Figure 1.
Microchip and experimental setup for evaluating stem cells towards osteogenesis under mechanical stimulation.
(A) The microchip is comprised of a cover, an air chamber, looped microvalves, and twelve cell culture chambers. These paired cell chambers share the inlet/outlet channel. The cells (hMSCs and hASCs) are loaded into half of the chip, individually. Scale bar = 1 cm. (B) Schematic diagram of top view (I) and simplified cross-sectional view (II) of the device. The device was designed to culture two different stem cells simultaneously and to apply mechanical stimulation using cyclic pneumatic force. (C) The experimental setup for mechanical stimulation, including a controlled nitrogen gas pressurized air chamber. The frequency of pneumatic pressure is controlled with a switching solenoid valve derived by a control circuit. During mechanical stimulation, microvalves are closed with higher pressure (P2>P1) to prevent undesired shear stress in the cell chambers.
Figure 2.
Osteogenesis characterizations of hMSCs and hASCs after 7 days.
hASCs and hMSCs cultured in the microchip with osteogenic medium for 7 days were stained with ALP and Alrizarin red. The stimulated group of BMSCs resulted in significantly enhanced ALP activity and calcium deposits. (Scale bars: ALP staining 100 (m, Alrizarin red staining 200 (m).
Figure 3.
Immunocytochemical staining of hMSCs and hACSs.
(A) The expression of osteogenic markers after 1 and 7 days. Bone sialoprotein (BSP), Osteopotin (OP), and Collagen type I (Col I) were stained with GFP and strongly expressed in the stimulated BMSCs. Blue = DAPI Nucleic Acid Stain. (Scale bars: 100 (m) Green fluruorecent expression intensity (B) and area (C) of ECMs in hMSCs and hASCS. Data presented in the line graph represent mean value with SD (n = 12). *p<0.05. Star (*) indicates comparison of statistical difference of stimulation to control and statistical difference between stimulated hMSCs and hASCs.
Figure 4.
Expressions of integrin CD11b and CD31.
(A) Fluorescent expression intensity and area of CD11b in hMSCs and hASCS. (B) Green fluorescent expression intensity and area of CD31 in hMSCs and hASCS. Star (*) indicates comparison of statistical difference of stimulation to control in the same cell type. *p<0.05. It also indicates statistical difference between stimulated hMSCs and hASCs.
Figure 5.
Osteogenesis related transcript levels and β1 integrin expression.
(A) Bone sialoprotein (BSP), (B) Osteopontin (OP), (C) Runt-related transcription factor 2 (Runx2), (D) β1 integrin (*p<0.05)