- Email: [email protected]
Intradiscal injections of osteogenic protein-1 restore the viscoelastic properties of degenerated intervertebral discs
The Spine Journal 6 (2006) 692–703
Clinical Study
Intradiscal injections of osteogenic protein-1 restore the viscoelastic properties of degenerated intervertebral discs Kei Miyamoto, MD, PhDa, Koichi Masuda, MDa,b,*, Jesse G. Kim, PhDa,c, Nozomu Inoue, MD, PhDa, Koji Akeda, MD, PhDa, Gunnar B.J. Andersson, MD, PhDa, Howard S. An, MDa Departments of aOrthopedic Surgery and bBiochemistry, Rush University Medical Center, 1735 W. Harrison Street, Chicago, IL 60612 USA c Department of Bioengineering, University of Illinois at Chicago, 851 S. Morgan Street, Chicago, IL 60607–7052, USA Received 14 January 2006; accepted 17 April 2006
Abstract
BACKGROUND CONTEXT: Using biochemical, histological, and radiological parameters in a rabbit model of intervertebral disc (IVD) degeneration, the intradiscal injection of a growth factor, such as osteogenic protein-1 (OP-1), has been shown to regenerate the IVD. However, very little is known about how such a biological therapeutic approach affects the biomechanical properties of the degenerated IVD. PURPOSE: To investigate the effects of an intradiscal injection of OP-1 on the biomechanical properties of IVDs in the rabbit annular-puncture disc degeneration model and to determine their relationship to biochemical properties. STUDY DESIGN/SETTING: In vivo study on the effects of intradiscally administered OP-1 on the biomechanical and biochemical properties of IVDs in the rabbit annular-puncture disc degeneration model. METHODS: New Zealand White rabbits (n516) underwent annulus fibrosus (AF) puncture, using an 18-gauge needle, at L2–L3 and L4–L5 (L3–L4: nonpunctured control). Four weeks later, the punctured discs received an injection of either 5% lactose (10 mL) or OP-1 (100 mg/10 mL of 5% lactose) into the nucleus pulposus (NP). The disc height was radiographically monitored biweekly. After sacrifice and removal of bone–disc–bone complexes 8 weeks postinjection, the dynamic viscoelastic properties of the IVDs were tested by applying a cycle of sinusoidal strain in uniaxial compression at six loading frequencies (0.05 to 2 Hz). The biochemical properties of the dissected IVDs were then analyzed and correlated with the biomechanical properties. RESULTS: A single injection of OP-1 significantly restored disc height when compared with the lactose-injected discs (OP-1 vs. lactose, p!.001). The elastic modulus of the IVDs in the OP-1injected discs was significantly higher than that in the lactose-injected discs at all frequencies (mean: þ43%, p!.001). The viscous modulus in the OP-1-injected discs was significantly higher at 0.05, 0.2, 0.5, and 1 Hz (mean: þ55%, p!.001) and showed higher tendencies at other frequencies (p5.08–.09). For both moduli, no significant differences were observed between the OP-1-injected and the nonpunctured control discs. The OP-1 injection significantly increased the proteoglycan (PG) content in the NP and AF, and the collagen content in the NP (p!.001–.05). Both elastic and viscous moduli showed significant positive correlations with PG content in the NP and collagen content in the NP and AF (Rho5.357–.466, p5.010–.047). CONCLUSIONS: We have shown for the first time that an injection of the growth factor, OP-1, restored the biomechanical properties of IVDs in a rabbit model of IVD degeneration. Comparing biomechanical with biochemical data suggests that the OP-1-induced biomechanical restoration
FDA device/drug status: investigational/not approved (osteogenic protein-1). This study was supported by NIH grants P50-AR-39239 and P01-AR48152. 1529-9430/06/$ – see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.spinee.2006.04.014
This paper is dedicated to the memory of Dr. Jesse G. Kim, friend and colleague, who passed away during review of the manuscript. * Corresponding author. 1735 W. Harrison St., Cohn 720, Chicago, IL 60612. Tel.: (312) 942-4661; fax: (312) 942-8828. E-mail address: [email protected] (K. Masuda)
K. Miyamoto et al. / The Spine Journal 6 (2006) 692–703
693
was a consequence of increased activities of anabolic pathways that resulted in biochemical changes in the IVD. Ó 2006 Elsevier Inc. All rights reserved. Keywords:
Intervertebral disc; Degeneration; Growth factor; Osteogenic protein-1; Biomechanics; Viscoelastic properties; Proteoglycan; Collagen; Animal model
Introduction Low back pain is rated as second only to upper respiratory problems among symptom-related reasons for visits to a physician, the fifth ranking cause of admission to hospitals, and the third most common reason for surgical procedures in the United States [1]. Generally, it is accepted that intervertebral disc (IVD) degeneration is, at least in part, one of the causes of discogenic low back pain [1]. Although several reports have presented evidence of the association between disc degeneration and low back pain [2–5], much remains unknown and controversy still exists. The unique structure of an IVD, which has a gelatinous proteoglycan (PG)-rich nucleus pulposus (NP) surrounded by a collagen-rich annulus fibrosus (AF), confers viscoelastic properties that are characteristic of the IVD. These properties enable the IVD to withstand mechanical forces, which is its most important function. Both the PG content, with its negatively charged hydrophilic nature, and collagen, which forms concentric lamellae, are known to contribute to the viscoelastic properties of the IVD. Biochemically, degenerative changes in IVDs are characterized by loss of the extracellular matrix (ECM) including PG and collagen [6,7]. Therefore, once disc degeneration develops, viscoelastic properties decrease considerably [8,9] and the IVDs can become susceptible to stages of intersegmental instability [10,11]. The biological repair of degenerated IVDs, such as that achieved by protein injection, cell-based and gene-delivering therapies [12–15], has recently been considered to be a minimally invasive and long-lasting treatment modality. Among the approaches aimed at regaining lost function of the IVD through biochemical, structural, and biomechanical perspectives, protein injection therapy has been considered to be the simplest approach. Several in vitro studies have indicated that various growth factors, such as insulin-like growth factor-1 [16–18], bone morphogenetic protein (BMP)-2 [19,20], BMP-7 [14,21,22], and transforming growth factor-b [18], stimulate the ECM metabolism of IVD cells. Osteogenic protein-1 (OP-1, also designated as BMP-7) is known to stimulate ECM production by disc cells in vitro [21,23], and its injection into the NP of normal adolescent rabbits has been shown to increase disc height as well as PG content of the NP [14]. Additionally, a single injection of OP-1 induced a significant restoration of disc height and an improvement in the histological and biochemical parameters of degenerated IVDs in the rabbit annular-puncture disc degeneration model [22,24]. However, the efficacy of biological treatments for degenerated IVDs, including the injection of OP-1, on the biomechanical function of IVDs,
which is essentially the main concern, has not been elucidated. Because OP-1 has strong anabolic effects on ECM production and accumulation, both in vitro and in vivo, it is reasonable to hypothesize that the injection of OP-1 could affect the viscoelastic properties of the IVD as a consequence of changes in its biochemical properties. Historically, biomechanical properties of the IVD have been explored using various biomechanical testing methods [25–30]. Because disc tissues exhibit nonlinear viscoelastic behaviors [31,32], methods testing only linear viscoelastic behavior might be limiting. Dynamic viscoelastic testing, with the advantage of being able to demonstrate the nonlinear behavior of tissues and dependency on frequency of load [33], was performed on articular cartilage [34], NP tissue [31], and the temporomandibular joint [35]. More recently, to achieve a better understanding of the function of the IVD as a whole, dynamic viscoelastic testing on the rabbit IVD as a functional unit was introduced as a reliable and valid method [36]. The present study investigated the effects of an intradiscal injection of OP-1 on both biomechanical and biochemical properties, and the possible relationship between these two properties, using the rabbit annular-puncture disc degeneration model [24] and the newly developed dynamic viscoelastic testing system for rabbit IVDs [36]. Methods Establishment of a degenerative IVD in the rabbit annular-puncture disc degeneration model and the injection of OP-1 All experimental protocols were approved by the authors’ Institutional Animal Care and Use Committee (IACUC #04-027). Under general anesthesia and using a right posterolateral retroperitoneal approach, New Zealand White rabbits (n516; 3–3.5 kg, 5 months old) underwent AF puncture with an 18-gauge needle at both the L2–L3 and L4–L5 discs to induce disc degeneration [24]. In all rabbits, the L3–L4 disc served as a nonpunctured control. Four weeks later, using a left posterolateral retroperitoneal approach, punctured discs in each animal (L2–L3 or L4–L5) received an injection of either the vehicle (5% lactose, 10 mL per disc) or recombinant human OP1 (rhOP-1); a gift from Stryker Biotech, Hopkinton, MA, 100 mg in 10 mL 5% lactose per disc using a 26-gauge needle. The disc level to receive either the lactose or rhOP-1 injection was randomly assigned. During the follow-up period, the disc height was monitored radiographically as described below. Eight weeks postinjection, rabbits were
694
K. Miyamoto et al. / The Spine Journal 6 (2006) 692–703
euthanized as previously described, [22] and the lumbar spines were harvested. Bone–disc–bone complexes, with the superior and inferior vertebra bodies perpendicular to the axis of the spine, were removed from L2–L3, L3–L4, and L4–L5 levels using a cutoff saw (McMaster-Carr, Chicago, IL) with adjustable-width parallel blades (Fig. 1, upper left).
