Tsai Tsung-Ting, Guttapalli Asha, Oguz Erbil, Chen Lih-Huei, Vaccaro Alexander R, Albert Todd J, Shapiro Irving M, Risbud Makarand V
Department of Orthopaedic Surgery and Graduate Program in Tissue Engineering and Regenerative Medicine, Thomas Jefferson University, Philadelphia, PA 19107, USA.
Spine (Phila Pa 1976). 2007 Mar 1;32(5):495-502. doi: 10.1097/01.brs.0000257341.88880.f1.
To investigate effects of FGF-2 on nucleus pulposus cell growth and differentiation.
To elucidate the phenotypic changes that occur during expansion of nucleus pulposus cells in monolayer culture, and to investigate the effects of fibroblast growth factor (FGF)-2 on cell growth and differentiation.
Nucleus pulposus cells would have a limited application for autologous cell transplantation if phenotypic dedifferentiation takes place during culture expansion. FGF-2 has been shown to retain the differentiation potential of monolayer expanded chondrocytic cells. However, its effect on nucleus pulposus cells is not known.
Bovine nucleus pulposus cells were serially passaged in the presence or absence of FGF-2 (1 and 10 ng/mL). After passage numbers 1 and 7, cells were immobilized in alginate beads and treated with transforming growth factor (TGF)-beta1 for 1 week to assess their differentiation.
During culture expansion in monolayer, nucleus pulposus cells maintained the expression of aggrecan messenger ribonucleic acid (mRNA). However, mRNA levels of collagen type I, collagen type II, Sox-9, and versican decreased with increasing passage number for both control (untreated) cells and FGF-2 treated cells. When grown in alginate with TFG-beta1, passage 7 cells that received FGF-2 during culture expansion restored the mRNA expression of type II collagen, Sox-9, COMP, chondroadherin, and fibromodulin. Moreover, FGF-2 treatment resulted in increased sulfated proteoglycan synthesis and lower aggrecan turnover compared to untreated controls under identical culture conditions. FGF-2 treated cells continued to express HIF-1alpha protein till passage 7, while MMP-2 expression was evident in cells treated with TGF-beta1. In addition, cells pretreated with FGF-2 showed higher induction of phospho ERK1/2 after treatment with TGF-beta1. Also, FGF-2 maintained smad 2/smad 3 mediated signaling in cells after TGF-beta treatment. FGF-2 action resulted in reduced actin stress fiber formation and migratory cell morphology, with no effect on cell proliferation.
The presence of FGF-2 during culture expansion of nucleus pulposus cells in monolayer can sustain a differentiated cell phenotype by maintaining responsiveness to TGF-beta1. Our results suggest that FGF-2 should be tested for its ability to maintain the reactivity of the nucleus pulposus cells to other morphogenic factors that may be used for cell-based transplantation therapy.
探讨成纤维细胞生长因子-2（FGF-2）对髓核细胞生长和分化的影响。
阐明单层培养中髓核细胞扩增过程中发生的表型变化，并研究成纤维细胞生长因子（FGF）-2对细胞生长和分化的影响。
如果在培养扩增过程中发生表型去分化，髓核细胞在自体细胞移植中的应用将受到限制。FGF-2已被证明能保留单层扩增软骨细胞的分化潜能。然而，其对髓核细胞的作用尚不清楚。
牛髓核细胞在有或无FGF-2（1和10 ng/mL）的情况下连续传代。在第1代和第7代传代后，将细胞固定在藻酸盐珠中，并用转化生长因子（TGF）-β1处理1周以评估其分化情况。
在单层培养扩增过程中，髓核细胞维持了聚集蛋白聚糖信使核糖核酸（mRNA）的表达。然而，对照（未处理）细胞和FGF-2处理细胞中，I型胶原、II型胶原、Sox-9和多功能蛋白聚糖的mRNA水平均随传代次数增加而降低。当在含有TFG-β1的藻酸盐中生长时，在培养扩增过程中接受FGF-2的第7代传代细胞恢复了II型胶原、Sox-9、软骨寡聚基质蛋白、软骨粘连蛋白和纤调蛋白的mRNA表达。此外，与相同培养条件下未处理的对照相比，FGF-2处理导致硫酸化蛋白聚糖合成增加，聚集蛋白聚糖周转降低。FGF-2处理的细胞在第7代传代前持续表达缺氧诱导因子-1α蛋白，而MMP-2表达在TGF-β1处理的细胞中明显。此外，用FGF-2预处理的细胞在用TGF-β1处理后显示出更高的磷酸化ERK1/2诱导。此外，FGF-2在TGF-β处理后维持细胞中smad 2/smad 3介导的信号传导。FGF-2的作用导致肌动蛋白应力纤维形成减少和细胞迁移形态改变，对细胞增殖无影响。
在单层培养的髓核细胞扩增过程中，FGF-2的存在可通过维持对TGF-β1的反应性来维持分化细胞表型。我们的结果表明，应测试FGF-2维持髓核细胞对其他可能用于基于细胞的移植治疗的形态发生因子反应性的能力。
