Control of the collagen fibril diameter in the equine superficial digital flexor tendon in horses by decorin

The distribution pattern of collagen fibril diameter in the equine superficial digital flexor tendon (SDFT) is known to differ in central and peripheral areas of some regions. This study reports the essence of collagen fibril differences among different regions of the equine SDFT by transmission electron microscopic (TEM) and high-voltage electron microscopic observations and biochemical analysis. The distribution of large collagen fibrils increased but the density of collagen fibrils decreased from the proximal metacarpal region to the distal metacarpal region. Large collagen fibrils with an irregular cross-sectional profile were found more frequently in the middle metacarpal region than in other regions. Three-dimensional reconstruction of images of irregularly shaped collagen fibrils revealed that these fibrils are formed through fusion of small collagen fibrils with large ones. The amount of decorin, which reportedly inhibits the lateral fusion of collagen fibrils, decreased in the direction of the distal metacarpal region. On the other hand, the size of decorin gradually increased in the direction of the distal metacarpal region. These results suggest that regional differences in collagen fibril distribution and density of collagen fibrils in the SDFT are due, at least in part, to fusion of collagen fibrils and the concomitant regional differences in the amount and size of decorin.     

Effect of exercise on age-related changes in collagen fibril diameter distributions in the common digital extensor tendons of young horses

OBJECTIVE: To determine whether specific treadmill exercise regimens would accelerate age-related changes in collagen fibril diameter distributions in the common digital extensor tendon (CDET) of the forelimbs of young Thoroughbreds. ANIMALS: 24 female Thoroughbreds. PROCEDURE: Horses were trained for 18 weeks (6 horses; short term) or 18 months (5 horses; long term) on a high-speed treadmill; 2 age-matched control groups (6 horses/group) performed walking exercise only. Horses were (mean +/- SD) 24 +/- 1 months and 39 +/- 1 months old at termination of the short-term and long-term regimens, respectively. Midmetacarpal CDET specimens were obtained and processed for transmission electron microscopy. Diameter and area of at least 1,000 collagen fibrils/specimen were measured by use of computerized image analysis. Mass-average diameter (MAD) of collagen fibrils and collagen fibril index were calculated for each horse. RESULTS: Collagen fibril MAD for the older horses was significantly less than that for the younger horses. Exercise did not significantly affect fibril diameter or distributions in either age group, and collagen fibril index did not differ significantly between groups. CONCLUSIONS AND CLINICAL RELEVANCE: Age-related reduction in collagen fibril MAD agreed with findings for other tendons and species. Training did not accelerate age-related change in the CDET in contrast to a reported decrease in collagen fibril MAD in the superficial digital flexor tendon of horses trained long term. Our results support the concept that the functionally distinct nature of the CDET and superficial digital flexor tendon in horses results in fundamentally different responses to high-speed exercise regimens.     

The collagen fibrils in fowl medullary bone

Medullary bone from the femurs of laying fowls was examined by electron microscopy, with particular reference to the nature and environment of its collagen fibrils. The collagen fibrils appeared to have a preferred orientation along the long axis of the bone. There were no clear‐cut cyclic changes in the fibril diameters during egg calcification. The tissue was rich in mucopolysaccharides, visualised as strands or sheets connecting the fibrils. Lysis of the fibrils appeared to be associated with osteoclasts rather than osteocytes.     

Observations on the ultrastructure of equatorial scleral collagen fibrils in sheep eyes

Objective To investigate collagen fibrils of the equatorial sclera in relation to the age‐related changes in eye size in sheep. Animals studied Lambs and outbred ewes. Procedures Sheep eyes (three lamb and three from adult outbred ewes), presumed disease‐free, were processed for transmission electron microscopy (TEM) immediately postmortem. Tissue blocks from the equatorial region were sectioned across fibril bundles orientated along the equator. Micrographs including at least 500 fibrils were projected at 22 000× magnification for measures of fibril diameters (FDs). Results Lamb eyes were smaller than those of adult ewes but equatorial scleral thickness was only marginally less at 0.232 ± 0.013 vs. 0.254 ± 0.012 mm (P value not significant). Scleral tissue was composed of compacted bundles of collagen fibers that tended to be rounder in outer compared to being flatter in inner regions. In typical (normal) appearing regions, FDs were distinctly larger (68–410 nm) in outer sclera compared to inner sclera (63–281 nm). Outer sclera FDs were bimodal averaging 192 ± 58 nm, compared to unimodal distributions at inner locations averaging 156 ± 48 nm (P < 0.001). Some atypical regions, especially at outer‐mid sclera locations, were also noted where the FD distribution was bimodal but also included numerous microfibrils (<50 nm diameter), with similar appearances being found for both lamb and adult ewe eyes. Conclusions The equatorial sclera is a mixture of rounder versus flatter collagen fiber bundles, the former being more likely to be made up of a mixture of both smaller and larger fibrils, as compared to slightly smaller fibrils.     

