Histological staining alters circular dichroism SHG measurements of collagen.

Circular dichroism second harmonic generation microscopy (CDSHG) is a powerful imaging technique, which allows three-dimensional visualization of collagen fibril orientation in tissues. However, recent publications have obtained contradictory results on whether CDSHG can be used to reveal the relative out-of-plane polarity of collagen fibrils. Here we compare CDSHG images of unstained tendon and tendon which has been stained with hematoxylin and eosin. We find significant differences in the CDSHG between these two conditions, which explain the recent contradictory results within the literature.

Production and physico-chemical characterization of nano-sized collagen from equine tendon.

In recent years, significant academic and commercial interest has focused on collagen derived from horse tendons, with potential applications across diverse sectors such as medicine, pharmaceuticals, and cosmetics. Nano collagen, with its enhanced wound penetration, improved cell contact, and heightened cellular regeneration and repair capabilities due to its high surface area, holds promise for a wide range of applications. In this study, we present a novel method for producing nano collagen from the equine tendon. Our approach is characterized by its speed, affordability, simplicity and environmentally friendly nature, with precise temperature-control to prevent collagen denaturation. We conducted a comprehensive characterization of the obtained samples, including assessments of morphology, chemical and thermal properties, particle size distribution and biocompatibility. Importantly, our results indicate improvements in thermal stability, and surface roughness of nano collagen, while preserving its molecular weight. These advancements expand the potential applications of nano collagen in various fields.

On the effect of pepsin incubation on type I collagen from horse tendon: Fine tuning of its physico-chemical and rheological properties.

Type I collagen is commonly recognized as the gold standard biomaterial for the manufacturing of medical devices for health-care related applications. In recent years, with the final aim of developing scaffolds with optimal bioactivity, even more studies focused on the influence of processing parameters on collagen properties, since processing can strongly affect the architecture of collagen at various length scales and, consequently, scaffolds macroscopic performances. The ability to finely tune scaffold properties in order to closely mimic the tissues' hierarchical features, preserving collagen's natural conformation, is actually of great interest. In this work, the effect of the pepsin-based extraction step on the material final properties was investigated. Thus, the physico-chemical properties of fibrillar type I collagens upon being extracted under various conditions were analyzed in depth. Correlations of collagen structure at the supramolecular scale with its microstructural properties were done, confirming the possibility of tuning rheological, viscoelastic and degradation properties of fibrillar type I collagen.

Modeling collagen fibril self-assembly from extracellular medium in embryonic tendon.

Collagen is a key structural component of multicellular organisms and is arranged in a highly organized manner. In structural tissues such as tendons, collagen forms bundles of parallel fibers between cells, which appear within a 24-h window between embryonic day 13.5 (E13.5) and E14.5 during mouse embryonic development. Current models assume that the organized structure of collagen requires direct cellular control, whereby cells actively lay down collagen fibrils from cell surfaces. However, such models appear incompatible with the time and length scales of fibril formation. We propose a phase-transition model to account for the rapid development of ordered fibrils in embryonic tendon, reducing reliance on active cellular processes. We develop phase-field crystal simulations of collagen fibrillogenesis in domains derived from electron micrographs of inter-cellular spaces in embryonic tendon and compare results qualitatively and quantitatively to observed patterns of fibril formation. To test the prediction of this phase-transition model that free protomeric collagen should exist in the inter-cellular spaces before the formation of observable fibrils, we use laser-capture microdissection, coupled with mass spectrometry, which demonstrates steadily increasing free collagen in inter-cellular spaces up to E13.5, followed by a rapid reduction of free collagen that coincides with the appearance of less-soluble collagen fibrils. The model and measurements together provide evidence for extracellular self-assembly of collagen fibrils in embryonic mouse tendon, supporting an additional mechanism for rapid collagen fibril formation during embryonic development.

Recombinant lysyl oxidase effects on embryonic tendon cell phenotype and behavior.

