Tenomodulin knockout mice exhibit worse late healing outcomes with augmented trauma-induced heterotopic ossification of Achilles tendon.

Heterotopic ossification (HO) represents a common problem after tendon injury with no effective treatment yet being developed. Tenomodulin (Tnmd), the best-known mature marker for tendon lineage cells, has important effects in tendon tissue aging and function. We have reported that loss of Tnmd leads to inferior early tendon repair characterized by fibrovascular scaring and therefore hypothesized that its lack will persistently cause deficient repair during later stages. Tnmd knockout (Tnmd-/-) and wild-type (WT) animals were subjected to complete Achilles tendon surgical transection followed by end-to-end suture. Lineage tracing revealed a reduction in tendon-lineage cells marked by ScleraxisGFP, but an increase in alpha smooth muscle actin myofibroblasts in Tnmd-/- tendon scars. At the proliferative stage, more pro-inflammatory M1 macrophages and larger collagen II cartilaginous template were detected in this group. At the remodeling stage, histological scoring revealed lower repair quality in the injured Tnmd-/- tendons, which was coupled with higher HO quantified by micro-CT. Tendon biomechanical properties were compromised in both groups upon injury, however we identified an abnormal stiffening of non-injured Tnmd-/- tendons, which possessed higher static and dynamic E-moduli. Pathologically thicker and abnormally shaped collagen fibrils were observed by TEM in Tnmd-/- tendons and this, together with augmented HO, resulted in diminished running capacity of Tnmd-/- mice. These novel findings demonstrate that Tnmd plays a protecting role against trauma-induced endochondral HO and can inspire the generation of novel therapeutics to accelerate repair.

Local anisotropy in mineralized fibrocartilage and subchondral bone beneath the tendon-bone interface.

The enthesis allows the insertion of tendon into bone thanks to several remarkable strategies. This complex and clinically relevant location often features a thin layer of fibrocartilage sandwiched between tendon and bone to cope with a highly heterogeneous mechanical environment. The main purpose of this study was to investigate whether mineralized fibrocartilage and bone close to the enthesis show distinctive three-dimensional microstructural features, possibly to enable load transfer from tendon to bone. As a model, the Achilles tendon-calcaneus bone system of adult rats was investigated with histology, backscattered electron imaging and micro-computed tomography. The microstructural porosity of bone and mineralized fibrocartilage in different locations including enthesis fibrocartilage, periosteal fibrocartilage and bone away from the enthesis was characterized. We showed that calcaneus bone presents a dedicated protrusion of low porosity where the tendon inserts. A spatially resolved analysis of the trabecular network suggests that such protrusion may promote force flow from the tendon to the plantar ligament, while partially relieving the trabecular bone from such a task. Focusing on the tuberosity, highly specific microstructural aspects were highlighted. Firstly, the interface between mineralized and unmineralized fibrocartilage showed the highest roughness at the tuberosity, possibly to increase failure resistance of a region carrying large stresses. Secondly, fibrochondrocyte lacunae inside mineralized fibrocartilage, in analogy with osteocyte lacunae in bone, had a predominant alignment at the enthesis and a rather random organization away from it. Finally, the network of subchondral channels inside the tuberosity was highly anisotropic when compared to contiguous regions. This dual anisotropy of subchondral channels and cell lacunae at the insertion may reflect the alignment of the underlying collagen network. Our findings suggest that the microstructure of fibrocartilage may be linked with the loading environment. Future studies should characterize those microstructural aspects in aged and or diseased conditions to elucidate the poorly understood role of bone and fibrocartilage in enthesis-related pathologies.

Visualization of microstructural change affected by mechanical stimulation in tendon healing with a novel tensionless model.