Ultrastructure of canine articular cartilage: comparison of normal and degenerative (osteoarthritic) hip joints.

Normal canine hip cartilage was compared with cartilage from the degenerative lesions found in young dogs with canine hip dysplasia. The upper 0.5 mm of normal cartilage was characterized. Four distinct layers or zones were found: a layer of fine fibrous material covering the surface, a layer (surface layer) of small (32 nm diameter or less) collagen fibrils tightly packed in bundles and oriented parallel to the surface, a layer (upper layer) or less tightly packed collagen fibrils oriented mostly parallel to the surface with about 33% of the fibrils 64 nm or more, and a layer (intermediate layer) of randomly oriented fibrils with more than 50% of the fibrils 64 nm or larger. Fibril density was high in the surface layer and decreased with depth into the cartilage. In a moderately advanced lesion of degenerative cartilage, there was a layer of amorphous material over the surface. The tightly packed surface layer of small fibrils was absent. The surface itself was uneven and fissued. At depths from the surface comparable to the upper and the intermediate layers in normal cartilage, the proportion of large fibrils was less than in normal cartilage. The overall density of fibrils in degenerative cartilage increased with depth into the tissue. Cells flattened parallel to the surface, with relatively large nuclei, were found in the upper layer of normal cartilage. Cells in the intermediate layers were larger and round. The oblong cells of the upper layer of normal cartilage were not found in any layer of degenerative cartilage. Differences between cells in other layers of normal and degenerative cartilages were minimal. A model for the arrangement of chondrocytes and collagen fibrils for normal and degenerative cartilage was proposed. Ultrastructural changes in degenerative cartilage were prominent in the upper 0.5 mm of cartilage. These changes were changes in the number of collagen fibrils/mum-2 and a change from a characteristic pattern of collagen fibril diameters and orientation found in normal tissue.     

Ultrastructure features of camel cornea--collagen fibril and proteoglycans

Introduction The uniform distribution of collagen fibrils and proteoglycans maintain the transparency of normal cornea. We describe the ultrastructural features of camel cornea including collagen fibrils and proteoglycans (PGs). Methods Camel corneas (of 6‐, 8‐, and 10‐month‐old animals) were fixed in 2.5% glutaraldehyde containing cuprolinic blue in sodium acetate buffer and processed for electron microscopy. The ‘AnalySIS LS Professional’ program was used to analyze the collagen fibril diameter. Results The camel cornea consists of four layers: the epithelium (227 μm), stroma (388 μm), Descemet’s membrane (DM), and endothelium. The epithelium constituted 36% of the camel cornea, whereas corneal stroma constituted 62% of the corneal thickness (629 μm). The PGs in the posterior stroma were significantly larger in number and size compared with the anterior and middle stroma. The collagen fibril diameter was 25 nm and interfibrillar spacing 40 nm. Fibrillar structures are present throughout the DM. Conclusion The structure of the camel cornea is very different from human and other animals. The unique structure of the cornea might be an adaptation to help the camel to survive in a hot and dry climate. The camel cornea may also be a good model to study the effect of hot and dry climates on the cornea.     

Comparative study of electron microscopical techniques for the detection of scrapie-associated fibrils

Samples of cervical spinal cord and four anatomical regions of the brains of 12 sheep with natural scrapie and six control sheep were examined by electron microscopy, after the tissues had been stored at 4°C and −20°C. The tissues were tested for the presence of scrapie-associated fibrils by a centrifugal extraction technique and by a touch-grid technique. The touch-grid technique was no better than the centrifugal extraction technique for the detection of fibrils. Structures which could have been classified as tubulofilaments were detected in touch-grid preparations without detergent treatment. With the centrifugal extraction technique there was a significant reduction of the fibril scores in some of the tissue extracts stored at −20°C, but not in any of the extracts stored at 4°C. There was, however, a reduction in the fibril scores when the final extracted pellets were stored at 4°C. The stability of the fibrils on the test grids was unaffected by six months storage at room temperature but the clarity of their ultrastructure did deteriorate. Poor hydrophilic spread of the sample on the test grids did not have a significant effect on the fibril scores.     

