Kobayashi K, Healey R M, Sah R L, Clark J J, Tu B P, Goomer R S, Akeson W H, Moriya H, Amiel D
Department of Orthopaedics, University of California-San Diego, La Jolla, California 92093-0630, USA.
Tissue Eng. 2000 Feb;6(1):29-38. doi: 10.1089/107632700320865.
A novel method of quantitating cell migration has been proposed for the potential utilization of tissue engineered scaffolds. Applying Alt's conservation law to describe the motion of first passage ACL and MCL cells, we have developed a quantitative method to assess innate differences in the motility of cells from these two ligamentous tissues. In this study, first passage ACL and MCL cells were cultured from four mature New Zealand white rabbits. One side of the cell monolayer was scraped completely away to create a wound model. The cell moved into the cell-free area, and cell density profiles were analyzed at 6 h and 12 h. Values of the random motility coefficient (mu) were then estimated by curve fitting the 6 h and 12 h data to a mathematical model, derived from the conservation law of cell flux. During 6 h of incubation in medium supplemented with 1% FBS, MCL cells (mu(MCL) = 4.63 +/- 0.65 X 10(-6) mm(2)/sec) were significantly (p < 0.05) more mobile than ACL cells (mu(ACL) = 2.51 +/- 0.31 X 10(-6) mm(2)/sec). At 12 h, the MCL cells also appeared to move faster (mu(ACL) = 4.39 +/- 0.63 X 10(-6) mm(2)/sec, mu(MCL) = 6.59 +/- 1.47 X 10(-6) mm(2)/sec), but the difference was not statistically significant (p = 0.18). Exposure of the cells to growth factors PDGF-BB or bFGF for 6 h had no significant effect on the migration of the ACL and MCL cells. However, exposure of the ACL cells (p < 0.05) and the MCL cells (p = 0.19) to 1 ng/mL of PDGFBB for 12 h enhanced their migration. Incubation with a high concentration (100 ng/mL) of PDGF-BB or bFGF at concentrations tested (1 or 100 ng/mL) for 12 h, produced little or no migratory stimulation on these ligament cells. Our findings support the previous qualitative observations made by numerous investigators. The novel methodology developed in this study may provide a basis for tissue engineering, and the results may be applied to tissue reconstruction techniques of the knee ligaments.
为了实现组织工程支架的潜在应用，人们提出了一种定量细胞迁移的新方法。应用阿尔特守恒定律来描述初代前交叉韧带（ACL）和内侧副韧带（MCL）细胞的运动，我们开发了一种定量方法，以评估来自这两种韧带组织的细胞在运动性方面的固有差异。在本研究中，从四只成年新西兰白兔身上培养初代ACL和MCL细胞。将细胞单层的一侧完全刮去以创建一个伤口模型。细胞迁移到无细胞区域，并在6小时和12小时时分析细胞密度分布。然后通过将6小时和12小时的数据拟合到一个基于细胞通量守恒定律推导出来的数学模型，来估计随机运动系数（μ）的值。在补充有1%胎牛血清（FBS）的培养基中孵育6小时期间，MCL细胞（μ（MCL）= 4.63±0.65×10⁻⁶ 平方毫米/秒）的运动性显著高于ACL细胞（μ（ACL）= 2.51±0.31×10⁻⁶ 平方毫米/秒）（p < 0.05）。在12小时时，MCL细胞似乎也移动得更快（μ（ACL）= 4.39±0.63×10⁻⁶ 平方毫米/秒，μ（MCL）= 6.59±1.47×10⁻⁶ 平方毫米/秒），但差异无统计学意义（p = 0.18）。将细胞暴露于生长因子血小板衍生生长因子-BB（PDGF-BB）或碱性成纤维细胞生长因子（bFGF）6小时，对ACL和MCL细胞的迁移没有显著影响。然而，将ACL细胞（p < 0.05）和MCL细胞（p = 0.19）暴露于1纳克/毫升的PDGF-BB 12小时可增强它们的迁移。在测试的浓度（1或100纳克/毫升）下，用高浓度（100纳克/毫升）的PDGF-BB或bFGF孵育12小时，对这些韧带细胞几乎没有或没有产生迁移刺激。我们的研究结果支持了众多研究者之前所做的定性观察。本研究中开发的新方法可能为组织工程提供基础，且研究结果可能应用于膝关节韧带的组织重建技术。