Since the majority of a tendon's dry weight is collagen fibers, tendon healing consists mainly of collagen repair and observing three-dimensional networks of collagen fibers with scanning electron microscopy (SEM) is optimal for investigating this process. In this report, a cell-maceration/SEM method was used to investigate extrasynovial tendon (unwrapped tendon in synovial tissue such as the tendon sheath) healing of an injured Achilles tendon in a rat model. In addition, since mechanical stimulation is important for tendon healing, a novel, tensionless, rat lower leg tendon injury model was established and verified by visualizing the structural change of collagen fibers under tensionless conditions by SEM. This new model was created by transplanting the leg of a rat with a tendon laceration to the back, removing mechanical stimulation. We then compared the process of tendon healing with and without tension using SEM. Under tension, collagen at the tendon stump shows axial alignment and repair that subsequently demarcates the paratenon (connective tissue on the surface of an extrasynovial tendon) border. In contrast, under tensionless conditions, the collagen remains randomly arranged. Our findings demonstrate that mechanical stimulation contributes to axial arrangement and reinforces the importance of tendon tension in wound healing.

Imbalanced cellular metabolism compromises cartilage homeostasis and joint function in a mouse model of mucolipidosis type III gamma.

Mucolipidosis type III (MLIII) gamma is a rare inherited lysosomal storage disorder caused by mutations in GNPTG encoding the γ-subunit of GlcNAc-1-phosphotransferase, the key enzyme ensuring proper intracellular location of multiple lysosomal enzymes. Patients with MLIII gamma typically present with osteoarthritis and joint stiffness, suggesting cartilage involvement. Using Gnptg knockout (Gnptgko ) mice as a model of the human disease, we showed that missorting of a number of lysosomal enzymes is associated with intracellular accumulation of chondroitin sulfate in Gnptgko chondrocytes and their impaired differentiation, as well as with altered microstructure of the cartilage extracellular matrix (ECM). We also demonstrated distinct functional and structural properties of the Achilles tendons isolated from Gnptgko and Gnptab knock-in (Gnptabki ) mice, the latter displaying a more severe phenotype resembling mucolipidosis type II (MLII) in humans. Together with comparative analyses of joint mobility in MLII and MLIII patients, these findings provide a basis for better understanding of the molecular reasons leading to joint pathology in these patients. Our data suggest that lack of GlcNAc-1-phosphotransferase activity due to defects in the γ-subunit causes structural changes within the ECM of connective and mechanosensitive tissues, such as cartilage and tendon, and eventually results in functional joint abnormalities typically observed in MLIII gamma patients. This idea was supported by a deficit of the limb motor function in Gnptgko mice challenged on a rotarod under fatigue-associated conditions, suggesting that the impaired motor performance of Gnptgko mice was caused by fatigue and/or pain at the joint.This article has an associated First Person interview with the first author of the paper.

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.

Development of a novel multiphysical approach for the characterization of mechanical properties of musculotendinous tissues.

At present, there is a lack of well-validated protocols that allow for the analysis of the mechanical properties of muscle and tendon tissues. Further, there are no reports regarding characterization of mouse skeletal muscle and tendon mechanical properties in vivo using elastography thereby limiting the ability to monitor changes in these tissues during disease progression or response to therapy. Therefore, we sought to develop novel protocols for the characterization of mechanical properties in musculotendinous tissues using atomic force microscopy (AFM) and ultrasound elastography. Given that TIEG1 knockout (KO) mice exhibit well characterized defects in the mechanical properties of skeletal muscle and tendon tissue, we have chosen to use this model system in the present study. Using TIEG1 knockout and wild-type mice, we have devised an AFM protocol that does not rely on the use of glue or chemical agents for muscle and tendon fiber immobilization during acquisition of transversal cartographies of elasticity and topography. Additionally, since AFM cannot be employed on live animals, we have also developed an ultrasound elastography protocol using a new linear transducer, SLH20-6 (resolution: 38 µm, footprint: 2.38 cm), to characterize the musculotendinous system in vivo. This protocol allows for the identification of changes in muscle and tendon elasticities. Such innovative technological approaches have no equivalent to date, promise to accelerate our understanding of musculotendinous mechanical properties and have numerous research and clinical applications.

Comparative multi-scale hierarchical structure of the tail, plantaris, and Achilles tendons in the rat.

