Study of centrifuge conditions for preparing rabbit leukocyte-poor platelet-rich plasma by single centrifugation_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

ZHANG Kefan ¹ , ZHANG Chenchen ² , REN Qiang ¹ , WANG Dening ¹ , SUN Li ¹ , WANG Xin ³ , WANG Yaomin ¹ ,  SHI Hui ¹

1. Department of Orthopedics and Sports Medicine, Binzhou Medical University Hospital, Binzhou Shandong, 256603, P. R. China;
2. Department of Operating Room, Binzhou Medical University Hospital, Binzhou Shandong, 256603, P. R. China;
3. Department of Orthopedics, Nanyang Central Hospital, Nanyang Henan, 473009, P. R. China;

Corresponding author：

SHI Hui, Email: 13475095025@163.com

Keywords：

Single centrifugation; leukocyte-poor platelet-rich plasma; centrifugal condition; rabbit

DOI：

10.7507/1002-1892.202311075

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Objective To explore the best centrifuge condition for preparing rabbit leukocyte-poor platelet-rich plasma (LP-PRP) by using single centrifugation method. Methods Sixteen healthy New Zealand rabbits, aged 3-4 months, were utilized in the investigation. A total of 15 mL anticoagulated blood was extracted from the central ear artery of each rabbit, with a repeat of the blood collection procedure after 1 and 2 months. The obtained blood specimens were individually subjected to centrifugation at a radius of 16.7 cm and speeds of 1 200, 1 300, 1 400, and 1 500 r/min (equivalent to centrifugal forces of 269×g, 315×g, 365×g, and 420×g) for durations of 2, 3, 4, and 5 minutes, resulting in a total of 16 groups. Following centrifugation, collect plasma from each group to a distance of 1.5 mL from the separation plane. The volumes, platelet enrichment coefficient, and platelet recovery rates of LP-PRP in each group, under varying centrifugation conditions, were methodically computed and subsequently compared. Results The volume of LP-PRP obtained under all centrifugation conditions ranged from 1.8 to 7.6 mL. At a consistent centrifugal speed, an extension of centrifugation time leaded to a significant increase in the volume of LP-PRP, accompanied by a declining trend in the platelet enrichment coefficient of LP-PRP. When centrifuged for 2 minutes, the volume of LP-PRP at speeds of 1 200 and 1 300 r/min was less than 2.0 mL, while the volume of LP-PRP obtained under other conditions was more than 2.0 mL. When centrifuged for 4 and 5 minutes, the volume of LP-PRP obtained at each speed was more than 4 mL. LP-PRP with a platelet enrichment coefficient more than 2.0 could be prepared by centrifuging at 1 200 r/min for each time group and 1 300 r/min for 2 and 3 minutes, and the highest LP-PRP platelet enrichment coefficient could be obtained by centrifugation for 2 minutes at a speed of 1 200 r/min. The platelet recovery rates of LP-PRP obtained by centrifugation at 1 200 r/min for 4 and 5 minutes, as well as centrifugation at 1 400 r/min for 5 minutes, were both greater than 60%. There was no significant difference between the groups when centrifuged at 1 200 r/min for 4 and 5 minutes (P>0.05). Conclusion In the process of preparing rabbit LP-PRP using a single centrifugation method, collecting 15 mL of blood and centrifuging at a radius of 16.7 cm and speed of 1 200 r/min for 4 minutes can prepare LP-PRP with a volume exceeding 2.0 mL, platelet enrichment coefficient exceeding 2.0, and platelet recovery rate exceeding 60%. This centrifugal condition can achieve the optimal LP-PRP action parameters in the shortest possible time.