Histology and ultrastructure of the developing superficial digital flexor tendon in rabbits

This study was designed to investigate the developmental changes in the superficial digital flexor tendon (SDFT) of the white New Zealand rabbit, from 5 to 7 days pre-natal to 112 days post-natal (PN), at histological and ultrastructural levels. The tendons changed from being highly cellular with 13079 ± 2538 cell/mm² and little directional organization to a longitudinally oriented, predominantly connective tissue structure with relatively few, mature tenocytes (2384 ± 365 cell/mm²). Fibrillogenesis was seen in isolated vacuoles of fibroblast cytoplasm during fetal life as well as after birth, but less frequently with increasing age. At the ultrastructural level, there was a progressive increase in the mean diameter of collagen fibrils with age throughout the population , until PN day 28. A bimodal distribution of collagen fibril size was first observed on PN day 56 while at day 112, the fibrils were fully differentiated and showed a multimodal size distribution. The development of elastic fibres preceded that of collagen fibres and accompanied progressively more marked sinusoidal crimping in collagen fibres on PN days 7 and 14. The tendons of older animals became less tightly crimped. The cells of the epi- and endotenon were less mature and relatively undifferentiated compared with the cells in the body of the tendon of the same age, which might explain their ability to initiate healing of tendon injuries in older animals. In conclusion, extensive changes were observed in tendons during growth and maturation, with diameters of collagen fibrils, tissue orientation and cellularity being the parameters affected, probably due largely to increased physical activity.     

The effect of cranial cruciate ligament insufficiency on caudal cruciate ligament morphology: An experimental study in dogs

OBJECTIVES: To investigate the effect of cranial cruciate ligament (CrCL) insufficiency on morphology of the canine caudal cruciate ligament (CdCL). STUDY DESIGN: In vivo experimental study. ANIMALS: Five adult foxhounds. METHODS: Two years after CrCL transection, the histologic appearance of CdCLs from CrCL-deficient and unoperated contralateral control (CrCL-intact) stifle joints were evaluated using light and transmission electron microscopy. RESULTS: CdCLs from CrCL-deficient joints had extracellular matrix changes, characterized by chondroid metaplasia and disruption of cell architecture. Percent of small-diameter fibrils in CdCLs from CrCL-deficient joints was significantly greater (P <.05) than that in CdCLs from CrCL-intact joints. Collagen fibril density in CdCLs from CrCL-deficient joints (41.09 +/- 5.39%) tended to be less than that in CdCLs from CrCL-intact joints (52.96 +/- 6.92%); however, this difference was not significant (P =.056). Mean eccentricity (ratio of minor to major diameters) of collagen fibrils was significantly (P <.0001) lower for CdCLs from CrCL-deficient joints (0.85 +/- 0.016) when compared with that for CdCLs from CrCL-intact joints (0.87 +/- 0.015). CONCLUSIONS: Significant alterations were found in the morphology of CdCLs from CrCL-deficient joints. These changes may be associated with repetitive microtrauma to the CdCL secondary to instability or enzymatic degradation in the hostile synovial environment of an unstable joint. CLINICAL RELEVANCE: Regardless of the cause, the switch to a predominantly small-diameter collagen fibril profile may reflect compromised material properties of the CdCL. This should be taken into account when considering surgical techniques that rely on the CdCL to stabilize CrCL-deficient stifles.     

