An automated method for tendon image segmentation on ultrasound using grey-level co-occurrence matrix features and hidden Gaussian Markov random fields.

BACKGROUND: Despite knowledge of qualitative changes that occur on ultrasound in tendinopathy, there is currently no objective and reliable means to quantify the severity or prognosis of tendinopathy on ultrasound. OBJECTIVE: The primary objective of this study is to produce a quantitative and automated means of inferring potential structural changes in tendinopathy by developing and implementing an algorithm which performs a texture based segmentation of tendon ultrasound (US) images. METHOD: A model-based segmentation approach is used which combines Gaussian mixture models, Markov random field theory and grey-level co-occurrence (GLCM) features. The algorithm is trained and tested on 49 longitudinal B-mode ultrasound images of the Achilles tendons which are labelled as tendinopathic (24) or healthy (25). Hyperparameters are tuned, using a training set of 25 images, to optimise a decision tree based classification of the images from texture class proportions. We segment and classify the remaining test images using the decision tree. RESULTS: Our approach successfully detects a difference in the texture profiles of tendinopathic and healthy tendons, with 22/24 of the test images accurately classified based on a simple texture proportion cut-off threshold. Results for the tendinopathic images are also collated to gain insight into the topology of structural changes that occur with tendinopathy. It is evident that distinct textures, which are predominantly present in tendinopathic tendons, appear most commonly near the transverse boundary of the tendon, though there was a large variability among diseased tendons. CONCLUSION: The GLCM based segmentation of tendons under ultrasound resulted in distinct segmentations between healthy and tendinopathic tendons and provides a potential tool to objectively quantify damage in tendinopathy.

Synergistic effects of mechanical stimulation and crimped topography to stimulate natural collagen development for tendon engineering.

Suitable scaffold structures and mechanical loading are essential for functional tendon engineering. However, the bipolar fibril structure of native tendon collagen is yet to be recaptured in engineered tendons. This study compared the development of Achilles tendons of postnatal rats with and without (via surgical section) mechanical loading to define the mechanism of mechanical stimulation-mediated tendon development. The results demonstrated that the severed tendons weakened mechanically and exhibited disorganization without a bipolar fibril superstructure. Proteomic analysis revealed differentially expressed key regulatory molecules related to the collagen assembly process, including decreased fibromodulin, keratocan, fibroblast growth factor-1, and increased lumican and collagen5a1 in the severed tendons with immunohistochemical verification. Additionally, a complex regulatory network of mechanical stimulation-mediated collagen assembly in a spatiotemporal manner was also revealed using bioinformatics analysis, wherein PI3K-Akt and HDAC4 may be the predominant signaling pathways. A wavy microgrooved surface (Y = 5.47sin(0.015x)) that biomimics tendon topography was observed to enhance the expression of collagen assembly molecules under mechanical loading, and the aforementioned pathways are particularly involved and verified with their respective inhibitors of LY-294002 and LMK-235. Furthermore, an electrospun crimped nanofiber scaffold (approximately 2 µm fiber diameter and 0.12 crimpness) was fabricated to biomimic the tenogenic niche environment; this was observed to be more effective on enhancing collagen production and assembly under mechanical stimulation. In conclusion, the synergistic effect between topographical niche and mechanical stimulation was observed to be essential for collagen assembly and maturation and should be applied to functional tendon engineering in the future. STATEMENT OF SIGNIFICANCE: In biomaterial-mediated tendon regeneration, mechanical stimulation is essential for tendon collagen assembly. However, the underlying mechanisms remain not fully defined, leading to the failure of the native-like collagen regeneration. In this study, a mechanical stimulation deprivation model of rat tendon was established to reveal the mechanisms in tendon development and define the key regulatory molecules including small leucine-rich proteoglycans, lysyl oxidase and collagen V. After ensuring the importance of biomimetic structure in tendon remodeling, crimped nanofibers were developed to verify these regulatory molecules, and demonstrated that mechanical stimulation significantly enhanced collagen assembly via PIK3 and HDAC4 pathways in biomaterial-regulated tendon regeneration. This study provides more insightful perspectives in the physiologically remodeling progression of tendon collagen and design of tendon scaffolds.