Citation： ZHANG Kefan, ZHANG Chenchen, REN Qiang, WANG Dening, SUN Li, WANG Xin, WANG Yaomin, SHI Hui. Study of centrifuge conditions for preparing rabbit leukocyte-poor platelet-rich plasma by single centrifugation. Chinese Journal of Reparative and Reconstructive Surgery, 2024, 38(2): 183-188. doi: 10.7507/1002-1892.202311075 Copy

1.	Marx RE. Platelet-rich plasma (PRP): what is PRP and what is not PRP? Implant Dent, 2001, 10(4): 225-228.
2.	Belk JW, Kraeutler MJ, Houck DA, et al. Platelet-rich plasma versus hyaluronic acid for knee osteoarthritis: A systematic review and meta-analysis of randomized controlled trials. Am J Sports Med, 2021, 49(1): 249-260.
3.	Dohan Ehrenfest DM, Rasmusson L, Albrektsson T. Classification of platelet concentrates: from pure platelet-rich plasma (P-PRP) to leucocyte- and platelet-rich fibrin (L-PRF). Trends Biotechnol, 2009, 27(3): 158-167.
4.	Le ADK, Enweze L, DeBaun MR, et al. Current clinical recommendations for use of platelet-rich plasma. Curr Rev Musculoskelet Med, 2018, 11(4): 624-634.
5.	Lana JFSD, Purita J, Paulus C, et al. Contributions for classification of platelet rich plasma-proposal of a new classification: MARSPILL. Regen Med, 2017, 12(5): 565-574.
6.	Riboh JC, Saltzman BM, Yanke AB, et al. Effect of leukocyte concentration on the efficacy of platelet-rich plasma in the treatment of knee osteoarthritis. Am J Sports Med, 2016, 44(3): 792-800.
7.	Everts P, Onishi K, Jayaram P, et al. Platelet-rich plasma: New performance understandings and therapeutic considerations in 2020. Int J Mol Sci, 2020, 21(20): 7794.
8.	Anitua E, Aguirre JJ, Algorta J, et al. Effectiveness of autologous preparation rich in growth factors for the treatment of chronic cutaneous ulcers. J Biomed Mater Res B Appl Biomater, 2008, 84(2): 415-421.
9.	Petrungaro PS. Using platelet-rich plasma to accelerate soft tissue maturation in esthetic periodontal surgery. Compend Contin Educ Dent, 2001, 22(9): 729-732, 734, 736 passim; quiz 746.
10.	Aghaloo TL, Moy PK, Freymiller EG. Investigation of platelet-rich plasma in rabbit cranial defects: A pilot study. J Oral Maxillofac Surg, 2002, 60(10): 1176-1181.
11.	Nagata MJH, Messora MR, Furlaneto FAC, et al. Effectiveness of two methods for preparation of autologous platelet-rich plasma: An experimental study in rabbits. Eur J Dent, 2010, 4(4): 395-402.
12.	Oudelaar BW, Peerbooms JC, Huis In’t Veld R, et al. Concentrations of blood components in commercial platelet-rich plasma separation systems: A review of the literature. Am J Sports Med, 2019, 47(2): 479-487.
13.	丁雷. 新西兰兔血液学参数的研究. 哈尔滨: 东北林业大学, 2010.
14.	Bocun L, Jing L, Jia L, et al. Effects of platelet-rich plasma injection for pain control and cartilage repair in knee osteoarthritis: A protocol for the systematic review and meta-analysis of randomized controlled trials in animal models. Medicine (Baltimore), 2021, 100(1): e24107.
15.	Huang P, Wang R, Pang X, et al. Platelet-rich plasma combined with ozone prevents cartilage destruction and improves weight-bearing asymmetry in a surgery-induced osteoarthritis rabbit model. Ann Palliat Med, 2022, 11(2): 442-451.
16.	钟阳. 富血小板血浆对兔颞下颌关节骨关节炎关节滑液中炎症介质表达水平影响的实验研究. 贵州: 遵义医科大学, 2021.
17.	Zhou M, Wang N, Wang G, et al. Parecoxib alleviates the inflammatory effect of leukocyte-rich platelet-rich plasma in normal rabbit tendons. BMC Musculoskelet Disord, 2020, 21(1): 831.
