PREPARATION OF PLATELET-RICH PLASMA GEL AND ITS EFFECT ON SKIN FLAP SURVIVAL OF RAT_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

LI Weiwei ¹ , FENG Rui. ²

Platelet-rich plasma Skin flap survival Growth factor Rat;

Corresponding author：

Keywords：

Platelet-rich plasma ，Skin flap survival ，Growth factor ，Rat

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【Abstract】 Objective To find out the best method to prepare platelet-rich plasma (PRP) and to evaluate the effect of PRP gel on skin flap survival and its mechanism. Methods Totally, 72 Wistar rats (aged 12 weeks, weighing 250-300 g) were used for the experiment. The arterial blood (8-10 mL) were collected from the hearts of 24 rats to prepare PRP with three kinds of centrifuge methods: in group A, 200 × g centrifuge for 15 minutes, and 500 × g centrifuge for 10 minutes；in group B, 312 × g centrifuge for 10 minutes, and 1 248 × g centrifuge for 10 minutes；and in group C, 200 × g centrifuge for 15 minutes, and 200 × g centrifuge for 10 minutes. The platelet was counted in the whole blood, PRP, and platelet-poor plasma (PPP) to determine an ideal centrifuge. PRP, PPP, and the serum after first centrifuge were collected. The concentrations of platelet-derived growth factor BB (PDGF-BB) and transforming growth factor β1 (TGF-β1) were measured in the PRP, PPP, and serum using the enzyme-linked immunosorbent assay method, and PRP and PPP gels were prepared. The flaps of 11 cm × 3 cm in size were elevated on the back of 48 rats, which were divided into 3 groups: PRP gel (PRP group, n=16) and PPP gel (PPP group, n=16) were injected, no treatment was given in the control group (n=16). The flap survival rate was measured at 7 days. Histological and real-time PCR were used to count the inflammatory cells and blood vessel density, and to detect the expressions of vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), PDGF-AA, and PDGF-BB mRNA at 8 hours, 24 hours, 3 days, and 7 days. Results Platelet counting showed platelet in group A was the highest. ELISA evaluation showed that the concentrations of TGF-β1 and PDGF-BB were significantly higher in PRP than in PPP and serum (P lt; 0.05). The flap survival rate was 61.2% ± 9.1% in PRP group, showing significant differences (P lt; 0.05) when compared with that in PPP group (35.8% ± 11.3%) and control group (28.0% ± 5.4%). The inflammatory cells were significantly lower and the blood vessel density was significantly higher in PRP group than in PPP group and control group (P lt; 0.05). When compared with PPP group and control group, the expressions of VEGF and PDGF-BB increased at all time after operation in PRP group; the expression of EGF increased within 24 hours; and the expression of PDGF-AA increased after 3 days. There were significant differences in PDGF-AA mRNA at 3 days and 7 days, PDGF-BB mRNA at 8 hours, VEGF mRNA at 24 hours and 3 days, and EGF mRNA at 24 hours between PRP group and PPP and control groups (P lt; 0.05). Conclusion 200 × g centrifuge for 15 minutes and 500 × g centrifuge for 10 minutes is the best PRP preparation method. PRP can improve the skin flap survival by regulating the genes involved in angiogenesis.

Citation： LI Weiwei,FENG Rui.. PREPARATION OF PLATELET-RICH PLASMA GEL AND ITS EFFECT ON SKIN FLAP SURVIVAL OF RAT. Chinese Journal of Reparative and Reconstructive Surgery, 2012, 26(1): 64-69. doi: Copy

