Glycosaminoglycans accelerate biomimetic collagen mineralization in a tissue-based in vitro model.

Mammalian teeth are attached to the jawbone through an exquisitely controlled mineralization process: unmineralized collagen fibers of the periodontal ligament anchor directly into the outer layer of adjoining mineralized tissues (cementum and bone). The sharp interface between mineralized and nonmineralized collagenous tissues makes this an excellent model to study the mechanisms by which extracellular matrix macromolecules control collagen mineralization. While acidic phosphoproteins, localized in the mineralized tissues, play key roles in control of mineralization, the role of glycosaminoglycans (GAGs) is less clear. As several proteoglycans are found only in the periodontal ligament, it has been hypothesized that these inhibit mineralization of collagen in this tissue. Here we used an in vitro model based on remineralization of mouse dental tissues to determine the role of matrix GAGs in control of mineralization. GAGs were selectively removed from demineralized mouse periodontal sections via enzymatic digestion. Proteomic analysis confirmed that enzymatic GAG removal does not significantly alter protein content. Analysis of remineralized tissue sections by transmission electron microscopy (TEM) shows that GAG removal reduced the rate of remineralization in mineralized tissues compared to the untreated control, while the ligament remained unmineralized. Protein removal with trypsin also reduced the rate of mineralization, but to a lesser extent than GAG removal, despite a much larger effect on protein content. These results indicate that GAGs promote mineralization in mineralized dental tissues rather than inhibiting mineral formation in the ligament, which may have broader implications for understanding control of collagen mineralization in connective tissues.

Cellular network across cementum and periodontal ligament elucidated by FIB/SEM tomography.

Cementocytes in cementum form a lacuna-canalicular network. However, the 3D ultrastructure and range of the cementocyte network are unclear. Here, the 3D ultrastructure of the cementocyte network at the interface between cementum and periodontal ligament (PDL) was investigated on the mesoscale using FIB/SEM tomography. The results revealed a cellular network of cementocytes and PDL cells. A previous histomorphological study revealed the osteocyte-osteoblast-PDL cellular network. We extended this knowledge and revealed the cementum-PDL-bone cellular network, which may orchestrate the remodeling and modification of periodontal tissue, using a suitable method for imaging of complex tissue.

Three-dimensional ultrastructural imaging and quantitative analysis of the periodontal ligament.

The periodontal ligament (PDL) is a unique connective tissue mainly comprising collagen fiber bundles and cells between the roots of teeth and inner walls of the alveolar-bone socket. PDL fiber bundles are arrayed between teeth and bone, with both ends embedded in the cementum or alveolar bone as Sharpey's fiber. These bundles, synthesized by PDL fibroblasts (PDLFs), form several distinct groups within the PDL which has important functions besides tooth anchoring including tooth nutrition, proprioception, sensory detection, homoeostasis, and repair of damaged tissue. However, little is known about how the regular-PDL fiber bundle arrays are formed, maintained, and remodeled over large distances from cementum to alveolar bone. Recently, novel instruments and 3D-imaging methods have been developed that have been applied to the investigation of hard tissues including the PDL. Work from our laboratory has revealed the three-dimensional (3D) ultrastructure of PDLFs and PDL collagen bundles by focused ion beam/scanning electron microscope tomography. We have shown that PDLFs have a flat shape with long processes or a wing-like shape, while PDL bundles are a multiple-branched structure wrapped in thin sheets of PDLF cytoplasm. Furthermore, PDLFs form an extensive cellular network between the cementum and alveolar bone. The PDL cellular network is presumed to synchronize PDL fiber bundles and regulate arrays of PDL fiber bundles via gap junctions. In this review, we summarize and discuss our current 3D-histomorphometric studies of the PDL at the mesoscale level.

Three-dimensional ultrastructural analysis and histomorphometry of collagen bundles in the periodontal ligament using focused ion beam/scanning electron microscope tomography.

