在旋转细胞培养模拟微重力环境中，成骨细胞分化增强。

Osteoblast differentiation is enhanced in rotary cell culture simulated microgravity environments.

作者信息

Ko Y Joon, Zaharias Rebecca S, Seabold Denise A, Lafoon John, Schneider Galen B

机构信息

Department of Prosthodontics, College of Dentistry, University of Iowa, Iowa City, IA, USA.

出版信息

J Prosthodont. 2007 Nov-Dec;16(6):431-8. doi: 10.1111/j.1532-849X.2007.00204.x. Epub 2007 Jun 9.

DOI:10.1111/j.1532-849X.2007.00204.x

PMID:17559538

Abstract

PURPOSE

As the aging population increases, more people will become reliant on regenerative dental medicine for implant therapy. The objective of this study was to test the hypothesis that 3D rotary cell culture (RCC) environments created by simulated microgravity would enhance osteogenic gene expression using integrin mediated pathways.

MATERIALS AND METHODS

Human embryonic palatal mesenchymal (HEPM, ATCC 1486) pre-osteoblasts were cultured in either RCC to create 3D environments or in 2D monolayers for 72 hours. Gross phenotypic analysis was performed using Alizarin Red S staining for calcium and microscopy. Real-time PCR analysis was used to detect differences in osteoblast gene expression. Aggregates developed in 3D RCC environments were treated with or without antibody to the collagen-I integrin receptor alpha2beta1 to determine whether this molecular pathway might contribute to the development of a mineralized matrix.

RESULTS

Microscopic analysis demonstrated that RCC environments promoted 3D aggregate formation by 72 hours without any scaffold. The mass appeared osseous-like with a white, shiny, translucent surface. The center was amorphous with areas of vacuolization, tubule-like structures, and fibrous-like extensions. Real-time PCR data showed that 3D environments enhanced osteogenic gene expression as compared with 2D monolayer culturing conditions. At 72 hours, changes in levels of osteogenic gene expression were noted. Cbfa1, a necessary transcription factor for osteoblast differentiation, was expressed 33% higher (p= 0.26); Collagen 1, 69% higher (p= 0.05); Osterix, 49% higher (p= 0.001); and BSPII, 54% higher (p= 0.001) than osteoblasts cultured for 72 hours in standard 2D monolayer conditions. When cultured in the presence of collagen alpha2beta1 integrin receptor antibody, 3D aggregates had decreased levels of mineralization as compared with non-treated aggregates.

CONCLUSION

RCC enhances osteoblast differentiation using integrin mediated pathways.

摘要

目的

随着老龄化人口的增加，越来越多的人将依赖再生牙科医学进行种植治疗。本研究的目的是验证以下假设：模拟微重力产生的三维旋转细胞培养（RCC）环境将通过整合素介导的途径增强成骨基因表达。

材料与方法

将人胚胎腭间充质（HEPM，ATCC 1486）前成骨细胞在RCC中培养以创建三维环境，或在二维单层中培养72小时。使用茜素红S染色法进行钙和显微镜的大体表型分析。实时PCR分析用于检测成骨细胞基因表达的差异。在三维RCC环境中形成的聚集体用或不用抗I型胶原整合素受体α2β1抗体处理，以确定该分子途径是否可能有助于矿化基质的形成。

结果

显微镜分析表明，RCC环境在72小时内促进了三维聚集体的形成，无需任何支架。聚集体外观呈骨样，表面白色、有光泽、半透明。中心为无定形，有空泡化区域、管状结构和纤维状延伸。实时PCR数据显示，与二维单层培养条件相比，三维环境增强了成骨基因表达。在72小时时，观察到成骨基因表达水平的变化。Cbfa1是成骨细胞分化所必需的转录因子，其表达比在标准二维单层条件下培养72小时的成骨细胞高33%（p = 0.26）；I型胶原高69%（p = 0.05）；osterix高49%（p = 0.001）；骨唾液蛋白II高54%（p = 0.001）。当在胶原α2β1整合素受体抗体存在下培养时，与未处理的聚集体相比，三维聚集体的矿化水平降低。