Tooth acellular extrinsic fibre cementum incremental lines in humans are formed by parallel branched Sharpey's fibres and not by its mineral phase.

In humans, the growth pattern of the acellular extrinsic fibre cementum (AEFC) has been useful to estimate the age-at-death. However, the structural organization behind such a pattern remains poorly understood. In this study tooth cementum from seven individuals from a Mexican modern skeletal series were analyzed with the aim of unveiling the AEFC collagenous and mineral structure using multimodal imaging approaches. The organization of collagen fibres was first determined using: light microscopy, transmission electron microscopy (TEM), electron tomography, and plasma FIB scanning electron microscopy (PFIB-SEM) tomography. The mineral properties were then investigated using: synchrotron small-angle X-ray scattering (SAXS) for T-parameter (correlation length between mineral particles); synchrotron X-ray diffraction (XRD) for L-parameter (mineral crystalline domain size estimation), alignment parameter (crystals preferred orientation) and lattice parameters a and c; as well as synchrotron X-ray fluorescence for spatial distribution of calcium, phosphorus and zinc. Results show that Sharpey's fibres branched out fibres that cover and uncover other collagen bundles forming aligned arched structures that are joined by these same fibres but in a parallel fashion. The parallel fibres are not set as a continuum on the same plane and when they are superimposed project the AEFC incremental lines due to the collagen birefringence. The orientation of the apatite crystallites is subject to the arrangement of the collagen fibres, and the obtained parameter values along with the elemental distribution maps, revealed this mineral tissue as relatively homogeneous. Therefore, no intrinsic characteristics of the mineral phase could be associated with the alternating AEFC incremental pattern.

Elevated activity levels do not influence extrinsic fiber attachment morphology on the surface of muscle-attachment sites.

Extrinsic fibers (EFs) are a type of penetrating collagenous fiber, closely related to the periodontal ligament, which help anchor soft tissue into bone. These fibers are associated with muscle attachment sites (entheses). Their size and grouping patterns are thought to be indicative of the loading history of the muscle. EFs are of particular significance in anthropology as potential tools for the reconstruction of behavior from skeletal remains and, specifically, entheses. In this study, we used a mouse model to experimentally test how activity level alters the morphology of EF insertion sites on the bone surface of a fibrocartilaginous enthesis, the biceps brachii insertion. Further, we adapted surface metrological techniques from studies of dental wear to perform automated, quantitative and non-destructive analysis of bone surface histology. Our results show that experimentally increased activity had no significant effect on the quantity or density of EF insertions at the enthesis, nor on the size of those insertions. Although EF presence does indicate muscle attachment, activity did not have an observable effect on EF morphology.

Quantitative characterizations of the Sharpey's fibers of rat molars.

BACKGROUND AND OBJECTIVE: The Sharpey's fibers of periodontal ligament (PDL) anchor the PDL to alveolar bone and cementum and are essential for the function of PDL. While qualitative analyses of the Sharpey's fibers have been widely explored, a comprehensive quantitative characterization of the Sharpey's fibers is not available. In this work, we selected rat molars as a model and comprehensively characterized the PDL Sharpey's fibers (diameter, density, length, embedding angle, and insertion angle). MATERIALS AND METHODS: A total of 24 rat mandibular molars, eight maxillary first molars, and their surrounding alveolar bone were harvested, fixed, rendered anorganic and observed under scanning electron microscopy (SEM). The mandibles and maxillae (n = 4) were harvested, processed, sectioned, and stained with Sirius red for histological observation. SEM images were used for quantitative analyses of diameters and densities of the Sharpey's fibers, while Sirius red staining images were used to measure lengths and angles. The Sharpey's fibers were comprehensively characterized in terms of positions (cervical, middle, and apical thirds), PDL fiber groups (alveolar crest, horizontal, oblique, apical, and interradicular groups), sides (cementum and bone sides), and teeth (mandibular first, second, third molars, and maxillary first molar). RESULTS: Our results showed that the characteristic parameters of the Sharpey's fibers varied in different positions, fiber groups, sides, and teeth. Specifically, the median diameter of the Sharpey's fibers on the bone side was significantly greater than that on the cementum side, while the median density of the Sharpey's fibers on the bone side was significantly lower than that on the cementum side, regardless of the positions and teeth. For the same tooth, the median length of the embedded Sharpey's fibers on the bone side was more than two times greater than that on the cementum side. Among all fiber groups, the alveolar crest group had the maximum length of the Sharpey's fibers on the bone side and the minimal length of the Sharpey's fibers on the cementum side. There is an approximate 5-15° difference between the embedding angle and the insertion angle in each group. The oblique group had the smallest embedding angles on both the bone and cementum sides. CONCLUSION: This study provides a comprehensive and quantitative characterization of the Sharpey's fibers using rat molars as a model. Overall, these parameters varied according to different vertical positions, fiber groups, teeth, and jawbones. The quantitative information of the Sharpey's fibers presented in this work facilitates our understanding of PDL functions and advances the development of biomimetic materials for periodontal tissue regeneration.