The data are reported as the IVD height expressed as the disc height index (DHI5IVD height/adjacent IVD body height) based on the method of Lu et al. [37] with a slight modification [14]. Changes in the DHI of injected discs are expressed as %DHI and normalized to the measured preoperative IVD height [%DHI5(postoperative DHI/preoperative DHI)100] [14]. Magnetic resonance imaging analyses
Radiographic analysis of disc height Radiographs were taken at 2-week intervals up to 12 weeks after the puncture. Extreme care was taken to maintain a consistent level of anesthesia (ketamine hydrochloride [25 mg/kg] and acepromazine maleate [1 mg/kg]) during radiography of each animal at each time point in order to obtain a similar degree of muscle relaxation, which may affect the disc height. The preoperative X-ray was used as a baseline measurement. All X-ray images were digitized and independently analyzed using a custom program for MATLAB software (Natick, MA) by an orthopedic researcher who was blinded to the treatment group.
Magnetic resonance imaging (MRI) analyses were performed in vitro on the spinal columns obtained from all rabbits using a 0.3-T imager (Airis II, version 4.0 A; Hitachi Medical System America, Inc.), with a quadrature extremity coil receiver as previously reported [24]. The MRIs were evaluated by a blinded observer using the modified Thompson classification [24] based on changes in the degree and area of signal intensity from grade 1 to 5 (15normal, 25minimal decrease of signal intensity but obvious narrowing of high signal area, 35moderate decrease of signal intensity, 45severe decrease of signal intensity but slight high signal area, 55no high signal area).
Fig. 1. Preparation of the specimen and measurement of the dynamic viscoelastic properties of rabbit intervertebral discs. The dynamic viscoelastic properties of rabbit intervertebral discs were measured using a custom-made testing system that consists of a linear actuator with optical encoder, a load cell (EL series, Entran), a custom-made loading frame, 316L stainless steel porous pucks (100 mm pore size), and a water chamber. A sinusoidal strain in uniaxial unconfined compression was applied.
K. Miyamoto et al. / The Spine Journal 6 (2006) 692–703
IVD preparation for dynamic viscoelastic testing After the anterior aspects of the L2–L3, L3–L4, and L4– L5 discs were exposed, each bone–disc–bone complex was obtained by cutting through the superior and inferior vertebral bodies perpendicular to a longitudinal axis of the spine using a modified compact cutoff saw (McMaster-Carr, Chicago, IL) with adjustable-width parallel blades (Fig. 1, upper left). Then, the posterior element was removed and the bone–disc–bone complex was obtained. The superior and inferior cut surfaces of the complex were rasped in order to align them parallel and to make both end plates thin enough (~2 mm) to maintain the integrity of the whole disc as a functional unit (Fig. 1, upper left). In samples with prominent osteophyte formation, the osteophyte was removed such that it would not cause impingement during biomechanical testing.
695
directions to assess in vitro disc height and cross-sectional area, respectively, using an X-ray machine (Faxitron, Faxitron X-ray Corporation, Wheeling, IL). The contact radiographs were scanned and disc height was measured as the area of the disc space divided by the mediolateral length from the anteroposterior view using image analysis software (Scion software, Scion Corporation, Frederick, MD). The cross-sectional area was measured as the area of the bony end plate surface from the craniocaudal view [36]. In addition, the cross-sectional area of the newly formed osteophyte was measured in each specimen before removal of osteophytes. The relative size of the osteophyte to the cross-sectional area of the bony end plate surface (%disc area) was compared between the lactose-injected and the OP-1-injected discs that previously underwent annular puncture. Analyses for dynamic viscoelastic properties
Radiographic measurement of IVD dimensions Contact radiographs of the bone–disc–bone complexes were taken in anteroposterior and craniocaudal (Fig. 2)
The dynamic viscoelastic properties of IVDs were measured by a custom-made dynamic viscoelastic biomechanical testing system [36]. This system consists of a linear
Fig. 2. Contact radiographs of bone–disc–bone complexes used in craniocaudal directions. Examples of an in vitro craniocaudal X-ray of the bone–disc– bone complex from lactose-injected and osteogenic protein-1 (OP-1)-injected discs are shown. In both lactose-injected and osteogenic protein-1-injected punctured discs, osteophyte formation was consistently observed at the right anterolateral side where the initial puncture using an 18-gauge needle was performed (upper row pictures, small arrows). Comparing the lactose-injected and the recombinant human osteogenic protein-1-injected discs, no significant differences were observed in the cross-sectional area of the osteophytes. Therefore, the osteophyte was removed such that it would not cause impingement during biomechanical testing.
696
K. Miyamoto et al. / The Spine Journal 6 (2006) 692–703
Fig. 3. (A) A sinusoidal compressive strain applied to the disc (e) and the out-of-phase resultant stress (s) with a phase angle (d). (B) The complex modulus (E*), the storage modulus (E0 ), and the loss modulus (E00 ), obtained by vectorial separations.
actuator with optical encoder (Digit series, UltraMotion, Mattituck, NY), a load cell (EL series, Entran, Hampton, VA), a custom-made loading frame, and a water chamber (Fig. 1). Each disc was placed on a 316L stainless steel porous puck (100 mm pore size; Mott Corporation; Farmington, CT) inside a water chamber that was filled with physiological saline (Fig. 1). The loading shaft with another porous puck was slowly loaded onto the disc (0.005 mm/s) until the contact criteria of 5 N compression (13% of the rabbit’s body weight) was reached to maintain contact with the disc during the unloading cycle of the test. Then, preconditioning was performed by applying 10 sinusoidal strain cycles with an amplitude of 10% at 1 Hz to achieve repeatable testing results. After a 3-minute recovery from preconditioning, six different physiologically relevant loading frequency tests (0.05, 0.1, 0.2, 0.5, 1, and 2 Hz) were performed with the same amplitude. Each frequency test was performed using one cycle of sinusoidal strain
followed by a 3-minute recovery. The loading frequencies and the strain amplitude were chosen to simulate dynamic in vivo physiological conditions. A dynamic compression was applied by a sinusoidal strain of 3(t)53oþ3a cos (ut) with an initial strain of 3o510%, oscillation amplitude of 3a510%, an angular frequency u (52pf) in radians/s, frequency f in cycle/s (Hz), and time t in seconds. Because of the viscoelastic properties, the resultant stress s(t)5soþsa cos (utd) oscillates sinusoidally with a phase angle of d (Fig. 3A). An elastic modulus (E0 ), a viscous modulus (E00 ), and a ratio of viscous-to-elastic moduli (tan d) were determined as the dynamic viscoelastic parameters of the IVD. The elastic modulus represents elastic behavior with its ability to store deformational energy, and the viscous modulus represents viscous behavior with the dissipation (loss) of energy during deformation in a viscoelastic material [35]. When the viscoelastic material is subjected to a sinusoidal strain, the resultant stress is out-of-phase with a time-lag
K. Miyamoto et al. / The Spine Journal 6 (2006) 692–703
697
Fig. 5. Magnetic resonance imaging (MRI) grading within each treatment group and a presentation of a sample MRI (mean6standard deviation). At 8 weeks postinjection, the MRI of the nucleus pulposus in the osteogenic protein (OP)-1-injected discs showed a slightly, but significantly, stronger T2 signal intensity than that of the lactose-injected discs (p!.05, MannWhitney test; graph on the left). This difference is visible on the sample MRI (image on the right).
