| Objective: Tendon injury is a common injury in the department of Hand Surgery. Reconstruction of tendon defects is a common clinical disease of Hand Surgery. Autograft tendon transplantation, transposition, extension and replacement with autograft fascia for tendon defects are commonly used, but the source of autograft tendon is limited. Multitude tendon defects in the clinical treatment is still an urgent problem to solve. As the study and preparation of allograft tendon blaze a new trail for tendon defects reconstruction with the following advantages: wide source, convenient drawing, shortened operation time, reduced new injury upon the patients and functional influences upon the donor site. However, the biggest problem confronting the wide clinical application of allograft tendon is the post-transplantation adhesion. Tendon is an issue with few blood supply and few cell bodies and a relatively low antigenicity. Collagen does not show the antigenicity and antigenicity of the tendon mainly exists in its cell components, i.e. histocompatibility antigen (HLA). The paper employs TBP chemical extraction to prepare the allograft tendon of which cells have been completely removed without changing its biomechanics. Immunity of the allograft tendon is better removed; exogenous healing is prevented and endogenous healing is promoted; post-transplantation adhesion is reduced.Methods: Totally 48 healthy male white 3-month-old Leghorn chickens with body mass of 1.9±0.053 Kg were randomly divided into three groups of A, B, C: Group A for TBP chemical extraction treatment, Group B for deep cryogenic treatment, Group C for blank controller. The III toes of both feet in the selected Leghorn chickens were employed to establish the tendon injury model. The incision of about 10cm was made longitudinally on toe volar.The tendons were sharply separated along the lateral plantar tendon sheath of shallow after skin opening. The flap was turned over at lateral side and exposed lateral plantar tendon sheath. The incision was longitudinal along the median in tendon sheath, about 10cm. The flexor digitorum profundus was cut at insertion of The flexor digitorum profundus under unbend position of interphalangeal joints. Long and short tendon vincula tendinum was respectively cut at the tendon junction and did not damage tendon end. Extend the incision to proximal end, carefully separated tendon symphysis, dissociated the III flexor digitorum profundus, cut it at the joints of its legs. Removed the tendons, rinsed in saline, and removed outer membrane. Group A of tendons: immersed in approximately 40 mL of an extraction solution (pH 8.0) consisting of Tris buffer, 5 mM EDTA, and 1% (v / v) TBP for 48 hours. All solutions were refreshed every 24 h. And the tendons were rinsed in deionized water (40 mL) for 24 h and finally washed in 70% ethyl alcohol (40 mL) for 24 h. (All steps were conducted at room temperature and with constant agitation.) Tendons were maintained in a standard tissue culture incubator (370C, 5% CO2) and bathed in tissue culture medium with fetal bovine serum [10%], HEPES [1%], Lglutamine [1%], gentamicin [0.4%], penicillin G [200U/mL], streptomycin [200 mg/mL], and amphotericin B[0.5 mg/mL]); Group B tendons: immersed in MEM solution (containing 10% glycerol) 10-15 minutes, packaged in sterile container, labeled the derived date and tendons type of items. Placed in a refrigerator controlled deep hypothermia, gradually cooling down to -80℃, 10 days later post-test; group C tendons: blank controller. The above-mentioned three groups of tendons were tested by the following three methods.①morphological and histological observation: respectively In general, HE staining and electron microscopy observation of the three aspects of morphological analysis②biomechanical testing: A Tytron-250 bio-mechanical test machine to the three groups were treated by maximum tensile breaking strength (Pmax), the maximum tensile tendon rupture power (Wmax), and the tendon tensile elongation at break (δmax) determination.