Rodent tendons are widely used to study human pathologies such as tendinopathy and repair, and to address fundamental physiological questions about development, growth, and remodeling. However, how the gross morphology and multi-scale hierarchical structure of rat tendons, such as the tail, plantaris, and Achilles tendons, compare with that of human tendons are unknown. In addition, there remains disagreement about terminology and definitions. Specifically, the definitions of fascicle and fiber are often dependent on diameter sizes, not their characteristic features, and these definitions impair the ability to compare hierarchical structure across species, where the sizes of the fiber and fascicle may change with animal size and tendon function. Thus, the objective of the study was to select a single species that is commonly used for tendon research (rat) and tendons with varying mechanical functions (tail, plantaris, Achilles) to evaluate the hierarchical structure at multiple length scales using histology, SEM, and confocal imaging. With the exception of the specialized rat tail tendon, we confirmed that in rat tendons there are no fascicles and the fiber is the largest subunit. In addition, we provided a structurally based definition of a fiber as a bundle of collagen fibrils that is surrounded by elongated cells, and this definition was supported by both histologically processed and unprocessed samples. In all rat tendons studied, the fiber diameters were consistently between 10 and 50 µm, and this diameter range appears to be conserved across larger species. Specific recommendations were made highlighting the strengths and limitations of each rat tendon as a research model. Understanding the hierarchical structure of tendon can advance the design and interpretation of experiments and development of tissue-engineered constructs.

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.

Effects of freeze-thaw on the biomechanical and structural properties of the rat Achilles tendon.

Rodent models are commonly used to investigate tendon healing, with the biomechanical and structural properties of the healed tendons being important outcome measures. Tendon storage for later testing becomes necessary when performing large experiments with multiple time-points. However, it is unclear whether freezing rodent tendons affects their material properties. Thus the aim of this study was to determine whether freezing rat Achilles tendons affects their biomechanical or structural properties. Tendons were frozen at either -20 °C or -80 °C directly after harvesting, or tested when freshly harvested. Groups of tendons were subjected to several freeze-thaw cycles (1, 2, and 5) within 3 months, or frozen for 9 months, after which the tendons were subjected to biomechanical testing. Additionally, fresh and thawed tendons were compared morphologically, histologically and by transmission electron microscopy. No major differences in biomechanical properties were found between fresh tendons and those frozen once or twice at -20 °C or -80 °C. However, deterioration of tendon properties was found for 5-cycle groups and both long-term freezing groups; after 9 months of freezing at -80 °C the tear resistance of the tendon was reduced from 125.4 ±â€¯16.4N to 74.3 ±â€¯18.4N (p = 0.0132). Moreover, tendons stored under these conditions showed major disruption of collagen fibrils when examined by transmission electron microscopy. When examined histologically, fresh samples exhibited the best cellularity and proteoglycan content of the enthesis. These properties were preserved better after freezing at -80 °C than after freezing at -20 °C, which resulted in markedly smaller chondrocytes and less proteoglycan content. Overall, the best preservation of histological integrity was seen with tendons frozen once at -80 °C. In conclusion, rat Achilles tendons can be frozen once or twice for short periods of time (up to 3 months) at -20 °C or -80 °C for later testing. However, freezing for 9 months at either -20 °C or -80 °C leads to deterioration of certain parameters.

Bone Marrow Stem Cells-Seeded Polyethylene Terephthalate Scaffold in Repair and Regeneration of Rabbit Achilles Tendon.

The objective of this study was to evaluate the effect of bone marrow stem cells (BMSCs)-seeded polyethylene terephthalate (PET) scaffold for Achilles tendon repair in a rabbit model. The allogeneic BMSCs were seeded onto the PET scaffold and cultured in vitro for 14 days. Sixteen mature New Zealand rabbits underwent surgery to establish a 2-cm Achilles tendon defect model. The BMSCs-seeded PET scaffold was implanted into the defect of one limb (BMSCs-PET group), while the PET scaffold without BMSCs was implanted into the defect of contralateral limb as the control (PET group). All rabbits were sacrificed at 6 and 12 weeks after surgery. At 12 weeks after surgery, macroscopic and histological results showed formation of tendon-like tissues, and the structure was more mature in the BMSCs-PET group. Immunohistochemical analysis and real-time polymerase chain reaction (RT-PCR) demonstrated that the collagen I and collagen III were significantly higher in the BMSCs-PET group compared with those in the PET group. Mechanically, both the failure load and the average stiffness were significantly higher in the BMSCs-PET group than those in the PET group. In conclusion, BMSCs-seeded PET scaffold could effectively facilitate the healing process after being implanted in a rabbit Achilles tendon defect model.