Fig. 4. Lateral radiogram of a rabbit lumbar spine before (preoperative) and after (4 weeks) an annular-puncture with an 18-gauge needle and 8 weeks postinjection (12 weeks after puncture) of either lactose or osteogenic protein-1 (OP-1). Changes in the intervertebral disc height index (DHI) after the annular-puncture and OP-1 injections (mean6standard error). The lateral radiographs show that the disc space, which showed narrowing 4 weeks after the annular needle puncture, is restored in the OP-1injected level, whereas the narrowing progressed in the lactose-injected level. The graph shows that an annular puncture with an 18-gauge needle decreased the disc height index (DHI) by approximately 32% when compared with the baseline %DHI values before the annular puncture, p!.0001). Then, the intradiscal injection of OP-1 significantly affected the disc height in the postinjection time course (p!.0001). When the DHI at each time point was assessed, at 4 weeks postinjection, the %DHI of the OP-1-injected discs showed an increase compared with the lactoseinjected discs with statistical significance (p!.001).
called the phase angle d of between 0 (purely elastic) and 90 (purely viscous) [38]. The complex modulus E* is vectorially separated into an elastic modulus E0 and a viscous 00 0 00 modulus pffiffiffiffiffiffiffi E in the complex plane by E*5E þiE , where i5 1 [38] (Fig. 3B). The magnitude of the complex modulus |E*| is determined by |E*|5s/e, where stress s5F (force)/A (cross-sectional area), strain e5DDH (change in disc height)/DH (disc height) [39,40]. The elastic modulus E0 , viscous modulus E00 , and a ratio of viscousto-elastic moduli tan d are determined by E0 5|E*| cos d, E00 5|E*| sin d, and tan d5E00 / E0 , respectively. In order to determine the biomechanical effects of the intradiscal injection of rhOP-1, the elastic and viscous
moduli and the ratio of viscous-to-elastic moduli were compared among the three IVD groups (nonpunctured control discs, OP-1-injected punctured discs, lactose-injected punctured discs). Additionally, a correlation between the elastic and viscous moduli was assessed. Biochemical analyses After completion of the biomechanical testing, the gelatinous NP tissue was sharply dissected out from each disc. A white fibrous tissue found in the punctured disc, which was observed in the area corresponding to the anatomical location of the normal NP, was also collected as NP. The remaining fibrous AF tissue was removed from the end plates and adjacent vertebral bodies. Wet and dry weights were obtained for all specimens before digestion with papain at 60 C for 48 hours [41]. Contents of deoxyribonucleic acid (DNA), PG, and collagen were analyzed as previously described [41]. To minimize the size differences in the IVDs found among the levels within a single animal and among the individual animals, all biochemical parameters were standardized by the cross-sectional area of the IVD (mm2), as determined by radiographs which had been digitized and measured. Data and statistical analyses The significance of differences among means was analyzed by two-way repeated measurement analysis of variance (ANOVA) or one-way ANOVA and Fisher’s
698
K. Miyamoto et al. / The Spine Journal 6 (2006) 692–703
protected least significant difference test as a post hoc test on data obtained from X-ray measurements, biomechanical parameters, and biochemical parameters. All data are expressed as the mean6standard deviation (SD) or the mean6standard error (SE). The Kruskal-Wallis test and Mann-Whitney U test were used to analyze nonparametric data (MRI grading) for the effect of treatment.
Correlations between biochemical properties and dynamic viscoelastic parameters and between biochemical and biomechanical properties were examined using the Spearman signed rank test. The elastic modulus and the viscous modulus at typical physiological loading frequency of 0.5 Hz (1 s loading and 1 s unloading) were correlated with the PG and collagen contents of NP and AF tissues, standardized by the cross-sectional area. All statistical analyses were performed using Statview, Version 5.0 (Windows version XP, Abacus Concepts, Berkeley, CA). A level of p!.05 was used to determine statistical significance.
Results Radiographic analysis of the in vivo disc height In both lactose and rhOP-1-injected discs, which had received an identical puncture with an 18-gauge needle, the %DHI at the 4-week time point after the AF puncture showed a similar decrease (lactose vs. OP-1, not significant [NS]) of approximately 32% (compared with the baseline %DHI values before the AF puncture; p!.0001, Fig. 4), whereas the %DHI of the control level (L3–L4) remained constant. Repeated ANOVA showed that the intradiscal injection of rhOP-1 significantly affected the disc height in the postinjection time course (p!.0001). Although no significant difference among the groups was observed at the 2-week postinjection time point, at 4 weeks postinjection, the %DHI of the rhOP-1-injected discs showed an increase compared with the lactose-injected discs with statistical significance (4 weeks, p!.001, 6 weeks, 8 weeks, p!.0001) (Fig. 4). At 8 weeks postinjection, the disc height of the OP-1-injected discs achieved approximately 88% of the DHI in the nonpunctured controls, which was not significantly different (OP-1 vs. nonpunctured control, NS)
= Fig. 6. Effects of osteogenic protein-1 (OP-1) on the elastic modulus (E0 ), the viscous modulus (E00 ) and the loss tangent (E00 /E0 ) of rabbit intervertebral discs after annular puncture (mean6standard error). In the OP-1injected discs (closed circles), at all loading frequencies the elastic modulus (E0 ) was significantly higher than that in the lactose-injected discs (triangles, mean: þ43%, p!.05) and approached that of the nonpunctured control discs (open circle) (OP-1 vs. nonpunctured control, not significant, upper graph). The viscous modulus was significantly higher in the OP-1injected discs (closed circle) than that in the lactose-injected discs (reverse triangle) at loading frequencies of 0.05, 0.2, 0.5, and 1 Hz (average: þ55%, p!.001), while showing a strong tendency to be higher at 0.1 (p5.077), and 2 Hz (p5.093) (middle graph). In both the elastic and the viscous moduli, no significant difference was observed between the OP1-injected discs and nonpunctured discs at all loading frequencies (upper and middle graphs). No significant difference was observed in the loss tangent among the nonpunctured control and the recombinant human osteogenic protein-1-injected and lactose-injected discs.
K. Miyamoto et al. / The Spine Journal 6 (2006) 692–703
699
intensity, with an average grade of 3.1, than that of the lactose-injected discs, with an average grade 3.6 (p!.05, Mann-Whitney test, Fig. 5). Gross observation of the retrieved discs It is important to note that there was a significant formation of osteophytes in both lactose-injected and OP-1-injected punctured discs; these were located mostly in the right anterolateral surface of the IVDs where the puncture was performed. When the cross-sectional areas of the osteophytes were compared between the lactose- and the OP-1-injected discs, no significant differences were observed (OP-1: 17.9610.1% of the disc area; lactose: 19.6612.5% of the disc area). Because these osteophytes might affect the biomechanical properties of the disc by bridging the vertebral bodies, all biomechanical analyses were performed with and without removing osteophytes to confirm the accuracy of assessment. Because the biomechanical results of a disc with or without osteophytes showed essentially the same trend, the results after removal of osteophytes are reported in the following section. Biomechanical properties
Fig. 7. Effects of the treatment with osteogenic protein-1 (OP-1) or lactose on wet weight, DNA, proteoglycan (PG) and collagen contents of the nucleus pulposus (NP) and annulus fibrosus (AF) of rabbit intervertebral discs (mean6standard deviation). In the NP, as can be observed in data from the lactose-injected discs, the annular needle puncture induced dramatic decreases in wet weight, DNA content, and PG content, whereas the collagen content was increased (left panel). In the AF, the annular needle puncture did not cause significant changes in any of the four parameters (right panel). The intradiscal injection of OP-1 significantly increased the wet weight and contents of DNA, PG, and collagen in the NP, when compared with the lactose-injected discs. OP-1 also increased the wet weight and PG content of the AF.