Liquid-Exfoliated Mesostructured Collagen from the Bovine Achilles Tendon as Building Blocks of Collagen Membranes.

Mesoscaled assemblies are organized in native collagen tissues to achieve remarkable and diverse performance and functions. In this work, a facile, low-cost, and controllable liquid exfoliation method was applied to directly extract these collagen mesostructures from bovine Achilles tendons using a sodium hydroxide (NaOH)/urea aqueous system with freeze-thaw cycles and sonication. A series of collagen fibrils with diameters of 26-230 nm were harvested using this process, and in situ observations under polarizing microscopy (POM) and using molecular dynamics simulations revealed the influence of the NaOH/urea system on the tendon collagen. FTIR and XRD results confirmed that these collagen fibrils preserved typical structural characteristics of type I collagen. These isolated collagen fibrils were then utilized as building blocks to fabricate free-standing collagen membranes, which exhibited good stability in solvents and outstanding mechanical properties and transparency, with potential for utility in optical and electronic sensors. Moreover, in vitro and vivo evaluations demonstrated that these new resulting collagen membranes had good cytocompatibility, biocompatibility, and degradability for potential applications in biomedicine. This work provides a new approach for collagen processing by liquid exfoliation with utility for the formation of robust collagen materials that consist of native collagen mesostructures as building blocks.

The effects of both jump/land phases and direction on Achilles tendon loading.

BACKGROUND: Athletes in jumping and running sports have a high incidence of Achilles tendon (AT) injuries. We compared AT loading during jumping and landing phases in anteroposterior (AP) and mediolateral (ML) directions. METHODS: Sixteen males (age: 21.6±1.8 years, height: 178.4±6.4 cm, weight: 76.4±11.2 kg) performed single leg AP and ML jump-landings during both propulsive (jump) and braking (land) phases. Inverse dynamics and static optimization were used to determine muscle forces. AT cross sectional area was measured with ultrasound. AT force was divided by cross sectional area to determine stress while strain was determined from previous data. Two-way repeated measures analysis of variance (α=0.05) compared several variables (vertical ground reaction force (VGRF), ankle and knee angle, ankle joint muscle moment arm, external ankle moment arm, AT tendon force, stress, and strain) between movements (jump-landings) and directions (AP/ML). RESULTS: AT loading was higher during jump than land in the ML compared to AP direction. VGRF was higher during land versus jump with no direction effect (AP/ML). An interaction showed a higher VGRF during the AP land and ML jump. The ankle joint moment arm was lower in jump and AP direction at peak tendon stress. External ankle moment arm at peak tendon stress was higher in jump and ML direction with an interaction. A larger external ankle moment arm occurred in ML but the change was less in the jump. CONCLUSIONS: Higher tendon loading occurred during the jump and ML direction. This may provide insight into both injuries and rehabilitation efforts.

Evaluation of an elastic decellularized tendon-derived scaffold for the vascular tissue engineering application.

Due to the limited success rate of currently available vascular replacements, tissue engineering has received tremendous attention in recent years. A main challenge in the field of regenerative medicine is creating a mechanically functional tissue with a well-organized extracellular matrix, particularly of collagen and elastin. In this study, the native collagen scaffold derived from decellularized tendon sections, as a scaffold having the potential to be used for vascular tissue engineering applications, was studied. We showed that the elasticity of the scaffolds was improved when crosslinked with the bovine elastin. The effect of different concentrations of elastin on mechanical properties of the collagen scaffolds was evaluated of which 15% elastin concentration was selected for further analysis based on the results. Addition of 15% elastin to collagen scaffolds significantly decreased Young's modulus and the tensile stress at the maximum load and increased the tensile strain at the maximum load of the constructs as compared to those of the collagen scaffolds or control samples. Moreover, tubular elastin modified collagen scaffolds showed significantly higher burst pressure compared to the control samples. Smooth muscle cells and endothelial cells cultured on the elastin modified collagen scaffolds showed high viability (>80%) after 1, 3, and 7 days. Furthermore, the cells showed a high tendency to align with the collagen fibers within the scaffold and produced their own extracellular matrix over time. In conclusion, the results show that the decellularized tendon sections have a great potential to be used as scaffolds for vascular tissue engineering applications. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1225-1234, 2019.