BACKGROUND AND OBJECTIVE: The periodontal ligament (PDL) is an essential tissue for tooth function. However, the 3-dimensional ultrastructure of these PDL collagen bundles on a mesoscale is not clear. We investigated the 3-dimensional ultrastructure of these collagen bundles and quantitatively analyzed their histomorphometry using focused ion beam/scanning electron microscope (FIB/SEM) tomography. MATERIAL AND METHODS: The PDLs of the first mandibular molar of male C57BL/6 mice were analyzed using FIB/SEM tomography. The serial images of the collagen bundles so obtained were reconstructed. The collagen bundles were analyzed quantitatively using 3-dimensional histomorphometry. RESULTS: Collagen bundles of the PDL demonstrated multiple branched structures, rather than a single rope-like structure, and were wrapped in cytoplasm sheets. The structure of the horizontal fiber of the collagen bundle was an extensive meshwork. In contrast, the oblique and apical fibers of the collagen bundle showed a chain-like structure. The area and the minor and major axis lengths of cross-sections of the horizontal fiber, as determined from 3-dimensional images, were significantly different from those of the oblique and apical fibers. CONCLUSION: These findings indicate that collagen bundles in horizontal fiber areas have high strength and that the tooth is firmly anchored to the alveolar bone by the horizontal fibers, but is not secured evenly to the alveolar bone. The tooth is firmly anchored around the cervical area, creating a "slingshot-like structure." This study has provided further insights into the structure of the PDL and forms the basis for the development of more effective therapies for periodontal tissue regeneration.

Elastic fiber system evaluated in the digestive organ of rats.

According to our previous reports, the intraperiodontal elastic fiber system comprises oxytalan fibers, whereas all types of elastic system fibers are present in the gingiva. Much remains to be elucidated regarding the topographic development of the elastic fiber system that constitutes the walls of the digestive organs. This study aimed to examine the topographic development of the elastic fiber system in the periodontal tissue, oral cavity and digestive tract of rats at light- and electron microscopic levels. At embryonic day 20, in situ hybridization revealed the mRNA expression of tropoelastin in the putative gingival lamina propria but not in the dental follicle. At the postnatal stage, the masticatory mucous membrane of the gingiva and hard palate comprised three different types of elastic system fibers (oxytalan, elaunin and elastic fibers). Conversely, the elastic fiber system comprised elaunin and elastic fibers in other oral mucosae and the lining mucosae of digestive tract organs (the esophagus, stomach and small intestine). The findings of our study suggest that the elastic fiber system is mainly related to tissue resistance in the periodontal ligament and tissue elasticity in the oral mucosae without masticatory mucosae and the overlying mucosa of digestive tracts and both functions in the gingiva and hard palate, respectively. The appearance of elaunin fibers in the periodontium of rats aged 14 weeks suggests the expression of tropoelastin induced by mechanical stressors such as mastication. The intraperiodontal difference in the distribution of elaunin fibers suggests heterogeneity among fibroblasts constituting the periodontium.

Three-dimensional ultrastructural and histomorphological analysis of the periodontal ligament with occlusal hypofunction via focused ion beam/scanning electron microscope tomography.

The periodontal ligament (PDL) maintains the environment and function of the periodontium. The PDL has been remodelled in accordance with changes in mechanical loading. Three-dimensional (3D) structural data provide essential information regarding PDL function and dysfunction. However, changes in mechanical loading associated with structural changes in the PDL are poorly understood at the mesoscale. This study aimed to investigate 3D ultrastructural and histomorphometric changes in PDL cells and fibres associated with unloading condition (occlusal hypofunction), using focused ion beam/scanning electron microscope tomography, and to quantitatively analyse the structural properties of PDL cells and fibres. PDL cells formed cellular networks upon morphological changes induced via changes in mechanical loading condition. Drastic changes were observed in a horizontal array of cells, with a sparse and disorganised area of collagen bundles. Furthermore, collagen bundles tended to be thinner than those in the control group. FIB/SEM tomography enables easier acquisition of serial ultrastructural images and quantitative 3D data. This method is powerful for revealing 3D architecture in complex tissues. Our results may help elucidate architectural changes in the PDL microenvironment during changes in mechanical loading condition and regeneration, and advance a wide variety of treatments in dentistry.

Histochemical examination on principal collagen fibers in periodontal ligaments of ascorbic acid-deficient ODS-od/od rats.

