12 Treatment of Degenerative Disc Disease/Disc Regeneration: Growth Factors and Platelet Rich Plasma
Koichi Masuda and Kenji Kato
Homeostasis of the intervertebral disc (IVD) is tightly regulated by a biological balance between anabolic and catabolic activities of disc cells. Various growth factors that stimulate disc cell matrix synthesis have been evaluated preclinically using animal and human disc cells in vitro. Effective growth factors have also been tested for intradiscal injection therapy in a variety of animal species with promising structural modification results. Some growth factors have been evaluated for safety in humans; however, further studies are needed for human applications. Because most primary outcome measures in clinical trials have had pain-related outcomes, to achieve future successful development of growth factor therapies it is essential to develop animal models that provide evidence for pain relief and to establish pharmacokinetic models.
As a point-of-care product, platelet rich plasma (PRP) has gained clinical attention in the orthopaedic field. A prospective, double-blind, randomized controlled study has been published showing encouraging results; future large-scale multicenter trials should reveal its clinical usefulness for patients with discogenic pain. Although limitations of growth factor therapy, including the lack of nutrients and hypoxic and acidic conditions, are well recognized, it can be anticipated that its application to meticulously selected patients will be beneficial for pain relief and prevention of the progression of disc degeneration.
Keywords: animal model, disc degeneration, disc injection, growth factor, platelet rich plasma
Degenerative disc diseases (DDDs) consist of various pathologies, such as intervertebral disc (IVD) herniation and discogenic low back pain, which cause various clinical symptoms. Generally, degenerated discs are characterized clinically by a reduction of disc height or IVD herniation as demonstrated by magnetic resonance imaging (MRI) or computed tomography (CT). These changes are considered to be caused by an imbalance of anabolism and catabolism, or homeostasis, of the disc cells residing in the nucleus pulposus (NP) and annulus fibrosus (AF).1 The modulation of disc cell metabolism involves a variety of molecules, including cytokines, enzymes, enzyme inhibitors, and growth factors.2 Although the pathogenesis of DDD is not yet fully understood, DDDs are characterized by a progressive process involving a combination of several factors, such as genetic background, aging, and mechanical stress. These changes result in the reduction of water content associated with the depletion of proteoglycans in the NP and structural changes or fissures in the AF. Genetic studies have shown relationships between DDDs and polymorphisms3 in genes encoding collagen type I,4 collagen type X,5,6 aggrecan,7 matrix metalloprotease-2 (MMP-2),8 MMP-3,9 interleukin-1 α (IL-1 α),10 IL-6,11 asporin,12 and cartilage intermediate layer protein, otherwise known as CILP.13,14
Considering these characteristics, to treat DDDs, many efforts have investigated therapeutic strategies that stimulate matrix synthesis by growth factors or inhibit inflammatory cytokines, such as IL-1 and tumor necrosis factor- α (TNF- α).15,16,17 Some efforts have developed therapeutic approaches to suppress disc degeneration by regulating the effects of proteolytic enzymes, such as MMP-1, MMP-3, and members of the disintegrin-like and metalloprotease with thrombospondin motifs (ADAMTS) family.18,19,20 However, if treatment is aimed at recovering disc structure damage, the direct stimulation of cells contributing to matrix metabolism may be required. Since the pioneering work by Thompson et al,21 the effects of a variety of growth factors have been tested (see reviews3,22,23) and have been shown to include an inhibitory effect on matrix degradation or cytokine expression.3,24,25
12.2 The Injection of Growth Factors into Intervertebral Discs (▶ Table 12.1)
The injection of growth factors was, for the first time, attempted in a mouse caudal disc degeneration model induced by static compression.26 Walsh et al26 reported that a single injection of growth and differentiation factor-5 (GDF-5), but not that of insulin-like growth factor-1 (IGF-1), transforming growth factor- β (TGF- β), or basic fibroblast growth factor (bFGF) was effective in stimulating disc regeneration. Furthermore, multiple injections (four injections, one per week) of TGF- β showed a stimulatory effect, although multiple injections of IGF-1, GDF-5, and bFGF did not significantly enhance their single dose effects.26 The discussion by Walsh et al26 suggested that a sustained delivery system, or a combined approach with a mechanical or cell-based device, might be essential to gain a beneficial therapeutic effect. Since the pioneering studies, a variety of growth factors have been studied in vitro and in vivo for disc regeneration (see reviews3,22). Osteogenic protein-1 (OP-1) and recombinant human GDF-5 (rhGDF-5) have been applied to patients as Phase Ib-II clinical trials following various preclinical safety and efficacy studies. Some evidence of efficacy of PRP has been shown in animal studies and has recently gained the attention of clinicians.