Lysyl oxidase (LOX) plays an important role in the elaboration of tendon mechanical properties during embryonic development by mediating enzymatic collagen crosslinking. We previously showed recombinant LOX (rLOX) treatment of developing tendon significantly increased LOX-mediated collagen crosslink density to enhance tendon mechanical properties at different stages of tissue formation. Working toward the future development of rLOX-based therapeutic strategies to enhance mechanical properties of tendons that are compromised, such as after injury or due to abnormal development, this study characterized the direct effects of rLOX treatment on embryonic tendon cells from different stages of tissue formation. Tendon cell morphology, proliferation rate, proliferative capacity, and metabolic activity were not affected by rLOX treatment. Tenogenic phenotype was stable with rLOX treatment, reflected by no change in cell morphology or tendon marker messenger RNA (mRNA) levels assessed by reverse-transcription polymerase chain reaction. Collagen mRNA levels also remained constant. Matrix metalloproteinase-9 expression levels were downregulated in later stage tendon cells, but not in earlier stage cells, whereas enzyme activity levels were undetected. Bone morphogenetic protein-1 (BMP-1) expression was upregulated in earlier stage tendon cells, but not in later stage cells. Furthermore, BMP-1 activity was unchanged when intracellular LOX enzyme activity levels were upregulated in both stage cells, suggesting exogenous rLOX may have entered the cells. Based on our data, rLOX treatment had minimal effects on tendon cell phenotype and behaviors. These findings will inform future development of LOX-focused treatments to enhance tendon mechanical properties without adverse effects on tendon cell phenotype and behaviors.

Multifilament Collagen Fiber Bundles with Tendon-like Structure and Mechanical Performance.

Collagen multifilament bundles comprised of thousands of monofilaments are prepared by multipin contact drawing of an entangled polymer solution consisting of collagen and poly(ethylene oxide) (PEO). The multifilament bundles are hydrated in graded concentrations of PEO and phosphate buffered saline (PBS) to promote assembly of collagen fibrils within each monofilament while preserving the structure of the multifilament bundle. Multiscale structural characterization reveals that the hydrated multifilament bundle contains properly folded collagen molecules packed in collagen fibrils containing microfibrils, staggered by exactly one-sixth of the microfibril D-band spacing to produce a periodicity of 11 nm. Sequence analysis predicts that in this structure, phenylalanine residues are close enough within and between microfibrils to become ultraviolet C (UVC) crosslinked. In agreement with this analysis, the ultimate tensile strength (UTS) and Young's modulus of the hydrated collagen multifilament bundles crosslinked by UVC radiation increase nonlinearly with total UVC energy to reach values in the range of native tendons without damage to the collagen molecules. This fabrication method recapitulates the structure of a tendon across multiple length scales and offers tunability in tensile properties using only collagen molecules and no other chemical additives in addition to PEO, which is almost entirely removed during the hydration process.

Biocompatibility-on-a-chip: Characterization and evaluation of decellularized tendon extracellular matrix (tdECM) hydrogel for 3D stem cell culture in a microfluidic device.

Researchers have always tried expensive in vitro tests to show the 3D usability of dECM. The use of tissue-specific hydrogels in a microfluidic device is rarely studied. In this study, we have used ECM obtained from goat digital flexor tendons by decellularization technique. The tdECM was characterized for its structural properties using Scanning Electron Microscopy (SEM). Collagen, dsDNA, GAGs, and protein contents were quantified using spectrophotometric assays. The cell viability and proliferation of human umbilical cord-derived mesenchymal stem cells (hUMSCs) encapsulated in the tdECM hydrogel inside the microfluidic device were checked using Calcein-AM/PI. The FTIR data showed prominent peaks of the amide group, indicating the presence of collagen. The SEM data showed intact fiber morphology after the decellularization process. There was a 95 % reduction in double-stranded DNA (dsDNA) content, proving the effectiveness of the decellularization technique. There was no significant difference in the collagen content of tdECM and the GAGs were also in the acceptable range compared to the native tissue. Over 90 % cell viability in hUMSCs was observed qualitatively and quantitatively in vitro and inside a microfluidic device. In conclusion, we characterized the tdECM hydrogel and demonstrated its compatibility with the microfluidic device.

Super-Strong, Nonswellable, and Biocompatible Hydrogels Inspired by Human Tendons.