Optimal number of chemical extraction treatments for maintaining the biological properties of an allogeneic tendon.

The aim of this study was to explore the biological effects of the amount of chemical extraction treatments performed on an allogeneic tendon through histomorphology, biological mechanics testing, and an immunogenicity assay. Sixteen New Zealand rabbits (body weight 2.5-3.0 kg) were randomly divided into four groups: group A (chemical extraction once), group B (chemical extraction twice), group C (chemical extraction three times), and group D (blank control group), with four rabbits in each group. The Achilles tendons of each rabbit were separated and subjected to a chemical extraction process with Triton X-100 and sodium deoxycholate, followed by hematoxylin and eosin staining, electron microscopy observation, biomechanical testing, and mixed lymphocyte culture. There were no significant differences in the surface color and fiber bundles between groups A and B and the blank control group, whereas group C showed clear differences from the blank control group with a rough surface, loose fibers, and poor tension. There were no significant differences in the biomechanics among the four groups. The four groups showed significant differences in the lymphocyte conversion ratio, with reduced rates of lymphocyte conversion along with increasing treatment numbers. Two chemical extractions of the tendon allowed for retaining most of the integrity of the original tendon fiber while removing immunogenicity with good biological properties. These findings lay a foundation for application of this method to human tendons so as to provide a good tissue source for tendon transplantation.

An epigenetic bioactive composite scaffold with well-aligned nanofibers for functional tendon tissue engineering.

Poor tendon repair is often a clinical challenge due to the lack of ideal biomaterials. Electrospun aligned fibers, resembling the ultrastructure of tendon, have been previously reported to promote tenogenesis. However, the underlying mechanism is unclear and the aligned fibers alone are not capable enough to commit teno-differentiation of stem cells. Here, based on our observation of reduced expression of histone deacetylases (HDACs) in tendon stem/progenitor cells (TSPCs) cultured on aligned fibers, we proposed a strategy to enhance the tenogenesis effect of aligned fibers by using a small molecule Trichostatin A (TSA), an HDAC inhibitor. Such a TSA-laden poly (l-lactic acid) (PLLA) aligned fiber (A-TSA) scaffold was successfully fabricated by a stable jet electrospinning method, and demonstrated its sustained capability in releasing TSA. We found that TSA incorporated aligned fibers of PLLA had an additive effect in directing tenogenic differentiation. Moreover, the in situ implantation study in rat model further confirmed that A-TSA scaffold promoted the structural and mechanical properties of the regenerated Achilles tendon. This study demonstrated that HDAC was involved in the teno-differentiation with aligned fiber topography, and the combination of HDAC with aligned topography might be a more efficient strategy to promote tenogenesis of stem cells. STATEMENT OF SIGNIFICANCE: Electrospun aligned fibers, resembling the ultrastructure of tendon, have been previously reported to promote tenogenesis. However, the underlying mechanism is unclear and the aligned fibers alone are not capable enough to commit teno-differentiation of stem cells. The uniqueness of our studies are as follows, based on our observation of reduced expression of histone deacetylases (HDACs) in tendon stem/progenitor cells (TSPCs) cultured on aligned fibers, we proposed a strategy to enhance the tenogenesis effect of aligned fibers by using a small molecule Trichostatin A (TSA), a HDAC inhibitor. Such a TSA-laden poly (l-lactic acid) (PLLA) aligned fiber (A-TSA) scaffold was successfully fabricated by a stable jet electrospinning method, and demonstrated its sustained capability in releasing TSA. The incorporation and subsequent release of bioactive small molecule TSA into electrospun aligned fibers allows a controllable manner for both biochemical and physical regulation of tenogenesis of stem cells both in vitro and in vivo. Collectively, the present study provides a model of "translating the biological knowledge learned from cell-material interaction into optimizing biomaterials (from Biomat-to-Biomat)".

Calcaneal Tendon Collagen Fiber Morphometry and Aging.