Comparative study of dermal components and plasma TGF-β1 levels in Slc39a13/Zip13-KO mice

Ehlers-Danlos syndrome (EDS) is a group of disorders caused by abnormalities that are identified in the extracellular matrix. Transforming growth factor-β1 (TGF-β1) plays a crucial role in formation of the extracellular matrix. It has been reported that the loss of function of zinc transporter ZRT/IRT-like protein 13 (ZIP13) causes the spondylocheiro dysplastic form of EDS (SCD-EDS: OMIM 612350), in which dysregulation of the TGF-β1 signaling pathway is observed, although the relationship between the dermis abnormalities and peripheral TGF-β1 level has been unclear. We investigated the characteristics of the dermis of the Zip13-knockout (KO) mouse, an animal model for SCD-EDS. Both the ratio of dermatan sulfate (DS) in glycosaminoglycan (GAG) components and the amount of collagen were decreased, and there were very few collagen fibrils with diameters of more than 150 nm in Zip13-KO mice dermis. We also found that the TGF-β1 level was significantly higher in Zip13-KO mice serum. These results suggest that collagen synthesis and collagen fibril fusion might be impaired in Zip13-KO mice and that the possible decrease of decorin level by reduction of the DS ratio probably caused an increase of free TGF-β1 in Zip13-KO mice. In conclusion, skin fragility due to defective ZIP13 protein may be attributable to impaired extracellular matrix synthesis accompanied by abnormal peripheral TGF-β homeostasis.     

Morphometric Analysis of Collagen: a Comparative Study in Cow and Pig Skins

Light and electron microscopic studies were conducted on dermis samples taken from different regions of adult cows and pigs to determine whether there is a correlation between subfibrillar architecture and collagen fibril diameter in dermis tissue. Dermis thicknesses, collagen fibril diameters, subfibrillar architecture diameters, and angles of the helical arrangement of subfibrillar architectures were measured in all dermis samples. The results showed that the differences between subfibrillar architecture diameters in dermis samples from different regions of the same species were not significant and that the diameters of subfibrillar architectures in dermis samples were similar (22-28 nm) in the two species (cow and pig) examined in this study. These findings therefore suggest that the collagen fibril diameter depends on the number of subfibrillar architectures in each fibril.     

A collagen dysplasia in a greyhound bitch

A 10-month-old greyhound bitch was referred to the Massey University Small Animal Clinic in January 1979, with a history of frequently occurring skin lacerations, especially of the feet and limbs. Clinical findings included multiple skin scars, lacerations and hyperextensibility of the skin.

Investigations showed an increased extensibility index of the skin and a sevenfold reduction in its tensile strength, as compared with that of a normal greyhound. On light microscopy dermal collagen fibres appeared to be slightly decreased in density and more whorled in appearance than that of normal sections. Electron microscopy showed dermal collagen fibrils and bundles which were largely normal, but areas could be found, in the papillary layer of the dermis, where malformed fibrils were prominent, and other areas showed disorganisation of fibrillar packing.

These findings indicated that this bitch had features of a collagen fibrillogenesis disorder similar to some of those described in the various forms of Ehlers-Danlos syndrome in man, and cats and mink. Whether the disease is inherited in the greyhound breed has not yet been determined.     

Effects of inflammatory periodontal disease ('broken mouth') on the ultrastructure of collagen fibrils in the sheep incisor periodontium

The ultrastructure of the matrix of the sheep central incisor periodontium showing clinical signs of severe periodontitis was analysed quantitatively. The distribution of collagen fibril diameters in the lower dental pad changed from a bimodal distribution seen in healthy periodontia to a unimodal distribution. Collagen fibrils with an abnormal morphology were seen in the connective tissue adjacent to the crest of the alveolar bone. These results suggest that the deepening periodontal pocket resulting from inflammation removes the major area of support for the tooth and abnormal loads are applied to fibres deeper within the tissue.     

Exercise modifies the age-related change in crimp pattern in the core region of the equine superficial digital flexor tendon

One of the current concepts with regard to equine superficial digital flexor tendonitis is that cumulative subclinical microscopic damage weakens the structure, predisposing the tendon to partial or complete rupture. This microtrauma is likely to affect the waveform or crimp of the collagen fibrils, which are the units of tensile strength. Collagen fibril crimp morphology characteristics were determined in superficial digital flexor tendons of 18 horses presented for necropsy. Horses were separated into exercised (n = 9) and non-exercised groups (n = 9), based on recent function. Five of the eight exercised horses below the age of 10 years showed a significantly lower crimp angle in the central region of the tendon in comparison with the periphery. Three of those five animals also showed a significantly lower crimp period length in the centre. No non-exercised horses in this age group showed such regional differences. Horses classified as non-exercised may have Horses classified as non-exercised may have undergone competitive galloping activity at an earlier age, implying that changes in central region crimp morphology did not occur at that time, or had reverted to normal values in the intervening period. A lower crimp angle in the core region in comparison with the periphery is abnormal in animals younger than 10 years, on the basis of previous data obtained from wild horses. It is hypothesised that imposed exercise regimens involving galloping modify the normal age-related reduction of crimp angle in the tendon core, probably as a result of the increased number of rapid high-strain cycles experienced by the collagen fibrils.     