Osteohistological correlates of muscular attachment in terrestrial and freshwater Testudines.

Sharpey's fibers are considered the anatomical structures integrated to the muscles. Since these fibers leave marks at the microscopic level, their presence and distribution are used as evidence of muscle attachment in extinct and extant forms. In recent years, studies have been focusing on muscle-bone and tendon-bone interaction mostly on mammals. The main objective of this work is to contribute to the morphological and histological knowledge of muscle attachment in other amniotes, such as reptiles, and their variation related to different locomotor habits. In this way, a study was performed on terrestrial and aquatic turtles. The musculature related to the movement of the humerus, and pectoral girdle in Chelonoidis chilensis, Phrynops hilarii and Hydromedusa tectifera was analyzed. Dissections were performed mapping the origins and insertions of each muscle and undecalcified thin sections were performed in specific muscular attachment sites. We found some differences which were not previously reported, related to the insertion of the m. pectoralis, the m. coracobrachialis magnus and the origin of the m. tractor radii. The osteohistology revealed the presence of Sharpey's fibers in the cortex of all the bone elements analyzed. Patterns were established in relation to the orientation and density of Sharpey's fibers, which were used for the categorization of each muscle attachment site. The comparative micro-anatomical study of these areas did not reveal any important differences between terrestrial and freshwater turtles in muscles involved with the rotation, abduction and adduction of the humerus. In this way, the preliminary results suggest an absence of correlation between the distribution and density of Sharpey's fibers between different habitat forms, at least in the bones and species analyzed.

The role of an octacalcium phosphate in the re-formation of infraspinatus tendon insertion.

BACKGROUND: To improve the success rate of rotator cuff repair, we investigated whether octacalcium phosphate (OCP) with gelatin (Gel) vehicle had a positive effect on tendon-to-bone healing. METHODS: We assessed the histologic characteristics of the tendon-to-bone healing using the rabbit rotator cuff repair model. We divided the shoulders into 3 groups: control (without OCP/Gel composite), OCP/Gel composite (OCP+group), and Gel alone without OCP (Gel group) to evaluate the effectiveness of gelatin. RESULTS: Both the number of newly formed tendon fibers and the Sharpey fibers at the repair site increased in the OCP+group compared with those in the other 2 groups on hematoxylin-eosin staining (P < .05). On immunohistochemical evaluation, both the bone and the fibers in the OCP+group demonstrated that type I collagen was picked up, whereas the newly formed tendon fibers and Sharpey fibers revealed type III collagen. CONCLUSION: Treatment with OCP made collagen fibers and the Sharpey fibers, constituted by type I and type III collagens, increase at the tendon-to-bone insertion. It might be beneficial for the healing of rotator cuff tendon to bone.

Entheseal surface (Sharpey's fiber insertion) alterations identify past trauma; bone base robusticity, level of routine activity.

