Kean Thomas J, Mera Hisashi, Whitney G Adam, MacKay Danielle L, Awadallah Amad, Fernandes Russell J, Dennis James E
a Matrix Biology Program , Benaroya Research Institute at Virginia Mason , Seattle , WA , USA.
b Department of Orthopedics , Case Western Reserve University , Cleveland , OH , USA.
Connect Tissue Res. 2016 Jul;57(4):319-33. doi: 10.1080/03008207.2016.1182996. Epub 2016 Apr 29.
PURPOSE/AIM: To determine the effect of reduced (5%) oxygen tension on chondrogenesis of auricular-derived chondrocytes. Currently, many cell and tissue culture experiments are performed at 20% oxygen with 5% carbon dioxide. Few cells in the body are subjected to this supra-physiological oxygen tension. Chondrocytes and their mesenchymal progenitors are widely reported to have greater chondrogenic expression when cultured at low, more physiological, oxygen tension (1-7%). Although generally accepted, there is still some controversy, and different culture methods, species, and outcome metrics cloud the field. These results are, however, articular chondrocyte biased and have not been reported for auricular-derived chondrocytes.
Auricular and articular chondrocytes were isolated from skeletally mature New Zealand White rabbits, expanded in culture and differentiated in high density cultures with serum-free chondrogenic media. Cartilage tissue derived from aggregate cultures or from the tissue engineered sheets were assessed for biomechanical, glycosaminoglycan, collagen, collagen cross-links, and lysyl oxidase activity and expression.
Our studies show increased proliferation rates for both auricular and articular chondrocytes at low (5%) O2 versus standard (20%) O2. In our scaffold-free chondrogenic cultures, low O2 was found to increase articular chondrocyte accumulation of glycosaminoglycan, but not cross-linked type II collagen, or total collagen. Conversely, auricular chondrocytes accumulated less glycosaminoglycan, cross-linked type II collagen and total collagen under low oxygen tension.
This study highlights the dramatic difference in response to low O2 of chondrocytes isolated from different anatomical sites. Low O2 is beneficial for articular-derived chondrogenesis but detrimental for auricular-derived chondrogenesis.
确定降低的(5%)氧张力对耳源性软骨细胞软骨形成的影响。目前,许多细胞和组织培养实验是在20%氧气和5%二氧化碳的条件下进行的。体内很少有细胞会受到这种超生理氧张力的影响。广泛报道称,软骨细胞及其间充质祖细胞在低氧、更接近生理水平的氧张力(1-7%)下培养时具有更强的软骨形成表达。尽管这一观点已被普遍接受,但仍存在一些争议,不同的培养方法、物种和结果指标使该领域变得模糊不清。然而,这些结果存在关节软骨细胞偏向性,耳源性软骨细胞的相关情况尚未见报道。
从骨骼成熟的新西兰白兔中分离出耳软骨细胞和关节软骨细胞,在培养中进行扩增,并在无血清软骨形成培养基的高密度培养中进行分化。对聚集培养或组织工程薄片衍生的软骨组织进行生物力学、糖胺聚糖、胶原蛋白、胶原交联以及赖氨酰氧化酶活性和表达的评估。
我们的研究表明,与标准(20%)氧相比,低(5%)氧条件下耳软骨细胞和关节软骨细胞的增殖率均有所提高。在我们无支架的软骨形成培养中,发现低氧可增加关节软骨细胞糖胺聚糖的积累,但不会增加交联II型胶原蛋白或总胶原蛋白的积累。相反,在低氧张力下,耳软骨细胞积累的糖胺聚糖、交联II型胶原蛋白和总胶原蛋白较少。
本研究突出了从不同解剖部位分离的软骨细胞对低氧反应的显著差异。低氧有利于关节源性软骨形成,但对耳源性软骨形成有害。