Abnormal skin fragility in a cat with cnolangio-carcinoma

An eight-year-old shorthaired cat which presented with a five-month history of bouts of anorexia and vomiting, developed a large spontaneous skin tear over the left shoulder when manipulated for clinical workup. The cat had no previous history of abnormal skin fragility and clinical examination of the skin revealed a general thinning but no scarring or hyperextensibility. After one day of hospitalisation the animal was euthanased because of its poor condition and on post mortem examination a cholangiocarcinoma with distant metastasis was identified. The skin biopsy specimens obtained from different sites revealed dermal atrophic changes, characterised in electron microscopy by disorganisation in the packing of both collagen fibrils and fibres, and by collagen fibrils with an abnormally wide range of diameters and with an irregular shape in cross section. Although no specific cause of the dermal lesions was suggested, this case differed greatly from the fragile skin conditions previously described in the cat.     

Bovine spongiform encephalopathy: detection and quantitation of fibrils, fibril protein (PrP) and vacuolation in brain

Bovine spongiform encephalopathy (BSE) is a new disease of cattle which has considerable homology with scrapie, the archetype of the transmissible spongiform encephalopathies. Abnormal brain fibrils, called scrapie associated fibrils (SAF), are specific ultrastructural markers for these diseases. Fibril detection was compared with histopathological diagnosis in the brains of 167 cattle; 157 clinically suspect BSE and 10 clinically normal. Fibrils were detected in samples of pooled brain regions of 67/144 in which vacuolar changes of BSE were confirmed, but absent in the remaining 23 brains, in which no vacuolation was found, including those from the clinically normal cattle and 13 with alternative neuropathological diagnoses. When eight defined anatomic regions from the brains of another 22 affected cows were examined, the sensitivity of fibril detection was greater than 90% for the brain stem areas. Fibril prevalence in these areas approximated to severity of vacuolar changes. When the same defined regions from four of the affected cows were assayed for fibril protein (PrP) by western blotting, the density of immuno-labelling generally correlated with the fibril prevalence. This study confirms the specificity of fibril detection for BSE, shows that the ease of fibril detection depends on anatomic region sampled and suggests an association between PrP accumulation and vacuolar changes in certain neuroanatomic areas.     

Comparative observations of the growth changes of the histochemical properties and collagen architecture of the iliotibialis lateralis muscle from Silky, layer and meat type cockerels

Growth‐related changes in the histochemical property and collagen architecture of the iliotibialis lateralis muscle were compared among Silky, layer and meat cockerels. Histochemical and immunohistochemical methods were employed to observe the collagen architecture. The total intramuscular collagen was also determined. The muscle consisted of type IIA, IIB and IIC myofibers, of which type IIB occurred at the highest frequency. The diameter of type IIB myofibers in each week was largest in the layer, followed by the meat, and was smallest in the Silky. The total amount of collagen reached 3.38 mg/g in the meat bird, 3.03 mg/g in the layer and 2.71 mg/g in the Silky by 30 weeks of age, respectively. In the perimysium, the collagen bundles increased in size and density of fibrils with growth. At 30 weeks of age the layer had compact collagen platelets while the Silky had loose collagen bundles. In the meat bird, the collagen bundles were moderately compact. The endomysial collagen network had a large mesh size at 1 week and thereafter accumulated many collagen fibrils to form a felt‐like fabric of fibrils at 30 weeks of age. From these results it appears that growth‐related changes in the iliotibialis lateralis muscle are not necessarily causally affected by the different growth rates of chicken breeds.     

