the protruded bone, i.e. bony protrusions are indicative of the directional stretch of original fibers, and may have prompted bone lining cells to produce mineral along the strained fibers. This of course should not come as a surprise because of the historic postulation that cortical bone formation occurs along the tension based strain profiles [4].
Within the bone-PDL-tooth fibrous joint, the pullout forces at the soft-hard tissue interfaces are the mechanobiologically active sites. Also known as enthesis organs in orthopedics, the interfaces have an integral role in maintaining organ-level biomechanics [8]. Most often a reductionist approach is sought, and interfaces by themselves are interrogated, with various state-of-the-art instrumentation. While the reductionist approach is necessary and can provide a clearer picture of the interfaces alone, this approach by itself fails to answer the impact of intrinsic characteristics of an interface on overall organ function. As stated by D’Arcy Thompson, “the beauty and strength of the mechanical construction lie not in one part or in another, but in the harmonious concatenation which all the parts, soft and hard,
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