Short and long terms healing of the experimentally transverse sectioned tendon in rabbits
© Oryan et al.; licensee BioMed Central Ltd. 2012
Received: 30 March 2011
Accepted: 10 April 2012
Published: 26 April 2012
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© Oryan et al.; licensee BioMed Central Ltd. 2012
Received: 30 March 2011
Accepted: 10 April 2012
Published: 26 April 2012
The incidences of tendon injuries in certain sections of human or animal populations such as athletes are high, but every human or animal, regardless of age or level of activity experiences some degree of tendon injury. In spite of the various investigations of injuries and treatment, comprehensive studies dealing with the histological, ultrastructural and biomechanical aspects of healing of load-bearing tendons are rare. This study was designed to compare the outcome of healing of the transverse sectioned superficial digital flexor tendon (SDFT) after 28 and 84 days post injury (DPI) in rabbits.
Forty white New Zealand mature female rabbits were randomly divided into two equal groups of 28 and 84 DPI After tenotomy and surgical repair of the left SDFT, the injured legs were casted for 14 days. The weight of the animals, tendon diameter, and clinical, radiographic and ultrasonographic evaluations were conducted at weekly intervals. The animals were euthanized on 28 and 84 DPI and the tendons were evaluated for histopathological, ultrastructural, biomechanical and percentage dry weight parameters.
Although the clinical, ultrastructural, morphological and biomechanical properties of the injured tendons on day 84 showed a significant improvement compared to those of the 28 DPI, these parameters were still significantly inferior to their normal contra-lateral tendons.
This study showed that tendon healing is very slow and at 84 days post-injury the morphological and biomechanical parameters were still inferior to the normal tendons and many collagen fibrils still had the same diameter as those seen at 28 DPI.
Tendon injuries present a significant clinical challenge to orthopedic surgeons and are a major problem in sports and occupational medicine [1–5]. This unique tissue plays an essential role in the biomechanical function of the musculoskeletal system by stabilizing and guiding the motion of diarthrodial joints [5–9]. Tendon is susceptible to both excessive tensile loads and compressive forces [2, 10, 11]. Injury represents a failure of the cell matrix to adapt to load exposure, which can be either acute or secondary to cyclic overuse [3–5, 12].
A healthy human tendon does not rupture accidentally [13–16], however, in cases of trauma, during surgery and similar conditions, it can be transected by a sharp instrument [12–14]. There are many traumatic cases in veterinary medicine such as car accidents, hitting and other related conditions in which sharp and hard metal materials penetrate the skin and cut the intact tendons, resulting in tendon rupture [2, 10, 13]. In another approach, in such cases of orthopedic surgery, for example, the internal fixation techniques to reduce fractures of the tibia or metatarsal bones, the surgeon should expose the fractured site in a manner that allows the fixation plate or other fixation materials to be implanted, implantation of the screws to be facilitated and the anatomical reduction of the fractured site to be achieved. During these procedures it is quite common to cut the intact tendons [13, 14].
Tendon ruptures are initially treated by direct suturing techniques as a standard of care [10, 12, 13, 17]. The suture technique is of primary importance in providing a stiff and strong repair throughout the early healing interval [10, 14, 18, 19]. However, despite improved surgical techniques and advances in rehabilitation techniques, early complications such as rupture of the repair site and restrictive postoperative adhesions are still encountered [2, 3, 20–22]. A repaired tendon needs to be protected for weeks until it gains enough strength to handle physiological loads . Thus, surgical results are unpredictable, with pre-injury functional levels difficult to attain [3, 13, 20–22].
With these challenges the knowledge of healing concepts in the acute transverse section in the experimental model are still unclear. Thus, it is apparent that a better understanding of the function of tendons, together with a knowledge of their biology and healing potential is necessary for investigators to develop novel strategies to accelerate and improve the process of healing of transverse sectioned tendons [4, 14].
The present study was designed to investigate the outcome of the healing response of two phases of early and late tendon healing to determine the effect of time on the biomechanical and morphological characteristics of the transverse sectioned superficial digital flexor tendon after primary repair and balanced post-operative rehabilitation in rabbits. The authors basic hypothesis was that the healing of the experimentally induced transverse sectioned of the SDFT, similar to other types of tendon injuries such as tendinopathy, is slow and the structural and functional properties of the injured tendon will somehow improve during the later stages of healing. However, the structural and physical performance of the injured tendon is significantly inferior to that of the normal contralateral tendon several months or even years after tendon injury. The collagen fibrils are still immature and the biomechanical performance is significantly inferior to the intact tendons, even for a long time after injury.