(Fig. 4). On the other hand, in the lactose-injected discs, the disc height loss was sustained at a level of approximately 69% of the DHI in the nonpunctured control for up to 8 weeks postinjection (Fig. 4).
MRI analyses At 8 weeks postinjection, the MRI of the NP in the OP-1-injected discs showed a slightly stronger T2 signal
The elastic modulus (E0 ) A two-way ANOVA demonstrated that the treatment of the IVD (punctured vs. nonpunctured; OP-1 injection vs. lactose injection) affected the elastic modulus significantly (p!.0001), whereas the loading frequency had no significant effect. The elastic modulus (E0 ) in the lactose-injected discs (triangle) was significantly lower than that in the nonpunctured control discs (open circle) at all loading frequencies (mean: 64% of the control, p!.01) (Fig. 6, upper graph). On the other hand, at all loading frequencies, the elastic modulus in the OP-1-injected discs (closed circle) was significantly higher than that in the lactose-injected discs (mean: þ43%, p!.05) and approached that of the nonpunctured control levels (OP-1 vs. nonpunctured control, NS) (Fig. 6, upper graph). The viscous modulus (E00 ) Two-way ANOVA demonstrated that both the treatment of the IVD (punctured vs. nonpunctured; OP-1 injection vs. lactose injection) and loading frequency affected the viscous modulus significantly (p!.0001), with no interactive effects. The viscous modulus had a trend to be low in the higher frequencies (Fig. 6, middle graph). When the effect of the treatment was assessed, the viscous modulus E00 ) in the lactose-injected discs (triangle) was significantly lower than that in the nonpunctured control discs (open circle) only at 0.05, and 0.2 Hz (mean: 65% of the control, p!.05; Fig. 6, middle graph). The viscous modulus in the OP-1injected discs (closed circle) was significantly higher than that in the lactose-injected discs at 0.05, 0.2, 0.5, and 1 Hz (average: þ55%, p!.001) and with a strong tendency
700
K. Miyamoto et al. / The Spine Journal 6 (2006) 692–703
Fig. 8. Correlations between the biomechanical properties at a typical physiological loading frequency of 0.5 Hz and the biochemical properties of rabbit intervertebral discs from lactose-injected and osteogenic protein-1 (OP-1)-injected rabbit intervertebral discs using the Spearman signed rank test. The elastic modulus (E0 ) was shown to have a significant positive correlation with the PG and collagen contents in the NP and the collagen content in the AF tissues. Similarly, the viscous modulus was also shown to have a significant correlation with the PG and collagen content in the NP and the collagen content in the AF.
to be higher at 0.1 (p5.077), and 2 Hz (p5.093) (Fig. 6, middle graph). In the viscous modulus, no significant differences were observed between the OP-1-injected discs and nonpunctured control discs at any loading frequencies. The ratio of viscous-to-elastic moduli (E00 / E0 ; loss tangent) A two-way ANOVA demonstrated that the loading frequency had a significant effect on the loss tangent (p!.0001) with smaller values at higher frequencies, whereas the treatment of the IVD (punctured vs. nonpunctured; OP-1 injection vs. lactose injection) had no significant effect (Fig. 6, lower graph).
In the NP, the intradiscal injection of OP-1 resulted in significant increases in wet weight (þ57%, p!.0001), DNA content (þ48%, p!.0001), PG content (þ83%, p!.0001), and collagen content (þ76%, p!.001), compared with the lactose-injected discs (Fig. 7, NP: left column). In the AF, significant increases in wet weight (þ20%, p!.01) and PG content (þ35%, p!.05) were also observed (Fig. 7, AF: right column). It is important to note that, after the intradiscal injection of OP-1, the PG content in the AF and the collagen content of the NP were significantly higher than those in the nonpunctured control IVDs (PG in the AF: þ28%, p!.05; collagen in the NP: þ234%, p!.001).
Biochemical properties In the NP, when comparing data between the nonpunctured control discs and the lactose-injected discs, the needle puncture induced dramatic decreases in wet weight (47%), DNA content (49%), and PG content (68%), whereas the collagen content was increased by the puncture (þ90%) (Fig. 7, NP: left column). In the AF, when comparing data between the nonpunctured control discs and the lactose-injected discs, the needle puncture did not produce significant changes in any of the four parameters (Fig. 7, AF: right column).
Correlations between biomechanical and biochemical properties using the Spearman rank correlation test When discs from all treatment groups were evaluated together, the elastic modulus (E0 ) was shown to have a significant positive correlation with the PG content in the NP (p5.014) and the PG and collagen content in the AF (PG: p5.01, collagen: p5.019) (Fig. 8, left panels). Similarly, the viscous modulus was shown to have a significant positive correlation with the PG content in the NP (p5.020)
K. Miyamoto et al. / The Spine Journal 6 (2006) 692–703
and the PG and collagen content in the AF (p5.007) (PG: p5.028, collagen: p5.047) (Fig. 8, right panels).