Spin locking in liquid entrapped in nanocavities: Application to study connective tissues.

Study of the spin-lattice relaxation in the spin-locking state offers important information about atomic and molecular motions, which cannot be obtained by spin lattice relaxation in strong external magnetic fields. The application of this technique for the investigation of the spin-lattice relaxation in biological samples with fibril structures reveals an anisotropy effect for the relaxation time under spin locking, T1ρ. To explain the anisotropy of the spin-lattice relaxation under spin-locking in connective tissue a model which represents a tissue by a set of nanocavities containing water is used. The developed model allows us to estimate the correlation time for water molecular motion in articular cartilage, τc=30µs and the averaged nanocavity volume, V≃5400nm3. Based on the developed model which represents a connective tissue by a set of nanocavities containing water, a good agreement with the experimental data from an articular cartilage and a tendon was demonstrated. The fitting parameters were obtained for each layer in each region of the articular cartilage. These parameters vary with the known anatomic microstructures of the tissue. Through Gaussian distributions to nanocavity directions, we have calculated the anisotropy of the relaxation time under spin locking T1ρ for a human Achilles tendon specimen and an articular cartilage. The value of the fitting parameters obtained at matching of calculation to experimental results can be used in future investigations for characterizing the fine fibril structure of biological samples.

Natural zeolite for adsorbing and release of functional materials.

Using multiphoton microscopy (MPM), we demonstrated that effective inducing of two-photon excited luminescence and second-harmonic generation signals in nano/microparticles of clinoptilolite type of zeolite (CZ) by femtosecond near-infrared laser excitation can be successfully utilized in multiphoton imaging of the drug adsorption processes. Adsorption of photodynamic active dyes (hypericin, chlorin e6, methylene blue, and fluorescein) and their release from CZ pores in the presence of biomolecules, such as collagen from bovine Achilles tendon, albumin, and hemoglobin, were investigated by absorption and fluorescence spectrometry. To quantify the experimental results on hypericin release, here we use a kinetic curves fitting approach and calculate hypericin release rates in different environments. This approach allows to compare various mathematical models and uses more parameters to better characterize drug release profiles. In addition, magnetic CZ particles were fabricated and proposed as a promising material for drug delivery and controlled release in biological systems. Optical spectrometry and MPM are effective approaches that may reveal potential of natural zeolites in controlled drug delivery and biomedical imaging.

Effects of maturation and advanced glycation on tensile mechanics of collagen fibrils from rat tail and Achilles tendons.

Connective tissues are ubiquitous throughout the body and consequently affect the function of many organs. In load bearing connective tissues like tendon, the mechanical functionality is provided almost exclusively by collagen fibrils that in turn are stabilized by covalent cross-links. Functionally distinct tendons display different cross-link patterns, which also change with maturation, but these differences have not been studied in detail at the fibril level. In the present study, a custom built nanomechanical test platform was designed and fabricated to measure tensile mechanics of individual fibrils from rat tendons. The influence of animal maturity (4 vs. 16 week old rats) and functionally different tendons (tail vs. Achilles tendons) were examined. Additionally the effect of methylglyoxal (MG) treatment in vitro to form advanced glycation end products (AGEs) was investigated. Age and tissue type had no significant effect on fibril mechanics, but MG treatment increased strength and stiffness without inducing brittleness and gave rise to a distinct three-phase mechanical response corroborating that previously reported in human patellar tendon fibrils. That age and tissue had little mechanical effect, tentatively suggest that variations in enzymatic cross-links may play a minor role after initial tissue formation. STATEMENT OF SIGNIFICANCE: Tendons are connective tissues that connect muscle to bone and carry some of the greatest mechanical loads in the body, which makes them common sites of injury. A tendon is essentially a biological rope formed by thin strands called fibrils made of the protein collagen. Tendon function relies on the strength of these fibrils, which in turn depends on naturally occurring cross-links between collagen molecules, but the mechanical influence of these cross-links have not been measured before. It is believed that beneficial cross-linking occurs with maturation while additional cross-linking with aging may lead to brittleness, but this study provides evidence that maturation has little effect on mechanical function and that age-related cross-linking does not result in brittle collagen fibrils.