18.	Kurnaz R, Balta O. Effect of platelet-rich plasma and platelet-rich fibrin matrix on healing of vertical meniscal tears in a rabbit model. Acta Orthop Traumatol Turc, 2020, 54(2): 186-195.
19.	Jiang G, Wu Y, Meng J, et al. Comparison of leukocyte-rich platelet-rich plasma and leukocyte-poor platelet-rich plasma on achilles tendinopathy at an early stage in a rabbit model. Am J Sports Med, 2020, 48(5): 1189-1199.
20.	Gelalis ID, Christoforou G, Charchanti A, et al. Autologous platelet-rich plasma (PRP) effect on intervertebral disc restoration: an experimental rabbit model. Eur J Orthop Surg Traumatol, 2019, 29(3): 545-551.
21.	Xue Y, Su X, Jiang M, et al. Pure platelet-rich plasma facilitates the repair of damaged cartilage and synovium in a rabbit hemorrhagic arthritis knee model. Arthritis Res Ther, 2020, 22(1): 68.
22.	Jo CH, Shin JS, Shin WH, et al. Platelet-rich plasma for arthroscopic repair of medium to large rotator cuff tears: a randomized controlled trial. Am J Sports Med, 2015, 43(9): 2102-2110.
23.	Hamid MS, Yusof A, Mohamed Ali MR. Platelet-rich plasma (PRP) for acute muscle injury: a systematic review. PLoS One, 2014, 9(2): e90538.
24.	Graziani F, Ivanovski S, Cei S, et al. The in vitro effect of different PRP concentrations on osteoblasts and fibroblasts. Clin Oral Implants Res, 2006, 17(2): 212-219.
25.	Weibrich G, Hansen T, Kleis W, et al. Effect of platelet concentration in platelet-rich plasma on peri-implant bone regeneration. Bone, 2004, 34(4): 665-671.
26.	Anitua E, Sánchez M, Zalduendo MM, et al. Fibroblastic response to treatment with different preparations rich in growth factors. Cell Prolif, 2009, 42(2): 162-170.
27.	Ozer K, Kankaya Y, Colak O, et al. The impact of duration and force of centrifugation on platelet content and mass in the preparation of platelet-rich plasma. Aesthetic Plast Surg, 2019, 43(4): 1078-1084.
28.	Perez AG, Lichy R, Lana JF, et al. Prediction and modulation of platelet recovery by discontinuous centrifugation of whole blood for the preparation of pure platelet-rich plasma. Biores Open Access, 2013, 2(4): 307-314.
29.	张昭远, 卫愉轩, 张长青, 等. 应用不同离心条件优化富白细胞富血小板血浆制作方案研究. 中国修复重建外科杂志, 2020, 34(8): 1025-1030.
30.	Dhurat R, Sukesh M. Principles and methods of preparation of platelet-rich plasma: A review and author’s perspective. J Cutan Aesthet Surg, 2014, 7(4): 189-197.
31.	Boswell SG, Schnabel LV, Mohammed HO, et al. Increasing platelet concentrations in leukocyte-reduced platelet-rich plasma decrease collagen gene synthesis in tendons. Am J Sports Med, 2014, 42(1): 42-49.
32.	Harrison TE, Bowler J, Levins TN, et al. Platelet yield and yield consistency for six single-spin methods of platelet rich plasma preparation. Platelets, 2020, 31(5): 661-666.
33.	Su F, Tong MW, Lansdown DA, et al. Leukocyte-poor platelet-rich plasma injections improve cartilage T1ρ and T2 and patient-reported outcomes in mild-to-moderate knee osteoarthritis. Arthrosc Sports Med Rehabil, 2023, 5(3): e817-e825.
34.	金哲, 吕刚, 王禹祥. 二次离心法制备富血小板血浆离心条件的比较. 中国医科大学学报, 2012, 41(3): 237-240.
35.	Piao L, Park H, Jo CH. Theoretical prediction and validation of cell recovery rates in preparing platelet-rich plasma through a centrifugation. PLoS One, 2017, 12(11): e0187509.

