Naranda Jakob, Gradišnik Lidija, Gorenjak Mario, Vogrin Matjaž, Maver Uroš
Department of Orthopaedics, University Medical Centre Maribor , Maribor , Slovenia.
Institute of Biomedical Sciences, University of Maribor, Faculty of Medicine , Maribor , Slovenia.
PeerJ. 2017 Mar 21;5:e3079. doi: 10.7717/peerj.3079. eCollection 2017.
Cartilage tissue engineering is a fast-evolving field of biomedical engineering, in which the chondrocytes represent the most commonly used cell type. Since research in tissue engineering always consumes a lot of cells, simple and cheap isolation methods could form a powerful basis to boost such studies and enable their faster progress to the clinics. Isolated chondrocytes can be used for autologous chondrocyte implantation in cartilage repair, and are the base for valuable models to investigate cartilage phenotype preservation, as well as enable studies of molecular features, nature and scales of cellular responses to alterations in the cartilage tissue.
Isolation and consequent cultivation of primary human adult articular chondrocytes from the surgical waste obtained during total knee arthroplasty (TKA) was performed. To evaluate the chondrogenic potential of the isolated cells, gene expression of collagen type 2 (COL2), collagen 1 (COL1) and aggrecan (ACAN) was evaluated. Immunocytochemical staining of all mentioned proteins was performed to evaluate chondrocyte specific production.
Cartilage specific gene expression of COL2 and ACAN has been shown that the proposed protocol leads to isolation of cells with a high chondrogenic potential, possibly even specific phenotype preservation up to the second passage. COL1 expression has confirmed the tendency of the isolated cells dedifferentiation into a fibroblast-like phenotype already in the second passage, which confirms previous findings that higher passages should be used with care in cartilage tissue engineering. To evaluate the effectiveness of our approach, immunocytochemical staining of the evaluated chondrocyte specific products was performed as well.
In this study, we developed a protocol for isolation and consequent cultivation of primary human adult articular chondrocytes with the desired phenotype from the surgical waste obtained during TKA. TKA is a common and very frequently performed orthopaedic surgery during which both femoral condyles are removed. The latter present the ideal source for a simple and relatively cheap isolation of chondrocytes as was confirmed in our study.
软骨组织工程是生物医学工程中一个快速发展的领域，其中软骨细胞是最常用的细胞类型。由于组织工程研究总是消耗大量细胞，简单且廉价的分离方法可为推动此类研究并使其更快进入临床提供有力基础。分离出的软骨细胞可用于软骨修复中的自体软骨细胞植入，并且是研究软骨表型维持的重要模型基础，还能用于研究细胞对软骨组织变化的分子特征、性质及反应程度。
从全膝关节置换术（TKA）过程中获得的手术废弃物中分离并培养原代成人关节软骨细胞。为评估分离细胞的软骨生成潜力，检测了Ⅱ型胶原蛋白（COL2）、Ⅰ型胶原蛋白（COL1）和聚集蛋白聚糖（ACAN）的基因表达。对所有上述蛋白进行免疫细胞化学染色以评估软骨细胞特异性产物。
COL2和ACAN的软骨特异性基因表达表明，所提出的方案可分离出具有高软骨生成潜力的细胞，甚至可能在传代至第二代时仍保持特定表型。COL1表达证实，分离出的细胞在传代至第二代时就已呈现出向成纤维细胞样表型去分化的趋势，这证实了先前的研究结果，即在软骨组织工程中使用较高传代细胞时应谨慎。为评估我们方法的有效性，还对评估的软骨细胞特异性产物进行了免疫细胞化学染色。
在本研究中，我们开发了一种从TKA过程中获得的手术废弃物中分离并培养具有所需表型的原代成人关节软骨细胞的方案。TKA是一种常见且经常进行的骨科手术，在此过程中双侧股骨髁均被切除。如我们的研究所证实，后者是简单且相对廉价地分离软骨细胞的理想来源。
