Section of Plastic Surgery, VA Palo Alto Health Care System and Division of Plastic Surgery, Stanford University Medical Center, Stanford, California 94305, USA.
Tissue Eng Part A. 2012 Apr;18(7-8):796-805. doi: 10.1089/ten.TEA.2011.0422. Epub 2011 Dec 2.
In mutilating hand injuries, tissue engineered tendon grafts may provide a reconstructive solution. We have previously described a method to decellularize cadaveric human flexor tendons while preserving mechanical properties and biocompatibility. The purpose of this study is to evaluate the immunogenicity and strength of these grafts when implanted into an immunocompetent rat model.
Cadaveric human flexor tendons were divided into two groups. Group 1 was untreated, and Group 2 was decellularized by treatment with sodium dodecyl sulfate (SDS), ethylenediaminetetraacetic acid (EDTA), and peracetic acid (PAA). Both groups were then analyzed for the presence of major histocompatibility complexes by immunohistochemistry (IHC). Pair-matched tendons from each group were then placed into the dorsal subcutaneous tissue and anchored to the spinal ligaments of Wistar rats for 2 or 4 weeks, and harvested. The infiltration of B-cells and macrophages was determined using IHC. The explants where then subjected to mechanical testing to determine the ultimate tensile stress (UTS) and elastic modulus (EM). Statistical analysis was performed using a paired Student's t-test.
The decellularization protocol successfully removed cells and MHC-1 complexes. At 2 weeks after implantation, there was increased infiltration of B-cells in Group 1 (untreated) compared with Group 2 (acellular), both in the capsule and tendon substance. There was improved ultimate tensile stress (UTS, 42.7 ± 8.3 vs. 22.8 ± 7.8 MPa, p<0.05) and EM (830.2 ± 206.7 vs. 421.2 ± 171.3 MPa, p<0.05) in tendons that were decellularized. At 4 weeks, there was continued B-cell infiltration in Group 1 (untreated) compared with Group 2 (acellular). There was no appreciable difference in macrophage infiltration at both time points. At 4 weeks Group 2 (acellular) demonstrated persistently greater UTS (40.5 ± 9.1 vs. 14.6 ± 4.2 MPa, p<0.05) and EM (454.05 ± 101.5 vs. 204.6 ± 91.3 MPa, p<0.05) compared with Group 1 (untreated).
Human flexor tendons that were decellularized with SDS, EDTA, and PAA resulted in removal of cellular antigens and a decreased immune response when placed into Wistar rats. These grafts showed better mechanical properties at 2 and 4 weeks when compared with control tendons. Decellularization is an important step toward the use of tissue engineered flexor tendons in upper extremity reconstruction.
在破坏性手部损伤中，组织工程肌腱移植物可能提供一种重建解决方案。我们之前描述了一种从尸体人屈肌腱中脱细胞化的方法，同时保留了机械性能和生物相容性。本研究的目的是评估这些移植物在免疫活性大鼠模型中的免疫原性和强度。
将尸体人屈肌腱分为两组。第 1 组未处理，第 2 组用十二烷基硫酸钠（SDS）、乙二胺四乙酸（EDTA）和过氧乙酸（PAA）处理脱细胞化。用免疫组织化学（IHC）分析两组的主要组织相容性复合物的存在情况。然后将每组配对的肌腱分别置于 Wistar 大鼠的背侧皮下组织中，并固定在脊柱韧带处 2 或 4 周，然后取出。用 IHC 测定 B 细胞和巨噬细胞的浸润情况。然后对标本进行力学测试，以确定极限拉伸应力（UTS）和弹性模量（EM）。使用配对学生 t 检验进行统计分析。
脱细胞化方案成功地去除了细胞和 MHC-1 复合物。在植入后 2 周时，与第 2 组（去细胞）相比，第 1 组（未处理）的胶囊和肌腱实质中 B 细胞浸润增加。去细胞化肌腱的极限拉伸应力（UTS，42.7 ± 8.3 对 22.8 ± 7.8 MPa，p<0.05）和 EM（830.2 ± 206.7 对 421.2 ± 171.3 MPa，p<0.05）均有所提高。在第 4 周时，与第 2 组（去细胞）相比，第 1 组（未处理）中仍持续存在 B 细胞浸润。在两个时间点，巨噬细胞浸润均无明显差异。在第 4 周时，第 2 组（去细胞）的 UTS（40.5 ± 9.1 对 14.6 ± 4.2 MPa，p<0.05）和 EM（454.05 ± 101.5 对 204.6 ± 91.3 MPa，p<0.05）仍明显高于第 1 组（未处理）。
用 SDS、EDTA 和 PAA 脱细胞化的人屈肌腱在植入 Wistar 大鼠后可去除细胞抗原，并降低免疫反应。与对照肌腱相比，这些移植物在 2 周和 4 周时表现出更好的机械性能。脱细胞化是组织工程屈肌腱在上肢重建中应用的重要步骤。
