Tissue Engineering Auricular Cartilage Using Late Passage Human Auricular Chondrocytes.
Tissue Engineering Auricular Cartilage Using Late Passage Human Auricular Chondrocytes.
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DOI：
10.1097/sap.0000000000001400
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发表时间：
2018-04
影响因子：
1.5
通讯作者：
Spector JA
中科院分区：
文献类型：
--
作者：
Bernstein JL;Cohen BP;Lin A;Harper A;Bonassar LJ;Spector JA
当前耳廓畸形自体重建方案的显著缺陷激发了人们对组织工程解决方案的极大兴趣。人耳软骨工程的一个主要障碍是能否获得足够的自体人软骨细胞。临床上可获得的耳软骨组织（即1克）只能产生大约1000万个细胞，而制造一个全尺寸的儿童耳朵需要25倍的细胞。人们认为，反复传代软骨细胞会导致去分化和软骨形成潜能的丧失。然而，关于人耳软骨细胞能够以最大化细胞扩张同时最小化去分化的方式传代的理想次数，几乎没有数据存在。从废弃的耳成形术标本中分离出人耳软骨细胞。然后扩增hac，将传代3、4、5的细胞包封在直径为8mm、细胞密度为2500万个/mL的I型胶原水凝胶制成的圆盘中。将构建体皮下植入裸鼠背部，在1个月和3个月后采集进行分析。含有传代3、4和5软骨细胞的构建体均保持其原始的圆柱形几何形状。3个月后，P3、P4和P5椎间盘直径分别为其初始直径的69±9%、67±10%和73±15%。无论通道数如何，所有构建体都呈现出光滑的白色软骨外观，类似于天然耳廓软骨。经Safranin-O染色和Verhoeff染色，组织学分析显示形成有组织的软骨膜，由胶原蛋白、丰富的蛋白聚糖基质、细胞腔隙和致密的弹性蛋白纤维蛋白网络组成。生化分析证实，与天然耳廓软骨相比，晚期传代构建的蛋白多糖和羟脯氨酸含量相似。这些数据表明，传代晚期的人耳穴软骨细胞（传代至5代）形成的弹性软骨在组织学、生化和生物力学上与天然人弹性软骨相似，具有用于耳穴软骨工程的潜力。
The significant shortcomings associated with current autologous reconstructive options for auricular deformities have inspired great interest in a tissue engineering solution. A major obstacle in the engineering of human auricular cartilage is the availability of sufficient autologous human chondrocytes. A clinically obtainable amount of auricular cartilage tissue (i.e. one gram) only yields approximately ten million cells, where 25 times this amount is needed for the fabrication of a full-scale pediatric ear. It is thought that repeated passaging of chondrocytes leads to de-differentiation and loss of the chondrogenic potential. However, little to no data exists regarding the ideal number of times that human auricular chondrocytes can be passaged in a manner that maximizes the cellular expansion while minimizing dedifferentiation. Human auricular chondrocytes (HAuCs) were isolated from discarded otoplasty specimens. The HAuCs were then expanded and cells from passage 3, 4, and 5 were encapsulated into 8mm diameter discs made from type I collagen hydrogels with a cell density of 25 million cells/mL. The constructs were implanted subcutaneously in the dorsa of nude mice, and harvested after 1 and 3 months for analysis. Constructs containing passage 3, 4, and 5 chondrocytes all maintained their original cylindrical geometry. After 3 months in vivo, the diameters of the P3, P4, and P5 discs were 69±9%, 67±10%, and 73±15% of their initial diameter, respectively. Regardless of the passage number, all constructs developed a glossy white cartilaginous appearance, similar to native auricular cartilage. Histologic analysis demonstrated development of an organized perichondrium composed of collagen, a rich proteoglycan matrix, cellular lacunae, and a dense elastin fibrin network by Safranin-O and Verhoeff’s stain. Biochemical analysis confirmed similar amounts of proteoglycan and hydroxyproline content in late passage constructs when compared to native auricular cartilage. These data indicate that late passage human auricular chondrocytes (up to passage 5) form elastic cartilage that is histologically, biochemically, and biomechanically similar to native human elastic cartilage and have the potential to be used for auricular cartilage engineering.