In this study, we aimed to clarify the role of ascorbic acid in collagen synthesis in periodontal ligaments using osteogenic disorder Shionogi (ODS)/ShiJcl-od/od rats lacking L-gulonolactone oxidase. These rats cannot synthesize ascorbic acid in vivo. Eight-week-old ODS/ShiJcl-od/od male rats were administered ascorbic acid solution at a concentration of 200 mg/dL (control group, n = 6) or ascorbic acid solution at concentration of 0.3 mg/dL (insufficient group, n = 12). Six rats of the insufficient group were then given with ascorbic acid solution at concentration of 200 mg/dL for additional 3 weeks (rescued group, n = 6), and then, their mandibles were histochemically examined. Consequently, the insufficient group specimens were seen to possess fewer collagen fibers, and silver impregnation revealed numerous fine, reticular fiber-like fibrils branching off from collagen in the periodontal ligaments. In control group, faint immunoreactivities for matrix metalloproteinase (MMP)2 and cathepsin H were seen in the periphery of blood vessels and throughout the ligament, respectively. In contrast, in the insufficient group, intense MMP2-immunoreactivity was observed to be associated with collagen fibrils in the periodontal ligaments, and cathepsin H-immunopositivity was seen in ligamentous cells. The rescued group showed abundant collagen fibers filling the periodontal ligament space. Under transmission electron microscopy, ligamentous fibroblasts incorporated collagen fibrils into tubular endosomes/lysosomes while simultaneously synthesizing collagen fibril bundles. Thus, ascorbic acid insufficiency affected the immunolocalization of cathepsin H and MMP2; however, ligamentous fibroblasts appear to possess the potential to synthesize collagen fibers when supplied with ascorbic acid.

An unusual disordered alveolar bone material in the upper furcation region of minipig mandibles: A 3D hierarchical structural study.

Teeth are subjected to compressive loads during mastication. Under small loads the soft tissue periodontal ligament (PDL) deforms most. However when the loads increase and the PDL is highly compressed, the tooth and the alveolar bone supporting the tooth, begin to deform. Here we report on the structure of this alveolar bone in the upper furcation region of the first molars of mature minipigs. Using light microscopy and scanning electron microscopy (SEM) of bone cross-sections, we show that this bone is hypermineralized, containing abundant small pores around 1-5 µm in diameter, lacunae around 10-20 µm as well as larger spaces. This bone does not possess the typical lamellar motif or other repeating structures normally found in cortical or trabecular mammalian bone. We also use high resolution focused ion beam scanning electron microscopy (FIB-SEM) in the serial surface mode to image the 3D organization of the demineralized bone matrix. We show that the upper furcation bone matrix has a disordered isotropic structure composed mainly of individual collagen fibrils with no preferred orientation, as well as highly staining material that is probably proteoglycans. Much larger aligned arrays of collagen fibers - presumably Sharpey's fibers - are embedded in this material. This unusual furcation bone material is similar to the disordered material found in human lamellar bone. In the upper furcation region this disordered bone comprises almost all the volume excluding Sharpey's fibers. We surmise that this most unusual bone type functions to resist the repeating compressive loads incurred by molars during mastication.

The effect of cyclic tensile force on the actin cytoskeleton organization and morphology of human periodontal ligament cells.

To explore Girdin/Akt pathway protein expression and morphology change by cyclic tension in the periodontal ligament cells. Human periodontal ligament cells were exposed to cyclic tension force at 4000 µstrain and 0.5 Hz for 6 h though a four-point bending system. Cyclic tension force upregulated F-actin, Girdin and Akt expression in hPDL. In transmission electron microscope assay showed that there are more and bigger mitochondria, more and longer cynapses, more cellular organisms after tension force stimulation than control. The actin filament was changed to be regular lines and pointed to poles of cells. However, we found that the Girdin-depleted cells are small and there are more micro-organisms including more lysosomes and matrix vesicles than control. These finding suggest that the STAT3/Girdin/Akt pathway in PDL to response to mechanical stimulation as well, and Girdin may play a significant role in triggering cell proliferation and migration during orthodontic treatment. It provided an insight into the molecular basis for development of a vitro cell model in studying orthodontic treatment.

Amelogenesis imperfecta: A novel FAM83H mutation and characteristics of periodontal ligament cells.