Type of experimental study: simple accidental interventional study.
Forty skeletally mature, 10-14 month-old, female white New Zealand rabbits of 1.66 ± 0.76 kg body weight were randomly divided into two equal groups and evaluated on 28 and 84 DPI. They were kept in individual cages at 25°C, 60% humidity and were maintained on the same standard rabbit diet with no limitation of access to food or water. Each animal served as its own control and the right SDFT was used as its normal control.
Before injury induction, the animals were weighed and the diameter of the right and left tendons and the covering skin were blindly measured as an index of the tendon swelling and post-surgical inflammation. The tendon and the covering skin diameter around the injury site, with a comparable area of the uninjured contra-lateral tendon, were measured using a micrometer measurement device (Samsung, Seocho-gu, Seoul, Korea). The weight of the animals and the tendon diameter were measured and analyzed before injury, and then at weekly intervals until the animals were euthanized. Each measurement was made three times to ensure that the repeatability of the measurements of the width was within 0.2 mm. From these, the average cross-sectional area of the tendon, together with the fascia and skin over it was calculated.
For the clinical investigations, two observers blindly determined the lameness and weight bearing capacity of the rabbits. The walking activity of each animal in the cage was checked 3 times a day (8 h intervals). The assessment was qualitative. Lameness and comfortable/uncomfortable physical activities were defined as tarsal flexion degree of each animal, both in the cage and on the floor, weight distribution of each animal on the hind limbs, both in the cage and on the floor, pain in palpation of the injured area, pain in complete extension of the hind paw and toe, and heel position of the injured leg .
The radiographic and ultrasonographic observations were blindly evaluated by a radiologist at weekly intervals for 12 weeks to define whether the tendon injury had altered the joints and bony structures of the hind paw. In addition, the maturity of the animals was confirmed by radiology. All animals were found to be mature. Lateral and dorsoventral position radiographs were provided from the whole body, using large film at 80 KVp and 6 MA. The cross-section echo texture of the SDFT of the rabbits due to their low diameter and view was not diagnostic; therefore, the animals were sonographed at longitudinal section with a 12 MHz linear probe (Simense SLR-400, Berlin, Germany; Echowave 3.23 software). The authors considered the following criteria to define the differences in the injured tendons with those of their normal contra-lateral ones: A) The ultra sonographical echogenicity of the tendon: 1) hyperechogenicity, 2) hypoechogenicity; B) The relation of the hyperechogenic area of the echotexture tendons compared to the hypoechogenic area of the echotexture tendons: 1) the tendon had a smooth echogenicty. This means that there was no diagnostic hyperechogenicity besides that of hypoechogenicty, 2) the tendon had no smooth echogenicity. This means that there were areas of hyperechogenicity besides those of hypoechogenicity known as an amputated view; C) The movement of the tendon with finger in ultrasonography: 1) The SDFT could be well moved transversely, 2) The SDFT movement in a transverse direction was not diagnostic; D) The diameter of the SDFT was calculated using the scale of the ultrasonography machine: 1) Thick, 2) Medium, 3) Thin [12, 17].
Twenty eight and 84 days after injury induction, the animals were euthanized by Na-thiopental (50 mg/Kg), Xylazin (20 mg/Kg) and Ketamin HCl (300 mg/Kg). The study was approved by the local ethics committee of our faculty, in accordance with the ethics standards of “Principles of Laboratory Animal Care”.
The specimens from each of the injured and uninjured SDFT of ten of the animals of each group were longitudinally sectioned in three pieces for light and electron microscopic studies and percentage dry weight analysis. In the remaining ten animals of each group, both injured and contra-lateral SDFT were carefully dissected from the surrounding tissues for biomechanical testing. The SDFT was cut and separated proximally to include 3 cm of the muscle belly and distally to the site of insertion of each phalangeal branch [7, 9, 25, 26].