1.	platelet-rich plasma: characterization in vitro and in the ischemic hind limb of the mouse. J Vasc Surg, 2009, 50(4): 870-879.
2.	bone in dogs. J Oral Maxillofac Surg, 2009, 67(9): 1850-1858.
3.	of flap tensile strength. J Periodontol, 2009, 80(6): 985-992.
4.	dermal fibroblasts. Plast Reconstr Surg, 2008, 122(5): 1352-1360.
5.	using thrombin receptor agonist peptide. J Oral Maxillofac Surg, 2005, 63(4): 529-535.
6.	J Oral Maxillofac Surg, 2000, 58(3): 297-300.
7.	chronic wounds. Transfus Apher Sci, 2004, 30(2): 145-151．.
8.	Surg, 2004, 6(4): 489-496.
9.	differentiation and regeneration during equine wound healing. Exp Mol Pathol, 2003, 74(3): 244-255.
10.	Plast Reconstr Surg, 2004, 114(6): 1502-1508．.
11.	of cultured smooth muscle cells from spontaneously hypertensive rats by platelet-derivled growth factor A-chain homodimer. Cell Regul, 1990, 1(11): 821-831.
12.	IUBMB Life, 2001, 52(1-2): 61-66.
13.	Saldalamacchia G, Lapice E, Cuomo V, et al. A controlled study of the use of autologous platelet gel for the treatment of diabetic foot ulcers. Nutr Metab Cardiovase Dis, 2004, 14(6): 395-396.
14.	Bir SC, Esaki J, Marui A, et al. Angiogenic properties of sustained release.
15.	Tajima N, Sotome S, Marukawa E. A three-dimensional cell-loading system using autologous plasma loaded into a porous β-tricalcium-phosphate block promotes bone formation at extraskeletal sites in rats. Materials Science and Engineering: C, 2007, 27(4): 625-632.
16.	Choi BH, Zhu SJ, Kim BY, et al. Effect of platelet-rich plasma (PRP) concentration on the viability and proliferation of alveolar bone cells: an in vitro study. Int J Oral Maxillofac Surg, 2005, 34(4): 420-424.
17.	Li NY, Yuan RT, Chen T. Effect of platelet-rich plasma and latissimus dorsi muscle flap on osteogenesis and vascularization of tissue-engineered.
18.	Powell CA, Bannister SR, Mackey SA, et al. Periodontal wound healing with and without platelet-rich plasma: histologic observations and assessment.
19.	Kakudo N, Minakata T, Mitsui T, et al. Proliferation-promoting effect of platelet-rich plasma on human adipose-derived stem cells and human.
20.	Landesberg R, Burke A, Pinsky D, et al. Activation of platelet-rich plasma.
21.	Landesberg R, Roy M, Glickman RS. Quantification of growth factor levels using a simplified method of platelet-rich plasma gel preparation.
22.	宋扬, 宫苹, 侯睿, 等. 传统二次离心法和改良法制备富血小板血浆的比较研究. 实用口腔医学杂志, 2005, 21(5): 629-632.
23.	Sclafani AP. Applications of platelet-rich fibrin matrix in facial plastic surgery. Facial Plast Surg, 2009, 25(4): 270-276.
24.	Crovetti G, Martinelli G, Issi M, et al. Platelet gel for healing cutaneous.
25.	冯扬, 唐休发. 富血小板血浆在颌面部非血管化骨移植中的应用. 中国修复重建外科杂志, 2006, 20(9): 944-946．.
26.	Mazor Z, Peleg M, Garg AK, et al. Platelet-rich plasma for bone graft enhancement in sinus floor augmentation with simultaneous implant placement: patient series study. Implant Dent, 2004, 13(1): 65-72．.
27.	Marx RE. Platelet-rich plasma: evidence to support its use. J Oral Maxillofac.
28.	Carter CA, Jolly DG, Worden CE Sr, et al. Platelet-rich plasma gel promotes.
29.	Epplev BL, Woodell JE, Higgins J. Platelet quantification and growth factor analysis from platelet-rich plasma: implications for wound healing.
30.	Griffin XL, Smith CM, Costa ML. The clinical use of platelet-rich plasma in the promotion of bone heating: a systematic review. Injury, 2009, 40(2): 158-162．.
31.	Werner S, Grose R. Regulation of wound healing by Growth factors and cytokines. Physiol Rev, 2003, 83(3): 835-870．.
32.	Resink TJ, Scott-Burden T, Hahh AW, et al. Specific growth stimulation.
33.	Kliche S, Waltenberg J. VEGF receptor signaling and endothelial function.
34.	Adelmann-Grill BC, Wach F, Cully Z, et al. Chemotactic migration of normal dermal fibroblasts towards epidermal growth factor and its modulation by platelet-derived growth factor and transforming growth factor-beta. Eur J Cell Biol, 1990, 51(2): 322-326.
35.	Eppley BL, Pietrzak WS, Blanton M. Platelet-rich plasma: a review of biology and applications in plastic surgery. Plast Reconstr Surg, 2006, 118(6): 147e-159e.
36.	Akeda K, An HS, Pichika R, et al. Platelet-rieh plasma (PRP) stimulates the extracellular matrix metabolism of porcine nucleus pulposus and anulus fibrosus cells cultured in alginate beads. Spine (Phila Pa 1976), 2006, 31(9): 959-966．.