OBJECTIVE: To delineate orodental features, dental mineral density, genetic aetiology and cellular characteristics associated with amelogenesis imperfecta (AI). MATERIALS AND METHODS: Three affected patients in a family were recruited. Whole-exome sequencing was used to identify mutations confirmed by Sanger sequencing. The proband's teeth were subjected for mineral density analysis by microcomputerised tomography and characterisation of periodontal ligament cells (PDLCs). RESULTS: The patients presented yellow-brown, pitted and irregular enamel. A novel nonsense mutation, c.1261G>T, p.E421*, in exon 5 of the FAM83H was identified. The mineral density of the enamel was significantly decreased in the proband. The patient's PDLCs (FAM83H cells) exhibited reduced ability of cell proliferation and colony-forming unit compared with controls. The formation of stress fibres was remarkably present. Upon cultured in osteogenic induction medium, FAM83H cells, at day 7 compared to day 3, had a significant reduction of BSP, COL1 and OCN mRNA expression and no significant change in RUNX2. The upregulation of ALP mRNA levels and mineral deposition were comparable between FAM83H and control cells. CONCLUSIONS: We identified the novel mutation in FAM83H associated with autosomal dominant hypocalcified AI. The FAM83H cells showed reduced cell proliferation and expression of osteogenic markers, suggesting altered PDLCs in FAM83H-associated AI.

Effect of preameloblast-conditioned medium and CPNE7 on root surfaces in dogs: a histologic and histomorphometric evaluation.

Preameloblast-conditioned medium (PACM) has been reported as a potent dentin regenerative material, but its effects as a mixture on periodontal regeneration and the role of CPNE7 in PACM are not known. The purpose of this study is to evaluate the histologic and histomorphometric effects of preameloblast-conditioned medium (PACM) and CPNE7 on periodontal tissue healing in dogs. Seventy-two mandibular premolar roots from ten dogs were extracted and randomly divided into six groups (n = 12 each): (1) positive control group; (2) negative control group; (3) cementum-removed and PACM-treated group; (4) cementum-preserved and PACM-treated group; (5) CPNE7-inactivated PACM-treated group; and (6) recombinant CPNE7-treated group. The extracted roots were replanted into extraction sockets for 4 and 8 weeks and analyzed histologically. Most of the root surfaces in the negative control group showed ankylosis; and those in the experimental groups showed newly formed PDL-like and cementum-like tissues. Histomorphometric analysis of horizontal sections showed that the mean length of the PDL on the roots of the positive controls was similar to those in cementum-removed or -preserved and PACM-treated group at 8 weeks (p = 1.08). Sagittal sections showed that the mean length of the new cementum on the roots in cementum-removed and PACM-treated group was significantly greater than that in CPNE7-inactivated PACM-treated group (p = 0.037). The mean length of the newly formed PDL on the roots in CPNE7- inactivated PACM-treated and rCPNE7-treated groups was significantly greater than that in the negative controls at 8 weeks (p = 0.037, p = 0.036). The use of PACM and CPNE7 in tooth replantation resulted in increased PDL and cementum formation, suggesting the beneficial role of PACM and CPNE7 in periodontal tissue healing.

Incompatible amount of 3-D and 2-D periodontal attachments on micro-CT scanned premolars.

Micro-computed tomography (micro-CT) was employed to relate the root surface area (RSA) to the periodontal attachment levels (PALs) of extracted premolars to diagnose periodontitis. Single-rooted human maxillary and mandibular premolars 31 and 36, respectively, were surveyed by micro-CT and its associated software. RSA levels from the 1st to 10th mm, corono-apically, were analyzed using statistical t tests. The average root length (RL) and RSA of the maxillary and mandibular premolars were significantly different (p < 0.05). Both premolars demonstrated a non-significant RSA percentage comparison at the evaluated PALs. For the 30% coronal 2-D radiographic RL, the 3-D RSAs 3.77 mm and 3.99 mm apical to the cementoenamel junction (CEJ) were 39.48% and 40.65% for maxillary and mandibular premolars, respectively. At the 15% coronal 2-D RL, the 3-D RSA 2 mm apical to the CEJ of the premolars was approximately 21%. At the 50% coronal 2-D RL level, approximately 62% coronal 3-D RSA and 6.5 mm RL decreased. The amount of decrease of the RSA attachment is significant in every 2-mm measurement for both premolars. Sampling periodontal microbial pathogens based on the condition of 2-D radiographic bone and clinical attachment losses without considering 3-D RSA is potentially inadequate and may underestimate the severity of the periodontitis.