Discussion The results of this study demonstrated that a single injection of OP-1 into the NP significantly ameliorated the dynamic viscoelastic properties of the degenerated disc in the rabbit annular-puncture disc degeneration model. The biochemical analysis of repaired tissues revealed that the OP-1 injection induced an increase in wet weight, DNA content, PG content, and collagen content of the NP, compared with the lactose-injected discs, whereas an increase was observed only in the wet weight and PG content of the AF. As has been primarily hypothesized, an increase in content of ECM components of the IVD tissues positively correlated with viscous and elastic moduli of the IVD, suggesting that biochemical changes induced by an injection of OP-1 may have resulted in the biomechanical restoration of the IVD. This is the first report providing evidence that a biological approach for disc repair is effective in restoring the biomechanical properties of degenerated IVDs. The biomechanical testing method used in this study enabled the measurement of the dynamic viscoelastic properties of the IVD over a broad range of loading frequencies (0.05–2 Hz), encompassing those occurring physiologically [36]. The analyses of the viscous and elastic response components of compressive stiffness have allowed detection of subtle variations in the biomechanical properties of IVDs from different levels (eg, L1–L2, L3–L4, L5–L6) [36]. Our previous study showed that annular puncture into the NP with an 18-gauge needle resulted, 4 weeks subsequently, in a marked decrease in both the elastic and viscous properties of the rabbit IVD [42]. Because our previous attempt, using static compression, failed to detect biomechanical changes in the degenerated disc in the rabbit stab model (data not shown), the method reported here, which tests dynamic viscoelastic properties, has proven useful to detect changes in biomechanical properties in a therapeutic modality. At the 12-week postpuncture time point analyzed in the current study, viscous and elastic moduli (Fig. 6) were similar to the corresponding values at 4 weeks postpuncture [42]; this suggests that the punctured and lactose-injected IVD does not have the capability of being restored in the short period used in this study, as seen radiographically (Fig. 4). The biomechanical analyses also provide insight into the therapeutic effects of the intradiscal administration of OP1. Such an injection, using a 26-gauge needle, restored those once lost viscoelastic properties by 8 weeks postinjection to levels that did not differ significantly from the nonpunctured control disc levels (Fig. 6). This indicates a functional biomechanical healing of the IVD damage during the 8-week time period. Interestingly, the loss tangent
701
(E00 /E0 ) was highest at the lowest frequency (0.05 Hz) tested in the present study, indicating that the viscous behavior of the rabbit IVD is most prominent at the lower frequency and that the frequency-dependence of compressive behavior in the radially unconfined configuration reflects decreased fluid exudation at the higher frequencies [43,44]. Similar loss tangents found among the nonpunctured control discs and both the OP-1-injected and lactose-injected punctured discs in this study may reflect an injury-associated decrease in the IVD compressive modulus and an increase in hydraulic permeability that are counterbalanced by a decrease in hydration with, thus, no overall effect on the characteristic response frequency. Also, similar loss tangents found between punctured OP-1-treated discs and lactose-injected discs provide further evidence for a full functional recovery associated with OP-1 injection. PGs and collagen both contribute to the mechanical properties of the NP and AF. PGs endow disc tissues with a fixed negative charge, which results in hydration and swelling of the tissue and the consequent ability to resist compressive loading [7]. The collagen network, which imparts tensile strength to the disc, entraps PG molecules in the tissues. In this study, a significant increase in wet weight and PG content was observed in both NP and AF tissues of the OP-1-injected discs, compared with the lactoseinjected discs, whereas an increase in collagen content was observed only in the NP. These results suggested that an increased PG content, induced by the injection of OP-1, resulted in tissue hydration in both the NP and AF. However, the possibility cannot be excluded that changes in collagen content contribute to changes in biomechanical properties. Although the biomechanical tests performed on the ‘‘whole disc’’ bone–disc–bone complex made correlation analyses difficult, several biochemical properties of the IVD, including the contents of two key ECM components, PGs and collagen, have been shown to correlate significantly with biomechanical parameters. This correlation supports our contention that stimulation of ECM metabolism by the in vivo application of growth factors is beneficial to the repair of degenerated IVDs [13]. The positive correlation between the collagen content of the AF with both the elastic and the viscous moduli especially suggests that a treatment which aims to increase the collagen content of the IVD might be one possible therapeutic approach to disc degeneration. The IVD samples harvested from punctured discs in this study at the 8-week postinjection time point were frequently associated with osteophytes, as has previously been reported [22,45]. In most punctured disc specimens, osteophytes were located on the right side where the initial annular puncture was made; this suggests that the etiology of the development of the osteophytes was due to extruded discs induced by the needle puncture. Our quantitative comparison of the cross-sectional area of osteophytes between the lactose-injected and OP-1-injected discs did not show any
702
K. Miyamoto et al. / The Spine Journal 6 (2006) 692–703
statistical differences. Because of concerns about the influence of the presence of osteophytes on the biomechanical properties of the IVDs caused by partial bridging between two vertebral bodies, the biomechanical test measurements were repeated before and after removal of osteophytes. The results showed that measurements after the removal of osteophytes provided results essentially similar to those before osteophyte removal, but with less variation (data not shown). There are several limitations to this study. First, adolescent rabbits (5 months old) still have notochordal cells present in their IVDs. However, adolescent rabbits were used in the belief that this is an acceptable model in which to start preliminary in vivo experiments. The use of mature animals or larger animals, which have a cellular phenotype closer to that of human IVD cells, might provide more critical information on the efficacy of OP-1 injections into the degenerated IVD. Second, this experiment to determine if there was an initial recovery of biomechanical properties after a single injection of OP-1 protein was done to test the feasibility of achieving a biological effect by growth factors on the biomechanical properties of a degenerated disc. In future research, longer follow-up data are needed to prove the long-term efficacy of this therapy. Third, this study did not address the biodistribution of rhOP-1 that was injected into the center of the NP. Because the presence of endogenous OP-1 made an experimental design to address this question difficult without the use of radiolabeled rhOP-1, a decision was made to elucidate the half-life of injected rhOP-1 in a separate experiment that is currently under way. The results of this biodistribution study may provide us with the frequency of injections needed for effective clinical treatment. Fourth, the biomechanical tests used in this study were performed on a bone–disc–bone complex. To reveal the involvement of the AF and NP tissues in the repair process, further biomechanical studies on individual tissues may be required. Finally, the biomechanical testing was performed applying uniaxial compression only. Because the human spine has multidirectional flexibility, supported by the mechanical properties of the IVDs, several variations in the biomechanical testing on in vivo rabbit IVDs need to be performed in the future. In conclusion, the findings of the current study provide preliminary evidence that an injection of OP-1 restored biomechanical properties of degenerated discs in the rabbit annular-puncture disc degeneration model. The biomechanical and biochemical data in this study suggested that OP-1induced biochemical changes by anabolic stimulation may have resulted in the biomechanical restoration of the IVD. Although further efficacy studies with larger animals and studies using pain as a primary endpoint for a therapeutic approach for disc degeneration are needed, the preliminary evidence in this rabbit study established that an injection of growth factor may be clinically applicable as a therapeutic approach to repair the degenerated IVD.
Acknowledgments The authors would like to acknowledge Stryker Biotech for providing the osteogenic protein-1. References [1] Andersson GB. Epidemiological features of chronic low-back pain. Lancet 1999;354:581–5. [2] Aprill C, Bogduk N. High-intensity zone: a diagnostic sign of painful lumbar disc on magnetic resonance imaging. Br J Radiol 1992;65: 361–9. [3] Freemont AJ, Peacock TE, Goupille P, Hoyland JA, O’Brien J, Jayson MI. Nerve ingrowth into diseased intervertebral disc in chronic back pain. Lancet 1997;350:178–81. [4] O’Neill CW, Kurgansky ME, Derby R, Ryan DP. Disc stimulation and patterns of referred pain. Spine 2002;27:2776–81. [5] Pye SR, Reid DM, Smith R, et al. Radiographic features of lumbar disc degeneration and self-reported back pain. J Rheumatol 2004; 31:753–8. [6] Antoniou J, Steffen T, Nelson F, et al. The human lumbar intervertebral disc: evidence for changes in the biosynthesis and denaturation of the extracellular matrix with growth, maturation, ageing, and degeneration. J Clin Invest 1996;98:996–1003. [7] Urban JP, Roberts S. Degeneration of the intervertebral disc. Arthritis Res Ther 2003;5:120–30. [8] Urban JP, McMullin JF. Swelling pressure of the lumbar intervertebral discs: influence of age, spinal level, composition, and degeneration. Spine 1988;13:179–87. [9] Adams MA, McNally DS, Dolan P. ’Stress’ distributions inside intervertebral discs: the effects of age and degeneration. J Bone Joint Surg Br 1996;78:965–72. [10] Fujiwara A, Lim TH, An HS, et al. The effect of disc degeneration and facet joint osteoarthritis on the segmental flexibility of the lumbar spine. Spine 2000;25:3036–44. [11] Zhao F, Pollintine P, Hole BD, Dolan P, Adams MA. Discogenic origins of spinal instability. Spine 2005;30:2621–30. [12] Nishida K, Kang JD, Suh JK, Robbins PD, Evans CH, Gilbertson LG. Adenovirus-mediated gene transfer to nucleus pulposus cells. Implications for the treatment of intervertebral disc degeneration. Spine 1998;23:2437–42. discussion 43. [13] Masuda K, Oegema TR Jr, An HS. Growth factors and treatment of intervertebral disc degeneration. Spine 2004;29:2757–69. [14] An HS, Takegami K, Kamada H, et al. Intradiscal administration of osteogenic protein-1 increases intervertebral disc height and proteoglycan content in the nucleus pulposus in normal adolescent rabbits. Spine 2005;30:25–31. [15] Sakai D, Mochida J, Iwashina T, et al. Differentiation of mesenchymal stem cells transplanted to a rabbit degenerative disc model: potential and limitations for stem cell therapy in disc regeneration. Spine 2005;30:2379–87. [16] Osada R, Ohshima H, Ishihara H, et al. Autocrine/paracrine mechanism of insulin-like growth factor-1 secretion, and the effect of insulin-like growth factor-1 on proteoglycan synthesis in bovine intervertebral discs. J Orthop Res 1996;14:690–9. [17] Okuda S, Myoui A, Ariga K, Nakase T, Yonenobu K, Yoshikawa H. Mechanisms of age-related decline in insulin-like growth factor-I dependent proteoglycan synthesis in rat intervertebral disc cells. Spine 2001;26:2421–6. [18] Gruber HE, Leslie K, Ingram J, Hoelscher G, Norton HJ, Hanley EN Jr. Colony formation and matrix production by human anulus cells: modulation in three-dimensional culture. Spine 2004;29: E267–74. [19] Tim Yoon S, Su Kim K, Li J, et al. The effect of bone morphogenetic protein-2 on rat intervertebral disc cells in vitro. Spine 2003;28: 1773–80.