Fabricating artificial materials that mimic the structures and properties of tendons is of great significance. Possessing a tensile stress of approximately 10.0 MPa and a water content of around 60%, human tendons exhibit excellent mechanical properties to support daily functions. In contrast to tendons, most synthetic hydrogels with similar water content typically exclude qualified strength, swelling resistance, and biocompatibility. Herein, a facile strategy based on poly(vinyl alcohol) (PVA) and tannic acid (TA) is demonstrated to tackle this problem via a combination of sequential steps including freezing-thawing PVA aqueous solutions to form crystalline regions, prestretching and air drying in confined conditions to induce anisotropic structures, soaking in TA solutions to form multiple hydrogen bondings between PVA and TA, and finally dialyzing against water for the removal of residual TA molecules and the rearrangements and homogenization of multiple hydrogen bonds. The obtained PVA hydrogels possess hierarchically anisotropic structures, where the alignment of PVA bundles promotes high modulus, while the hydrogen bonding between PVA and TA endows them with an energy dissipation mechanism. Benefitting from the synergy of material composition and structural engineering, the obtained hydrogel displays super-strong mechanics (a tensile stress of 19.3 MPa and a toughness of 32.1 MJ/m3), outperforming most tough hydrogels. Remarkably, this hydrogel demonstrates excellent swelling resistance. It barely expands after immersion in deionized water, phosphate-buffered saline (PBS), and SBF aqueous solutions for 7 days with the strength and volume nearly the same as their initial values. All of the features, combined with excellent cytocompatibility, make it an ideal material for biotechnological and biomedical applications.

Picrosirius Red Staining: Revisiting Its Application to the Qualitative and Quantitative Assessment of Collagen Type I and Type III in Tendon.

Collagen has a major role in the structural organization of tendons. Picrosirius red (PSR) staining viewed under polarized light microscopy is the standard method to evaluate the organization of collagen fibers in tissues. It is also used to distinguish between type I and type III collagen in tissue sections. However, accurate analysis and interpretation of PSR images are challenging because of technical factors and historical misconceptions. The aim of this study was to clarify whether collagen types I and III can be distinguished by PSR staining in rat Achilles tendons, using double immunohistochemistry as the positive control. Our findings showed that PSR staining viewed with polarized light microscopy was suitable for qualitative and quantitative assessment of total collagen but was not able to distinguish collagen types. We found it critical to use a polarizing microscope equipped with a rotating stage; tendon section orientation at 45° with respect to crossed polarizers was optimal for the qualitative and quantitative assessment of collagen organization. Immunohistochemistry was superior to PSR staining for detection of collagen type III. We also compared formalin and Bouin solution as fixatives. Both produced similar birefringence, but formalin-fixed tendons provided higher quality histological detail with both hematoxylin-eosin and immunostaining.

Measuring collagen fibril diameter with differential interference contrast microscopy.

Collagen fibrils, linear arrangements of collagen monomers, 20-500 nm in diameter, comprising hundreds of molecules in their cross-section, are the fundamental structural unit in a variety of load-bearing tissues such as tendons, ligaments, skin, cornea, and bone. These fibrils often assemble into more complex structures, providing mechanical stability, strength, or toughness to the host tissue. Unfortunately, there is little information available on individual fibril dynamics, mechanics, growth, aggregation and remodeling because they are difficult to image using visible light as a probe. The principle quantity of interest is the fibril diameter, which is difficult to extract accurately, dynamically, in situ and non-destructively. An optical method, differential interference contrast (DIC) microscopy has been used to visualize dynamic structures that are as small as microtubules (25 nm diameter) and has been shown to be sensitive to the size of objects smaller than the wavelength of light. In this investigation, we take advantage of DIC microscopy's ability to report dimensions of nanometer scale objects to generate a curve that relates collagen diameter to DIC edge intensity shift (DIC-EIS). We further calibrate the curve using electron microscopy and demonstrate a linear correlation between fibril diameter and the DIC-EIS. Using a non-oil immersion, 40x objective (NA 0.6), collagen fibril diameters between ~100 nm to ~ 300 nm could be obtained with ±11 and ±4 nm accuracy for dehydrated and hydrated fibrils, respectively. This simple, nondestructive, label free method should advance our ability to directly examine fibril dynamics under experimental conditions that are physiologically relevant.

The influence of animal species, gender and tissue on the structural, biophysical, biochemical and biological properties of collagen sponges.

