Chick Tendon Research Articles

Structural determinants of tendon multiscale mechanics and their sensitivity to mechanical stimulation during development in an embryonic chick model

There is an abrupt increase in the multiscale mechanical properties and load-bearing capabilities of tendon during development. While prior work has identified numerous changes that occur within the collagenous structure during this developmental period, the primary structural elements that give rise to this abrupt increase in mechanical functionality, and their mechanobiological sensitivity, remain unclear. To address this knowledge gap, we used a shear lag model along with ultrastructural imaging, biochemical/thermodynamic assays, and multiscale mechanical testing to investigate the dynamic structure-function relationships during late-stage embryonic chick development and to establish their sensitivity to mechanical stimulation. Mechanical testing and modeling suggested that the rapid increase in multiscale mechanics can be explained by increases in fibril length, intrafibrillar crosslinking, and fibril area fraction. To partially test this, we inhibited collagen crosslinking during development and observed a drastic reduction in multiscale mechanical behavior that was explained by a reduction in both fibril modulus and length. Using muscle paralysis to investigate mechanosensitivity, we observed a significantly impaired multiscale mechanical response despite minimal changes in fibril diameter and fibril area fraction. Additionally, the shear lag model found a trend toward lower fibril lengths with paralysis and experimental data found decreased crosslinking and fibril modulus values following flaccid paralysis. Together, these data suggest that both intrafibrillar crosslink formation and fibril elongation are critical to the formation of load-bearing capabilities in tenogenesis and are sensitive to mechanical loading. These findings provide critical insights into the biological and structural mechanisms that give rise to tensile load-bearing soft tissue. Statement of SignificanceDespite prior work investigating the structural and mechanical changes that occur during tendon development, there has not been a comprehensive analysis of how these simultaneous changes in structure and function are connected. In this study, we performed a comprehensive battery of mechanical and structural assessments of embryonic chick tendons and input these data into a shear lag model to estimate the individual importance of each structural change to the tendon mechanical properties. Additionally, we inhibited muscle activity in the embryos to evaluate the impact of mechanical stimulation on these evolving structure-function relationships during tendon development. These data provide insight into the primary structural elements that produce the tensile load-bearing capabilities of tendon, which will inform efforts to produce tissue engineered tendon replacements.

Templated freezing: a simple method may increase gripping force of the clamp on the tendon

PurposeTo evaluate the effectiveness of combining a customized mold with frozen conventional clamps against other freezing and non-freezing methods. MethodsForty-five porcine and 45 chicken tendons were evenly divided into five groups (n = 9 + 9/group): control group, non-freezing with gauze placed between tendon and clamp (gauze), non-freezing with suture fixation at tendon ends (suture), freezing with dry ice pocket placed at the clamps (pocket), and freezing using a templated liquid nitrogen clamp with a customized mold (mold). Tension tests were used to measure failure modes and loads.ResultSlippage and avulsion were observed in non-freezing groups with significantly lower failure loads compared to freezing methods. With freezing, rupture occurred near the central point only in the mold group. The failure loads for porcine tendons in the mold group were higher (2121.651 ± 73.101 N) than the pocket group (1746.337 ± 68.849 N). The failure loads of chicken tendons in the mold (243.552 ± 15.881 N) and pocket groups (260.647 ± 22.161 N) were not statistically different.ConclusionFreezing clamps represent the better choice for soft tissue clamping. The customized mold method could improve gripping effectiveness.

Intravascular polarization-sensitive optical coherence tomography based on polarization mode delay

Intravascular polarization-sensitive optical coherence tomography (IV-PSOCT) provides depth-resolved tissue birefringence which can be used to evaluate the mechanical stability of a plaque. In our previous study, we reported a new strategy to construct polarization-sensitive optical coherence tomography in a microscope platform. Here, we demonstrated that this technology can be implemented in an endoscope platform, which has many clinical applications. A conventional intravascular OCT system can be modified for IV-PSOCT by introducing a 12-m polarization-maintaining fiber-based imaging probe. Its two polarization modes separately produce OCT images of polarization detection channels spatially distinguished by an image separation of 2.7 mm. We experimentally validated our IV-PSOCT with chicken tendon, chicken breast, and coronary artery as the image samples. We found that the birefringent properties can be successfully visualized by our IV-PSOCT.