K. Miyamoto et al. / The Spine Journal 6 (2006) 692–703 [20] Li J, Yoon ST, Hutton WC. Effect of bone morphogenetic protein-2 (BMP-2) on matrix production, other BMPs, and BMP receptors in rat intervertebral disc cells. J Spinal Disord Tech 2004;17:423–8. [21] Masuda K, Takegami K, An H, et al. Recombinant osteogenic protein-1 upregulates extracellular matrix metabolism by rabbit annulus fibrosus and nucleus pulposus cells cultured in alginate beads. J Orthop Res 2003;21:922–30. [22] Masuda K, Imai Y, Okuma M, et al. Osteogenic protein-1 (OP-1) injection into a degenerated disc induces the restoration of disc height and structural changes in the rabbit annular puncture model. Spine 2006;31:742–54. [23] Takegami K, An HS, Kumano F, et al. Osteogenic protein-1 is most effective in stimulating nucleus pulposus and annulus fibrosus cells to repair their matrix after chondroitinase ABC-induced in vitro chemonucleolysis. Spine J 2005;5:231–8. [24] Masuda K, Aota Y, Muehleman C, et al. A novel rabbit model of mild, reproducible disc degeneration by an anulus needle puncture: correlation between the degree of disc injury and radiological and histological appearances of disc degeneration. Spine 2005;30:5–14. [25] Bradford DS, Oegema TR Jr, Cooper KM, Wakano K, Chao EY. Chymopapain, chemonucleolysis, and nucleus pulposus regeneration: a biochemical and biomechanical study. Spine 1984;9:135–47. [26] Best BA, Guilak F, Setton LA, et al. Compressive mechanical properties of the human anulus fibrosus and their relationship to biochemical composition. Spine 1994;19:212–21. [27] Leahy JC, Hukins DW. Viscoelastic properties of the nucleus pulposus of the intervertebral disk in compression. J Mater Sci Mater Med 2001;12:689–92. [28] Sarver JJ, Elliott DM. Altered disc mechanics in mice genetically engineered for reduced type I collagen. Spine 2004;29:1094–8. [29] Elliott DM, Sarver JJ. Young investigator award winner: validation of the mouse and rat disc as mechanical models of the human lumbar disc. Spine 2004;29:713–22. [30] Johannessen W, Elliott DM. Effects of degeneration on the biphasic material properties of human nucleus pulposus in confined compression. Spine 2005;30:E724–9. [31] Iatridis JC, Weidenbaum M, Setton LA, Mow VC. Is the nucleus pulposus a solid or a fluid? Mechanical behaviors of the nucleus pulposus of the human intervertebral disc. Spine 1996;21:1174–84. [32] Iatridis JC, Setton LA, Weidenbaum M, Mow VC. Alterations in the mechanical behavior of the human lumbar nucleus pulposus with degeneration and aging. J Orthop Res 1997;15:318–22.
703
[33] Beek M, Aarnts MP, Koolstra JH, Feilzer AJ, van Eijden TM. Dynamic properties of the human temporomandibular joint disc. J Dent Res 2001;80:876–80. [34] Zhu W, Mow VC, Koob TJ, Eyre DR. Viscoelastic shear properties of articular cartilage and the effects of glycosidase treatments. J Orthop Res 1993;11:771–81. [35] Tanaka E, Aoyama J, Tanaka M, Murata H, Hamada T, Tanne K. Dynamic properties of bovine temporomandibular joint disks change with age. J Dent Res 2002;81:618–22. [36] Kim JG, An HS, Masuda K, Inoue N. Dynamic viscoelastic properties of rabbit lumbar discs. Trans Orthop Res Soc 2005;3:1280. [37] Lu DS, Shono Y, Oda I, Abumi K, Kaneda K. Effects of chondroitinase ABC and chymopapain on spinal motion segment biomechanics: an in vivo biomechanical, radiologic, and histologic canine study. Spine 1997;22:1828–34. [38] Ferry JD. The nature of viscoelastic behavior. In: Ferry JD, editor. Viscoelastic properties of polymers. 1st ed. New York: John Wiley & Sons, 1961:1–20. [39] Ferry JD. The nature of viscoelastic behavior. In: Ferry JD, editor. Viscoelastic properties of polymers. 3rd ed. New York: John Wiley & Sons, 1980:1–32. [40] Tanaka E, Aoyama J, Tanaka M, et al. The proteoglycan contents of the temporomandibular joint disc influence its dynamic viscoelastic properties. J Biomed Mater Res A 2003;65:386–92. [41] Chiba K, Andersson GBJ, Masuda K, Momohara S, Williams JM, Thonar EJ-MA. A new culture system to study the metabolism of the intervertebral disc in vitro. Spine 1998;23:1821–8. [42] Kim JG, Miyamoto K, Masuda K, An HS, Andersson G, Inoue N. Correlations among biomechanical, biochemical and structural properties of the degenerated intervertebral disc. Trans Orthop Res Soc 2006;31:49. [43] Mow VC, Kuei SC, Lai WM, Armstrong CG. Biphasic creep and stress relaxation of articular cartilage in compression? Theory and experiments. J Biomech Eng 1980;102:73–84. [44] Kim YJ, Bonassar LJ, Grodzinsky AJ. The role of cartilage streaming potential, fluid flow and pressure in the stimulation of chondrocyte biosynthesis during dynamic compression. J Biomech 1995;28: 1055–66. [45] Sobajima S, Kompel JF, Kim JS, et al. A slowly progressive and reproducible animal model of intervertebral disc degeneration characterized by MRI, X-ray, and histology. Spine 2005;30: 15–24.