Effects of tissue fixation and dehydration on tendon collagen nanostructure.

Collagen is the most prominent protein in biological tissues. Tissue fixation is often required for preservation or sectioning of the tissue. This may affect collagen nanostructure and potentially provide incorrect information when analyzed after fixation. We aimed to unravel the effect of 1) ethanol and formalin fixation and 2) 24h air-dehydration on the organization and structure of collagen fibers at the nano-scale using small and wide angle X-ray scattering. Samples were divided into 4 groups: ethanol fixed, formalin fixed, and two untreated sample groups. Samples were allowed to air-dehydrate in handmade Kapton pockets during the measurements (24h) except for one untreated group. Ethanol fixation affected the collagen organization and nanostructure substantially and during 24h of dehydration dramatic changes were evident. Formalin fixation had minor effects on the collagen organization but after 12h of air-dehydration the spatial variation increased substantially, not evident in the untreated samples. Generally, collagen shrinkage and loss of alignment was evident in all samples during 24h of dehydration but the changes were subtle in all groups except the ethanol fixed samples. This study shows that tissue fixation needs to be chosen carefully in order to preserve the features of interest in the tissue.

Amino acid composition in determination of collagen origin and assessment of physical factors effects.

The amino acid composition of collagen is a characteristic feature of this protein. Collagen, irrespective of its origin, contains 19 amino acids, including hydroxyproline which does not occur in other proteins. Its atypical amino acid composition is characterized by high content of proline and glycine, as well as the absence of cysteine. This paper shows the comparison of qualitative composition of amino acids of fish skin (FS) collagen, bovine Achilles tendon (BAT) collagen, and bone collagen. Results demonstrate that FS collagen as well as BAT collagen contains no cysteine and significantly different amount of hydroxyproline. In BAT collagen hydroxyproline content is 30% higher than hydroxyproline content of FS collagen. In bone collagen the amount of hydroxyproline is two times more than in FS collagen. Furthermore, it is shown that sensitivity to radiation of individual amino acids varies and depends on the absorbed dose of ionizing radiation. The changes observed in the amino acid composition become very intense for the doses of 500kGy and 1000kGy.

Manipulation of Foot Strike and Footwear Increases Achilles Tendon Loading During Running.

BACKGROUND: The Achilles tendon is the most common site of tendon overuse injury in humans. Running with a forefoot strike pattern and in minimal shoes is a topic of recent interest, yet evidence is currently limited regarding the combined influence of foot strike and footwear on Achilles tendon loading. PURPOSE: To investigate the influence of both foot strike and footwear on Achilles tendon loading in habitual rearfoot strike runners. STUDY DESIGN: Controlled laboratory study. METHODS: Synchronized kinematic and force data were collected from 22 habitual rearfoot strikers (11 male), who habitually ran in nonminimal running shoes, during overground running at 3.6 m·s-1. Participants ran in 3 different footwear conditions (standard running shoe, minimal running shoe, and barefoot) with both a rearfoot strike (RFS) and an imposed forefoot strike (FFS) in each footwear condition. Achilles tendon loading was estimated by use of inverse dynamics, where the Achilles tendon moment arm was determined with a regression equation. A 2-way, repeated-measures analysis of variance was used to compare conditions. RESULTS: Achilles tendon impulse was greater when subjects ran with an FFS rather than an RFS in minimal shoes. Achilles tendon loading rates were higher when subjects ran either in minimal shoes or barefoot than in standard shoes, regardless of foot strike. CONCLUSION: In runners who habitually rearfoot strike in standard running shoes, running in minimal shoes or barefoot increased the rate of tendon loading, and running with a forefoot strike in minimal shoes increased the magnitude of tendon loading. CLINICAL RELEVANCE: Transitioning to these running conditions may increase the risk of tendinopathy.