Three-Dimensional Tendon Scaffold Loaded with TGF-β1 Gene Silencing Plasmid Prevents Tendon Adhesion and Promotes Tendon Repair.

Tendon adhesion formation is associated with the aberrant expression of many genes, and interfering with the expression of these genes can prevent adhesion and promote tendon repair. Recent studies have found that silencing the transforming growth factor β-1 (TGF-β1) gene can reduce the occurrence of tendon adhesions. The development of tissue engineering and three-dimensional (3D) printing technology have provided new solutions for tendon repair. In this study, TGF-β1 gene silencing microRNA (miRNA) based RNAi plasmid was loaded on a 3D tendon scaffold using 3D printing technology. In vitro experiments confirmed the sustained release of plasmid and the good biocompatibility of the printed tendon scaffold. Subsequently, the TGF-β1 gene silencing plasmid loaded tendon scaffold was implanted in a chicken tendon defect model to evaluate the effect of the scaffold in vivo. The results from biomechanical tests and histological examinations showed that the scaffold not only promoted tendon regeneration but also prevented tendon adhesion, which was conducive to the recovery of biofunction. Evaluation of protein expression showed that the loaded plasmids prevented tendon adhesion and promoted tendon functional repair via silencing of the TGF-β1 gene.

Instant polarized light microscopy for imaging collagen microarchitecture and dynamics.

Collagen fibers are a primary load-bearing component of connective tissues and are therefore central to tissue biomechanics and pathophysiology. Understanding collagen architecture and behavior under dynamic loading requires a quantitative imaging technique with simultaneously high spatial and temporal resolutions. Suitable techniques are thus rare and often inaccessible. In this study, we present instant polarized light microscopy (IPOL), in which a single snapshot image encodes information on fiber orientation and retardance, thus fulfilling the requirement. We utilized both simulation and experimental data from collagenous tissues of chicken tendon, sheep eye, and porcine heart to evaluate the effectiveness of IPOL as a quantitative imaging technique. We demonstrate that IPOL allows quantitative characterization of micron-scale collagen fiber architecture at full camera frame rates (156 frames/second herein).

Processing conditions using electric discharge and optical characteristics for a short multimode fiber imaging probe

Using a short multimode fiber (SMMF) imaging probe with a diameter of 125μm and a length of 7.4 mm, the full-field optical coherence microscopy (FF OCM) to measure sectional images of in vivo rat brains deeper than 4 mm have been reported (Sato, 2019). However, since the tip of the SMMF was as cut, the process should be studied for smooth insertions into tissues and appropriate contacts to tissues. The SMMF processing conditions using electric discharge and the optical characteristics of the processed SMMF were investigated. Using a high voltage of 12,000 V for discharge duration of 11 s, the fiber tips of SMMF were processed to fabricate a spherical shape with a 77μm curvature radius. The measured imaging conditions approximate the optical model calculations of the parabolic refractive index and the spherical surface. Local blur and distortions in the measured images were identified in detail. Using the processed SMMF with a length of 7.35 mm and a diameter of 125μm, the transmission image of a chicken tendon samples closely corresponded to the image recorded with an optical microscope.

Ultrastructural study of the three-dimensional tenocyte network in newly hatched chick Achilles tendons using serial block face-scanning electron microscopy.

The lateral cytoplasmic processes of tenocytes extend to form three-dimensional network surrounding collagen fibers. It is unknown whether connections between two cytoplasmic processes involve overlapping of the processes or merely surface contact. In this study, the two-dimensional and three-dimensional structure of tenocytes in the Achilles tendons of the newly hatched chicks were studied using transmission electron microscopy and serial block face-scanning electron microscopy. Observation of the two-dimensional structures revealed various forms of cellular connections, including connections between the cytoplasmic processes of adjacent tenocytes and between the cytoplasmic process of tenocytes and fibroblasts. Three-dimensional observation showed spike-like cytoplasmic processes extending from one tenocyte that interlocked with cytoplasmic processes from other tenocytes. Cytoplasmic processes from each tenocyte within the chick tendons interlocked to ensure a tight cell-to-cell connection around growing collagen fibers. A cellular network formed by these cytoplasmic processes surrounds each collagen fiber. Cell-cell junctions, which were suggested to be gap junctions, observed at sites of cytoplasmic process overlap most likely represent the major route for communication between tenocytes associated with fibroblasts, enabling vital signals important for maintaining the cell and tendon integrity to be transmitted.