12.2.1 Injection of OP-1 into Intervertebral Discs
OP-1, also designated as bone morphogenetic protein-7 (BMP-7), was originally found to stimulate bone formation and is now known to also stimulate proteoglycan and collagen synthesis in chondrocytes27 and IVD cells.28,29,30,31 Therefore, we investigated the safety and efficacy of a single injection of OP-1 in the treatment of DDD, first using healthy rabbit IVDs31 and then degenerated IVDs in the annular-puncture disc degeneration rabbit model.32 A single injection of OP-1 into the NP of a disc 4 weeks after puncture resulted in a significant restoration of disc height and an increased signal intensity of the NP in T2-weighted MRIs at the 6-week time point after the OP-1 injection; these effects were sustained for the entire experimental period, up to 24 weeks. The degeneration grades of the punctured discs in the OP-1-injected group were significantly lower than those of the control lactose-injected group with a higher proteoglycan (PG) content of the NP and AF.32 Biomechanical studies using similar conditions revealed that the injection of OP-1 restored dynamic viscoelastic biomechanical properties, such as elastic and viscous moduli, of puncture-degenerated IVDs.33 Similar reparative effects on disc structures were observed in the chondroitinase-ABC (C-ABC) and OP-1 co-injected model34 and in the C-ABC-induced matrix depletion model of disc degeneration in the rabbit.35 A clinical trial of OP-1 was initiated in 2007, but the results of the Phase I trial have not been published due to the withdrawal of the trial.
12.2.2 Injection of GDF-5 into Intervertebral Discs
GDF-5, originally found as a factor responsible for skeletal alternations in brachypodism mice,36 is a member of the BMP family known for its role in bone formation. The gene-deletion of GDF-5 is reported to be associated with chondro-dysplasia in humans37,38 and IVD degeneration in mice.39 rhGDF-5 is another promising growth factor whose efficacy has been evaluated in in vitro and in animal models.26,40,41 In a preclinical study, an injection of rhGDF-5 in the rabbit annular-puncture model resulted in a restoration of disc height and improvements in MRI and histological grading scores with statistical significance for 12 weeks (▶ Fig. 12.1).41 In the rat tail disc puncture model, an injection of rhGDF-5 encapsulated in polylactic glycolic acid (PLGA) microspheres restored disc height, improved glycosa-minoglycan (GAG) content, and increased collagen type II messenger ribonucleic acid (mRNA) levels.42
Fig. 12.1 Changes in the intervertebral disc (IVD) disc height index (DHI) after annular puncture and recombinant human growth and differentiation factor-5 (rhGDF-5) injection. (a) In a needle-puncture model of disc degeneration in adolescent (5–6 month-old) rabbits, a single injection of rhGDF-5 restored disc height after 4 weeks. Four weeks after an annular needle puncture in New Zealand White rabbits (weighing 3.5–4 kg), the rabbits received an injection of phosphate-buffered saline (PBS; 10 μL) or rhGDF-5 (10 ng, 1 or 100μg, in 10 μL PBS) and were monitored radiographically for up to 12 weeks after the injections. By 4 weeks after the rhGDF-5 injection, the mean %DHI of injected discs in the rhGDF-5 group (100 μg) was significantly higher than that in the PBS group (p<0.05). (b) After sacrifice, the spines were assessed by magnetic resonance imaging (MRI). The MRI of the nucleus pulposus (NP) in the rhGDF-5 group showed increased T2 signal intensity compared with that of the control group. (Modified with permission from Chujo et al.41)
Since 2009, several Phase Ib and Phase II studies have been conducted (ClinicalTrials.gov, NCT00813813, United States; NCT01158924, Australia; NCT01182337, Korea) including a double-blinded study (NCT01124006, United States). The inclusion and exclusion criteria and outcome measures are listed in ▶ Table 12.2 and detailed results can be obtained at trials website.