Although collagen type I is extensively used in biomedicine, no study to-date has assessed how the properties of the produced scaffolds are affected as a function of species, gender and tissue from which the collagen was extracted. Herein, we extracted and characterised collagen from porcine and bovine, male and female and skin and tendon tissues and we subsequently fabricated and assessed the structural, biophysical, biochemical and biological properties of collagen sponges. All collagen preparations were of similar purity and free-amine content (p > 0.05). In general, the porcine groups yielded more collagen; had higher (p < 0.05) denaturation temperature and resistance to enzymatic degradation; and lower (p < 0.05) swelling ratio and compression stress and modulus than the bovine groups of the same gender and tissue. All collagen preparations supported growth of human dermal fibroblasts and exhibited similar biological response to human THP-1 monocytes. These results further illustrate the need for standardisation of collagen preparations for the development of reproducible collagen-based devices. Assessment of the physicochemical and biological properties of collagen sponges as a function of animal species (bovine versus porcine), gender (male versus female) and tissue (skin versus tendon).

Adoption of a Newly Introduced Dermal Matrix: Preliminary Experience and Future Directions.

INTRODUCTION: Acellular dermal matrix (ADM) products are adopted in the management of injuries to soft tissues. ADMs have been increasingly employed for their clinical advantages, and they are acquiring relevance in the future of plastic surgery. The aim of our study is to evaluate the application of ADMs in our patients who could not undergo fast reconstruction. MATERIALS AND METHODS: We performed a retrospective study on 12 patients who underwent ADM placement for scalp and limb surgical reconstructions at the Humanitas Research Hospital, Rozzano (Milano), Italy. Wounds resulted from 9 tumor resections and 3 chronic ulcers. The ADM substrate used to treat these lesions was PELNAC™ (Gunze, Japan), a double-layered matrix composed of atelocollagen porcine tendon and silicon reinforcement. All patients underwent a second surgical operation to complete the treatment with a full-thickness skin graft to cover the lesion. RESULTS: In this study, 12 patients were treated with PELNAC™: 11 out of 12 patients showed a good attachment over a median time of 21.3 days (range 14-27). After almost 23 days, all patients were ready to undergo a full-thickness skin grafting. CONCLUSION: This study assesses the benefits of PELNAC™ and proposes this method as an alternative to traditional approaches, especially in situations where the latter techniques cannot be applied.

Implementation of a logarithmic division-of-focal-plane polarimeter to quantify changes in collagen alignment at varying levels of illumination.

We examine the impact of illumination, aperture, and sample thickness on two division-of-focal-plane (DoFP) polarimeters, one created using a standard 3 T pixel and the other with a forward-biased, logarithmic pixel. Across all measured metrics the logarithmic DoFP polarimeter was better able to track real-time changes in collagen alignment than the standard DoFP polarimeter.

Orientation Matters: Polarization Dependent IR Spectroscopy of Collagen from Intact Tendon Down to the Single Fibril Level.

Infrared (IR) spectroscopy has been used for decades to study collagen in mammalian tissues. While many changes in the spectral profiles appear under polarized IR light, the absorption bands are naturally broad because of tissue heterogeneity. A better understanding of the spectra of ordered collagen will aid in the evaluation of disorder in damaged collagen and in scar tissue. To that end, collagen spectra have been acquired with polarized far-field (FF) Fourier Transform Infrared (FTIR) imaging with a Focal Plane Array detector, with the relatively new method of FF optical photothermal IR (O-PTIR), and with nano-FTIR spectroscopy based on scattering-type scanning near-field optical microscopy (s-SNOM). The FF methods were applied to sections of intact tendon with fibers aligned parallel and perpendicular to the polarized light. The O-PTIR and nano-FTIR methods were applied to individual fibrils of 100-500 nm diameter, yielding the first confirmatory and complementary results on a biopolymer. We observed that the Amide I and II bands from the fibrils were narrower than those from the intact tendon, and that both relative intensities and band shapes were altered. These spectra represent reliable profiles for normal collagen type I fibrils of this dimension, under polarized IR light, and can serve as a benchmark for the study of collagenous tissues.

Biochemical differences between distal limb extensor and flexor tendons among equine breeds selected for racing and sport.

