Preliminary separation of the growth factors in platelet-rich plasma: effects on the proliferation of human marrow-derived mesenchymal stem cells.
BACKGROUND
Platelet-rich plasma (PRP) as a storage vehicle of growth factors has been successfully used in clinical applications, but in most cases the platelets were autologous. However, the large volume of blood withdrawn has detrimental effects on patients with anemia or poor general health. To overcome these limitations, this study was designed to separate the growth factors in homologous platelet-rich plasma.


METHODS
The gel chromatography with Superdex-75 column was applied to separate PRP supernatants into 4 major fractions. Then the four fractions were vacuumed freeze-dried and re-dissolved in phosphate buffered saline. Proteins concentrations in PRP and in four fractions were detected by bicinchoninic acid protein assay; platelet derived growth factor-AB (PDGF-AB) and transforming growth factor beta1 (TGF-beta1) levels were determined by sandwich enzyme-linked immunosorbent assays. The effects of fractions on the proliferation of human marrow-derived mesenchymal stem cells (MSCs) were determined by 3-(4, 5- dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay.


RESULTS
PRP supernatants were separated into four major fractions by gel chromatography. The proteins recovery was 96.72%. Of the four fractions, fraction B contained the highest TGF-beta1 and PDGF-AB levels, and the highest proteins concentrations. Cell proliferation curves of MSC demonstrated that fraction B and C induced a remarkable increase of MTT values compared to the untreated culture (P < 0.05), and the effects of fraction B and C showed no significant difference compared to the PRP group (P > 0.05). Fraction A and D showed no significant difference to the negative control group (P > 0.05).


CONCLUSIONS
The growth factors in PRP supernatants could be preliminarily separated into four fractions by gel chromatography, and the freeze-drying fractions retained the biological activity of growth factors. The growth factors were mostly presented in fraction B and C, and they promoted cell proliferation effectively.
Huang
Qian
Q
Department of Orthopedics and Traumatology, Nanfang Hospital, Southern Medical University, Guangdong, China.
Wang
Yun-dan
YD
Wu
Tao
T
Jiang
Shan
S
Hu
Yan-ling
YL
Pei
Guo-xian
GX
D001854:Bone Marrow Cells / Q000166:cytology*; D049109:Cell Proliferation / Q000187:drug effects; D002850:Chromatography, Gel; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000187:drug effects; D010976:Platelet Count; D010982:Platelet-Derived Growth Factor / Q000302:isolation & purification* / Q000494:pharmacology; D053657:Platelet-Rich Plasma / Q000737:chemistry*; D053773:Transforming Growth Factor beta1 / Q000302:isolation & purification* / Q000494:pharmacology
D010982:Platelet-Derived Growth Factor; D053773:Transforming Growth Factor beta1; C095477:platelet-derived growth factor AB