Sharpey's fiber alterations, referred to as entheseal reaction or enthesopathy, have long been considered an indicator of daily activities. Such semantic transformation seems to conflate processes which alter the characteristics of tendonous and ligamentous attachments to bone with the rugosity and extent of their base/footprint. Rather than reflecting normal activities, it is suggested that surface reactions are actually the response to the application of sudden or unconditioned repetitive stresses-analogous to stress fractures. Thus, they are distinct from enlargement of the base/footprint, the bone remodeling process responsible for the robusticity of the area to which the enthesis attaches, which is actually a measure of actual muscle activity. Surface reactions in attachment areas represent injury, be it mechanical stress fracture-equivalents or inflammation-derived. Bone base/footprint is the reaction of the enthesis to stresses of routine physical activities. The character of underlying bone supporting Sharpey's fibers may be augmented by applied stress, but there is neither a physiologic mechanism nor is there evidence for significant addition of Sharpey's fibers beyond ontogeny. Behavior is responsible for the physiologic response of robusticity; spiculation, pathology.

Structural characteristics of periodontal ligaments in the pig model with experimental periodontitis.

To investigate how the structure of Sharpey's fibers in the periodontal ligaments (PDL) were affected by experimental periodontitis in a young pig model, 7 were periodically inoculated with four types of bacteria and a ligature around the last maxillary molar for 8 weeks to induce periodontitis (PG), and 10 served as controls (CG). The harvested molar blocks were sectioned coronally and stained with either hematoxylin & eosin (H&E) or Sirius Red (SR). The H&E-stained images were first reviewed. Then, images of each adjacent SR-stained sections were captured at the region close to the apex of mesial roots under polarizing light microscopy. Sharpey's fiber numbers in each bundle, total bundle numbers, connection of the bundle to the cementum and alveoli, as well as bundle angulations to the molar long axis were quantified in the defined area 500µm apical to the root apex. Compared to CG, PG showed the following features: 15.13% less total bundle number; 17.8% less bundle size; significantly less connected fiber bundles; 9.2% more interrupted fiber bundles; and 3.85% more oriented toward the cervical in the bundle angulation. These results suggest that experimental periodontitis alters the PDL structure, leading to more interruptions of Sharpey's fiber attachments to the cementum.

Osteophytes: The product of convergent evolution.

The very reasonable suggestion, that diarthrodial joint and juxta-discal (vertebral centra-marginal) bony overgrowths (referred to as osteophytes) have different etiologies, has eluded previous confirmation. The prevailing perspective is that diarthrodial osteophytes represent the product of compressive forces and that those on the margins of vertebral centra result from traction and therefore are enthesial in derivation. If diarthrodial joint osteophytes result from intrinsic pressures, any surface responses would require transcortical nutritional support, easily recognized by en face microscopic examination. This contrasts with enthesially derived growth, the surface of which is characterized by Sharpey's fiber insertions. These are recognized as inverted cones with a central protrusion on examination of related bone surfaces. We hypothesize that diarthrodial and disc-adjacent osteophytes have a different pathophysiology, distinguishable on the basis of microscopic surface appearance. We pursued microscopic examination of the surfaces of osteophytes present on diarthrodial joints (hip, knee, elbow, costovertebral) and vertebrae (cervical, thoracic, and lumbar) from the CAL Milano Cemetery Skeletal Collection for presence of transcortical channels and the inverted cones of Sharpey's fiber insertions. Examination of 22 diarthrodial joint osteophytes reveals the presence solely of transcortical channels, while examination of 35 vertebral centra marginal osteophytes reveals the presence only of inverted cones. Findings are independent of age, gender, joint affected, position in the spinal column and osteophyte "severity." It is now evidenced that all osteophytes are not created equal. Diarthrodial joint osteophytes are endochondrally derived; vertebral centra osteophytes, enthesial in derivation. Different pathophysiology at least partially explain the clinical character of these processes.

Histological analysis of ankylothecodonty in Silesauridae (Archosauria: Dinosauriformes) and its implications for the evolution of dinosaur tooth attachment.