Table 12.2 A multicenter, randomized, double-blind, placebo-controlled, clinical trial to evaluate the safety, tolerability, and preliminary effectiveness of two doses of intradiscal rhGDF-5 (single administration) for the treatment of early-stage lumbar disc degeneration (summary of the protocol, NCT01124006)
Study type: Interventional
Study design: Phase I, Phase II
•Endpoint classification: Safety/Efficacy study
•Intervention model: Parallel assignment
•Masking: Double-blind (subject, investigator)
•Primary purpose: Treatment
•Persistent low back pain with at least 3 months of nonsurgical therapy at one suspected symptomatic lumbar level (L3/L4-L5/S1) confirmed using a standardized provocative discography protocol
•Oswestry Disability Index of ≥ 30
•Low back pain score ≥ 4 cm as measured by Visual Analog Scale (VAS), at Visit 1 baseline
•Persons unable to have a discogram, CT, or MRI
•Abnormal neurological exam at baseline (e.g., chronic radiculopathy)
•Active radicular pain due to anatomical compression
•Extravasation of contrast agent into the epidural space during discography
•Suspected symptomatic facet joints and/or severe facet joint degeneration at the index level or adjacent segments
Primary outcome measures (12 mo):
•Neurological assessment for motor function and reflexes/sensory
•Treatment-emergent adverse events: Relationship to study drug
•Oswestry Disability Index (ODI) change
•Physical component summary of quality-of life-measure assessed by SF-36
•Mental component summary quality-of-life measure assessed by SF-36
Abbreviations: CT, computerized tomography; MRI, magnetic resonance imaging; rhGDF-5, recombinant human growth and differentiation factor-5; SF-36, 36-item Short Form Health Survey.
*Additional follow-up by telephone at 24 and 36 months. Full description and results are at clinicaltrials.gov/ct2/show/NCT01124006.
12.2.3 Injection of Platelet Rich Plasma into Intervertebral Discs
PRP, a plasma fraction containing multiple growth factors concentrated at high levels, can be produced by centrifugal separation of whole blood in the operating room. However, there are several methods to activate platelets and some publications lack descriptions about the activation method used; therefore, differences in biological activities can be expected. A detailed review of in vitro and in vivo effects of PRP on the IVD has been published.43 Akeda et al44 first reported that PRP is an effective stimulator of cell proliferation and PG and collagen synthesis, as well as PG accumulation, by porcine NP and AF cells cultured in alginate beads. Kim et al,45 Liu et al,46 and Cho et al47 have reported the anti-inflammatory effects of PRP on various cytokines and metalloproteinases under inflammatory conditions.
In vivo experiments using rabbits have shown PRP prepared in gelatin hydrogel microspheres to be effective in the suppression of the progression of IVD degeneration induced by the partial removal of the NP.48,49 Chen et al50 showed an increased expression of aggrecan and collagen II mRNA 2 months after an injection of PRP. Gullung et al51 showed that the immediate injection of PRP retained normal morphological features and high fluid content, as well as disc height, in the rat lumbar-disc needle-puncture model. Moreover, using the rabbit annular-puncture model, the active soluble releasate isolated following platelet activation of PRP (PRP-releasate) induced a reparative effect on rabbit degenerated IVDs.52 Gui et al53 reported similar results in the rabbit annular-puncture model at both 2 and 4 weeks postinjury.