Throughout the ages, humans have selected different horse breeds for their locomotor capacities. Consequently, the properties of equine locomotor tissues could have diversified because of the specific requirements of different disciplines. Therefore, this study aimed to compare biochemical properties of tendons in different equine breeds traditionally selected for racing or sports performance. We hypothesised that tendons in racing breeds would have biochemical properties that would increase strength, whereas those in sporting breeds would have more elastic properties. An ex vivo tendon tissue study comparing the common digital extensor tendon (CDET) and superficial digital flexor tendon (SDFT) of sports horses (Friesian horse, Warmblood horse) and racehorses (Thoroughbred horse; the oldest, reference standard breed) was performed. The SDFT and CDET from middle-aged Friesian (n = 12), Warmblood (n = 12) and Thoroughbred horses (n = 8) were harvested, and their biochemical properties were compared. The biochemical analysis demonstrated significantly higher water percentage, lower collagen concentrations/glycosaminoglycan content and higher crosslink concentrations in the SDFT of sports horses compared to racing breed horses (P < 0.05); DNA content was also significantly lower in sports horses than racehorses (P < 0.05). Racehorses had mainly extra fibrillar collagen support, whereas sports horses had mainly extra crosslink collagen support. From a functional perspective, the racing Thoroughbred relied on stronger tendons, while the sporting Friesians and Warmbloods relied on less stiff, more elastic tendons. In conclusion, there were significant biochemical differences in tendon properties between breeds, possibly related to their intended locomotor performance, although this requires further biomechanical and ultimately genetic confirmation.

A microfluidics-derived growth factor gradient in a scaffold regulates stem cell activities for tendon-to-bone interface healing.

Treatment of tendon-to-bone interface injury has long been challenging in sports medicine. The major obstacle lies with the complicated three-layer structure of the tissue that consists of a bone region with osteocytes, a tendon region with tenocytes and a transitional region with chondrocytes. Conventional tissue engineering approaches using simply biomaterial scaffolds, stem cells and combinations of them had limited abilities to reconstruct the gradient structure with normal biomechanical properties. We herein aim to construct a three-layer structure with bone marrow-derived stem cells and tendon stem cells cultured in a decellularized tendon scaffold, through application of a gradient of biological cues in the longitudinal direction of the scaffold that guides the stem cells to differentiate and remodel the extracellular matrix in response to different medium concentrations in different regions. A microfluidic chip, on which a tree-like flow pattern was implemented, was adopted to create the concentration gradient in a dichotomous manner. We screened for an optimized seeding ratio between the two stem cell types before incubation of the scaffold in the medium concentration gradient and surgical implantation. Histology and immunohistochemistry assessments, both qualitatively and semi-quantitatively, showed that the microfluidic system provided desired guidance to the seeded stem cells that the healing at 8-week post-implantation presented a similar structure to that of a normal tendon-to-bone interface, which was outstanding compared to treatments without gradient guidance, stem cells or scaffolds where chaotic and fibrotic structures were obtained. This strategy offers a potentially translational tissue engineering approach for better outcomes in tendon-to-bone healing.

Mechanoradicals in tensed tendon collagen as a source of oxidative stress.

As established nearly a century ago, mechanoradicals originate from homolytic bond scission in polymers. The existence, nature and biological relevance of mechanoradicals in proteins, instead, are unknown. We here show that mechanical stress on collagen produces radicals and subsequently reactive oxygen species, essential biological signaling molecules. Electron-paramagnetic resonance (EPR) spectroscopy of stretched rat tail tendon, atomistic molecular dynamics simulations and quantum-chemical calculations show that the radicals form by bond scission in the direct vicinity of crosslinks in collagen. Radicals migrate to adjacent clusters of aromatic residues and stabilize on oxidized tyrosyl radicals, giving rise to a distinct EPR spectrum consistent with a stable dihydroxyphenylalanine (DOPA) radical. The protein mechanoradicals, as a yet undiscovered source of oxidative stress, finally convert into hydrogen peroxide. Our study suggests collagen I to have evolved as a radical sponge against mechano-oxidative damage and proposes a mechanism for exercise-induced oxidative stress and redox-mediated pathophysiological processes.

Quantitative Assessment of Tendon Hierarchical Structure by Combined Second Harmonic Generation and Immunofluorescence Microscopy.

