Interventional effects of whole blood and platelet-rich plasma with different preparation methods on nucleus pulposus cells
Lian Shilin1, 2, Zhang Yan2, Jiang Qiang2, Zhang Hanshuo2, Li Tusheng2, Ding Yu1, 2
1School of Medicine, South China University of Technology, Guangzhou 510006, Guangdong Province, China; 2Orthopedics of TCM Senior Department, the Sixth Medical Center of PLA General Hospital, Beijing 100048, China
Abstract: BACKGROUND: Platelet-rich plasma therapy for disc degeneration has received increasing attention. Platelet-rich plasma preparation methods for intervertebral disc repair include manual preparation using one-spin centrifugation and kit preparation using two-spin centrifugation. There was insufficient evidence of which preparation method is better.
OBJECTIVE: To compare the effects of intervention of porine nucleus pulposus cells with manually prepared platelet-rich plasma, kit-prepared platelet-rich plasma and whole blood and to explore the better preparation method of platelet-rich plasma for intervertebral disc repair.
METHODS: The porcine venous blood was extracted to prepare platelet-rich plasma using the manual preparation method of one-spin centrifugation and OSP kit preparation method of two-spin centrifugation. Some whole blood was reserved as control. And PBS was used to be blank control. The concentration of platelets and leukocytes in platelet-rich plasma was detected to calculate fold enrichment. ELISA was used to detect the concentration of transforming growth factor-β1, platelet-derived growth factor-BB, tumor necrosis factor-α and interleukin-1β in platelet-rich plasma lysate. Porcine nucleus pulposus cells were cultivated to P3 and intervened with prepared platelet-rich plasma and whole blood lysate. Cell counting kit-8 was used to detect the proliferation of nucleus pulposus cells after intervention with platelet-rich plasma and whole blood lysate. Western blot was used to detect the anabolic effects of platelet-rich plasma on type II collagen and aggrecan in nucleus pulposus cells.
RESULTS AND CONCLUSION: Compared with the manual group, the OSP group showed significantly longer preparation time and higher platelet and leukocyte concentrations in the preparation of platelet-rich plasma. The fold enrichment of platelet-rich plasma was 5.6 in the OSP group and 3.0 in the manual group, while the fold enrichment of leukocytes was 2.21 and 1.36 respectively. The concentrations of transforming growth factor-β1, platelet-derived growth factor-BB, tumor necrosis factor-α and interleukin-1β of the OSP group were significantly higher than those of the manual group (P < 0.05). In the cell counting kit-8 detection of cell proliferation, the absorbance value at 450 nm of the OSP group was significantly higher than that of the other groups (P < 0.000 1); there was no significant difference in the absorbance value at 450 nm of the manual group compared with the whole blood and control groups (P=0.192 7). The relative expression of type II collagen in the OSP group was significantly higher than that in the other groups (P < 0.000 1). As for the synthesis of aggrecan, the relative expression of aggrecan in the manual group was lower than that in the remaining groups (P=0.040 5). These findings indicate that compared with the manual preparation method, the kit preparation method represented by OSP consumes more time and can acquire significantly higher concentration of leukocytes and proinflammatory cytokines. However, the kit preparation method can obtain higher concentrations of platelets and growth factors and has better cell proliferation effect and synthesis effect of extracellular matrix. For a better therapeutic effect of platelet-rich plasma, it is suggested to enhance the isolation of leukocytes when using the kit preparation method.
Key words: nucleus pulposus cell, platelet-rich plasma, manual preparation, OSP kit preparation, intervertebral disc degeneration, tissue repair