1. Marx RE. Platelet-rich plasma (PRP): what is PRP and what is not PRP? Implant Dent, 2001, 10(4): 225-228.
2. Belk JW, Kraeutler MJ, Houck DA, et al. Platelet-rich plasma versus hyaluronic acid for knee osteoarthritis: A systematic review and meta-analysis of randomized controlled trials. Am J Sports Med, 2021, 49(1): 249-260.
3. Dohan Ehrenfest DM, Rasmusson L, Albrektsson T. Classification of platelet concentrates: from pure platelet-rich plasma (P-PRP) to leucocyte- and platelet-rich fibrin (L-PRF). Trends Biotechnol, 2009, 27(3): 158-167.
4. Le ADK, Enweze L, DeBaun MR, et al. Current clinical recommendations for use of platelet-rich plasma. Curr Rev Musculoskelet Med, 2018, 11(4): 624-634.
5. Lana JFSD, Purita J, Paulus C, et al. Contributions for classification of platelet rich plasma-proposal of a new classification: MARSPILL. Regen Med, 2017, 12(5): 565-574.
6. Riboh JC, Saltzman BM, Yanke AB, et al. Effect of leukocyte concentration on the efficacy of platelet-rich plasma in the treatment of knee osteoarthritis. Am J Sports Med, 2016, 44(3): 792-800.
7. Everts P, Onishi K, Jayaram P, et al. Platelet-rich plasma: New performance understandings and therapeutic considerations in 2020. Int J Mol Sci, 2020, 21(20): 7794.
8. Anitua E, Aguirre JJ, Algorta J, et al. Effectiveness of autologous preparation rich in growth factors for the treatment of chronic cutaneous ulcers. J Biomed Mater Res B Appl Biomater, 2008, 84(2): 415-421.
9. Petrungaro PS. Using platelet-rich plasma to accelerate soft tissue maturation in esthetic periodontal surgery. Compend Contin Educ Dent, 2001, 22(9): 729-732, 734, 736 passim; quiz 746.
10. Aghaloo TL, Moy PK, Freymiller EG. Investigation of platelet-rich plasma in rabbit cranial defects: A pilot study. J Oral Maxillofac Surg, 2002, 60(10): 1176-1181.
11. Nagata MJH, Messora MR, Furlaneto FAC, et al. Effectiveness of two methods for preparation of autologous platelet-rich plasma: An experimental study in rabbits. Eur J Dent, 2010, 4(4): 395-402.
12. Oudelaar BW, Peerbooms JC, Huis In’t Veld R, et al. Concentrations of blood components in commercial platelet-rich plasma separation systems: A review of the literature. Am J Sports Med, 2019, 47(2): 479-487.
13. 丁雷. 新西兰兔血液学参数的研究. 哈尔滨: 东北林业大学, 2010.
14. Bocun L, Jing L, Jia L, et al. Effects of platelet-rich plasma injection for pain control and cartilage repair in knee osteoarthritis: A protocol for the systematic review and meta-analysis of randomized controlled trials in animal models. Medicine (Baltimore), 2021, 100(1): e24107.
15. Huang P, Wang R, Pang X, et al. Platelet-rich plasma combined with ozone prevents cartilage destruction and improves weight-bearing asymmetry in a surgery-induced osteoarthritis rabbit model. Ann Palliat Med, 2022, 11(2): 442-451.
16. 钟阳. 富血小板血浆对兔颞下颌关节骨关节炎关节滑液中炎症介质表达水平影响的实验研究. 贵州: 遵义医科大学, 2021.
17. Zhou M, Wang N, Wang G, et al. Parecoxib alleviates the inflammatory effect of leukocyte-rich platelet-rich plasma in normal rabbit tendons. BMC Musculoskelet Disord, 2020, 21(1): 831.
18. Kurnaz R, Balta O. Effect of platelet-rich plasma and platelet-rich fibrin matrix on healing of vertical meniscal tears in a rabbit model. Acta Orthop Traumatol Turc, 2020, 54(2): 186-195.