After fixation in 10% neutral buffered formalin, the tendon samples were washed, dehydrated, cleared, embedded in paraffin, sectioned at 4–5 μm, stained with haematoxylin and eosin and examined by a light microscope (Olympus, Tokyo, Japan). The cells and vascular populations of each section were estimated using an eye piece graticule. An average was then taken from five different microscopic fields for each cell type. Duplicate counts were carried out by double blind method. In addition, using a digital camera (Sony, T-700, Tokyo, Japan), the pictures from each slide were transferred to a computer for morphometric analysis. Maturity of the tenoblasts together with the density of the collagen fibers and blood vessels on the normal and inverted photomicrographs were determined using Adobe Photoshop cs-3 10 final . The mesenchymal cells at the injury site were divided into three categories based on their diameter, cytoplasmic granules and cell staining capacity. The largest elliptical cells with high granular and basophilic cytoplasm were determined as immature tenoblasts (fibroblasts). The long, cigar-shaped cells with less granulated but eosinophilic cytoplasm were estimated as tenocytes, while the medium sized cells with neutral cytoplasm and medium amounts of cytoplasmic granules were accounted as mature tenoblasts (fibroblast) . Additionally, the crimp pattern, tissue maturation, alignment and density, together with the types of degeneration and foreign body reactions on each sample, were qualitatively and semi-quantitatively analyzed and scored. The number of vessels was evaluated in 5 fields of each histopathologic section with x200 magnification. The mean of the data for each animal and the mean of the histopathologic sections of the animals of each group were then determined .
The samples from the injured site and a comparable area of the normal contra-lateral tendon were fixed in cold 4% glutaraldehyde, dehydrated and embedded in Epon resin 812. Thin sections of 80-90 nm in diameter were cut and standard methods were employed for production of the transmission electron micrographs (Philips CM 10 transmission electron microscope, Eindhoven, Netherlands) . Ultra-micrographs of different final magnifications (5,200-158,000) were taken for studying the collagen and elastic fibrillar morphology, inflammatory cell constituents and tenoblast’s maturity. For fibrillar density, ten pictures were captured from ten horizontal and vertical fields; for each sample the surface area of the collagen fibrils regarding their category dependency were measured and analyzed. The number of collagen fibrils and their diameters in five different fields of each tissue section was measured. The collagen fibrils were divided, based on their diameter, into 5 different categories of 33-64, 65-102, 103-153, 154-256 and 257-307 nm (nm) respectively . The number and diameter of the collagen fibrils were measured, and their mean diameter was calculated by a computerized morphometric technique, using Adobe Photoshop CS4. In addition, the number of elastic fibers of each field was counted and their maturity was qualitatively evaluated.
After application of the standard preservation methods, biomechanical tests were performed using a tensile testing machine (Instron Tensile Testing Machine, London, U.K.) . The specimens were mounted between the two metal clamps and were subjected to tensile deformation at a strain rate of 10 mm/min and the load deformation and stress–strain curves were recorded by a personal computer. The complete method has previously been described . The ultimate tensile strength, yield strength, ultimate strain, yield stain, stress and stiffness were determined.
After application of the normality distribution test, the injured tendon of each animal was compared with the normal contra-lateral tendon of the same animal, using paired sample t-Test. The right and left tendons of the 28 DPI animals were compared with the right and left tendons of the 84 DPI animals, using the independent sample t-Test. Nonparametric tests were applied to check the results again. Statistics were performed using the computer software SPSS version 17 for windows (SPSS Inc., Chicago, IL, USA). Differences of p < 0.05 were considered significant [9,17].
In the first two weeks post-injury, the animals showed lameness with lower amounts of physical activity and the injured area was hyperemic and warm. From day 14 post-injury these abnormalities gradually started to relieve so that the physical activity of the animals returned back to an almost normal level on day 35 post-injury. At this stage, the hyperemia was resolved and the incision site on the skin was sealed with new granulation tissue. At five weeks post injury the weight bearing capacity on the injured leg was comparable to the normal contra-lateral leg.
No lesions such as soft tissue swelling, calcification, osteoarthritis, bone fracture and abnormal signs of radiolucency or radio opacity were observed in the radiographs of either group at any post-surgical interval.