Demonstration of flat-top beam illumination in widefield multiphoton microscopy.

.Multiphoton microscopy provides a suitable technique for imaging biological tissues with submicrometer resolution. Usually a Gaussian beam (GB) is used for illumination, leading to a reduced power efficiency in the multiphoton response and vignetting for a square-shaped imaging area. A flat-top beam (FTB) provides a uniform spatial intensity distribution that equalizes the probability of a multiphoton effect across the imaging area. We employ a customized widefield multiphoton microscope to compare the performance of a square-shaped FTB illumination with that based on using a GB, for both two-photon fluorescence (TPF) and second-harmonic generation (SHG) imaging. The variation in signal-to-noise ratio across TPF images of fluorescent dyes spans for the GB and for the FTB illumination, respectively. For the GB modality, TPF images of mouse colon and Convallaria root, and SHG images of chicken tendon and human breast biopsy tissue showcase area that are not imaged due to either insufficient or lack of illumination. For quantitative analysis that depends on the illuminated area, this effect can potentially lead to inaccuracies. This work emphasizes the applicability of FTB illumination to multiphoton applications.

The influence of tantalum on human cell lineages important for healing in soft-tissue reattachment surgery: an in-vitro analysis

BackgroundPorous tantalum is currently used in orthopaedic surgery for a variety of indications including soft tissue re-attachment. However, the clinical results have been variable and a previous laboratory study has suggested that tantalum may actually inhibit chick tendon fibroblasts. The influence of tantalum on human cell-types involved in soft tissue re-attachment has not been defined.MethodsHuman fibroblasts, human osteoblasts and human mesenchymal stem cells were plated on glass cover slips, half of which were coated with tantalum. Cell numbers were assessed at 1, 2, 7 and 14 days using Cyquant® assay. Cell adhesion and morphology were assessed using light microscopy at 7, 14 and 28 days. To reduce the effect of an expected rate of error, n = 4 was utilised for each cell type and the experiment was repeated twice.ResultsStatistically similar numbers of human osteoblasts and human mesenchymal stem cells were present at 14 days on tantalum-coated and uncoated glass cover slips, revealing no inhibitory effect on cell proliferation. More than double the number of human fibroblasts was seen on tantalum-coated cover slips at that time point (compared to controls), which was statistically significant (p < 0.0001). Morphological assessment revealed normal cell spreading and adhesion on both substrates at all time points.ConclusionsIn vitro study demonstrates that Tantalum causes a significant increase in the proliferation of human fibroblasts with no quantifiable negative effects seen on fibroblast behaviour after 28 days culture. Furthermore, tantalum does not exert any inhibitory effects on the proliferation or behaviour of human osteoblasts or human mesenchymal stem cells. Tantalum could be an appropriate biomaterial for use in situations where soft tissue requires direct reattachment to implants and may stimulate soft tissue healing.

Orientation-dependent anisotropy of acoustic properties of tendons at micrometer scale

Tendons are bands of fibrous connective tissue connecting muscles to bones. They are composed of parallel arrays of collagen fibers closely packed together which makes them highly anisotropic. The anisotropy of the acoustic properties of tendons was investigated at an ultra-fine resolution (< 7 μm) using quantitative acoustic microscopy (QAM). Chicken tendons were fixed (formalin) while loaded longitudinally, then cryosectioned (16-µm) at several orientations (every 15 deg) from parallel (i.e., 0 deg) to perpendicular to the fibers (i.e., 90 deg). Two regions of two sections per angle were scanned using a QAM system operating at a center frequency of 250 MHz yielding a total of 28 QAM datasets which were processed to yield two-dimensional (2D) maps of the bulk modulus, mass density, acoustic impedance, and speed of sound for each scanned region. Acoustic parameters were averaged within each 2D map and mean and standard deviations computed at each angle. Results demonstrated a strong acoustical anisotropy. For instance acoustic impedance increased from 1.68 ± 0.08 to 1.87 ± 0.19 MRayl between 0 and 75 deg. Similarly, the speed of sound increased from 1686 ± 90 to 1958 ± 186 m/s between 0 and 75 deg. These results demonstrate the value of QAM to investigate the anisotropy of tissue microstructure and pave the way for using it to characterize other soft tissues with complex three-dimensional fiber orientations. [Work supported in part by NIH Grants EY023966 and EY028662.]