Fabrication and characterization of a decellularized bovine tendon sheet for tendon reconstruction.

Obtaining a performing decellularized tendon scaffold with proper dimensions and adequate availability is highly desirable. However, the combined study of complete decellularization and detailed characterization of native tendon extracellular matrix (ECM) from large animals is still lacking. In the present study, we developed a new decellularization protocol, including physical methods and enzymatic solutions for processing bovine Achilles tendons, and produced a decellularized bovine tendon sheet (DBTS) scaffold for tendon reconstruction. The decellularization effectiveness was demonstrated by DNA quantification and histological qualification. The removal of the alpha-gal epitopes was confirmed by ELISA analysis and immunohistochemical staining. After decellularization, there were no significant alterations of the native tendon extracellular matrix (ECM) properties, including the internal ultrastructure, biochemical compositions such as collagen, glycosaminoglycans (GAGs), basic fibroblast growth factor (bFGF) and transforming growth factor-ß1 (TGF-ß1), fibronectin and decorin, as well as substantial mechanical strength. Furthermore, the DBTS scaffold showed no cytotoxic and promoted the proliferation of NIH-3T3 fibroblasts in vitro. When implanted into rat subcutaneous tissue, the DBTS scaffold displayed excellent histocompatibility in vivo. Our results, while offering a new decellularization protocol for large tendons, can provide a promising biologic scaffold with a combination of mechanical strength and tendon ECM bioactive factors that may have many potential applications in tendon reconstruction. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2299-2311, 2017.

Three dimensional microstructural network of elastin, collagen, and cells in Achilles tendons.

Similar to most biological tissues, the biomechanical, and functional characteristics of the Achilles tendon are closely related to its composition and microstructure. It is commonly reported that type I collagen is the predominant component of tendons and is mainly responsible for the tissue's function. Although elastin has been found in varying proportions in other connective tissues, previous studies report that tendons contain very small quantities of elastin. However, the morphology and the microstructural relationship among the elastic fibres, collagen, and cells in tendon tissue have not been well examined. We hypothesize the elastic fibres, as another fibrillar component in the extracellular matrix, have a unique role in mechanical function and microstructural arrangement in Achilles tendons. It has been shown that elastic fibres present a close connection with the tenocytes. The close relationship of the three components has been revealed as a distinct, integrated and complex microstructural network. Notably, a "spiral" structure within fibril bundles in Achilles tendons was observed in some samples in specialized regions. This study substantiates the hierarchical system of the spatial microstructure of tendon, including the mapping of collagen, elastin and tenocytes, with 3-dimensional confocal images. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1203-1214, 2017.

The combined effects of grain supplementation and tenderstretching on alpaca (Vicugna pacos) meat quality.

This study investigated the effects of feeding a mixed grain supplement and tenderstretching (TS) alpaca carcasses on meat quality. A total of 56 castrated 24month old alpacas were divided into two treatments (pasture-only, and pasture plus supplementation). Supplemented groups were fed a mixed grain ration in addition to ad lib pasture for 10weeks. Animals were slaughtered across two kill days (n=28). One half of each carcass was suspended by the pelvis (TS) prior to chilling, and the other half was Achilles tendon hung (AH). After 24h, muscles were removed and aged for 10 and 25d. TS significantly increased sarcomere length and reduced shear force and cooking loss in the m. semimembranosus. This trend was not observed in other muscles including the m. longissimus thoracis et lumborum (LL) and m. psoas major. Ageing period resulted in a marginal improvement in LL tenderness. There is clear evidence that TS improves tenderness in the hindquarter of alpacas.

Proteomic Analysis of Human Tendon and Ligament: Solubilization and Analysis of Insoluble Extracellular Matrix in Connective Tissues.