Due to the small diameter of the SDFT, there was no diagnostic imaging at cross sectional ultrasonography, but the longitudinal sections at ultrasonographic levels, were diagnostic, in order to compare the two stages with the normal tendon. Amputated view with irregular echogenicity in the lesions was evident in the first 6 weeks post injury. However, from day 49 up to the end of the experiment, there was no evidence of amputated view and the echogenicity of the injured tendon proper showed considerable improvement; however, at this stage they were still inferior to their normal contra-lateral tendons (Figure 3A-C).
The uninjured contra-lateral tendons exhibited parallel bundles of collagen fibers and aligned tenocytes. The cells had dark spindle-shaped nuclei with small amounts of eosinophilic cytoplasm. In the longitudinal section the collagen fibers displayed a characteristic "crimp" pattern.
The injured area at day 28 post-injury was edematous and hypercellular and high degrees of cellularity consisting of fibroblasts, lymphocytes, plasma cells and macrophages were present in their lesions. The cells and collagen fibers showed no sign of crimp pattern and were arranged in a random orientation, resulting in peritendinous adhesion. The remnant of the suture material was still present in the lesions. The new reparative cells had pale vesicular nuclei and were larger than normal mature tenocytes. The cytoplasm of the regenerative tenoblasts was basophilic in contrast to that of the mature tenocytes. Numerous capillaries were evident in the lesions at this stage.
Histopathology: number of cells with their differentiation and vascularity (mean and Standard deviation), (28 DPI vs. 84 DPI vs. their normal contralateral).
Four weeks post injury
Twelve weeks post injury
237.75 ± 26.58
6.00 ± 12.00
211 ± 22.22
33.00 ± 49.65
29.00 ± 7.52
14.00 ± 7.43
34.25 ± 12.03
6.50 ± 1.64
3.25 ± 1.50
15.75 ± 7.84
12.25 ± 3.68
8.50 ± 2.51
15.50 ± 3.00
28.25 ± 5.56
15.00 ± 4.83
3.75 ± 3.30
2.00 ± 1.63
Ultrastructural fibrillar count and morphometric analysis of the injured tendons and their normal contralateral tendons on days 28 and 84 post injury and surgical intervention
Four weeks post injury
Twelve weeks post injury
Number of collagen fibrils at different range of collagen fibrils diameter
617.50 ± 91.61
49.75 ± 6.13
378.00 ± 51.55
43.25 ± 7.36
9.50 ± 2.08
88.00 ± 25.85
17.50 ± 1.29
12.25 ± 2.50
14.50 ± 2.08
20.25 ± 2.21
10.00 ± 1.41
8.50 ± 0.577
617.50 ± 91.61
91.75 ± 1.70
466.00 ± 67.14
92.75 ± 8.65
Diameter of collagen fibrils at different range of collagen fibrils diameter
37.07 ± 4.75
38.20 ± 4.33
46.79 ± 4.37
37.73 ± 2.25
98.51 ± 2.65
70.50 ± 4.91
98.61 ± 3.04
149.56 ± 1.89
152.12 ± 0.74
234.39 ± 8.13
236.55 ± 9.07
278.28 ± 4.73
37.07 ± 4.75
177.50 ± 15.71
51.12 ± 4.11
193.25 ± 2.50
Number of elastic fibers
0.50 ± 0.57
0.50 ± 1.00
51.67 ± 9.50
94.59 ± 3.00
84.56 ± 3.16
95.66 ± 2.77
The injured tendons of the 28 days post-injury consisted of a homogenous population of small-sized, new, regenerated, haphazardly organized fibrils of 33-64 nm, and mean diameter of 37.07 ± 4.75 nm, which made up all of the tendon bulk. The maximum diameter of the collagen fibrils at the injured site of the 28 days post-injury lesions was 15.8% of those of their uninjured normal contra-lateral tendons.
Compared to the tenoblasts of the 28 days post injury lesions that were granulated and contained more rough endoplasmic reticulum, mitochondria, golgi apparatus, lysosomes, mitotic indices and collagen production capability in their cytoplasm, the tenoblasts and tenocytes of the 84 days post-injury lesions were more mature, contained lower amounts of cytoplasmic granules, rarely showed collagen production capacity, and many of them were almost comparable to those of the normal contra-lateral tenocytes (Table 2).