Clinically, a prospective randomized study54 showed that the intradiscal PRP-injection group showed a significant improvement in pain (only in the numerical rating scale [NRS] best pain score), function, and patient satisfaction compared with the control group at the 8-week time point. Because this was a relatively small study with only 47 patients, and not all pain parameters were improved and pain relieve was modest, further large-scale studies may be required to confirm the long-term efficacy of PRP for low back pain due to DDD as a point-of-care treatment.
12.2.4 Other Growth Factors or Growth Factor-related Molecules at Preclinical Stages
GDF-6 (BMP-13; also known as cartilage morphogenetic protein-2 [CDMP2]) is another member of the BMP family that is important for disc development55; it is expressed in human NP cells and induced discogenic differentiation of mesenchymal stem cells (MSCs).56 The injection of BMP-13 at the time of surgery in an ovine annular-stab model prevented or retarded disc degeneration with higher cell numbers and matrix retention 12 months after the injection.57 An injection of BMP-2 in the rabbit disc degeneration annular-stab model induced more degeneration and an increased vascularity; however, this study was intended to obtain fusion with and without coral and no clear quantitative outcome measures were presented.58 Recent studies on BMP-2 or BMP-2/-7 heterodimers in the C-ABC-induced goat disc degeneration model showed no adverse effects, but also no significant improvement.59 An injection of PLGA-polyethylene glycol (PEG)-PLGA loaded with hepatocyte growth factor (HGF) in the rat tail disc puncture model showed retardation of disc degeneration confirmed by increased MRI T2-weighted signal and the maintenance of disc height in the treatment group.60 Thus, the effect of BMP-2 remains to be revealed in in vivo studies. Link N peptide, which stimulates matrix synthesis similar to that of growth factors,61 was effective in recovering disc height with elevated expression of matrix genes in a rabbit disc degeneration model.62 Instead of the direct application of growth factors into IVDs, the intradiscal administration of simvastatin63,64 or lovastatin65 in a rat disc degeneration model has been shown to stimulate BMP-2 or SRY (sex determining region Y) box 9 (SOX9) and shows some promise.
12.3 Limitations of the Therapeutic Application of the Injection of Growth Factors into Intervertebral Discs
12.3.1 The Half-Life of Growth Factors Injected into Intervertebral Discs
It is still not well known how long injected compounds are retained in the disc space after a single injection. Some authors have suggested a short half-life in the order of minutes,66 and stressed the requirement for a drug delivery mechanism to provide the prolonged retention of injected drugs; however, a study of the half-life of OP-1 using radio-labeling indicated much greater times, likely more than 1 month (▶ Fig. 12.2).67 This long elimination of substances from discs may be due to the enclosed structure by the AF and slow diffusion from the NP to the end plate, as well as their specific binding to collagen molecules.68 The elimination time of injected growth factors depends on the binding capacity to the extracellular matrix (ECM), various conditions of the disc, (i.e., the degree of disc degeneration and/or vascularization), the injection procedure itself (i.e., needle size, injection location, and volume), and characteristics of the vehicle or carrier. In addition, the effect of growth factor on cells is dependent on how long activity is retained after binding to their receptors. Further research to determine the mechanism of elimination using in vitro modeling analysis may be helpful.
Fig. 12.2 Retention of radiolabeled bone morphogenetic protein 7 (BMP-7) in a rabbit disc. 125I-labeled BMP-7 (otherwise known as osteogenic protein-1) was injected into the L3–4 disc of normal rabbits. The rabbits were subjected to whole body quantitative autoradiography (WBQA). At 6 hours after injection (top), 125I-labeled BMP-7 was detected at the injection site, and in several organs, including the bladder and thyroid. The signal from the radiolabeled BMP-7 is prominent even after 14 days (bottom). The results of quantification of autography images indicated that the half-life of 125I-labeled BMP-7 was longer than 1 month. (Modified with permission from Pierce et al.67)