This study evaluated the radiological and histological recovery of rabbit IVDs induced by intradiscal injection of PRPr after condoliase injection. Radiological, MRI, and histological assessments revealed that PRPr injection caused a significantly greater regenerative effect than saline injection on the degenerated disc induced by condoliase.

Yang et al. reported that condoliase injection at 0.015 U/disc gradually decreased rabbit DH during the follow-up, with a 40% decrease at 16 weeks [22]. Imai et al. showed that injecting condoliase at 0.01 U/disc into rabbit IVDs significantly decreased DH by approximately 34% at the 4-week time point and did not recover until 16 weeks [23]. The current study used a pharmaceutical-grade condoliase (Hernicore®, 0.0125 U/disc, the same dose clinically used for patients with LDH) for intradiscal injection into rabbit IVDs. DH was decreased to approximately 52% of baseline height at 4 weeks post-injection. However, contrary to earlier studies [22, 23], DH began to recover and reached approximately 76% at week 16, even in the saline-injected discs. Previous studies evaluated rabbit DH using the disc height index (DHI: ratio of disc height to vertebral height), and changes in the DH were expressed as %DHI that normalized to the baseline DHI [22, 23]. However, in the current study, the actual value of DH was directly measured by calibrating the radiological measurement using a 50-mm metal wire plated at the height of the spinal process of the rabbit lumbar spine, and the changes were expressed as %DH that normalized to the baseline. Differences in the measurement methods of disc height may have led to inconsistent results.

Several clinical studies on patients with LDH have shown spontaneous recovery of DH after injection of condoliase [9, 24, 25]. Banno et al. reported the 1-year outcomes of condoliase-induced chemonucleolysis for LDH in 60 patients. They showed significant DH recovery at 1 year following a substantial decrease in DH at 3 months post-injection [10]. Therefore, DH recovery in the rabbit animal model supports the spontaneous recovery observed during the clinical course of patients with LDH after the injection of condoliase (Hernicore®). Kobayashi et al. [26] reported that the age of LDH patients injected with condoliase of whom the remarkable restoration of DH was identified was significantly younger than those with poor DH restoration, suggesting that the regenerative potential of IVD against condoliase may be higher in the younger generation. The present study used 16-week-old rabbits corresponding to the late teens in humans, implying that young rabbit IVDs may have great regenerative potential to counteract condoliase injection.

Previous studies have reported the typical histological changes of IVD degeneration after the injection of condoliase, including matrix loss in the NP, reduction of cell number, and loss of distinction between the AF and NP border [22, 23]. In accordance with previous studies, the histology in the current study showed progressive degenerative changes, including distortion of NP shape, reduction of the NP area, condensation of the matrix, decrease in NP cell number, and loss of distinction between AF and NP in the saline groups at 16 weeks post-injection of condoliase. No histological changes were observed in the EP area, as previously reported [27].

Park et al. [28] also reported a decrease in chondrocyte-like cells in the NP with proteoglycan depletion seven days after the injection of condoliase, and electron microscopy revealed that collapsed cells (necrosis) were distributed in the NP. The results of the present and previous studies suggest that condoliase can enzymatically degrade the NP matrix, accompanied by cytotoxic effects that lead to degenerative changes in rabbit IVDs.

The present study showed that the intradiscal injection of PRPr at 4 weeks after condoliase injection significantly stimulated DH recovery (95.5% recovery to baseline value) compared to saline injection. In accordance with our results, Imai et al. [23] showed that intradiscal injection of recombinant human osteogenic protein 1 (rOP-1) into degenerated rabbit discs at 4 weeks after condoliase injection resulted in a significant increase in DH at 6 weeks after rhOP-1 injection; this change was sustained up to 16 weeks (> 90% recovery to baseline value). This suggests that the intradiscal administration of PRPr has an effect equivalent to that of rhOP-1 on DH restoration.

