The Effect Of Cell Spreading Shape And Area On Osteoblastic Apoptosis

In a biological body, not only cytokines can mediate the physiological behaviors of cells, but also the physical environment around cells plays critical roles in regulating the proliferation and differentiation of cells. For example, the spatial structure of extracellular matrix outside cells is able to influence cell spreading shape, area and cell-matrix interaction. Osteoblasts are derived from bone and finally differentiate into osteocytes. Two notable phenomena were observed during this differentiation process. First, the cellular shape changes from spindle or triangle (osteoblasts) to dendrites (osteocytes). In addition, osteoblasts closely cover on the trabeculae but mature osteocytes sparsely distribute inside lacunae, which implies that there must be a large number of osteoblasts entering apoptosis. Basing on the above phenomena, we hypothesize that cell spreading shape and area may affect the apoptosis of osteoblasts.Osteoblastic cell MC3T3-E1 was seeded on the micropatterned surface and five patterns were designed including circle, circle with rectangular branch, circle with triangular branch, triangle and square. After measuring the real spreading areas of osteoblasts, the pattern areas varied as 113, 201, 314, 1256μm2. Three branch lengths were designed for the specific patterns. At 12, 24 and 48 hours after seeding osteoblasts on the above patterns, the apoptosis of osteoblasts were detected by TUNEL assay kits (Roche). Then immunofluorescent staining was used to investigate the expression and distribution of actin, and vinculin, a component protein of focal adhesion complex. Themain experiments and results are as follows:1) Cell spreading shape and area influenced the apoptosis of osteoblasts:①For all kinds of islands, the apoptosis rate of osteoblasts decreased along with the increase of islands'area, for example, the apoptosis of osteoblasts on 1256μm2 island was 50% lower than the cells on 113μm2 isoland;②For different pattern of islands with same area, the apoptosis was significantly reduced in the osteoblast cells cultured on islands with longer branches. For example, only 20% of cells cultured on circular islands with longest branches were dead, but dead cells increased to about 55% on circular island with same spreading area of 314μm 2;③Rectangular branch had more important effect on the inhibition of osteoblasts apoptosis than triangular branch.2) Cell spreading shape and area affected the expression and distribution of actin and vinculin in osteoblasts:①Cell spreading shape significantly influenced the distribution of cytoskeleton, for example, actin could form clearer stress fiber in the cells on 314μm2 islands with longest branches.②Contrasting with the vinculin distribution in ostoeblasts in circular, most of the vinculin accumulated in the branches in the circular island with rectangular branches.In conclusion, our researches showed that cell spreading shape and area significantly influence the apoptosis of osteoblastic cells and the preliminary study on cytoskeleton and focal adhesion proteins will give insight into the cellular mechanotransduction.