Improving resolution of second harmonic generation microscopy via scanning structured illumination.

Second harmonic generation microscopy (SHGM) is a well-known technique for examining the noncentrosymmetric structures in biomedical research. However, without real-state transitions, fluorescence-based superresolution methods cannot be applied. To improve the resolution, fringe-scanning SHGM (FS-SHGM), which combines SHGM with structured illumination based on point-scanning, is introduced in this paper. The scanning path was modulated to generate illumination patterns. For the coherent parts of SHG signals, a mathematical model of image formation and reconstruction was established. Both simulations and experiments showed a resolution improvement factor of ~1.4 in the lateral and 1.56 in the axial directions for chicken tendons and mouse skin.

The effect of increasing the contact surface on tendon healing.

BackgroundThe most common complication after tendon repair is the development of adhesion, with subsequent rupture.MethodsIn this study, we present a new method in which the tendon healing contact surface is increased to reduce these complications. The tendons of chickens in groups 1, 3, and 5 were transversely cut and repaired with in the traditional fashion with double-modified Kessler method and 5/0 polypropylene. In the other groups, 3 mm of the tendon was removed from the proximal half of the upper end and from the distal half of the lower end of the tendon, and they were repaired with the modified Kessler method. The tendons of the chickens in groups 1 and 2 were evaluated immediatelly after surgery. Groups 3 and 4 were evaluated at 4 weeks after surgery. Groups 5 and 6 were evaluated at 6 weeks.ResultsIncreases in transient inflammation and connective tissue formation were observed more clearly in the group treated with the new method in histopathological investigations at weeks 4 and 6. The stretching test showed statistically significant differences between groups 3 and 4 (P<0.05) and groups 5 and 6 (P<0.05).ConclusionsWhen repairing tendons with the new method, the healing surface increases and the direction of collagen fibers at the surface changes. Because of these effects, the strength of the tendon healing line increases; we therefore expect that this technique will enable patients to safely engage in early active exercise after the operation, with less risk of tendon rupture.

Effects of replacing soybean meal with chickpea seeds in the diet on mechanical and thermal properties of tendon tissue in broiler chicken

The efficiency of the musculoskeletal system of broiler chickens, in particular during locomotion and in ensuring its supportive function, depends directly on the adequate function and mechanical endurance of soft tissues, including tendons. However, little is known whether the properties of musculoskeletal soft tissues can be influenced by changes of dietary protein. We substituted soybean meal with raw chickpea seeds as the primary protein source in the diet and studied the effects it had on the mechanical and thermal properties of drumstick tendons in broiler Ross 308 chickens. In the experiment, 160 chicks were divided into 2 groups, receiving in their diet either soybean meal (n = 80) or chickpea seeds (n = 80). The experiment lasted 42 days. The physical condition of the drumstick tendons was analyzed on the basis of a tensile test and the results of thermal denaturation as measured by a differential scanning calorimetry. The mechanical evaluation of tendon tensile strength of the broilers fed with chickpea seeds demonstrated an increase in the ultimate strain (for over 22%, P < 0.04) and total energy absorbed by the tendon until rupture (for over 57%, P < 0.05) as when compared to the group fed with soybean meal. Thermal analysis demonstrated alterations in tendon collagen cross-linking as transition onset temperature decreased (from 63.8 to 61.8°C, P < 0.001), whereas the calorimetric enthalpy increased (from 16.2 to 22.1 J⋅g-1, P < 0.05) in the group fed with chickpea seeds. In summary, this study demonstrated that dietary protein source can impact the physical properties of tendons and showed that thermal analysis can be a useful tool for studying the effect of nutrition on the development and structural changes in tendons of broiler chickens.