Fibrillar collagen in tendons and its natural development in rabbits are discussed in this paper. Achilles tendons from newborn (~7 days) to elderly (~38 months) rabbits were monitored in intact (n tendons=24) and microtome sectioned (n tendons=11) states with label-free second harmonic generation microscopy. After sectioning, the collagen fiber pattern was irregular for the younger animals and remained oriented parallel to the load axis of the tendon for the older animals. In contrast, the collagen fiber pattern in the intact samples followed the load axis for all the age groups. However, there was a significant difference in the tendon crimp pattern appearance between the age groups. The crimp amplitude (A) and wavelength (Λ) started at very low values (A=2.0±0.6 µm, Λ=19±4 µm) for the newborn animals. Both parameters increased for the sexually mature animals (>5 months old). When the animals were fully mature the amplitude decreased but the wavelength kept increasing. The results revealed that the microtome sectioning artifacts depend on the age of animals and that the collagen crimp pattern reflects the physical growth and development.

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.

Biological incorporation of human acellular dermal matrix used in Achilles tendon repair.

Human acellular dermal matrices (ADMs) are used successfully in a variety of procedures, including sports medicine related, wound repair, and breast reconstructions, but the mechanism of repair is still not fully understood. An opportunity to explore this mechanism presented itself when a patient experienced a rerupture of the native tendon due to a fall that occurred 2 months after undergoing an Achilles tendon repair using Matracell treated ADM. The ADM was removed and an extensive histology analysis was performed on the tissue. Additionally, a literature review was conducted to determine the mechanism of ADM integration into the tendon structure and explore if differences in this mechanism exist for different types of human ADMS. The histology analysis demonstrated that the healing process during a tendon reconstruction procedure is similar to that of wound healing. Furthermore, the literature review showed that differences exist in the mechanism for integration among various human ADMs and that these differences may be due to variances in the methods and technologies that manufactures use to process human ADMs.

Reconstruction of Ligament and Tendon Defects Using Cell Technologies.

We studied the possibility of restoring the integrity of the Achilles tendon in rabbits using autologous multipotent stromal cells. Collagen or gelatin sponges populated with cells were placed in a resorbable Vicryl mesh tube and this tissue-engineered construct was introduced into a defect of the middle part of the Achilles tendon. In 4 months, histological analysis showed complete regeneration of the tendon with the formation of parallel collagen fibers, spindle-shaped tenocytes, and newly formed vessels.

High-resolution study of the 3D collagen fibrillary matrix of Achilles tendons without tissue labelling and dehydrating.

Knowledge of the collagen structure of an Achilles tendon is critical to comprehend the physiology, biomechanics, homeostasis and remodelling of the tissue. Despite intensive studies, there are still uncertainties regarding the microstructure. The majority of studies have examined the longitudinally arranged collagen fibrils as they are primarily attributed to the principal tensile strength of the tendon. Few studies have considered the structural integrity of the entire three-dimensional (3D) collagen meshwork, and how the longitudinal collagen fibrils are integrated as a strong unit in a 3D domain to provide the tendons with the essential tensile properties. Using second harmonic generation imaging, a 3D imaging technique was developed and used to study the 3D collagen matrix in the midportion of Achilles tendons without tissue labelling and dehydration. Therefore, the 3D collagen structure is presented in a condition closely representative of the in vivo status. Atomic force microscopy studies have confirmed that second harmonic generation reveals the internal collagen matrix of tendons in 3D at a fibril level. Achilles tendons primarily contain longitudinal collagen fibrils that braid spatially into a dense rope-like collagen meshwork and are encapsulated or wound tightly by the oblique collagen fibrils emanating from the epitenon region. The arrangement of the collagen fibrils provides the longitudinal fibrils with essential structural integrity and endows the tendon with the unique mechanical function for withstanding tensile stresses. A novel 3D microscopic method has been developed to examine the 3D collagen microstructure of tendons without tissue dehydrating and labelling. The study also provides new knowledge about the collagen microstructure in an Achilles tendon, which enables understanding of the function of the tissue. The knowledge may be important for applying surgical and tissue engineering techniques to tendon reconstruction.

* Comparison of Autologous, Allogeneic, and Cell-Free Scaffold Approaches for Engineered Tendon Repair in a Rabbit Model-A Pilot Study.