Dinosaurs possess a form of tooth attachment wherein an unmineralized periodontal ligament suspends each tooth within a socket, similar to the condition in mammals and crocodylians. However, little information is known about tooth attachment and implantation in their close relatives, the silesaurids. We conducted a histological survey of several silesaurid taxa to determine the nature of tooth attachment in this phylogenetically and paleoecologically important group of archosaurs. Our histological data demonstrate that these early dinosauriforms do not exhibit the crocodilian/dinosaur condition of a permanent gomphosis, nor the rapid ankylosis that is plesiomorphic for amniotes. Instead, all sampled silesaurids exhibit delayed ankylosis, a condition in which teeth pass through a prolonged stage where the teeth are suspended in sockets by a periodontal ligament, followed by eventual mineralization and fusion of the tooth to the jaws. This suggests that tooth attachment in crocodylians and dinosaurs represent the further retention of an early ontogenetic stage compared to silesaurids, a paedomorphic trend that is mirrored in the evolution of synapsid tooth attachment. It also suggests that the dinosaur and crocodylian gomphosis was convergently acquired via heterochrony or, less likely, that the silesaurid condition represents a reversal to a plesiomorphic state. Moreover, if Silesauridae is nested within Ornithischia, a permanent gomphosis could be convergent between the two main dinosaur lineages, Ornithischia and Saurischia. These results demonstrate that dental characters in early archosaur phylogenies must be chosen and defined carefully, taking into account the relative duration of the different phases of dental ontogeny.

Bioinspired Collagen/Gelatin Nanopillared Films as a Potential Implant Coating Material.

Collagen-based Sharpey's fibers are naturally located between alveolar bone and tooth, and they have critical roles in a well-functioning tooth such as mechanical stability, facile differentiation, and disease protection. The success of Sharpey's fibers in these important roles is due to their unique location, vertical alignment with respect to tooth surface, as well as their micronanofiber architecture. Inspired by these structures, herein, we introduce the use of nanoporous anodic aluminum oxide molds in a drop-casting setup to fabricate biopolymeric films possessing arrays of uniform Collagen:Gelatin (Col:Gel) nanopillars. Obtained structures have diameters of ∼90 nm and heights of ∼300 nm, yielding significantly higher surface roughness values compared to their flat counterparts. More importantly, the nanostructures were parallel to each other but perpendicular to the underlying film surface imitating the natural collagenous structures of Sharpey's fibers regarding nanoscale morphology, geometrical orientation, as well as biochemical content. Viability testing showed that the nanopillared Col:Gel films have high cell viabilities (over 90%), and they display significantly improved attachment (ca. ∼ 2 times) and mineralization for Saos-2 cells when compared to flat Col:Gel films and Tissue Culture Polystyrene (TCPS) controls, plausibly due to their largely increased surface roughness and area. Hence, such Sharpey's fiber-inspired bioactive nanopillared Col:Gel films can be used as a dental implant coating material or tissue engineering platform with enhanced cellular and osteogenic properties.

The effect of femoral bone tunnel configuration on tendon-bone healing in an anterior cruciate ligament reconstruction: An animal study.

