Tissue Engineering Laboratory, Department of Mechanical Engineering, The University of Hong Kong, Pokfulam Rd, Hong Kong Special Administrative Region, China.
Spine J. 2011 Oct;11(10):947-60. doi: 10.1016/j.spinee.2011.07.004. Epub 2011 Aug 16.
Degenerative disc disease poses an increasing threat to our quality of life as we age. Existing treatments have limitations. New treatment modalities focusing on biologic rather than surgical approach would be appealing.
Culturing intervertebral disc cells in a three-dimensional (3D) model that can retain cellular characteristics and phenotype is a critical step toward understanding how the disc cells respond to and interact with extrinsic signals before better therapeutics can be derived.
In this work, we studied the culture of rabbit nucleus pulposus (NP) cells in a collagen microsphere system and compared their cell morphology and expression of a few potential phenotypic markers with that in monolayer culture.
Specifically, rabbit NP cells isolated from both young and old animals were encapsulated and cultured in collagen microspheres with different monomeric concentrations and with different cell encapsulation density for different period of time. Evaluation on the growth kinetics, the viability, the cell morphology, the expression of Types I and II collagen, glycosaminoglycans (GAGs), and Keratin 19, and the ultrastructure of the fiber meshwork were conducted to compare the microsphere 3D culture system and the traditional monolayer cultures.
Nucleus pulposus cells in two-dimensional culture lost the phenotypic expression of Type II collagen and keratin 19 and expressed Type I collagen. In contrast, the 3D collagen microsphere culture system consistently outperformed the traditional monolayer culture in maintaining a round morphology and preserving the phenotypes of NP cells with persistent expression of Type II collagen and Keratin 19. These cells also remodeled the template collagen matrix in the microspheres by depositing new matrices, including collagen Type II and GAGs in a cell seeding density and collagen concentration dependent manner.
This study demonstrates the appeal of the 3D collagen microsphere system for NP cell culture over traditional monolayer culture because it preserves the phenotypic characteristics of NP cells. This system also enables the NP cells to remodel the template collagen matrix by depositing new matrices, suggesting an innovative way to reconstitute cell-specific and native tissue-like environment in vitro for future studies on stem cell matrix niche and interactions of NP cell with extrinsic factors.
随着年龄的增长，退行性椎间盘疾病对我们的生活质量构成了越来越大的威胁。现有的治疗方法存在局限性。新的治疗方法侧重于生物方法而非手术方法，这将非常有吸引力。
在三维（3D）模型中培养椎间盘细胞，该模型可以保留细胞特征和表型，这是理解椎间盘细胞如何对外界信号做出反应和相互作用的关键步骤，以便在此基础上开发更好的治疗方法。
在这项工作中，我们研究了兔髓核细胞在胶原微球系统中的培养，并将其与单层培养相比，比较了细胞形态和少数潜在表型标志物的表达。
具体来说，从年轻和老年动物中分离出的兔 NP 细胞被包封并在具有不同单体浓度的胶原微球中培养，不同的细胞包封密度和不同的时间段。评估生长动力学、活力、细胞形态、I 型和 II 型胶原、糖胺聚糖（GAGs）和角蛋白 19 的表达以及纤维网的超微结构，以比较微球 3D 培养系统和传统的单层培养。
二维培养的髓核细胞失去了 II 型胶原和角蛋白 19 的表型表达，表达了 I 型胶原。相比之下，3D 胶原微球培养系统在维持圆形形态和保持 NP 细胞表型方面始终优于传统的单层培养，NP 细胞持续表达 II 型胶原和角蛋白 19。这些细胞还通过以细胞接种密度和胶原浓度依赖的方式沉积新的基质，包括 II 型胶原和 GAGs，重塑微球中的模板胶原基质。
这项研究表明，3D 胶原微球系统在 NP 细胞培养方面优于传统的单层培养，因为它保留了 NP 细胞的表型特征。该系统还使 NP 细胞能够通过沉积新的基质重塑模板胶原基质，这为未来研究干细胞基质生态位和 NP 细胞与外源性因素的相互作用提供了一种创新的方法，以在体外重建具有细胞特异性和类似天然组织的环境。