Histological evaluation of healing tendons is primarily focused on monitoring restoration of longitudinal collagen alignment, although the elastic property of energy-storing flexor tendons is largely attributed to interfascicular sliding facilitated by the interfascicular matrix (IFM). The objectives of this study were to explore the utility of second harmonic generation (SHG) imaging to objectively assess cross-sectional tendon fascicle architecture, to combine SHG microscopy with elastin immunofluorescence to assess the ultrastructure of collagen and elastin in longitudinal and transverse sections, and lastly, to quantify changes in IFM elastin and fascicle collagen alignment of normal and collagenase-injured flexor tendons. Paraffin-embedded transverse and longitudinal histological sections (10-µm thickness) derived from normal and collagenase-injured (6- and 16-week time-points) equine superficial digital flexor tendons were de-paraffinized, treated with Tris EDTA at 80°C for epitope retrieval, and incubated with mouse monoclonal anti-elastin antibody (1:100 dilution) overnight. Anti-mouse IgG Alexa Flour 546 secondary antibody was applied, and sections were mounted with ProLong Gold reagent with 4',6-diamidino-2-phenylindole (DAPI). Nuclei (DAPI) and elastin (Alexa Fluor 546) signals were captured by using standard confocal imaging with 405 and 543 nm excitation wavelengths, respectively. The SHG signal was captured by using a tunable Ti:Sapphire laser tuned to 950 nm to visualize type I collagen. Quantitative measurements of fascicle cross-sectional area (CSA), IFM thickness in transverse SHG-DAPI merged z-stacks, fascicle/IFM elastin area fraction (%), and elastin-collagen alignment in longitudinal SHG-elastin merged z-stacks were conducted by using ImageJ software. Using this methodology, fascicle CSA, IFM thickness, and IFM elastin area fraction (%) at 6 weeks (∼2.25-fold; ∼2.8-fold; 60% decrease; p < 0.001) and 16 weeks (∼2-fold; ∼1.5-fold; 70% decrease; p < 0.001) after collagenase injection, respectively, were found to be significantly different from normal tendon. IFM elastin and fascicle collagen alignment characterized via fast Fourier transform (FFT) frequency plots at 16 weeks demonstrated that collagen re-alignment was more advanced than that of elastin. The integration of SHG-derived quantitative measurements in transverse and longitudinal tendon sections supports comprehensive assessment of tendon structure. Our findings demonstrate the importance of including IFM and non-collagenous proteins in tendon histological evaluations, tasks that can be effectively carried out by using SHG and immunofluorescence microscopy. Impact statement This work demonstrated that second harmonic generation microscopy in conjunction with elastin immunofluorescence provided a comprehensive assessment of multiscale structural re-organization in healing tendon than when restricted to longitudinal collagen fiber alignment alone. Utilizing this approach for tendon histomorphometry is ideal not only to improve our understanding of hierarchical structural changes that occur after tendon injury and during remodeling but also to monitor the efficacy of therapeutic approaches.

An insight on type I collagen from horse tendon for the manufacture of implantable devices.

Type I collagen is the most abundant protein of the human body. Due to its favourable properties, collagen extracted from animal tissues is adopted to manufacture a wide range of devices for biomedical applications. Compared to bovine and porcine collagens, which are the most largely used, equine collagen is free from the risk of zoonosis, has no reported immune reactions, and has not religious constraints. In this work, a recently available type I collagen extracted from horse tendon was evaluated and compared with a commercially available collagen isoform derived from the same species and tissue. Detailed physical, chemical and biological investigations were performed, in agreement with the requirements of the current standard for the characterization of type I collagen to be used for the manufacture of Tissue Engineering Medical Products. To the best of our knowledge, this is the first report on the complete primary structure of the investigated collagen.

Micro- and nano-bone analyses of the human mandible coronoid process and tendon-bone entheses.

The coronoid process provides attachment to temporalis and masseter muscles, and thus plays an important role in mastication. Tendons connect muscles and bones, mediating the transmission of functional loads to bones. Thus, tendon-bone entheses govern mechanical stress in bones. The preferential orientation of biological apatite (BAp) crystallites, the main mineral component in bones, is an important index for bone quality and function, and is largely influenced by locally applied stress. In this study, we analyzed BAp orientation, Young's modulus, and bone mineral density (BMD) at different sites in the human coronoid process. No differences in BMD were found among the analyzed sites, but BAp crystal orientation was observed to differ. BAp crystallites showed a uni-directional orientation in the mesiodistal direction at the coronoid process apex, but were oriented in the direction vertical to the occlusal plane at other sites. Young's modulus tended to vary according to the BAp orientation. At the apex, a tendon form with characteristics different from those at other sites, including the presence of a fibrocartilaginous layer that may act as a stretching brake to control stress concentration, was observed. These findings suggest that the functional pressure of the temporalis muscle affects bone quality and strength.