Connective tissues such as tendon, ligament and cartilage are mostly composed of extracellular matrix (ECM). These tissues are insoluble, mainly due to the highly cross-linked ECM proteins such as collagens. Difficulties obtaining suitable samples for mass spectrometric analysis render the application of modern proteomic technologies difficult. Complete solubilization of them would not only elucidate protein composition of normal tissues but also reveal pathophysiology of pathological tissues. Here we report complete solubilization of human Achilles tendon and yellow ligament, which is achieved by chemical digestion combined with successive protease treatment including elastase. The digestion mixture was subjected to liquid chromatography-mass spectrometry. The low specificity of elastase was overcome by accurate mass analysis achieved using FT-ICR-MS. In addition to the detailed proteome of both tissues, we also quantitatively determine the major protein composition of samples, by measuring peak area of some characteristic peptides detected in tissue samples and in purified proteins. As a result, differences between human Achilles tendon and yellow ligament were elucidated at molecular level.

Ring-Mesh Model of Proteoglycan Glycosaminoglycan Chains in Tendon based on Three-dimensional Reconstruction by Focused Ion Beam Scanning Electron Microscopy.

Tendons are composed of collagen fibrils and proteoglycan predominantly consisting of decorin. Decorin is located on the d-band of collagen fibrils, and its glycosaminoglycan (GAG) chains have been observed between collagen fibrils with transmission electron microscopy. GAG chains have been proposed to interact with each other or with collagen fibrils, but its three-dimensional organization remains unclear. In this report, we used focused ion beam scanning electron microscopy to examine the three-dimensional organization of the GAG chain in the Achilles tendon of mature rats embedded in epoxy resin after staining with Cupromeronic blue, which specifically stains GAG chains. We used 250 serial back-scattered electron images of longitudinal sections with a 10-nm interval for reconstruction. Three-dimensional images revealed that GAG chains form a ring mesh-like structure with each ring surrounding a collagen fibril at the d-band and fusing with adjacent rings to form the planar network. This ring mesh model of GAG chains suggests that more than two GAG chains may interact with each other around collagen fibrils, which could provide new insights into the roles of GAG chains.

Ovine tendon collagen: Extraction, characterisation and fabrication of thin films for tissue engineering applications.

Collagen is the most abundant extracellular matrix (ECM) protein in the human body, thus widely used in tissue engineering and subsequent clinical applications. This study aimed to extract collagen from ovine (Ovis aries) Achilles tendon (OTC), and to evaluate its physicochemical properties and its potential to fabricate thin film with collagen fibrils in a random or aligned orientation. Acid-solubilized protein was extracted from ovine Achilles tendon using 0.35M acetic acid, and 80% of extracted protein was measured as collagen. SDS-PAGE and mass spectrometry analysis revealed the presence of alpha 1 and alpha 2 chain of collagen type I (col I). Further analysis with Fourier transform infrared spectrometry (FTIR), X-ray diffraction (XRD) and energy dispersive X-ray spectroscopy (EDS) confirms the presence of triple helix structure of col I, similar to commercially available rat tail col I. Drying the OTC solution at 37°C resulted in formation of a thin film with randomly orientated collagen fibrils (random collagen film; RCF). Introduction of unidirectional mechanical intervention using a platform rocker prior to drying facilitated the fabrication of a film with aligned orientation of collagen fibril (aligned collagen film; ACF). It was shown that both RCF and ACF significantly enhanced human dermal fibroblast (HDF) attachment and proliferation than that on plastic surface. Moreover, cells were distributed randomly on RCF, but aligned with the direction of mechanical intervention on ACF. In conclusion, ovine tendon could be an alternative source of col I to fabricate scaffold for tissue engineering applications.

Can ultrashort-TE (UTE) MRI sequences on a 3-T clinical scanner detect signal directly from collagen protons: freeze-dry and D2 O exchange studies of cortical bone and Achilles tendon specimens.

