Yao Shiyi, Liang Zuru, Lee Yuk Wa, Yung Patrick Shu Hang, Lui Pauline Po Yee
Department of Orthopaedics and Traumatology, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, China.
Am J Sports Med. 2023 Jan;51(1):66-80. doi: 10.1177/03635465221135770.
Stem cell sheets provide a scaffold-free option for the promotion of graft healing after anterior cruciate ligament reconstruction (ACLR). However, cell viability, stability, and potential uncontrolled actions create challenges for clinical translation. The decellularization of cell sheets may overcome these problems as studies have shown that the natural extracellular matrix of stem cells is bioactive and can promote tissue repair.
The decellularized tendon-derived stem cell (dTDSC) sheet can promote graft healing after ACLR.
Controlled laboratory study.
An optimized decellularization protocol was developed to decellularize the TDSC sheets. A total of 64 Sprague-Dawley rats underwent ACLR with or without the dTDSC sheet wrapping the tendon graft (n = 32/group). At 2 and 6 weeks after surgery, graft healing was assessed by micro-computed tomography, histology, and biomechanical testing. The accumulation of iNOS and CD206 cells and the expression of metalloproteinase 1 (MMP-1), MMP-13, and tissue inhibitor of metalloprotease 1 (TIMP-1) were assessed by immunohistochemistry.
The decellularization was successful, with the removal of 98.4% nucleic acid while preserving the collagenous proteins and bioactive factors. The expression of bone morphogenetic protein 2 (BMP-2) and VEGF in the dTDSC sheet was comparable with the TDSC sheet ( > .05). Micro-computed tomography showed significantly more tunnel bone formation in the dTDSC sheet group. The dTDSC sheet group demonstrated better graft osteointegration and higher integrity of graft midsubstance with significantly higher ultimate failure load (16.58 ± 7.24 vs 8.93 ± 2.45 N; = .002) and stiffness (11.97 ± 5.21 vs 6.73 ± 2.20 N/mm; = .027). Significantly fewer iNOS cells but more CD206 cells, as well as lower MMP-1 and MMP-13 but higher TIMP-1 expression, were detected at the tendon-bone interface and graft midsubstance in the dTDSC sheet group.
An optimized decellularization protocol for producing bioactive dTDSC sheets was developed. Wrapping tendon graft with a dTDSC sheet promoted graft healing after ACLR, likely via enhancing bone formation and angiogenesis by BMP-2 and VEGF, modulating macrophage polarization and MMP/TIMP expression, and physically protecting the tendon graft.
dTDSC sheets alleviate the quality control and safety concerns of cell transplantation and can be used as a cell-free alternative for the promotion of graft healing in ACLR.
干细胞片为促进前交叉韧带重建（ACLR）术后移植物愈合提供了一种无支架选择。然而，细胞活力、稳定性以及潜在的不受控制的作用给临床转化带来了挑战。细胞片的脱细胞处理可能会克服这些问题，因为研究表明干细胞的天然细胞外基质具有生物活性，能够促进组织修复。
脱细胞肌腱来源干细胞（dTDSC）片可促进ACLR术后移植物愈合。
对照实验室研究。
开发了一种优化的脱细胞方案来对TDSC片进行脱细胞处理。总共64只Sprague-Dawley大鼠接受了ACLR，其中一组的肌腱移植物包裹有dTDSC片，另一组没有（每组n = 32）。在术后2周和6周，通过微型计算机断层扫描、组织学和生物力学测试评估移植物愈合情况。通过免疫组织化学评估诱导型一氧化氮合酶（iNOS）和CD206细胞的积聚以及金属蛋白酶1（MMP-1）、MMP-13和金属蛋白酶组织抑制剂1（TIMP-1）的表达。
脱细胞处理成功，去除了98.4%的核酸，同时保留了胶原蛋白和生物活性因子。dTDSC片中骨形态发生蛋白2（BMP-2）和血管内皮生长因子（VEGF）的表达与TDSC片相当（P >.05）。微型计算机断层扫描显示dTDSC片组的隧道骨形成明显更多。dTDSC片组表现出更好的移植物骨整合以及移植物中部更高的完整性，其极限破坏载荷显著更高（16.58±7.24 vs 8.93±2.45 N；P =.002），刚度也更高（11.97±5.21 vs 6.73±2.20 N/mm；P =.027）。在dTDSC片组的肌腱-骨界面和移植物中部检测到的iNOS细胞明显更少，但CD206细胞更多，同时MMP-1和MMP-13更低，但TIMP-1表达更高。
开发了一种用于生产具有生物活性的dTDSC片的优化脱细胞方案。用dTDSC片包裹肌腱移植物可促进ACLR术后移植物愈合，可能是通过BMP-2和VEGF增强骨形成和血管生成、调节巨噬细胞极化以及MMP/TIMP表达，并在物理上保护肌腱移植物。
dTDSC片减轻了细胞移植的质量控制和安全问题，可作为一种无细胞替代物用于促进ACLR术中移植物的愈合。