Periodontal ligament-like tissue regeneration with drilled porous decalcified dentin matrix sheet composite.

OBJECTIVE: In this study, we constructed a composite by combining the human dental follicle cell sheet and a manual drilled porous decalcified dentin matrix that was used to construct ectopic tissue-engineered periodontal ligament-like tissues in renal capsules of nude mice. MATERIALS AND METHODS: Human dental follicle cells were harvested from human lower third molars and then embedded into a temperature-sensitive culture dish. These cells were then placed into frozen porous decalcified dentin matrix sheets and induced by 50 g/ml ascorbic acid. This established a "sandwich structure" in vitro implant that was placed in nude mice under the renal capsule. The mice were sacrificed at 4 and 8 weeks after implantation, and the implants were assessed after haematoxylin-eosin staining, Masson staining and immunohistochemical staining. RESULTS: The experimental group showed a fibre structure between the dentin and HA-TCP after 4 weeks. After 8 weeks, the collagen fibres increased, and the direction was perpendicular to the dentin. Immunohistochemistry showed positive staining in the osteopontin and periostin. CONCLUSION: The composite can induce ectopic bone and fibre formation, and the fibre had a certain directionality. Besides, the composite can maintain the stability of the periodontal ligament width.

The intricate anatomy of the periodontal ligament and its development: Lessons for periodontal regeneration.

The periodontal ligament (PDL) connects the tooth root and alveolar bone. It is an aligned fibrous network that is interposed between, and anchored to, both mineralized surfaces. Periodontal disease is common and reduces the ability of the PDL to act as a shock absorber, a barrier for pathogens and a sensor of mastication. Although disease progression can be stopped, current therapies do not primarily focus on tissue regeneration. Functional regeneration of PDL may be achieved using innovative techniques, such as tissue engineering. However, the complex fibrillar architecture of the PDL, essential to withstand high forces, makes PDL tissue engineering very challenging. This challenge may be met by studying PDL anatomy and development. Understanding PDL anatomy, development and maintenance provides clues regarding the specific events that need to be mimicked for the formation of this intricate tissue. Owing to the specific composition of the PDL, which develops by self-organization, a different approach than the typical combination of biomaterials, growth factors and regenerative cells is necessary for functional PDL engineering. Most specifically, the architecture of the new PDL to be formed does not need to be dictated by textured biomaterials but can emerge from the local mechanical loading conditions. Elastic hydrogels are optimal to fill the space properly between tooth and bone, may house cells and growth factors to enhance regeneration and allow self-optimization by the alignment to local stresses. We suggest that cells and materials should be placed in a proper mechanical environment to initiate a process of self-organization resulting in a functional architecture of the PDL.

Mechanobiology of the tooth movement during the orthodontic treatment: a literature review.

Orthodontic tooth movement differs significantly from the physiological tooth movement, as it determines a biological response of the surrounding tissues of the teeth, resulting in a remodelling of the periodontal ligament and the alveolar bone. The result is a biochemical adaptive response to the application of the orthodontic force with the reorganization of the intracellular and the extracellular matrix, in addition to a change of the local vascularization. This in turn leads to the synthesis and the release of arachidonic acid, growth factors, metabolites, cytokines and various enzymes. Biologically, not only the intensity of the force, but also its duration and the tissue response to the application of the same are important for tooth movement. Having these insights it will possible to examine the concept of optimal orthodontic force, a determining factor for the success of orthodontic treatment. The purpose of this revision was to describe the biological processes and future perspective of the application of orthodontic force, by providing relevant information to understand the changes at the molecular and cellular level occurring when the tissues are subjected to such forces. Knowledge on the subject of mechanics and biology in orthodontics is constantly growing, producing an increasingly strong basis for clinical success.

Apical External Root Resorption and Repair in Orthodontic Tooth Movement: Biological Events.

Some degree of external root resorption is a frequent, unpredictable, and unavoidable consequence of orthodontic tooth movement mediated by odontoclasts/cementoclasts originating from circulating precursor cells in the periodontal ligament. Its pathogenesis involves mechanical forces initiating complex interactions between signalling pathways activated by various biological agents. Resorption of cementum is regulated by mechanisms similar to those controlling osteoclastogenesis and bone resorption. Following root resorption there is repair by cellular cementum, but factors mediating the transition from resorption to repair are not clear. In this paper we review some of the biological events associated with orthodontically induced external root resorption.

