納米復合羥基磷灰石接枝聚乳酸(HA/PLA)為支架材料,探討體外動態培養構建人工骨組織的效果.方法 將大鼠骨髓間充質干細胞(MSC)接種在生物活性納米復合HA/PLA三維多孔支架上,實驗組用體外灌注動態培養體系構建骨組織;對照組用常規靜態培養構建人工骨.分別探討細胞接種密度、灌注流速對細胞增殖、形態和堿性磷酸酶(ALP)的影響.結果 兩組均能體外構建骨組織,實驗組新生成骨細胞數量更多、密度更大,組織結構排列更規整
Effect of the Shear Stress in the Human Bone Marrow Mesenchymal Stem Cell Behavior
Clara Alcaine1,2, Sonia Santander1,2, Ignacio Ochoa1,2, Jose Manuel Garcia-Aznar1,2, Manuel Doblare1,2
Corresponding Author: iochgar@unizar.es
1 Group of Structural Mechanics and Materials Modelling (GEMM). Aragón Institute of Engineering
Research (I3A). Universidad de Zaragoza, Spain 2 Centro de Investigación Biomédica en Red en
Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN) Aragón Institute of Health Sciences, Spain
Introduction
Shear stress has been previously reported to be a
powerful differentiation stimuli (1,2). However,
other effects (proliferation, migration) on Human
Bone Marrow Mesenchymal Stem Cells (MSCs)
have not been deeply studied. Our main goal is
to determine if the shear stress is able to affect
not only differentiation but also other important
cell processes such as cell proliferation,
migration or cell adhesion.
Materials and Methods
Bone Marrow Mesenchymal Stem Cells have
been seeded in μ-Slide I flow kit (Ibidi) and
cultured under a shear stress of 5 dynes /cm2 in a
perfusion bioreactor (TEB-1000, EBERS) for 7
days. Proliferation was determined after cell
counting and apoptosis (Annexin V) was
measured with flow citometry techniques.
Migration experiments were performed in a
multidimensional microscope for 24 hours.
Inmunofluorescent staining to determine the cell
area and cytoskeleton organization was carried
out in a confocal microscope. Expression of the
cell adhesion molecules was determined by RTPCR.
ANOVA and t-student tests were carried
out to determine statistical differences.
Results
Significant differences in cell proliferation have
been observed after 7 days between static and
shear stimulated cells (Fig.1). However, no
significant differences in apoptosis were
obtained between both studied groups (Fig.2).
Differences in cell area, speed of migration,
cytoskeleton organization and adhesion
molecules has been also observed.
In a future, we will try to determine the effect of
different shear stress ratios on the previously
described variables. |
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