Quexada-Rodríguez Diego, Messelmani Taha, Jellali Rachid, Le Goff Anne, Ho Ba Tho Marie-Christine, Garzón-Alvarado Diego, Trabelsi Olfa
Université de technologie de Compiègne, CNRS, Biomechanics and Bioengineering, Centre de Recherche Royallieu, CS 60 319 - 60 203, Compiègne Cedex, France.
Numerical Methods and Modeling Research Group (GNUM), Universidad Nacional de Colombia, Colombia.
J Orthop. 2025 May 31;70:173-182. doi: 10.1016/j.jor.2025.05.058. eCollection 2025 Dec.
Matrix mineralization is a key process in endochondral ossification and cartilage maturation. While optimized biochemical protocols using ATDC5 chondrogenic cells have shortened mineralization timelines, the role of mechanical stimulation in enhancing this process remains underexplored. This study investigates whether dynamic mechanical stimulation in 2D monolayer cultures can accelerate mineralization and influence extracellular matrix (ECM) composition.
ATDC5 cells were cultured on PDMS membranes coated with collagen and subjected to cyclic tensile strain (12 % at 0.05 Hz for 1 h/day) using a uniaxial bioreactor over 5 days. The culture protocol included supplementation with β-glycerophosphate and ascorbic acid. Mineralization and ECM production were assessed at days 7, 17, and 23 using Alizarin Red and Alcian Blue staining. SEM/EDX confirmed calcium-phosphate deposition. A phenomenological finite element model was developed to correlate mechanical stimuli with hypertrophy using the osteogenic index.
Mechanical stimulation led to a 32 % increase in Alizarin Red staining compared to controls (P < 0.001), indicating faster mineralization. GAG production was reduced under mechanical loading (P < 0.05), consistent with early mineralization. SEM/EDX confirmed more uniform mineral deposition in stimulated samples. Morphologically, stimulated cells aligned along the loading axis and displayed a nearly 10-fold increase in hypertrophic cell area. The numerical model showed elevated osteogenic index values and stress peaks in the chondrocyte membrane under mechanical loading.
Dynamic tensile stimulation in 2D culture significantly accelerates ECM mineralization in ATDC5 cells. This effect appears to be mediated through enhanced chondrocyte hypertrophy and alignment of ECM components. The combined use of mechanical and biochemical cues provides a promising strategy for optimizing in vitro models of endochondral ossification and developing tissue engineering therapies.
基质矿化是软骨内成骨和软骨成熟的关键过程。虽然使用ATDC5软骨生成细胞的优化生化方案缩短了矿化时间，但机械刺激在增强这一过程中的作用仍未得到充分探索。本研究调查二维单层培养中的动态机械刺激是否能加速矿化并影响细胞外基质（ECM）组成。
将ATDC5细胞培养在涂有胶原蛋白的聚二甲基硅氧烷（PDMS）膜上，并使用单轴生物反应器在5天内施加循环拉伸应变（0.05Hz下12%，每天1小时）。培养方案包括添加β-甘油磷酸酯和抗坏血酸。在第7天、第17天和第23天使用茜素红和阿尔新蓝染色评估矿化和ECM产生。扫描电子显微镜/能谱仪（SEM/EDX）证实了磷酸钙沉积。开发了一个唯象有限元模型，以使用成骨指数将机械刺激与肥大相关联。
与对照组相比，机械刺激导致茜素红染色增加32%（P<0.001），表明矿化更快。在机械加载下糖胺聚糖（GAG）产生减少（P<0.05），与早期矿化一致。SEM/EDX证实在受刺激的样本中矿物质沉积更均匀。在形态学上，受刺激的细胞沿加载轴排列，肥大细胞面积增加近10倍。数值模型显示在机械加载下软骨细胞膜中的成骨指数值和应力峰值升高。
二维培养中的动态拉伸刺激显著加速了ATDC5细胞中的ECM矿化。这种效应似乎是通过增强软骨细胞肥大和ECM成分排列来介导的。机械和生化信号的联合使用为优化软骨内成骨的体外模型和开发组织工程疗法提供了一种有前景的策略。