While the elastic fibers of the 28 days post-injury lesions were immature and consisted solely of parallel microfibrils, they were more mature on day 84 post-injury and a dense matrix began to be deposited from the center of the fiber and, in some instances, covered the central microfibrils. However, there was no significant difference in the number of elastic fibers on 28 and 84 days post-injury.
All samples failed from the mid part of the tendon during the tensile testing. The ultimate strength of the injured tendons on day 28 was significantly inferior to those of day 84 post-injury (P = 0.001) and they were 44.08% of their normal contralateral tendon (P = 0.001). Although, the ultimate strength of the injured tendons of the 84 days post injury animals showed 25.0% improvement compared to the 28 days post-injury ones, the ultimate strength of the injured tendons on day 84 post injury was still significantly inferior to their normal contra-lateral normal tendons (P = 0.003) and was only 59.1% of the normal tendon.
On day 28 post-injury, the amount of dry weight content of the injured tendon was significantly inferior to those of the 84 day post-injury (P = 0.001) and the injured tendons of both groups were still significantly inferior to their normal contra-lateral normal tendons respectively (P = 0.001, P = 0.015) (Figure 2B).
The results of the present study demonstrated that while the surgical protocol and time of immobilization was effective in improving the structural and physical characteristics of the experimentally induced transverse section SDFT after 12 weeks post-injury, it strongly elucidated that the healing of the sharp ruptured tendons is quite slow and, despite intensive remodeling over the following months, complete regeneration of the tendon was not achieved.
Normal tendon is presented as an array of parallel waveform fibers and tenoblasts [12, 17]. After injury, collagen fibrils and fibroblasts are laid down in a random pattern without a preferred orientation, and no obvious waveform pattern ("crimp") can be seen [9, 14]. Random orientation of the collagen fibers and tenoblasts at 28 days post injury, with little signs of crimp formation and high degree of cellularity consisting of numerous immature fibroblasts, lymphocytes, plasma cells and macrophages together with a homogenous population of small-sized, new regenerated collagen fibrils of 33-64 nm, resulted in a lower biomechanical property wound at this stage. In addition, low echogenicity with amputated view on ultrasonography with higher water content and higher tendon diameter of the injured tissue at 28 days post injury are other reasons why the biomechanical performance is still low at this stage of healing. The newly regenerated collagen fibrils are mostly of type III collagen fibrils, which is considered a weak and embryonated type of collagen [7, 27], as they do not have axial periodicity and are not able to resist violent physical activities or higher biomechanical loads [1, 8, 28]. The ultimate strength of the injured tendons at this stage was 44.0% of their normal contra-laterals. Possibly, part of the biomechanical property of the injured tendon may still be due to the presence of the suture material that made part of their biomechanical performance at this stage. It has been stated that the suture material has an important role in tendon stability in the earlier stages of healing [14, 17, 18], and it is reported that after interosseous and extraosseous flexor tendon repair following tenotomy in rabbits, at biomechanical testing, 38 of the 40 injured tendons failed at the suture site .
At the later stages of healing, the edema diminishes, the tenoblasts start to mature to tenocytes and their number decreases [12, 14]. Corresponding to a diminution in the number of tenoblasts, the collagen synthesis and degradation reaches equilibrium between 3 and 6 weeks after injury, the immature type III collagen decreases, the mature type I enhances, and the total collagen content becomes stable [11, 30, 31]. The newly formed tissue starts to mature, and the collagen fibrils are increasingly orientated along the direction of force through the tendon [9, 14]. At the earlier stages of healing, the unorganized tissue lacks crimp pattern [1, 27, 28], but as the collagen fibrils are organized, they are bundled into large fibers that are evident throughout the tendon under light and polarized microscopes as a crimped pattern facilitating 1-3% elongation of the tendon . This elongation of the individual fibers serves to buffer the tendon from sudden mechanical loading . Interactions between collagen fibrils at the macromolecular level leading to the fiber unit architecture dictate the strength of the tendon [6, 27, 33]. Improvement in the alignment of the collagen fibers and tissue maturation together with an increase in the diameter and cross-linking of the collagen fibrils clearly explains why the biomechanical properties and physical performance of the injured tendons were more advanced at 84 days post injury compared to those of the 28 days post-injury.
