[Experimental research of the promotion effect of autogeneic PRP on osteogenic differentiation of human adipose-derived stem cells in vitro].

OBJECTIVE: To study the effect of autogeneic PRP on proliferation and osteogenetic differentiation of human adipose-derived stem cells (ADSCs) in vitro.

METHODS: ADSCs were isolated from adipose tissue obtained from donor undergoing liposuction and were cultured, and growth condition of the cells was observed by inverted microscope. ADSCs at passage 3 were cultured in adipogenic or chondrogenic medium and underwent identification, immunofluorescence staining observations for CD29 and CD44 were performed. ADSCs at passage 3 were divided into 2 groups: PRP group cultured by osteogenic induction culture medium containing 10 mL/L PRP, and control group cultured by osteogenic induction culture medium without PRP. Then growth condition of the cells was observed by inverted microscope. MTT method was used to observe cell proliferation activity 1, 2, 3, 4 and 5 days after culture. ALP activity detection was conducted 7, 14, 21 and 28 days after culture. ALP staining was performed on PRP group 7 and 14 days after culture. Alizarin red staining was performed on PRP group 14 days after culture to detect the formation of calcium nodule.

RESULTS: Under the inverted microscope, most ADSCs at passage 3 were spindle-shaped and the doubling time was about 35 hours. Adipogenic and chondrogenic differentiation were confirmed, and the cells were positive for CD29 and CD44 immunofluorescence staining. MTT method revealed the absorbance value of PRP group at 1, 2, 3, 4 and 5 days was 0.137 +/- 0.015, 0.219 +/- 0.023, 0.367 +/- 0.031, 0.586 +/- 0.039 and 0.948 +/- 0.046, respectively, and in the control group, it was 0.081 +/- 0.009, 0.115 +/- 0.012, 0.162 +/- 0.017, 0.242 +/- 0.025 and 0.356 +/- 0.032, respectively, suggesting there were significant differences between two groups (P < 0.01). At 7 days after osteogenic induction, PRP group was positive for ALP staining, grey-black cell plasma and black precipitate were evident; the positive cells increased 14 days after osteogenic induction. ALP activity detection demonstrated the cell activity value of PRP group at 7, 14, 21 and 28 days was 23.96 +/- 2.05, 41.26 +/- 3.38, 38.12 +/- 3.03 and 35.89 +/- 2.24, respectively, while in the control group, it was 17.83 +/- 1.62, 26.64 +/- 2.37, 23.85 +/- 1.99 and 20.78 +/- 1.81, respectively, indicating there were significant differences between two groups (P < 0.01). At 14 days after osteogenic induction, Alizarin red staining showed the formation of calcium nodule in PRP group.

CONCLUSION: Autogeneic PRP can improve the proliferation of human ADSCs and induce their osteogenic differentiation in vitro, providing a new source of seed cells for bone tissue engineering.