Effects of cell density on proliferation and osteogenic differentiation of human mesenchymal stem cells during subculturing in vitro

Xiaojun DUAN; Liu YANG; Guangxing CHEN; Zhong LI; Hongxin ZHANG

ABSTRACT

BACKGROUND:

Rapid development of tissue engineering is the new hope of repairing bone defect so as to obtain complete bone regeneration,and rapid proliferation of seed cells is one of the important factors in it.

OBJECTIVE:

To evaluate the proliferation and osteogenic differentiation of human mesenchymal stem cells (MSCs) plated at different cell density during subculturing period.

DESIGN:

Repetitive measurement.

PARTICIPANTS:

The experiment was conducted in the Trauma, Burn and Combined Injury Laboratory of the Third Military Medical University of Chinese PLA between January 2003 and March 2004. MSCs were obtained from iliac bone bone marrow of the 5 healthy male volunteers by separation and proliferation.

METHODS:

The MSCs of second generation were inoculated at 8×103/cm2,3×103/cm2, and 8×102/cm2, respectively, and then the growth curve was drawn and analyzed. After 18 days of proliferation and culture, the increased amount of MSCs was recorded to analyze the post-proliferation growth curve and osteogenic ability.MAIN OUTCOME

MEASURES:

①Growth curve of cells plated at different cell density; ②Results of alkaline phosphatase(ALP) staining and immunohistochemical staining of osteocalcin.

RESULTS:

Human MSCs from bone marrow were CD34 negative and expressed low level of ALP. Population doubling time was 40 hours after MSCs were plated at 8×103/cm2 and the number of the cells was expanded (51±13) times after 18 days. MSCs were expanded (28±6) times 18 days after plated at 3×103/cm2. While plated at 8×102/cm2, the number of the cells was only expanded (5±3) times. Proliferative ability of cells cultured at a low density was stronger that that at a high density. MSCs could be induced to differentiate into the osteocytic lineages after plated at not only 8×103/cm2 but also 8×102/cm2.

CONCLUSION:

Cell density plays an evident role in the proliferation of MSCs. Cell density of 8×103/cm2 is proper to the rapid proliferation of MSCs as seed cells in bone tissue engineering.