6. Label-free selective plane illumination microscopy of tissue samples

Abstract type Basic Science Research. Presentation type Poster Presentation. Introduction There is an increasing demand for non-invasive, label-free, three-dimensional imaging techniques for characterisation of optically turbid samples such as tissue engineered constructs, used in Regenerative Medicine, and native tissue for medical diagnosis. Selective plane provide both high temporal and spatial information, deliver rapid results, are capable of assessing quality control criteria of engineered products and are applicable to a clinical environment. Many existing optical imaging techniques are not suitable for imaging samples that are non-transparent and turbid. In this project a unique technique which is Label-free Selective Plane Microscopy will be demonstrated. The imaging capabilities of this new method will be compared with existing techniques including bright field microscopy, fluorescence microscopy and confocal microscopy. Methodology Test samples of polystyrene microbeads were prepared to simulate tissue. Red (6 micron in diameter) and clear microbeads (8 micron in diameter) were used to represent tissues that are and are not highly perfused with blood. For comparison with fluorescence imaging techniques, samples containing fluorescent beads were also prepared. The samples were imaged using bright field microscopy, Label-free Selective Plane Microscopy, fluorescence microscopy and confocal microscopy. Native tissue samples were also studied and as above samples with different optical absorption and scattering properties. Chicken tendon and sheep kidney were used. Results Label-free Selective Plane Microscopy have the capability to image highly scattering and absorbing tissue in 3D. Label-free Selective Plane Microscopy (SPIM) is a fast method for imaging tissue samples with high lateral and axial resolution and good depth penetration. Conclusion Overall this method meets the demands of the current needs for 3D imaging tissue samples in a label-free manner. Label-free Selective Plane Microscopy directly provides excellent information about the structure of the tissue samples. This work has highlighted the superiority of Label-free Selective Plane Microscopy to current approaches to label-free 3D imaging of tissue.

Phenotypic, genotypic and antigenic characterization of emerging avian reoviruses isolated from clinical cases of arthritis in broilers in Saskatchewan, Canada

In recent years, emerging strains of pathogenic arthrogenic avian reovirus (ARV) have become a challenge to the chicken industry across USA and Canada causing significant economic impact. In this study, we characterized emerging variant ARV strains and examined their genetic and antigenic relationship with reference strains. We isolated 37 emerging variant ARV strains from tendons of broiler chickens with clinical cases of arthritis/tenosynovitis at commercial farms in Saskatchewan, Canada. Viral characterization using immunocytochemistry, gold-immunolabeling and electron microscopy revealed distinct features characteristic of ARV. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analyses of the viral Sigma C gene revealed genetic heterogeneity between the field isolates. On phylogenetic analyses, the Sigma C amino acid sequences of the isolates were clustered into four distinct genotypic groups. These ARV field strains were genetically diverse and quite distant from the vaccine and vaccine related field strains. Antibodies produced against a commercial Reo 2177 ® vaccine did not neutralize these variants. Moreover, structure based analysis of the Sigma C protein revealed significant antigenic variability between the cluster groups and the vaccine strains. To the best of our knowledge, this is the first report on the genetic, phenotypic and antigenic characterization of emerging ARVs in Canada.

Turkey model for flexor tendon research: in vitro comparison of human, canine, turkey, and chicken tendons

BackgroundFlexor tendon injuries are one of the most common hand injuries and remain clinically challenging for functional restoration. Canine and chicken have been the most commonly used animal models for flexor tendon–related research but possess several disadvantages. The purpose of this study was to explore a potential turkey model for flexor tendon research. MethodsThe third digit from human cadaveric hands, canine forepaws, turkey foot, and chicken foot were used for this study. Six digits in each of four species were studied in detail, comparing anatomy of the flexor apparatus, joint range of motioņ tendon excursion, tendon cross-sectional area, work of flexion, gliding resistance at the level of the A2 pulley, modulus of elasticity, suture retention strength, and histology across species. ResultsAnatomically, the third digit in the four species displayed structural similarities; however, the tendon cross-sectional area of the turkey and human were similar and larger than canine and chicken. Furthermore, the turkey digit resembles the human's finger with the lack of webbing between digits, similar vascularization, tendon excursion, work of flexion, gliding resistance, mechanical properties, and suture holding strength. More importantly, human and turkey tendons were most similar in histological appearance. ConclusionsTurkey flexor tendons have many properties that are comparable to human flexor tendons which would provide a clinically relevant, economical, nonhuman companion large animal model for flexor tendon research.