③measured mixed lymphocyte culture: RPM I1640 liquid containing 0.2% collagenase digested three groups tendons which were chopped. Separated access to cellular components.Regulated the concentration of 1×106/ml, as an incentive cells. while taked receptors chicken vein blood. separated lymphocytes Used lymphocyte separation medium. Regulated the concentration of 1×106/ml as a reaction cell. The stimulate cells were mixed with the same as reaction cell culture. Each group made three double-pored, mixed culture 5 days. Drawed cell suspension and centrifuged to push the deposition films, Sweden Giemsa staining. Calculated the number of total lymphocyte count (about 500) and conversion cells in light microscope. Conversion rates are as follows: lymphocyte transformation rate = the number of transformed cells / total lymphocyte cells×100%. Calculated the mean of three holes each group conversion rate as this group conversion rate.10 toe flexor tendon were collected from the chicken, and 10 receptor chicken blood were tested. All statistical results used analysis of variance.Results:①Morphological and histological observation: general situation: chemically extracted tendon was porcelain white, glossy on surface, soft, tenacious, intact and non-defect; deep frozen tendon was milky white, non-glossy on surface, soft, tenacious, intact and non-defect; The blank controller was milky white, non-glossy on surface, soft, tenacious, intact and non-defect; HE staining: between the tendon bundles of loose connective tissues were seen in chemical extraction treatment. Collagen fibers paralleled with each other in bundles without tendon cells. Between the tendon bundles of loose connective tissues were seen in deep frozen tendons. Collagen fibers paralleled with each other in bundles. Tendon cells were seen between the clusters arrangement. Between the tendon bundles of loose connective tissues were seen in blank controller. Collagen fibers arranged in parallel in bundles. Between the clusters arrangement of tendon cells were seen. TEM: Chemically extracted tendons: no residual cells, collagen fibers neatly arranged, multi-wave-shaped fiber cross-linked with each other, fiber on the cyclical nature of light and dark stripes alternating. Deep frozen tendons: no swollen cells, the unclear membrane, no degenerated nucleus, intact nuclear membrane, cytoplasmic little, rough endoplasmic reticulum, no obviously swollen mitochondria. collagen fibers neatly arranged, mostly wavy fibers cross-linked with each other, some swollen fibers, increased fiber gap, the fiber has a cyclical alternating light and dark stripes and stripes unclear. Blank controller tendons: No swollen cells, clear membrane, no degenerated nucleus, intact nuclear membrane, rough endoplasmic reticulum and no swollen mitochondria. Collagen fibers arranged neat, close. Fiber thickness uniformity, mostly wavy, cross-linked with each other, on the cyclical nature of alternating light and dark stripes and a clear cross striations.②Biomechanical test: The maximum tensile tendon rupture strength (Pmax): differences among the three groups show no statistical significance (P = 0.527); The maximum tensile tendon rupture power (Wmax): difference among the three groups show no statistical significance (P = 0.275); Tendon tensile elongation at break (δmax): differences among the three groups have no statistical significance (P = 0.545). Comprehensive three-pronged test results that differences among the three groups of tendon biomechanics have no statistical significance.③Mixed lymphocyte culture test: Experimental results suggested difference among the three group the statistical significance (F = 4.652, P = 0.018). The lymphocyte transformation rate of chemical extraction treatment method (1.85±0.63%) significantly lower than those of deep cryogenic treatment group (6.89±1.05%) and untreated group (14.96±1.81%) (p value<0.05); lymphocyte transformation rate of deep cryogenic treatment group is also lower than that of untreated group (p value<0.05). Chemical extraction treatment method virtually eliminates the tendon immunogenicity, and the deep freezing treatment method significantly reduces the tendon immunity.Conclusion: TBP chemical extraction treatment method can completely remove tendon cells without destroying the intercellular matrix. It almost eradicates the allograft tendon immunity as a new approach for tendon allograft transplantation.TBP chemical extraction treatment method can remove the tendon immunogenicity more efficiently than the deep freezing treatment method without affecting its cell matrix and collagen fibers. It is more conducive to reimplant cultured tendon cells on the scaffold.It promotes post-transplantation endogenous tendon healing and reduces exogenous healing to reduce the adhesion occurrence.
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