The Role Of TGF-β1in Platelet-rich Plasma In Retarding Intervertebral Disc Degeneration

Objective: To explore the effect of platelet-rich plasma (PRP) in retardingintervertebral disc degeneration (IDD), and the potential mechanism of this effect, in orderto provide feasible experimental support and reference method for clinical treatment ofintervertebral disc degeneration.Methods: Experiment was divided into two parts, in vitro and in vivo experiments.The in vitro part: determine the level of platelet and TGF-β1in PRP by counting andELISA method. Culture the rabbit nucleus pulposus cells with PRP with a concentrationgradient of10%,5%,2.5%and1%respectively, than determine the daily absorbance byCCK-8assay from1to7days after the cells were cultured, to evaluate the effects ofdifferent concentrations of PRP in rabbit nucleus pulposus cell proliferation. We choose theoptimal concentration of PRP and TGF-β1inhibitor to treat nucleus pulposus cells, detectthe changes in TGF-β1, collagen II and aggrecan gene mRNA expression levels byreal-time quantitative PCR assay.The in vivo part: select12adult New Zealand white rabbits in normal weight, theywere randomly divided into model group, the injection of PRP group and the injection ofPRP and TGF-β1inhibitor group, using the18G needle trocar puncture discs, after theconfirmation of disc degeneration with MRI examination four weeks later, then injectautologous PRP and mixture of PRP and TGF-β1inhibitor(SB431542) to the degenerateddiscs, respectively.4weeks and8weeks after injection, we observe the MRI signalchanges again, all of the animals were sacrificed in the latest point, remove the discs andconduct histology and immunohistochemical analysis.Results: The platelet count in PRP is (1170.25±56.08)×109/L, which is (4.15±0.28) times than whole blood, TGF-β1content in PRP is (1180.13±45.88)pg/mL, which is (3.65±0.60) times than whole blood. PRP can enhance the activity of the nucleus pulposus cellsand promote proliferation, the strongest effect reveals at the fourth day after culture, thengradually weakened, the concentration of2.5%PRP has the best effect in proliferation andanabolism. After the treatment of PRP, TGF-β1, collagen II and aggrecan gene mRNAexpression of nucleus pulposus cells are (19.38±1.52),(4.43±0.35) and (11.81±1.87)times than before, respectively (P<0.05). The results decrease to (5.59±0.34),(2.43±0.39) and (4.19±0.50) times after the role of TGF-β1inhibitor. After the injection of PRPto the degenerated intervertebral discs, MRI shows that the disc signal improvessignificantly (P <0.05), while after the injection of a mixture of PRP and TGF-β1inhibitor,the improvement is not obvious. Immunohistochemistry results show that injection of PRPwithin the nucleus lead to the significantly increase of type II collagen content in theextracellular matrix, and, TGF-β1positive cells are also significantly increased (P <0.05).Conclusion: Platelet-rich plasma can promote nucleus pulposus cells’ proliferationand biological function in vitro, the effect might be decreased by TGF-β1inhibitor.Autologous platelet-rich plasma can retard the development of rabbits’ disc degeneration,and even has a reverse effect, in which TGF-β1plays an important role.