Clinical Study
Intradiscal injections of osteogenic protein-1 restore the viscoelastic properties of degenerated intervertebral discs Kei Miyamoto, MD, PhDa, Koichi Masuda, MDa,b,*, Jesse G. Kim, PhDa,c, Nozomu Inoue, MD, PhDa, Koji Akeda, MD, PhDa, Gunnar B.J. Andersson, MD, PhDa, Howard S. An, MDa Departments of aOrthopedic Surgery and bBiochemistry, Rush University Medical Center, 1735 W. Harrison Street, Chicago, IL 60612 USA c Department of Bioengineering, University of Illinois at Chicago, 851 S. Morgan Street, Chicago, IL 60607–7052, USA Received 14 January 2006; accepted 17 April 2006
Abstract
BACKGROUND CONTEXT: Using biochemical, histological, and radiological parameters in a rabbit model of intervertebral disc (IVD) degeneration, the intradiscal injection of a growth factor, such as osteogenic protein-1 (OP-1), has been shown to regenerate the IVD. However, very little is known about how such a biological therapeutic approach affects the biomechanical properties of the degenerated IVD. PURPOSE: To investigate the effects of an intradiscal injection of OP-1 on the biomechanical properties of IVDs in the rabbit annular-puncture disc degeneration model and to determine their relationship to biochemical properties. STUDY DESIGN/SETTING: In vivo study on the effects of intradiscally administered OP-1 on the biomechanical and biochemical properties of IVDs in the rabbit annular-puncture disc degeneration model. METHODS: New Zealand White rabbits (n516) underwent annulus fibrosus (AF) puncture, using an 18-gauge needle, at L2–L3 and L4–L5 (L3–L4: nonpunctured control). Four weeks later, the punctured discs received an injection of either 5% lactose (10 mL) or OP-1 (100 mg/10 mL of 5% lactose) into the nucleus pulposus (NP). The disc height was radiographically monitored biweekly. After sacrifice and removal of bone–disc–bone complexes 8 weeks postinjection, the dynamic viscoelastic properties of the IVDs were tested by applying a cycle of sinusoidal strain in uniaxial compression at six loading frequencies (0.05 to 2 Hz). The biochemical properties of the dissected IVDs were then analyzed and correlated with the biomechanical properties. RESULTS: A single injection of OP-1 significantly restored disc height when compared with the lactose-injected discs (OP-1 vs. lactose, p!.001). The elastic modulus of the IVDs in the OP-1injected discs was significantly higher than that in the lactose-injected discs at all frequencies (mean: þ43%, p!.001). The viscous modulus in the OP-1-injected discs was significantly higher at 0.05, 0.2, 0.5, and 1 Hz (mean: þ55%, p!.001) and showed higher tendencies at other frequencies (p5.08–.09). For both moduli, no significant differences were observed between the OP-1-injected and the nonpunctured control discs. The OP-1 injection significantly increased the proteoglycan (PG) content in the NP and AF, and the collagen content in the NP (p!.001–.05). Both elastic and viscous moduli showed significant positive correlations with PG content in the NP and collagen content in the NP and AF (Rho5.357–.466, p5.010–.047). CONCLUSIONS: We have shown for the first time that an injection of the growth factor, OP-1, restored the biomechanical properties of IVDs in a rabbit model of IVD degeneration. Comparing biomechanical with biochemical data suggests that the OP-1-induced biomechanical restoration
FDA device/drug status: investigational/not approved (osteogenic protein-1). This study was supported by NIH grants P50-AR-39239 and P01-AR48152. 1529-9430/06/$ – see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.spinee.2006.04.014
This paper is dedicated to the memory of Dr. Jesse G. Kim, friend and colleague, who passed away during review of the manuscript. * Corresponding author. 1735 W. Harrison St., Cohn 720, Chicago, IL 60612. Tel.: (312) 942-4661; fax: (312) 942-8828. E-mail address: [email protected] (K. Masuda)
K. Miyamoto et al. / The Spine Journal 6 (2006) 692–703
693
was a consequence of increased activities of anabolic pathways that resulted in biochemical changes in the IVD. Ó 2006 Elsevier Inc. All rights reserved. Keywords:
Intervertebral disc; Degeneration; Growth factor; Osteogenic protein-1; Biomechanics; Viscoelastic properties; Proteoglycan; Collagen; Animal model
Introduction Low back pain is rated as second only to upper respiratory problems among symptom-related reasons for visits to a physician, the fifth ranking cause of admission to hospitals, and the third most common reason for surgical procedures in the United States [1]. Generally, it is accepted that intervertebral disc (IVD) degeneration is, at least in part, one of the causes of discogenic low back pain [1]. Although several reports have presented evidence of the association between disc degeneration and low back pain [2–5], much remains unknown and controversy still exists. The unique structure of an IVD, which has a gelatinous proteoglycan (PG)-rich nucleus pulposus (NP) surrounded by a collagen-rich annulus fibrosus (AF), confers viscoelastic properties that are characteristic of the IVD. These properties enable the IVD to withstand mechanical forces, which is its most important function. Both the PG content, with its negatively charged hydrophilic nature, and collagen, which forms concentric lamellae, are known to contribute to the viscoelastic properties of the IVD. Biochemically, degenerative changes in IVDs are characterized by loss of the extracellular matrix (ECM) including PG and collagen [6,7]. Therefore, once disc degeneration develops, viscoelastic properties decrease considerably [8,9] and the IVDs can become susceptible to stages of intersegmental instability [10,11]. The biological repair of degenerated IVDs, such as that achieved by protein injection, cell-based and gene-delivering therapies [12–15], has recently been considered to be a minimally invasive and long-lasting treatment modality. Among the approaches aimed at regaining lost function of the IVD through biochemical, structural, and biomechanical perspectives, protein injection therapy has been considered to be the simplest approach. Several in vitro studies have indicated that various growth factors, such as insulin-like growth factor-1 [16–18], bone morphogenetic protein (BMP)-2 [19,20], BMP-7 [14,21,22], and transforming growth factor-b [18], stimulate the ECM metabolism of IVD cells. Osteogenic protein-1 (OP-1, also designated as BMP-7) is known to stimulate ECM production by disc cells in vitro [21,23], and its injection into the NP of normal adolescent rabbits has been shown to increase disc height as well as PG content of the NP [14]. Additionally, a single injection of OP-1 induced a significant restoration of disc height and an improvement in the histological and biochemical parameters of degenerated IVDs in the rabbit annular-puncture disc degeneration model [22,24]. However, the efficacy of biological treatments for degenerated IVDs, including the injection of OP-1, on the biomechanical function of IVDs,
which is essentially the main concern, has not been elucidated. Because OP-1 has strong anabolic effects on ECM production and accumulation, both in vitro and in vivo, it is reasonable to hypothesize that the injection of OP-1 could affect the viscoelastic properties of the IVD as a consequence of changes in its biochemical properties. Historically, biomechanical properties of the IVD have been explored using various biomechanical testing methods [25–30]. Because disc tissues exhibit nonlinear viscoelastic behaviors [31,32], methods testing only linear viscoelastic behavior might be limiting. Dynamic viscoelastic testing, with the advantage of being able to demonstrate the nonlinear behavior of tissues and dependency on frequency of load [33], was performed on articular cartilage [34], NP tissue [31], and the temporomandibular joint [35]. More recently, to achieve a better understanding of the function of the IVD as a whole, dynamic viscoelastic testing on the rabbit IVD as a functional unit was introduced as a reliable and valid method [36]. The present study investigated the effects of an intradiscal injection of OP-1 on both biomechanical and biochemical properties, and the possible relationship between these two properties, using the rabbit annular-puncture disc degeneration model [24] and the newly developed dynamic viscoelastic testing system for rabbit IVDs [36]. Methods Establishment of a degenerative IVD in the rabbit annular-puncture disc degeneration model and the injection of OP-1 All experimental protocols were approved by the authors’ Institutional Animal Care and Use Committee (IACUC #04-027). Under general anesthesia and using a right posterolateral retroperitoneal approach, New Zealand White rabbits (n516; 3–3.5 kg, 5 months old) underwent AF puncture with an 18-gauge needle at both the L2–L3 and L4–L5 discs to induce disc degeneration [24]. In all rabbits, the L3–L4 disc served as a nonpunctured control. Four weeks later, using a left posterolateral retroperitoneal approach, punctured discs in each animal (L2–L3 or L4–L5) received an injection of either the vehicle (5% lactose, 10 mL per disc) or recombinant human OP1 (rhOP-1); a gift from Stryker Biotech, Hopkinton, MA, 100 mg in 10 mL 5% lactose per disc using a 26-gauge needle. The disc level to receive either the lactose or rhOP-1 injection was randomly assigned. During the follow-up period, the disc height was monitored radiographically as described below. Eight weeks postinjection, rabbits were
694
K. Miyamoto et al. / The Spine Journal 6 (2006) 692–703
euthanized as previously described, [22] and the lumbar spines were harvested. Bone–disc–bone complexes, with the superior and inferior vertebra bodies perpendicular to the axis of the spine, were removed from L2–L3, L3–L4, and L4–L5 levels using a cutoff saw (McMaster-Carr, Chicago, IL) with adjustable-width parallel blades (Fig. 1, upper left).
