This study examined the efficacy of the injection of PRP-releasate into degenerated discs in the well-established rabbit anular needle puncture model. The results of this study demonstrated that the intradiscal injection of PRP-releasate is effective for restoring disc height in this animal model. The histological analysis also revealed a reparative effect of PRP-releasate on the degenerated IVD.
Platelets have three major granule storage compartments: alpha granules, dense granules, and lysosomes. Alpha granules contain a large number of different secretory proteins, such as growth factors, coagulation proteins, adhesion molecules, cytokines, cell-activating agents, and angiogenic factors . Platelets must be activated to release the contents of alpha granules to the external milieu where they exert potent biological effects. In many in vitro or in vivo studies (or both), activation of PRP is accomplished by adding bovine or human thrombin with calcium chloride [8, 21, 22, 25–28]. However, it would be more feasible for clinical use if the concern about immunogenic reactions or disease transmission in using xenogeneic or allogeneic blood products were eliminated. In addition, the use of thrombin was of concern to us because thrombin has been shown to degrade cartilage tissues  and to cleave the same site as plasmin [30, 31]. Therefore, in this study, we used a mixture of autologous serum and calcium chloride for activation of PRP.
The administration of PRP-releasate into degenerated IVDs greatly restored the decrease in disc height following anular needle puncture in this in vivo rabbit model. In a previous study using the same animal model, the injection of OP-1  or growth differentiation factor-5 (GDF-5)  induced restoration of disc height to a level approaching 90% of that of a normal non-punctured disc. The results of our study suggest that the administration of PRP-releasate had an effect on the structural restoration of disc height similar to those of OP-1 or GDF-5 or both.
The major advantage of using PRP over purified growth factors is that PRP can be isolated from an autologous source, thus eliminating concerns about the potential for cancer  or autoantibodies. In addition, the preparation of PRP can be performed in a regular hospital setting with a minimum requirement for equipment and less vigorous regulatory oversight. However, the inter-individual variability in the concentration of growth factors found in PRP, compared with the well-controlled recombinant growth factors currently in clinical trials (OP-1 and GDF-5), may result in inconsistent effects.
MRI has recently been used not only for morphological evaluation but also for characterization of the structural organization and matrix content of the IVD. The T2 mapping technique has shown its potential to quantitatively evaluate changes in the molecular composition and structural organization of the IVD [33–36]. In our study, the T2 mapping technique was used to detect and quantify changes in the matrix structure and integrity of degenerated rabbit IVDs injected with PBS and PPP- and PRP-releasate. Our results showed that the mean T2 value in the AF and NP region of the PRP group was not increased significantly over other groups, even though a minor increase was seen. Additional samples may be needed to show treatment effects on T2 values, which may have been subtle.
The histological analyses showed that the injection of PRP-releasate induced an increase in the number of chondrocyte-like cells in the NP and the anterior inner AF, and this suggests tissue phenotype changes from fibrotic tissue to cartilaginous tissue. No inflammatory reactions, such as invasion of inflammatory cells or microvessels (or both), or ossification within the IVD tissue was found, suggesting that PRP-releasate had no adverse effects on disc tissues.
In the radiographic and histological analyses, PPP-releasate also induced a reparative effect on degenerated IVDs, although its effect was less than those of the PRP-releasate. Previous in vitro studies [21, 22] have shown that PPP has effects on cell proliferation and matrix metabolism similar to those of fetal bovine serum, which is the most widely used serum supplement for in vitro cell culture; this suggests that PPP also contains growth factors and has the potential to stimulate the matrix metabolism of degenerated IVDs.
In the past, two different groups have performed in vivo intradiscal injection studies by using PRP to evaluate its effect on disc degeneration in animal models. Chen and colleagues  reported the effect of PRP on disc degeneration both in an ex vivo organ culture system and an in vivo porcine disc degeneration model. The releasate isolated from clotted PRP, which was induced by addition of bovine thrombin, was injected into the degenerated disc induced by chymopapain. In their study, PRP, which was shown to promote NP regeneration, also resulted in the upregulation of chondrogenesis and extracellular matrix accumulation. In the in vivo model, the recovery of disc height was not significant (P = 0.5). This may be due to the use of thrombin. With consideration for a future clinical application, we have decided not to use thrombin, but rather autologous serum and calcium, to activate PRP.
The other group [37, 38] reported in vivo intradiscal injection studies using PRP-impregnated biodegradable gelatin hydrogel microspheres (PRP-GHMs) in a rabbit disc degeneration model induced by the partial aspiration of the NP with a 21-gauge needle. Two weeks after the initial surgery, autologous PRP alone or PRP-GHMs were injected into the degenerated discs. Contrary to the results of our study, the progression of disc degeneration was significantly suppressed by the combined administration of PRP-GHMs but not by PRP alone. Possible explanations for the conflicting findings concerning the effect of PRP would be, first, the differing methods used in the processing the injectable agents. We injected PRP-releasate isolated from activated platelets into the degenerated disc, whereas in the study by Nagae and colleagues , the PRP alone injected into the degenerated disc was prepared without platelet activation (without coagulation). Indeed, in an article just published, DeLong and colleagues  state that the manner in which platelet activation occurs is a variable that determines the efficacy of PRP; they propose a classification system for PRP publications that includes 'platelet activation manner'. Second, differences in the disc degeneration animal models and evaluation methods could be reflected in the conflicting results. The anular puncture model has been shown to induce controlled and progressive disc degeneration and effectively detect the therapeutic effects [11, 24, 40]. A controlled degree of disc degeneration may be required to test the effects of biological therapies.
A limitation of this study is that the well-characterized rabbit anular puncture model  used is a short-term (1-month) injury animal model of disc degeneration. Although this model does not truly reflect the course of human disc degeneration, similar histological and biomechanical changes have been previously reported [11, 23, 24] and confirmed in this study. Therefore, the results of this study may contribute to the understanding of the cellular responses and the mechanism of the repair process after PRP application in degenerative discs.