Despite the relative improvement in the structural and biomechanical indices on day 84 post injury, compared to those of day 28 post-injury, the hierarchical organization and physical characteristics of the injured tendons were still significantly inferior to their normal tendons at this stage. The morphological and biomechanical findings of the present study showed that the remodeling phase of the tendon healing is possibly extremely prolonged and the area remains abnormal in several histological, ultrastructural, biomechanical and biochemical characteristics for a long period after the original injury is induced.
Maturation and differentiation of the collagen fibrils in the wound area is much slower than in the tendon of the normal growing animals . It has been reported that the mean diameter of the collagen fibrils of a normal SDFT of a 28 day-old rabbit was 38.1 ± 2.3 nm, however, their diameter increased very quickly thereafter, so that the collagen fibrils of 162 nm and mean diameter of 124.5 ± 6.1 nm were seen in the 112 day-old rabbits , while in the present study, compared to the mean diameter of 193.3 ± 2.5 nm in the normal contra-lateral tendons, the collagen fibrils diameter of the injured area of the tendons at 84 days post-injury was 51.1 ± 4.1 nm. However, other tissue hierarchical organization was partly retrieved at this stage and most of the mesenchymal cells were mature tenoblasts. Nevertheless, the tissue was hypercellular and its percentage dry weight content was also significantly inferior to those of their normal contra-lateral tendons. Concomitant with the structural insufficiencies, the biomechanical properties of the injured tendons were not comparable to those of their normal contra-lateral ones so that the ultimate strength of the injured tendons on day 84 post-injury were only 59.1% of their normal contra-lateral tendons (P =0.003). In addition, the yield strength, stiffness, ultimate strain, yield strain and maximum stress of the injured tendons on day 84 post-injury were still significantly inferior to their normal contra-laterals (P=0.002) at this stage. Possibly, the ultimate strength on the earlier stages of healing in the present study is mostly dependent upon the fibrillogenesis which is demonstrated as the presence of numerous small regenerated collagen fibrils in the lesion, while at the later stages of healing the increase in the diameter of the fibrils and improvement in the collagen cross-linking together with tissue alignment are more important in gaining advanced biomechanical performance at this stage.
Potentially one of the blind points of the present study could be the time of the investigation.
However, if the experiment were to be continued for a longer time, the small collagen fibrils may enlarge and differentiate to a multimodal distribution pattern. This structural improvement could enhance the physical characteristics of the tendon at later stages of healing. In addition, determination of some of the biochemical criteria such as collagen type III and matrix metalloproteinases could also be helpful in explanation of various stages of tendon healing.
Therefore, this study could strongly demonstrate that the healing of the transverse sectioned tendons is substantially slow and, despite intensive remodeling over the following months, complete regeneration of the tendon is not achieved for a long time. The tissue replacing the defect remains hypercellular and the diameter of the collagen fibrils is altered, favoring small-diameter embryonic fibrils, and the biomechanical strength is significantly lower than the normal tendon. Tendon healing, even when successful, does not result in normal tendon, however, the biomechanical performance is possibly enough for routine life locomotion activities. Longer term ultrastructural, biochemical, molecular and biomechanical studies could demonstrate whether the morphological, physical and metabolic activities of the injured tendons finally coincide those of the normal contra-lateral tendons or not.
The authors would like to thank the authorities of the Veterinary School, Shiraz University for their support and cooperation. They also appreciate the Iranian National Science Foundation for partial financial support of this project. We are grateful to Dr. A. Tabatabaie, Dr. M. Kafi, Dr. A. Raayat Jahromi and Dr. N. Golestani from the Department of Clinical Sciences, School of Veterinary Medicine, Shiraz University, Shiraz, Iran for their cooperation and advice. We are also grateful to Mr. G. Yousefi, Mr. L. Shirvani and Mr. M. Zareh from the Department of Pathobiology, Mr. M. Safavi from the electron microscopy laboratory, School of Veterinary Medicine, Shiraz University, Shiraz, Iran for their technical assistance. We also thank Dr. R. Ebrahimi and Mr. A.R. Solhpour from the Department of Materials Science and Engineering, Shiraz University, Shiraz, Iran for their help in the biomechanical testing of the specimens.
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