19. Jiang G, Wu Y, Meng J, et al. Comparison of leukocyte-rich platelet-rich plasma and leukocyte-poor platelet-rich plasma on achilles tendinopathy at an early stage in a rabbit model. Am J Sports Med, 2020, 48(5): 1189-1199.
20. Gelalis ID, Christoforou G, Charchanti A, et al. Autologous platelet-rich plasma (PRP) effect on intervertebral disc restoration: an experimental rabbit model. Eur J Orthop Surg Traumatol, 2019, 29(3): 545-551.
21. Xue Y, Su X, Jiang M, et al. Pure platelet-rich plasma facilitates the repair of damaged cartilage and synovium in a rabbit hemorrhagic arthritis knee model. Arthritis Res Ther, 2020, 22(1): 68.
22. Jo CH, Shin JS, Shin WH, et al. Platelet-rich plasma for arthroscopic repair of medium to large rotator cuff tears: a randomized controlled trial. Am J Sports Med, 2015, 43(9): 2102-2110.
23. Hamid MS, Yusof A, Mohamed Ali MR. Platelet-rich plasma (PRP) for acute muscle injury: a systematic review. PLoS One, 2014, 9(2): e90538.
24. Graziani F, Ivanovski S, Cei S, et al. The in vitro effect of different PRP concentrations on osteoblasts and fibroblasts. Clin Oral Implants Res, 2006, 17(2): 212-219.
25. Weibrich G, Hansen T, Kleis W, et al. Effect of platelet concentration in platelet-rich plasma on peri-implant bone regeneration. Bone, 2004, 34(4): 665-671.
26. Anitua E, Sánchez M, Zalduendo MM, et al. Fibroblastic response to treatment with different preparations rich in growth factors. Cell Prolif, 2009, 42(2): 162-170.
27. Ozer K, Kankaya Y, Colak O, et al. The impact of duration and force of centrifugation on platelet content and mass in the preparation of platelet-rich plasma. Aesthetic Plast Surg, 2019, 43(4): 1078-1084.
28. Perez AG, Lichy R, Lana JF, et al. Prediction and modulation of platelet recovery by discontinuous centrifugation of whole blood for the preparation of pure platelet-rich plasma. Biores Open Access, 2013, 2(4): 307-314.
29. 张昭远, 卫愉轩, 张长青, 等. 应用不同离心条件优化富白细胞富血小板血浆制作方案研究. 中国修复重建外科杂志, 2020, 34(8): 1025-1030.
30. Dhurat R, Sukesh M. Principles and methods of preparation of platelet-rich plasma: A review and author’s perspective. J Cutan Aesthet Surg, 2014, 7(4): 189-197.
31. Boswell SG, Schnabel LV, Mohammed HO, et al. Increasing platelet concentrations in leukocyte-reduced platelet-rich plasma decrease collagen gene synthesis in tendons. Am J Sports Med, 2014, 42(1): 42-49.
32. Harrison TE, Bowler J, Levins TN, et al. Platelet yield and yield consistency for six single-spin methods of platelet rich plasma preparation. Platelets, 2020, 31(5): 661-666.
33. Su F, Tong MW, Lansdown DA, et al. Leukocyte-poor platelet-rich plasma injections improve cartilage T1ρ and T2 and patient-reported outcomes in mild-to-moderate knee osteoarthritis. Arthrosc Sports Med Rehabil, 2023, 5(3): e817-e825.
34. 金哲, 吕刚, 王禹祥. 二次离心法制备富血小板血浆离心条件的比较. 中国医科大学学报, 2012, 41(3): 237-240.
35. Piao L, Park H, Jo CH. Theoretical prediction and validation of cell recovery rates in preparing platelet-rich plasma through a centrifugation. PLoS One, 2017, 12(11): e0187509.

Chinese Journal of Reparative and Reconstructive Surgery

Study of centrifuge conditions for preparing rabbit leukocyte-poor platelet-rich plasma by single centrifugation

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