The extent of disc degeneration by MRI grading was milder in condoliase-induced degenerated discs injected with PRPr than in those injected with saline. Interestingly, the MRI grading score [20] showed a significant and strong correlation with the histological score of rabbit disc degeneration [21]. Histological analysis revealed that the NP area and cellularity scores were significantly better in the PRPr group than in the saline group. Furthermore, the cell cloning scores in the NP and inner AF were significantly higher in the PRPr-injected discs than in the saline-injected discs. Similar to the histology in the present study, Obata et al. [18] reported that the number of chondrocyte-like cells in the anterior AF and NP was significantly higher in discs injected with PRPr than in those injected with PBS in a rabbit annular puncture model [29]. The current histological results suggest that the intradiscal administration of PRPr may enhance cell proliferative activity and matrix remodeling within rabbit degenerated discs induced by condoliase, leading to the improvement in MRI grading and structural restoration of DH.

Two previous randomized controlled studies that determined the efficacy and safety of condoliase in patients with LDH showed that condoliase significantly improved neurological symptoms in patients with LDH [12, 13]. However, back pain, Modic type I changes, and a decrease in DH were frequently observed in the condoliase group. Recently, Banno et al. [9] reported the 2-year follow-up results of 67 patients with LDH who received condoliase. They reported that condoliase therapy was effective in 76.1% of the patients. However, 11.9% of the patients required additional surgery because of the ineffectiveness of this therapy. Progression of MRI-graded disc degeneration was observed in 57.1% of patients at three months; however, 30% recovered to baseline values at 2 years. These results suggest that condoliase therapy is effective in many patients with LDH; however, approximately 20% of patients may have worse clinical outcomes, poor DH recovery, or progression of MRI-graded disc degeneration.

The present preclinical study showed that the intradiscal injection of PRPr significantly enhanced DH recovery and MRI-graded disc degeneration. Hence, PRPr injection therapy may be indicated for patients with poor recovery from disc degeneration or worse clinical outcomes after condoliase treatment. PRP and PRPr have been reported to have significant anti-inflammatory effects on IVD cells in vitro [14]. A previous clinical trial reported that the intradiscal injection of PRPr relieved LBP and improved disability and quality of life during 60 weeks of observation in patients with discogenic LBP [17], suggesting that PRPr injection may also improve the clinical symptoms of patients with LDH in whom condoliase was ineffective.

Previous histological and biochemical studies showed that the enzymatic activity of condoliase lasts up to 4 weeks after the intradiscal injection in rabbit IVDs [24]. Therefore, PRPr was intradiscally administrated 4 weeks after the injection of condoliase in the current study. In the clinical setting, PRPr should be injected into the degenerated discs 4 weeks after the condoliase treatment in patients with lumbar disc herniation.

The current histological study showed that intradiscal injection of PRPr following condoliase treatment did not induce disc herniation in the rabbit IVDs. The previous randomized clinical trial showed that intradiscal injection of PRPr was safe and maintained significant improvement in pain, disability, and quality of life without disc herniation or worsening disc bulging during 60 weeks of follow-up in patients with discogenic LBP [17, 30]. Therefore, the authors speculated that the intradiscal injection of PRPr following condoliase treatment in patients with lumbar disc herniation is also safe and does not lead to the recurrence of disc herniation. Nevertheless, clinicians should consider the possibility of hernia recurrence following PRPr injection.

This study has several limitations. First, this study compared the saline and PRPr groups; however, intradiscal injection of saline 4 weeks post-injection of condoliase may have some effects on disc cells. Therefore, only condoliase-injected discs should be assessed to evaluate the natural history of condoliase injection. Second, the observation period of this study was 16 weeks after the injection of condoliase. A long-term follow-up study is needed to evaluate the long-term results of condoliase and subsequent PRPr injections in future research. Third, immunohistochemical analysis of the expression of anabolic, catabolic, and matrix molecules would help further understand the biological effects of PRPr in degenerated rabbit IVDs, which should be evaluated in future studies. Lastly, potential biomechanical and anatomic differences exist between the rabbit (quadrupedal animal) and human (bipedal animal) lumbar spines. Sixteen-week-old New Zealand white rabbits used in this study are equivalent to a 10- to 12-year-old human. Hence, the regenerative capacity of young rabbit IVDs used in this study might be higher than that of human IVDs of 30–50 years of age, at which lumbar disc herniation usually occurs. Therefore, the regenerative effects of PRPr identified in the rabbit IVDs may not directly reflect the responses of PRPr in human degenerated discs.