OBJECTIVES: To compare the effect of femoral bone tunnel configuration on tendon-bone healing in an anterior cruciate ligament (ACL) reconstruction animal model. METHODS: Anterior cruciate ligament reconstruction using the plantaris tendon as graft material was performed on both knees of 24 rabbits (48 knees) to mimic ACL reconstruction by two different suspensory fixation devices for graft fixation. For the adjustable fixation device model (Socket group; group S), a 5 mm deep socket was created in the lateral femoral condyle (LFC) of the right knee. For the fixed-loop model (Tunnel group; group T), a femoral tunnel penetrating the LFC was created in the left knee. Animals were sacrificed at four and eight weeks after surgery for histological evaluation and biomechanical testing. RESULTS: Histologically, both groups showed a mixture of direct and indirect healing patterns at four weeks, whereas only indirect healing patterns were observed in both groups at eight weeks. No significant histological differences were seen between the two groups at four and eight weeks in the roof zone (four weeks, S: mean 4.8 sd 1.7, T: mean 4.5 sd 0.5, p = 0.14; eight weeks, S: mean 5.8 sd 0.8, T: mean 4.8 sd 1.8, p = 0.88, Mann-Whitney U test) or side zone (four weeks, S: mean 5.0 sd 1.2, T: mean 4.8 sd 0.4, p = 0.43; eight weeks, S: mean 5.3 sd 0.8,T: mean 5.5 sd 0.8, p = 0.61, Mann-Whitney U test) . Similarly, no significant difference was seen in the maximum failure load between group S and group T at four (15.6 sd 9.0N and 13.1 sd 5.6N) or eight weeks (12.6 sd 3.6N and 17.1 sd 6.4N, respectively). CONCLUSION: Regardless of bone tunnel configuration, tendon-bone healing after ACL reconstruction primarily occurred through indirect healing. No significant histological or mechanical differences were observed between adjustable and fixed-loop femoral cortical suspension methods.Cite this article: Y. Sato, R. Akagi, Y. Akatsu, Y. Matsuura, S. Takahashi, S. Yamaguchi, T. Enomoto, R. Nakagawa, H. Hoshi, T. Sasaki, S. Kimura, Y. Ogawa, A. Sadamasu, S. Ohtori, T. Sasho. The effect of femoral bone tunnel configuration on tendon-bone healing in an anterior cruciate ligament reconstruction: An animal study. Bone Joint Res 2018;7:327-335. DOI: 10.1302/2046-3758.75.BJR-2017-0238.R2.

Effects of tendon-bone healing of anterior cruciate ligament reconstruction by osteoprotegerin combined with deproteinized bovine bone.

BACKGROUND: The healing of a tendon graft in a bone tunnel depends on bone ingrowth into the interface between tendon and bone, or that can enhance tendon-bone healing, which is important to reduce the failure rate after ACL reconstruction. METHODS: Sixty skeletally mature, New Zealand white rabbits underwent left ACL reconstruction. OPG/DBB compound (concentration ratio of 30%, 60%, 100%) was delivered to the tendon-bone interface with use of a DBB carrier, and nothing as control group. Twenty animals were killed at 4, 8 and 12 weeks after surgery. I-IV levels of semi-quantitative and Sharpey fibers at the healing tendon-bone interface were evaluated, and the biomechanical properties were tested. RESULTS: A significantly greater amount of Sharpey fibers at the healing tendon-bone interface in the concentration ratio of 100% OPG/DBB-treated group was found compared with the others at all time-points (P<0.05), and it is the same to the Grade Scores at 12 weeks (P<0.05). The femur-ACL-tibia complex of the concentration ratio of 100% OPG/DBB-treated group has significantly increased stiffness compared with the others at 12 weeks (P<0.05). CONCLUSION: The concentration ratio of 100% OPG/DBB compound significantly improve bone formation around the grafted tendon and improve the stiffness at the healing tendon-bone junction in a rabbit model.

Chronological changes in the collagen-type composition at tendon-bone interface in rabbits.

OBJECTIVES: The purpose of this study was to evaluate chronological changes in the collagen-type composition at tendon-bone interface during tendon-bone healing and to clarify the continuity between Sharpey-like fibres and inner fibres of the tendon. METHODS: Male white rabbits were used to create an extra-articular bone-tendon graft model by grafting the extensor digitorum longus into a bone tunnel. Three rabbits were killed at two, four, eight, 12 and 26 weeks post-operatively. Elastica van Gieson staining was used to colour 5 µm coronal sections, which were examined under optical and polarised light microscopy. Immunostaining for type I, II and III collagen was also performed. RESULTS: Sharpey-like fibres comprised of type III collagen in the early phase were gradually replaced by type I collagen from 12 weeks onwards, until continuity between the Sharpey-like fibres and inner fibres of the tendon was achieved by 26 weeks. CONCLUSIONS: Even in rabbits, which heal faster than humans, an observation period of at least 12 to 26 weeks is required, because the collagen-type composition of the Sharpey-like fibre bone-tendon connection may have insufficient pullout strength during this period. These results suggest that caution is necessary when permitting post-operative activity in humans who have undergone intra-bone tunnel grafts.