الفهرس 
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Abstract
T
endon injuries are considered one of the most common injuries of human body. They may be either complete tendon ruptures or incomplete injuries such as tendinitis. These injuries cause large health care costs and individual disability. Although there are current medical treatments for most of these conditions, tendon injuries treatment is still among the challenges in medicine. Continuous efforts are still needed to enhance the effectiveness of the treatments and to accelerate recovery to the normal biochemical properties of the tendon.
The human amniotic membrane is the inner-most layer of the placenta. It is non-vascular, non-immunogenic tissue that contains extracellular collagen matrix. It has been recently used in wound healing, and tissue re‐modulation or regeneration. It was reported to promote cellular migration and trophic factors secretion at the site of injury which consequently might promote healing.
The current experiment was carried out to study the effect of human amniotic membrane graft application on the repair of induced Achilles tendon tear in adult male albino rats.
Three human placentas were harvested from the obstetrics and gynecology hospital for preparation of the human amniotic membrane grafts. The grafts were examined by the light microscope after being stained by Hematoxylin and Eosin, Masson’s trichrome, VEGF immunohistochemical staining and toluidine blue-staining for semithin sections. They were also examined by the transmission electron microscope.
Forty-two adult male albino rats of weight range 200-250 grams were used in this experiment.
The animals were divided into three groups:
group I: served as control group (n=12). It was further subdivided into two subgroups. Subgroup IA served as negative control (n=3). Subgroup IB served as sham operation group (n=9), in which incision in the midline of the distal portion of the right hindlimb skin was done. Then the skin was closed by sutures without tendon tear injury.
group II (n=15): served as tendon tear untreated group. They underwent tendon tear surgery in the right hindlimb Achilles tendon.
group III (n=15): served as amniotic membrane (AM) treated group. Rats underwent surgery as in group II followed by circumferentially applying an amniotic membrane fragment to the tendon at the site of injury.
Rats of all groups were sacrificed 3, 14 and 28 days post induction of tear.
Tendon specimens were collected and fixed in 10 % formalin to form paraffin blocks. Five µm serial longitudinal sections were cut and stained by Hematoxylin and Eosin, Masson’s trichrome staining for demonstration of collagen fibers. Immunohistochemical staining for detection of vascular endothelial growth factor (VEGF) was also done. Very small tendon specimens (1 mm3) were fixed in 2.5 % glutaraldehyde for transmission electron microscopic examination.
Morphometric study of the results was done for mean area percentage of collagen fibers, collagen fibril diameter, mean count of tenocytes/ high power field, mean area percentage of VEGF reaction and mean optical density of VEGF reaction. The morphometric data were further analyzed by statistical analysis.
Examination of the amniotic membrane (AM) grafts by light and transmission electron microscope showed the characteristics of its two surfaces: epithelial and mesenchymal surfaces. The epithelial side appeared as a sheet of closely packed monolayer of epithelial cells. Epithelial cells appeared as high cuboidal cells resting on a basement membrane with euchromatic nuclei and few short microvilli. They also showed positive cytoplasmic reaction to VEGF. The mesenchymal side appeared as a connective tissue that included a compact connective tissue layer, that was followed by the stromal layer with its spindle shaped fibroblast like stromal cells and an outermost thin spongy layer of amorphous connective tissue.
The results of the present study revealed that the mean area percentage of collagen fibers and collagen fibrils diameter were significantly increased in the amniotic membrane treated group (group III) in comparison to the tendon tear untreated group (group II). There was no significant difference between mean area percentage of collagen or collagen fibril diameter after 28 days in the AM treated group III and the control group.
Regarding the cellular components in the different subgroups, there was a significant increase of tenocytes count in group III after application of AM compared to group II except after 28 days where there was a non-significant decrease in tenocyte count in subgroup III compared to its matching subgroup II. Twenty-eight days post induction of tear and application of AM still showed significant increase in tenocytes count compared to control group.
There were also apparent larger areas of heavy mononuclear cellular infiltration in group II as compared to those seen in group III.
Vascularity of the tendon represented by both the mean area percentage and mean optical density of VEGF antibody were aligned. They revealed significant increase in VEGF reaction mean area percentage in group II compared to group III. Moreover, non-significant difference was detected after 28 days in group III in comparison to control group.
It was concluded that the application of human amniotic membrane grafts in experimentally induced full thickness Achilles tendon tear in rats was effective in promoting tendon repair. This was demonstrated by enhancement of collagen fibers reconstruction, remodeling, and organization.