Ultrashort-TE (UTE) sequences can obtain signal directly from short-T2 , collagen-rich tissues. It is generally accepted that bound and free water can be detected with UTE techniques, but the ability to detect protons directly on the collagen molecule remains controversial. In this study, we investigated the potential of UTE sequences on a 3-T clinical scanner to detect collagen protons via freeze-drying and D2 O-H2 O exchange studies. Experiments were performed on bovine cortical bone and human Achilles tendon specimens, which were either subject to freeze-drying for over 66 h or D2 O-H2 O exchange for 6 days. Specimens were imaged using two- and three-dimensional UTE with Cones trajectory techniques with a minimum TE of 8 µs at 3 T. UTE images before treatment showed high signal from all specimens with bi-component T2 * behavior. Bovine cortical bone showed a shorter T2 * component of 0.36 ms and a longer T2 * component of 2.30 ms with fractions of 78.2% and 21.8% by volume, respectively. Achilles tendon showed a shorter T2 * component of 1.22 ms and a longer T2 * component of 15.1 ms with fractions of 81.1% and 18.9% by volume, respectively. Imaging after freeze-drying or D2 O-H2 O exchange resulted in either the absence or near-absence of signal. These results indicate that bound and free water are the sole sources of UTE signal in bovine cortical bone and human Achilles tendon samples on a clinical 3-T scanner. Protons on the native collagen molecule are not directly visible when imaged using UTE sequences. Copyright © 2016 John Wiley & Sons, Ltd.

Birefringence of collagen fibres in rat calcaneal tendons treated with acupuncture during three phases of healing.

BACKGROUND: Birefringence is an optical anisotropy that is investigated by polarisation microscopy, and has been valuable for the study of the oriented organisation of collagen fibres in tendons. However, the application of this technology to evaluate the effect of different acupuncture points during tendon healing has not yet been described. OBJECTIVES: To evaluate the concentration of non-collagenous proteins (NCP) and birefringence in rat calcaneal tendons following injury during the three different phases of healing: inflammatory (7th day), proliferative (14th day), and remodelling (21st day). METHODS: Tendons of 120 Wistar rats were tenotomised and left untreated (teno group, n=24), treated with manual acupuncture at ST36 (ST36 group, n=24), BL57 (BL57 group, n=24) or ST36+BL57 (SB group, n=24), or treated with electroacupuncture at ST36+BL57 (EA group, n=24). Tendon samples were collected at 7, 14 and 21 days after injury (n=8 per group). NCP concentrations were measured using the Bradford method (n=4 each) and birefringence was examined using polarisation microscopy and image analysis (n=4 each). Comparison was also made with healthy (non-tenotomised) tendons in a subgroup of rats (n=4 each). RESULTS: Manual acupuncture at ST36 and BL57 increased molecular organisation of collagen fibres on day 14 and 21 after injury. Isolated use of BL57 and ST36 also increased collagen fibre organisation when examined on day 14 and 21, respectively. No significant increase in NCP concentration was observed in any of the treated tenotomised groups. CONCLUSIONS: Acupuncture, through putative anti-inflammatory and mechanotransductor effects, may have a role in strengthening tendons and increasing resistance to re-rupture.

Automatic Evaluation of Collagen Fiber Directions from Polarized Light Microscopy Images.

Mechanical properties of the arterial wall depend largely on orientation and density of collagen fiber bundles. Several methods have been developed for observation of collagen orientation and density; the most frequently applied collagen-specific manual approach is based on polarized light (PL). However, it is very time consuming and the results are operator dependent. We have proposed a new automated method for evaluation of collagen fiber direction from two-dimensional polarized light microscopy images (2D PLM). The algorithm has been verified against artificial images and validated against manual measurements. Finally the collagen content has been estimated. The proposed algorithm was capable of estimating orientation of some 35 k points in 15 min when applied to aortic tissue and over 500 k points in 35 min for Achilles tendon. The average angular disagreement between each operator and the algorithm was -9.3±8.6° and -3.8±8.6° in the case of aortic tissue and -1.6±6.4° and 2.6±7.8° for Achilles tendon. Estimated mean collagen content was 30.3±5.8% and 94.3±2.7% for aortic media and Achilles tendon, respectively. The proposed automated approach is operator independent and several orders faster than manual measurements and therefore has the potential to replace manual measurements of collagen orientation via PLM.