Mesenchymal stem cells reside in anterior cruciate ligament remnants in situ
Mesenchymal stem cells reside in anterior cruciate ligament remnants in situ
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DOI：
10.1007/s00264-015-2925-1
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发表时间：
2016-07-01
影响因子：
2.7
通讯作者：
Li, Jian
中科院分区：
文献类型：
--
作者：
Fu, Weili;Li, Qi;Li, Jian
目的有报道称，前十字韧带在急性损伤或一期缝合修复后具有一定的自愈能力。许多研究证实，结合前交叉韧带重建的残端保存技术有助于前交叉韧带愈合的生物强化。然而，尚不清楚间充质干细胞(MSC)是否存在于原位残留的前交叉韧带中。本研究的目的是探讨前交叉韧带断裂患者残留前交叉韧带来源的MSC的培养和鉴定方法，并对其特性进行分析。方法采用酶消化法分离前交叉韧带损伤后残留的前交叉韧带细胞，在显微镜下体外培养至第3代，观察其形态和生长情况。结果体外培养第3~5天，可见少量长梭形细胞出现，并贴壁贴壁。培养第6~9天，细胞聚集，可见集落。第3代细胞形态均匀，增殖良好，出现典型的表面标志MSC、CD29、CD44、CD90和CD105。造血干细胞表面标志CD34、CD45不表达。在适宜的体外培养条件下，分离的细胞可分化为沉积矿化基质并表达早期成骨标志物的成骨细胞、胞浆内聚集脂滴的脂肪细胞、分泌软骨细胞特异性基质聚集素和II型胶原的软骨细胞。实时定量聚合酶链式反应分析表明，成骨、成脂和软骨形成的特异性mRNA表达在培养第0d显著高于对照组。根据细胞的贴壁能力、形态、表面标志和多向分化潜能鉴定为MSC。前交叉韧带残留物来源的MSC可作为韧带再生的种子细胞来源。
Purpose It has been reported that the anterior cruciate ligament (ACL) has certain self-healing ability after acute injury or with primary suture repair. Many studies have confirmed that a remnant preservation technique with ACL reconstruction contributes to biological augmentation for ACL healing. However, it remains unclear whether mesenchymal stem cells (MSC) reside in ACL remnants in situ. The aim of this study was to investigate the methods of culture and identification of MSC derived from the remnants of ACL rupture patients and to analyse these MSC's properties.Methods The cells of ACL remnants from the ACL rupture patients were isolated by the methods of enzymatic digestion and cultured in vitro to the third passage under the microscope to observe their morphology and growth status. The third passage of isolated cells was analysed for the identification of immunophenotype, osteogenic, adipogenic and chondrogenic differentiation.Results On the third to fifth days of in vitro culture, a few cells of long fusiform shape appeared and were adherent to the plastic walls. On the sixth to ninth days, cells clustered and colonies were observed. The third passage cells showed uniform cell morphology and good proliferation, with appearance of the typical surface markers of MSC, CD29, CD44, CD90 and CD105. The surface markers of CD34 and CD45 of haematopoietic stem cells were not expressed. Under appropriate conditions of in vitro culture, isolated cells could be differentiated into osteoblasts that deposit mineralised matrix and express early osteogenic markers, adipocytes that accumulate lipid droplets in cytoplasm and chondrocytes that secrete chondrogenic-specific matrix aggrecan and collagen II. Real-time polymerase chain reaction (PCR) analysis demonstrated that the specific mRNA expression of osteogenesis, adipogenesis and chondrogenesis increased significantly compared with the control groups at day zero.Conclusions Stem cells derived in situ from the human ACL stump were successfully isolated and characterised. Those isolated cells were identified as MSC according to their adherent ability, morphology, surface markers and multilineage differentiation potential. MSC derived from ACL remnants could be a potential source of seeding cells for ligament regeneration.