Tendons are subjected to high strength dynamic mechanical forces in vivo. Mechanical strength is an essential requirement for tendon scaffold materials. A composite scaffold was used in this study to provide mechanical strength, which was composed of an inter part of nonwoven polyglycolic acid (PGA) fibers and an outer part of the net knitted with PGA and polylactic acid (PLA) fibers in a ratio of 4:2. This study compared three different approaches for in vivo tendon engineering, that is, cell-free scaffold and allogeneic and autologous cell seeded scaffolds, using a rabbit Achilles tendon repair model. Dermal fibroblasts were, respectively, isolated from the dermis of regular rabbits or green fluorescence protein transgenic rabbits as the autologous and the allogeneic cell sources, respectively. The cell scaffolds and cell-free scaffolds were implanted to bridge a partial segmental defect of rabbit Achilles tendon. The engineered tendons were harvested at 7 and 13 months postsurgery for various examinations. The results showed that all three groups could achieve in vivo tendon regeneration similarly with slightly better tissue formation in autologous group than in other two groups, including better scaffold degradation and relatively thicker collagen fibrils. There were no statistically significant differences in mechanical parameters among three groups. This work demonstrated that allogeneic fibroblasts and scaffold alone are likely to be used for tendon tissue engineering.

Experimental Diabetes Alters the Morphology and Nano-Structure of the Achilles Tendon.

Although of several studies that associate chronic hyperglycemia with tendinopathy, the connection between morphometric changes as witnessed by magnetic resonance (MR) images, nanostructural changes, and inflammatory markers have not yet been fully established. Therefore, the present study has as a hypothesis that the Achilles tendons of rats with diabetes mellitus (DM) exhibit structural changes. The animals were randomly divided into two experimental groups: Control Group (n = 06) injected with a vehicle (sodium citrate buffer solution) and Diabetic Group (n = 06) consisting of rats submitted to intraperitoneal administration of streptozotocin. MR was performed 24 days after the induction of diabetes and images were used for morphometry using ImageJ software. Morphology of the collagen fibers within tendons was examined using Atomic Force microscopy (AFM). An increase in the dimension of the coronal plane area was observed in the diabetic group (8.583 ± 0.646 mm2/100g) when compared to the control group (4.823 ± 0.267 mm2/100g) resulting in a significant difference (p = 0.003) upon evaluating the Achilles tendons. Similarly, our analysis found an increase in the size of the transverse section area in the diabetic group (1.328 ± 0.103 mm2/100g) in comparison to the control group (0.940 ± 0.01 mm2/100g) p = 0.021. The tendons of the diabetic group showed great irregularity in fiber bundles, including modified grain direction and jagged junctions and deformities in the form of collagen fibrils bulges. Despite the morphological changes observed in the Achilles tendon of diabetic animals, IL1 and TNF-α did not change. Our results suggest that DM promotes changes to the Achilles tendon with important structural modifications as seen by MR and AFM, excluding major inflammatory changes.

Quantification of collagen fiber orientation in human tendons with the coefficient of variation of echogenicity.

The grayscale distribution on the ultrasonic images of tendons may be reduced with alignment of collagen fibers, because ultrasound signal intensity changes with alterations in tendon collagen fiber orientation due to acoustic anisotropy in the tendons. The purpose of this study was to investigate changes in the coefficient of variation (CV) of echogenicity in the Achilles tendon during passive dorsiflexion (the angle task) and isometric plantar flexion (the contraction task). Achilles tendon transverse ultrasonic images were collected from 14 healthy individuals every 10° from 20° to -20° (positive values for plantar flexion) in the angle task and every 10% maximum voluntary contraction (MVC) from 0% to 70% MVC in the contraction task. The CV of echogenicity was measured in each image. In addition, relative changes in the measured variables between the former half (20-0° in the angle task, 0% to 30% MVC in the contraction task) and the latter half (0° to -20° in the angle task, 40% to 70% MVC in the contraction task) of each task were compared. The CV of echogenicity decreased with increases in the dorsiflexion angle and intensity of isometric contractions. Furthermore, relative changes in the CV of echogenicity were greater at more dorsiflexed positions in the angle task and at lower torque levels in the contraction task. These results suggested that decreases in the CV of echogenicity were partially related to the alignment of the tendon collagen fibers with tendon stretching.