Role of Microtubules in Osteogenic Differentiation of Mesenchymal Stem Cells on 3D Nanofibrous Scaffolds

被引:21
作者
Meka, Sai Rama Krishna [1 ]
Chacko, Leeba Ann [2 ,3 ]
Ravi, Ashwini [2 ]
Chatterjee, Kaushik [1 ,2 ]
Ananthanarayanan, Vaishnavi [2 ]
机构
[1] Indian Inst Sci, Dept Mat Engn, Bangalore 560012, Karnataka, India
[2] Indian Inst Sci, Ctr BioSyst Sci & Engn, Bangalore 560012, Karnataka, India
[3] Natl Univ Singapore, Machanobiol, Singapore, Singapore
来源
ACS BIOMATERIALS SCIENCE & ENGINEERING | 2017年 / 3卷 / 04期
关键词
cytoskeleton; stem cells; osteogenesis; nanofibers; tissue scaffolds; FOCAL ADHESION; CYTOSKELETAL; SHAPE; ORGANIZATION; PLURIPOTENT; MORPHOLOGY; STIFFNESS; MICROENVIRONMENTS; PROLIFERATION; MODULATION;
D O I
10.1021/acsbiomaterials.6b00725
中图分类号
TB3 [工程材料学]; R318.08 [生物材料学];
学科分类号
0805 ; 080501 ; 080502 ;
摘要
Human bone marrow mesenchymal stem cells (MSCs) cultured on three-dimensional (3D) nanofibrous scaffolds are known to undergo osteogenic differentiation even in the absence of soluble osteoinductive factors. Although this process of differentiation has been attributed to the shape that cells assume on the fibrous scaffolds, it is unclear how constriction of cell shape would contribute to the differentiation phenotype. Here, we quantitatively compared cell and nuclear morphologies of cells cultured on 3D poly(ecaprolactone) (PCL) nanofibers (NF) and two-dimensional (2D) flat films using confocal fluorescence microscopy. We discovered that while cells on the 2D films exhibited cellular and nuclear morphologies similar to those cultured on tissue culture polystyrene, cells cultured on the 3D NF showed distinct cell and nuclear morphologies, with lower areas and perimeters, but higher aspect ratios. We next tested the effect of treatment of cells with actin-depolymerizing cytochalasin D and microtubule-depolymerizing nocodazole on these morphologies. In both 2D and 3D scaffolds, actin depolymerization brought about gross changes in cell and nuclear morphologies. Remarkably, microtubule depolymerization resulted in a phenotype similar to actin depolymerization in cells cultured on 3D NF alone, indicating a significant role for the microtubule cytoskeleton in the maintenance of cell shape and structure in 3D. The morphological changes of the nucleus that were apparent upon cytoskeletal perturbation were reflected in the organization of heterochromatin in the nucleus, with MSCs on 3D alone exhibiting a differentiation phenotype. Finally, we tested the effect of cytoskeletal depolymerization on mineralization of cells. Again, we observed higher mineralization in cells cultured on 3D NF, which was lost in cells treated with either cytochalasin D or nocodazole. Taken together, our results suggest that both the actin and microtubule cytoskeletons contribute significantly toward maintenance of cell and nuclear shape in cells cultured on 3D scaffolds, and consequently to their osteogenic differentiation.
引用
收藏
页码:551 / 559
页数:9
相关论文
共 43 条
[1]   Deconstructing the third dimension - how 3D culture microenvironments alter cellular cues [J].
Baker, Brendon M. ;
Chen, Christopher S. .
JOURNAL OF CELL SCIENCE, 2012, 125 (13) :3015-3024
[2]   Gelatin nanoparticles loaded poly(ε-caprolactone) nanofibrous semi-synthetic scaffolds for bone tissue engineering [J].
Binulal, N. S. ;
Natarajan, Amrita ;
Menon, Deepthy ;
Bhaskaran, V. K. ;
Mony, Ullas ;
Nair, S. V. .
BIOMEDICAL MATERIALS, 2012, 7 (06)
[3]   Substrate Elastic Modulus Regulates the Morphology, Focal Adhesions, and α-Smooth Muscle Actin Expression of Retinal Muller Cells [J].
Bu, Shao-Chong ;
Kuijer, Roel ;
van der Worp, Roelofje J. ;
van Putten, Sander M. ;
Wouters, Olaf ;
Li, Xiao-Rong ;
Hooymans, Johanna M. M. ;
Los, Leonoor I. .
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE, 2015, 56 (10) :5974-5982
[4]   GEF-H1 couples nocodazole-induced microtubule disassembly to cell contractility via RhoA [J].
Chang, Yuan-Chen ;
Nalbant, Perihan ;
Birkenfeld, Joerg ;
Chang, Zee-Fen ;
Bokoch, Gary M. .
MOLECULAR BIOLOGY OF THE CELL, 2008, 19 (05) :2147-2153
[5]   Cell shape provides global control of focal adhesion assembly [J].
Chen, CS ;
Alonso, JL ;
Ostuni, E ;
Whitesides, GM ;
Ingber, DE .
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 2003, 307 (02) :355-361
[6]   Tensegrity and mechanoregulation: from skeleton to cytoskeleton [J].
Chen, CS ;
Ingber, DE .
OSTEOARTHRITIS AND CARTILAGE, 1999, 7 (01) :81-94
[7]   Geometric control of cell life and death [J].
Chen, CS ;
Mrksich, M ;
Huang, S ;
Whitesides, GM ;
Ingber, DE .
SCIENCE, 1997, 276 (5317) :1425-1428
[8]   Nanopatterning reveals an ECM area threshold for focal adhesion assembly and force transmission that is regulated by integrin activation and cytoskeleton tension [J].
Coyer, Sean R. ;
Singh, Ankur ;
Dumbauld, David W. ;
Calderwood, David A. ;
Craig, Susan W. ;
Delamarche, Emmanuel ;
Garcia, Andres J. .
JOURNAL OF CELL SCIENCE, 2012, 125 (21) :5110-5123
[9]   Modulating the Substrate Stiffness to Manipulate Differentiation of Resident Liver Stem Cells and to Improve the Differentiation State of Hepatocytes [J].
Cozzolino, Angela Maria ;
Noce, Valeria ;
Battistelli, Cecilia ;
Marchetti, Alessandra ;
Grassi, Germana ;
Cicchini, Carla ;
Tripodi, Marco ;
Amicone, Laura .
STEM CELLS INTERNATIONAL, 2016, 2016
[10]   Microtubules Mechanically Regulate Cell Adhesion Strengthening Via Cell Shape [J].
Elineni, Kranthi Kumar ;
Gallant, Nathan D. .
CELLULAR AND MOLECULAR BIOENGINEERING, 2014, 7 (01) :136-144