The effect of connective tissue growth factor (CTGF) and CTGF antibody on collagen secretion of chicken tendon synovial cells

Objective To investigate the effect of CTGF and CTGF antibody in different concentration on collagen secretion of tendon synovial cells. Methods From February, 2014 to August, 2014, tendon synovial cells were obtained from LegHorn chicken tendon synovial tissues and cultured in Petri dish with incremental concentration of CTGF or CTGF antibody, ELISA was used to detect secretion of collagen Ⅰ/Ⅲ. Results The secretion of collagenⅠincreased gradually as the concentration of CTGF elevating from 5 ng/ml to 50 ng/ml, there were significant differences between the groups (P < 0.05) . While the secretion of collagen Ⅲincreased gradually as the concentration of CTGF elevating from 10 ng/ml to 200 ng/ml, no significant difference was observed between the other groups. CollagenⅠof synovial cells showed a downward trend on secretion when cultured with CTGF antibody as the concentration elevating from 8 μg/ml to 32 μg/ml, there were significant differences between the groups (P < 0.05) . While the secretion of collagen Ⅲ showed a downward trend when cultured with CTGF antibody of the concentration 4 μg/ml, there was no significant difference between the other groups. Conclusion CTGF can promote collagen secretion of synovial cells in vitro with an optimal concentration, the concentration was 50 ng/ml (collagen I) , 200 ng/ml (collagen III) . CTGF antibody can inhibit collagen secretion of synovial cell. Key words: Connective tissue growth factor; Synovial cells; Collagen; Chicken

Imaging characteristics of an 8.8 mm long and 125 μm thick graded-index short multimode fiber probe.

We demonstrated the feasibility of an ultrathin imaging probe with a 50-μm core diameter, a 125 μm total diameter, and an 8.8 mm length, which is a typical graded-index multimode fiber for optical communications. We used an ABCD matrix to analyze the imaging conditions and magnification, which corresponded closely to the measured results. The lateral resolution was calculated at 1.2 μm with a wavelength of 730 nm, which reflects the image test pattern where a period of 4.38 μm was measured with a wavelength of 730 nm. In the numerical aperture of the objective lens, we experimentally evaluated the tradeoff between the magnification and the coupling efficiency. At four wavelengths of 540 nm, 632 nm, 730 nm, and 852 nm, the contrast and signal intensity versus the wavelength were investigated to show that the contrast at 632 and 730 nm is relatively high. By using a thin random phase screen model, we explained that as the wavelength decreases the greater the decrease in the optical transfer function at higher spatial frequencies. Using a 635 nm LED light source, we imaged the surfaces of chicken tendons in contact and the surface roughness was visible.

Chick tendon fibroblast transcriptome and shape depend on whether the cell has made its own collagen matrix.

Collagen- and fibrin-based gels are extensively used to study cell behaviour. However, 2D–3D and collagen-fibrin comparisons of gene expression, cell shape and mechanotransduction, with an in vivo reference, have not been reported. Here we compared chick tendon fibroblasts (CTFs) at three stages of embryonic development with CTFs cultured in collagen- or fibrin-based tissue engineered constructs (TECs). CTFs synthesised their own collagen matrix in fibrin-based TECs and better recapitulated the gene expression, collagen fibril alignment and cell shape seen in vivo. In contrast, cells in 3D collagen gels exhibited a 2D-like morphology and expressed fewer of the genes expressed in vivo. Analysis of YAP/TAZ target genes showed that collagen gels desensitise mechanotransduction pathways. In conclusion, gene expression and cell shape are similar on plastic and 3D collagen whereas cells in 3D fibrin have a shape and transcriptome better resembling the in vivo situation. Implications for wound healing are discussed.

High-throughput multiphoton-induced three-dimensional ablation and imaging for biotissues

In this study, a temporal focusing-based high-throughput multiphoton-induced ablation system with axially-resolved widefield multiphoton excitation has been successfully applied to rapidly disrupt biotissues. Experimental results demonstrate that this technique features high efficiency for achieving large-area laser ablation without causing serious photothermal damage in non-ablated regions. Furthermore, the rate of tissue processing can reach around 1.6 × 10(6) μm(3)/s in chicken tendon. Moreover, the temporal focusing-based multiphoton system can be efficiently utilized in optical imaging through iterating high-throughput multiphoton-induced ablation machining followed by widefield optical sectioning; hence, it has the potential to obtain molecular images for a whole bio-specimen.