1. platelet-rich plasma: characterization in vitro and in the ischemic hind limb of the mouse. J Vasc Surg, 2009, 50(4): 870-879.
2. bone in dogs. J Oral Maxillofac Surg, 2009, 67(9): 1850-1858.
3. of flap tensile strength. J Periodontol, 2009, 80(6): 985-992.
4. dermal fibroblasts. Plast Reconstr Surg, 2008, 122(5): 1352-1360.
5. using thrombin receptor agonist peptide. J Oral Maxillofac Surg, 2005, 63(4): 529-535.
6. J Oral Maxillofac Surg, 2000, 58(3): 297-300.
7. chronic wounds. Transfus Apher Sci, 2004, 30(2): 145-151．.
8. Surg, 2004, 6(4): 489-496.
9. differentiation and regeneration during equine wound healing. Exp Mol Pathol, 2003, 74(3): 244-255.
10. Plast Reconstr Surg, 2004, 114(6): 1502-1508．.
11. of cultured smooth muscle cells from spontaneously hypertensive rats by platelet-derivled growth factor A-chain homodimer. Cell Regul, 1990, 1(11): 821-831.
12. IUBMB Life, 2001, 52(1-2): 61-66.
13. Saldalamacchia G, Lapice E, Cuomo V, et al. A controlled study of the use of autologous platelet gel for the treatment of diabetic foot ulcers. Nutr Metab Cardiovase Dis, 2004, 14(6): 395-396.
14. Bir SC, Esaki J, Marui A, et al. Angiogenic properties of sustained release.
15. Tajima N, Sotome S, Marukawa E. A three-dimensional cell-loading system using autologous plasma loaded into a porous β-tricalcium-phosphate block promotes bone formation at extraskeletal sites in rats. Materials Science and Engineering: C, 2007, 27(4): 625-632.
16. Choi BH, Zhu SJ, Kim BY, et al. Effect of platelet-rich plasma (PRP) concentration on the viability and proliferation of alveolar bone cells: an in vitro study. Int J Oral Maxillofac Surg, 2005, 34(4): 420-424.
17. Li NY, Yuan RT, Chen T. Effect of platelet-rich plasma and latissimus dorsi muscle flap on osteogenesis and vascularization of tissue-engineered.
18. Powell CA, Bannister SR, Mackey SA, et al. Periodontal wound healing with and without platelet-rich plasma: histologic observations and assessment.
19. Kakudo N, Minakata T, Mitsui T, et al. Proliferation-promoting effect of platelet-rich plasma on human adipose-derived stem cells and human.
20. Landesberg R, Burke A, Pinsky D, et al. Activation of platelet-rich plasma.
21. Landesberg R, Roy M, Glickman RS. Quantification of growth factor levels using a simplified method of platelet-rich plasma gel preparation.
22. 宋扬, 宫苹, 侯睿, 等. 传统二次离心法和改良法制备富血小板血浆的比较研究. 实用口腔医学杂志, 2005, 21(5): 629-632.
23. Sclafani AP. Applications of platelet-rich fibrin matrix in facial plastic surgery. Facial Plast Surg, 2009, 25(4): 270-276.
24. Crovetti G, Martinelli G, Issi M, et al. Platelet gel for healing cutaneous.
25. 冯扬, 唐休发. 富血小板血浆在颌面部非血管化骨移植中的应用. 中国修复重建外科杂志, 2006, 20(9): 944-946．.
26. Mazor Z, Peleg M, Garg AK, et al. Platelet-rich plasma for bone graft enhancement in sinus floor augmentation with simultaneous implant placement: patient series study. Implant Dent, 2004, 13(1): 65-72．.
27. Marx RE. Platelet-rich plasma: evidence to support its use. J Oral Maxillofac.
28. Carter CA, Jolly DG, Worden CE Sr, et al. Platelet-rich plasma gel promotes.
29. Epplev BL, Woodell JE, Higgins J. Platelet quantification and growth factor analysis from platelet-rich plasma: implications for wound healing.
30. Griffin XL, Smith CM, Costa ML. The clinical use of platelet-rich plasma in the promotion of bone heating: a systematic review. Injury, 2009, 40(2): 158-162．.
31. Werner S, Grose R. Regulation of wound healing by Growth factors and cytokines. Physiol Rev, 2003, 83(3): 835-870．.
32. Resink TJ, Scott-Burden T, Hahh AW, et al. Specific growth stimulation.
33. Kliche S, Waltenberg J. VEGF receptor signaling and endothelial function.
34. Adelmann-Grill BC, Wach F, Cully Z, et al. Chemotactic migration of normal dermal fibroblasts towards epidermal growth factor and its modulation by platelet-derived growth factor and transforming growth factor-beta. Eur J Cell Biol, 1990, 51(2): 322-326.
35. Eppley BL, Pietrzak WS, Blanton M. Platelet-rich plasma: a review of biology and applications in plastic surgery. Plast Reconstr Surg, 2006, 118(6): 147e-159e.
36. Akeda K, An HS, Pichika R, et al. Platelet-rieh plasma (PRP) stimulates the extracellular matrix metabolism of porcine nucleus pulposus and anulus fibrosus cells cultured in alginate beads. Spine (Phila Pa 1976), 2006, 31(9): 959-966．.

Chinese Journal of Reparative and Reconstructive Surgery

PREPARATION OF PLATELET-RICH PLASMA GEL AND ITS EFFECT ON SKIN FLAP SURVIVAL OF RAT

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