Comparison of human dental follicle cells and human periodontal ligament cells for dentin tissue regeneration.

AIM: To compare the odontogenic potential of human dental follicle cells (DFCs) and periodontal ligament cells (PDLCs). MATERIALS & METHODS: In vitro and in vivo characterization studies of DFCs and PDLCs were performed comparatively. DFCs and PDLCs were subcutaneously implanted into the dorsum of mice for 8 weeks after combined with treated dentin matrix scaffolds respectively. RESULTS: Proteomic analysis identified 32 differentially expressed proteins in DFCs and PDLCs. Examination of the harvested grafts showed PDLCs could form the dentin-like tissues as DFCs did. However, the structure of dentin tissues generated by DFCs was more complete. CONCLUSION: PDLCs could contribute to regenerate dentin-like tissues in the inductive microenvironment of treated dentin matrix. DFCs presented more remarkable dentinogenic capability than PDLCs did.

Fibromodulin and Biglycan Modulate Periodontium through TGFß/BMP Signaling.

A full understanding of the key regulators controlling periodontal development and homeostasis is necessary for the design of improved periodontal regenerative therapies. Small leucine-rich proteoglycans (SLRPs) are extracellular matrix molecules suggested to regulate collagen organization and cell signaling. Mice with double-deficiency of 2 SLRPs, fibromodulin and biglycan (dKO), acquire skeletal abnormalities, but their roles in regulating the periodontium remain undefined and were the focus of our studies. Transmission electron microscopy studies showed abnormal collagen fibrils in the periodontal ligament (PDL) and altered remodeling of alveolar bone in dKO mice. Immunohistochemistry (IHC) revealed increased staining of SLRPs (asporin, lumican, and decorin) and dentin matrix protein-1 (DMP1, a mechanosensory/osteocyte marker), while osteoblast markers, bone sialoprotein and osteopontin, remained unchanged. Disruption of homeostasis was further evidenced by increased expression of receptor-activator of nuclear factor-κB ligand (RANKL) and elevated numbers of osteoclasts, especially noted around the alveolar bone of molars (buccal side) and incisors. Polymerase chain reaction (PCR) array revealed hyperactive transforming growth factors beta/bone morphogenetic protein (TGFß/BMP) signaling in dKO PDL tissues, which was further confirmed by elevated expression of phosphorylated Smad5 (p-Smad5) by IHC in dKO PDL. These studies highlight the importance of SLRPs in maintaining periodontal homeostasis through regulation of TGFß/BMP signaling, matrix turnover, and collagen organization.

Cryo-TEM analysis of collagen fibrillar structure.

Fibrillar collagens are important structural proteins and are known to be closely associated with mineral in the case of mineralized tissues. However, the precise role of collagen in the mineralization process remains unclear, and the evaluation of structural differences in collagen from mineralized and nonmineralized tissues may be instructive in this regard. Here, we review the use of cryo-transmission electron microscopy to investigate the axial structure of collagen fibrils in tissue sections from both mineralizing and nonmineralizing tissues. By examining collagen fibrillar structure in an unstained frozen-hydrated state, it is possible to avoid artifacts normally associated with staining and dehydration that are required for conventional TEM. We describe both sample preparation and image analysis with emphasis on the particular challenges of using image averaging techniques, which can be used to overcome the low signal-to-noise ratio that is inherent in this technique. Detailed banding patterns can be obtained from averaged images, and these can be analyzed to obtain quantitative information on fibril periodicity and structure.

The plastic nature of the human bone-periodontal ligament-tooth fibrous joint.

This study investigates bony protrusions within a narrowed periodontal ligament space (PDL-space) 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 (nano-TXM), and microtomography (MicroXCT™). 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 (2-8GPa) compared to lamellar bone (0.8-6GPa), and increased quantities of Ca, P and Zn as revealed by µ-XRF. Interestingly, the hygroscopic 10-30µm interface between protruded BB and lamellar bone exhibited higher X-ray attenuation similar to cement lines and lamellae within 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.