Elashry Mohamed I, Gegnaw Shumet T, Klymiuk Michele C, Wenisch Sabine, Arnhold Stefan
Institute of Veterinary Anatomy-, Histology and -Embryology, University of Giessen, 35392, Giessen, Germany; Anatomy and Embryology Department, Faculty of Veterinary Medicine, University of Mansoura, 35516, Egypt.
Institute of Veterinary Anatomy-, Histology and -Embryology, University of Giessen, 35392, Giessen, Germany; Institute des Neurosciences Cellulaires et Integratives (INCI), University of Strasbourg, 67084, Strasbourg, France.
Acta Histochem. 2019 Apr;121(3):344-353. doi: 10.1016/j.acthis.2019.02.002. Epub 2019 Feb 23.
Cell-based therapies have become a promising approach to promote tissue regeneration and the treatment of musculoskeletal disorders. Bone regeneration maintains bone homeostasis, mechanical stability and physical performance. Mechanical stimulation showed to induce stem cell differentiation into the osteogenic fate. However, the effect of various osteogenic protocols on the osteogenic commitment of equine adipose-derived stem cells is not fully elucidated. Here we examined the influence of fluid-based shear stress (FSS) via mechanical rocking to assess whether mechanical stimulation promotes osteogenic differentiation of equine adipose-derived stem cells (ASCs). ASCs were induced into osteogenic fate using osteogenic differentiation medium (ODM) protocol or additional supplementation of 5 mM CaCl and 7.5 mM CaCl protocol compared to cells cultivated in basal medium (BM) up to 21 day. The ASCs proliferation pattern was evaluated using the sulforhodamine B (SRB) protein assay. Osteogenic differentiation examined via semi-quantification of alizarin red staining (ARS) and alkaline phosphatase activity (ALP) as well as, via quantification of osteocalcin (OC), alkaline phosphatase (ALP), osteopontin (OP), and collagen type-1 (COL1) gene expression using RT-qPCR. We show that mechanical FSS increased the proliferation pattern of ASCs compared to the static conditions. Mechanical FSS together with 5 mM CaCl and 7.5 mM CaCl promoted osteogenic nodule formation and increased ARS intensity compared to the standard osteogenic protocols. We observed that combined mechanical FSS with ODM protocol increase ALP activity compared to static culture conditions. We report that ALP and OC osteogenic markers expression were upregulated under mechanical FSS culture condition particularly with the ODM protocol. Taken together, it can be assumed that mechanical stress using FSS promotes the efficiency of the osteogenic differentiation protocols of ASCs through independent mechanisms.
基于细胞的疗法已成为促进组织再生和治疗肌肉骨骼疾病的一种有前景的方法。骨再生维持骨稳态、机械稳定性和身体机能。机械刺激已显示可诱导干细胞向成骨命运分化。然而，各种成骨方案对马脂肪来源干细胞成骨定向分化的影响尚未完全阐明。在此，我们通过机械摇晃研究了基于流体的剪切应力（FSS）的影响，以评估机械刺激是否促进马脂肪来源干细胞（ASCs）的成骨分化。与在基础培养基（BM）中培养长达21天的细胞相比，使用成骨分化培养基（ODM）方案或额外补充5 mM氯化钙和7.5 mM氯化钙方案将ASCs诱导为成骨命运。使用磺酰罗丹明B（SRB）蛋白测定法评估ASCs的增殖模式。通过茜素红染色（ARS）和碱性磷酸酶活性（ALP）的半定量以及使用RT-qPCR对骨钙素（OC）、碱性磷酸酶（ALP）、骨桥蛋白（OP）和I型胶原蛋白（COL1）基因表达进行定量来检测成骨分化。我们表明，与静态条件相比，机械FSS增加了ASCs的增殖模式。与标准成骨方案相比，机械FSS与5 mM氯化钙和7.5 mM氯化钙共同促进了成骨结节形成并增加了ARS强度。我们观察到，与静态培养条件相比，机械FSS与ODM方案联合可增加ALP活性。我们报告说，在机械FSS培养条件下，特别是使用ODM方案时，ALP和OC成骨标志物的表达上调。综上所述，可以假设使用FSS的机械应力通过独立机制提高了ASCs成骨分化方案的效率。