Age-related differences in collagen crimp patterns in the superficial digital flexor tendon core region of untrained horses

Objective To measure collagen fibril crimp angles and lengths as well as collagen fibril mass-average diameters in central and peripheral regions of the superficial digital flexor tendon of wild horses, to ascertain any age-related changes in either region in the absence of imposed galloping exercise.
Design Measurements from a random cull of wild horses.
Sample population Superficial digital flexor tendon samples were taken from 23 wild horses ranging in age from two to ten years.
Procedure Horses were divided into 'young' (< 5 years, n = 10), 'middle-aged' (5 to < 10 years, n = 9) and 'old' groups (10+ years, n = 4) and the mean crimp angle, mean crimp length and collagen fibril mass-average diameter calculated for the central region, and for the peripheral region of each group. Differences between groups and regions were analysed using two-tailed t tests.
Results The crimp angle for the central region was found to decrease with age, so that in old horses it was smaller than that for the tendon periphery (P < 0.05). The crimp angle for the latter region did not alter significantly with age. Crimp period lengths and collagen fibril mass-average diameters did not show significant changes with age in either region.
Conclusion Reduction of the crimp angle in the core of the superficial digital flexor tendon occurs normally with age, as tendons of older animals would have undergone a higher number of loading cycles. It is possible that athletic training increases the frequency and/or the magnitude of high loading cycles experienced by the tendon, and may accelerate and worsen the normal load-related ageing process in the superficial digital flexor tendons of young performance horses, particularly in the central regions where lesions usually occur.     

Microstructure and glycosaminoglycan ratio of canine cornea after reconstructive transplantation with glycerin-preserved porcine amniotic membranes

Objective  Although amniotic membranes of canine, feline, and equine species have some advantages as corneal transplantation material in many canine ocular diseases, their softness, thinness, and low availability can pose problems. As an alternative, the more abundant porcine amniotic membranes may be used. This paper describes the use of glycerin-preserved porcine amniotic membranes in corneal transplantation in eight normal dogs.
Method  A 0.4-mm deep recipient bed in the axial cornea of the OS of all dogs was created using an 8-mm Barron radial vacuum trephine. The recipient bed was then filled with amnion, and the entire cornea was covered with another piece of the glycerin-preserved membrane. The ocular signs evaluated were corneal opacity and corneal vascularization. The dogs were euthanized on days 5, 10, 20, or 40 after surgery, and samples were collected to evaluate corneal thickness, parenchymal cell number, mean collagen fibril diameter, collagen fibril content and the glycosaminoglycan (GAG) ratio.
Results  Corneal opacity was observed immediately after surgery. Restoration of corneal transparency, regression of corneal vascularization, and visualization of the pupil and iris were noted on day 40.
Conclusions  The clinical observations were supported histologically by regained corneal thickness, parenchymal cell number, mean collagen fibril diameter, collagen fibril content, and GAG ratio, suggesting that this technique may be a novel method for the treatment of ocular surface disorders.     

Comparison of collagen content, distribution and architecture among the pectoralis, iliotibialis lateralis and puboischiofemoralis muscles with different myofiber composition in Silky cocks

The total amount of collagen, the relative distributions of types I and III collagens in perimysium and endomysium, and the collagen fiber architecture were compared among the pectoralis (PT), iliotibialis lateralis (ITL) and puboischiofemoralis (PIF) muscles in Silky cocks. All of the myofibers in the PT muscle were type IIB, the myofibers in the ITL muscle were divided into type IIA, 41.7% and IIB, 58.3%, and the PIF muscle was composed of type I, 24.6%; IIA, 64.6%; and transitional, 10.8%. The total amount of collagen differed significantly among the PT (2.92 mg/g), PIF (4.20 mg/g) and ITL (8.06 mg/g) material, where only the PIF was a whole muscle with epimysium. On the image analysis of the immunohistochemical preparations, the percentage area of perimysial collagen to the total area in each type differed significantly among the PIF, PT and ITL muscles, where it was 26.8, 50.0 and 74.4% for the type I collagen and 27.4, 32.9 and 61.7% for the type III collagen, respectively. In the scanning electron micrography of the perimysium in macerated preparations, thick bundles of collagen fibers were observed in the ITL muscle, thinner but broad platelets in the PT muscle, and a coarse tissue of thinner collagen fibers in the PIF muscle. However, the endomysial fabric of collagen fibrils was similar among the muscles. Small, transverse collagen fibers, which branched off from the thicker perimysia, occupied narrow interendomysial spaces and separated the primary myofiber fasciculi. The results indicate that the ITL muscle, localized in the distorted and overextended part of the leg and subject to strong external forces, had highly developed perimysial collagen fiber bundles, but the ITL endomysial collagen architecture was similar to that of the PT and PIF muscles.