Inhibitory effect of ultrasonic wave on the proliferation and differentiation of porcine preadipocyte cultured in vitro

Ailin LI; Xuejie CHEN; Xiaowei WU

ABSTRACT

BACKGROUND:

Soft tissue filling is a problem in clinic. It has been proved that ultrasonic wave-treated mature adipocytes cannot be used for cell transplantation.It is hopeful to solve the problem with adipose tissue engineering.

OBJECTIVE:

To observe the effect of ultrasonic wave on the in vitro culture and proliferation of preadipocytes, and validate the possibility of preadipocyte as seed cell in adipose tissue engineering following ultrasound-assisted liposuction.

DESIGN:

Controlled observation experiment.

SETTING:

Department of Plastic Surgery, Rennin Hospital, Wuhan University. MATERIALS This experiment was carried out in the Medical College of Wuhan University from July 2003 to September 2004. One local 3-month-old hybridized pig was provided by the Experimental Animal Center of Medical College of Wuhan University. After the pig was anesthetized with ketamine, an area of 10 cm×20 cm was labeled on both sides of back respectively, and the tissue in the labeled area was harvested. The tissue on the right side served as experimental group and that on the left side served as control group.

METHODS:

The tissue of the expedmental group was pretreated for 8 minutes by ultrasonic wave with the energy of 3W/cm2. Under aseptic condition, the skin layer was open, and 100 g subcutaneous adipose tissue was resected from each side and then placed in prepared container containing cold D-hanks solution for later use. The ultrasonic wave-treated porcine preadipocytes of adipose tissue of experimental group were isolated and cultured in vitro, and the number and lipid content of preadipocytes were measured every other 2 days. Results were presented as the mean val ue of the number of cells of three wells. In the control group, pretreatment of ultrasonic wave was omitted. The growth curves of two groups were drawn. Intracellular adipose content was measured by oil red O staining. Absorbance (A) was measured with spectrophotometer (HITACHI G2000), which was regulated at the wavelength of 510 nm. MAIN OUTCOME

MEASURES:

① Morphological observation of culture of porcine preadipocytes. ②The growth curves of experimental and control groups. ③3 Change in the lipid content of experimental and control groups.

RESULTS:

① Cells in the control group adhered to the wall within 6 to 24 hours, and those in the experimental group basically adhered to the wall after 1 to 2 days, and there were many non-adhesive cells in the exoerimental group. Accumulation of adipose granules in the cells was found in the control group after 4 days and that was found in the experimental group after 6 days. On day 12, the cells in the control group basically differentiated into the cells with single lipid drop or multiple lipid drops, and a small amount of natant mature adipocytes were found, while few cells with lipid drop were found in the experimental group. ②Under the condition of the same amount of cells, the cell doubling time of experimental group was about 72 hours and that of control group was 36 hours. After 9-day culture, the number of cells in the experimental group and control group was 13×104 cells/well and 18×104 cells/well, respectively. The rate of cell proliferation of experimental group was very slow. ③Lipid of the experimental group appeared on the 6th day after culture, and that of the control group appeared on the 4th day after culture. The peak value of A was 0.32 and 0.68 in e penmental group and control group respectively.

CONCLUSION:

Ultrasonic wave has obviously inhibitory effect on the proliferation and differentiation of preadipocytes.The preadipocytes treated by ultrasonic wave are not suitable as seed cells.