The data are reported as the IVD height expressed as the disc height index (DHI5IVD height/adjacent IVD body height) based on the method of Lu et al. [37] with a slight modification [14]. Changes in the DHI of injected discs are expressed as %DHI and normalized to the measured preoperative IVD height [%DHI5(postoperative DHI/preoperative DHI)100] [14]. Magnetic resonance imaging analyses
Radiographic analysis of disc height Radiographs were taken at 2-week intervals up to 12 weeks after the puncture. Extreme care was taken to maintain a consistent level of anesthesia (ketamine hydrochloride [25 mg/kg] and acepromazine maleate [1 mg/kg]) during radiography of each animal at each time point in order to obtain a similar degree of muscle relaxation, which may affect the disc height. The preoperative X-ray was used as a baseline measurement. All X-ray images were digitized and independently analyzed using a custom program for MATLAB software (Natick, MA) by an orthopedic researcher who was blinded to the treatment group.
Magnetic resonance imaging (MRI) analyses were performed in vitro on the spinal columns obtained from all rabbits using a 0.3-T imager (Airis II, version 4.0 A; Hitachi Medical System America, Inc.), with a quadrature extremity coil receiver as previously reported [24]. The MRIs were evaluated by a blinded observer using the modified Thompson classification [24] based on changes in the degree and area of signal intensity from grade 1 to 5 (15normal, 25minimal decrease of signal intensity but obvious narrowing of high signal area, 35moderate decrease of signal intensity, 45severe decrease of signal intensity but slight high signal area, 55no high signal area).
Fig. 1. Preparation of the specimen and measurement of the dynamic viscoelastic properties of rabbit intervertebral discs. The dynamic viscoelastic properties of rabbit intervertebral discs were measured using a custom-made testing system that consists of a linear actuator with optical encoder, a load cell (EL series, Entran), a custom-made loading frame, 316L stainless steel porous pucks (100 mm pore size), and a water chamber. A sinusoidal strain in uniaxial unconfined compression was applied.
K. Miyamoto et al. / The Spine Journal 6 (2006) 692–703
IVD preparation for dynamic viscoelastic testing After the anterior aspects of the L2–L3, L3–L4, and L4– L5 discs were exposed, each bone–disc–bone complex was obtained by cutting through the superior and inferior vertebral bodies perpendicular to a longitudinal axis of the spine using a modified compact cutoff saw (McMaster-Carr, Chicago, IL) with adjustable-width parallel blades (Fig. 1, upper left). Then, the posterior element was removed and the bone–disc–bone complex was obtained. The superior and inferior cut surfaces of the complex were rasped in order to align them parallel and to make both end plates thin enough (~2 mm) to maintain the integrity of the whole disc as a functional unit (Fig. 1, upper left). In samples with prominent osteophyte formation, the osteophyte was removed such that it would not cause impingement during biomechanical testing.
695
directions to assess in vitro disc height and cross-sectional area, respectively, using an X-ray machine (Faxitron, Faxitron X-ray Corporation, Wheeling, IL). The contact radiographs were scanned and disc height was measured as the area of the disc space divided by the mediolateral length from the anteroposterior view using image analysis software (Scion software, Scion Corporation, Frederick, MD). The cross-sectional area was measured as the area of the bony end plate surface from the craniocaudal view [36]. In addition, the cross-sectional area of the newly formed osteophyte was measured in each specimen before removal of osteophytes. The relative size of the osteophyte to the cross-sectional area of the bony end plate surface (%disc area) was compared between the lactose-injected and the OP-1-injected discs that previously underwent annular puncture. Analyses for dynamic viscoelastic properties
Radiographic measurement of IVD dimensions Contact radiographs of the bone–disc–bone complexes were taken in anteroposterior and craniocaudal (Fig. 2)
The dynamic viscoelastic properties of IVDs were measured by a custom-made dynamic viscoelastic biomechanical testing system [36]. This system consists of a linear
Fig. 2. Contact radiographs of bone–disc–bone complexes used in craniocaudal directions. Examples of an in vitro craniocaudal X-ray of the bone–disc– bone complex from lactose-injected and osteogenic protein-1 (OP-1)-injected discs are shown. In both lactose-injected and osteogenic protein-1-injected punctured discs, osteophyte formation was consistently observed at the right anterolateral side where the initial puncture using an 18-gauge needle was performed (upper row pictures, small arrows). Comparing the lactose-injected and the recombinant human osteogenic protein-1-injected discs, no significant differences were observed in the cross-sectional area of the osteophytes. Therefore, the osteophyte was removed such that it would not cause impingement during biomechanical testing.
696
K. Miyamoto et al. / The Spine Journal 6 (2006) 692–703
Fig. 3. (A) A sinusoidal compressive strain applied to the disc (e) and the out-of-phase resultant stress (s) with a phase angle (d). (B) The complex modulus (E*), the storage modulus (E0 ), and the loss modulus (E00 ), obtained by vectorial separations.
actuator with optical encoder (Digit series, UltraMotion, Mattituck, NY), a load cell (EL series, Entran, Hampton, VA), a custom-made loading frame, and a water chamber (Fig. 1). Each disc was placed on a 316L stainless steel porous puck (100 mm pore size; Mott Corporation; Farmington, CT) inside a water chamber that was filled with physiological saline (Fig. 1). The loading shaft with another porous puck was slowly loaded onto the disc (0.005 mm/s) until the contact criteria of 5 N compression (13% of the rabbit’s body weight) was reached to maintain contact with the disc during the unloading cycle of the test. Then, preconditioning was performed by applying 10 sinusoidal strain cycles with an amplitude of 10% at 1 Hz to achieve repeatable testing results. After a 3-minute recovery from preconditioning, six different physiologically relevant loading frequency tests (0.05, 0.1, 0.2, 0.5, 1, and 2 Hz) were performed with the same amplitude. Each frequency test was performed using one cycle of sinusoidal strain
followed by a 3-minute recovery. The loading frequencies and the strain amplitude were chosen to simulate dynamic in vivo physiological conditions. A dynamic compression was applied by a sinusoidal strain of 3(t)53oþ3a cos (ut) with an initial strain of 3o510%, oscillation amplitude of 3a510%, an angular frequency u (52pf) in radians/s, frequency f in cycle/s (Hz), and time t in seconds. Because of the viscoelastic properties, the resultant stress s(t)5soþsa cos (utd) oscillates sinusoidally with a phase angle of d (Fig. 3A). An elastic modulus (E0 ), a viscous modulus (E00 ), and a ratio of viscous-to-elastic moduli (tan d) were determined as the dynamic viscoelastic parameters of the IVD. The elastic modulus represents elastic behavior with its ability to store deformational energy, and the viscous modulus represents viscous behavior with the dissipation (loss) of energy during deformation in a viscoelastic material [35]. When the viscoelastic material is subjected to a sinusoidal strain, the resultant stress is out-of-phase with a time-lag
K. Miyamoto et al. / The Spine Journal 6 (2006) 692–703
697
Fig. 5. Magnetic resonance imaging (MRI) grading within each treatment group and a presentation of a sample MRI (mean6standard deviation). At 8 weeks postinjection, the MRI of the nucleus pulposus in the osteogenic protein (OP)-1-injected discs showed a slightly, but significantly, stronger T2 signal intensity than that of the lactose-injected discs (p!.05, MannWhitney test; graph on the left). This difference is visible on the sample MRI (image on the right).
Fig. 4. Lateral radiogram of a rabbit lumbar spine before (preoperative) and after (4 weeks) an annular-puncture with an 18-gauge needle and 8 weeks postinjection (12 weeks after puncture) of either lactose or osteogenic protein-1 (OP-1). Changes in the intervertebral disc height index (DHI) after the annular-puncture and OP-1 injections (mean6standard error). The lateral radiographs show that the disc space, which showed narrowing 4 weeks after the annular needle puncture, is restored in the OP-1injected level, whereas the narrowing progressed in the lactose-injected level. The graph shows that an annular puncture with an 18-gauge needle decreased the disc height index (DHI) by approximately 32% when compared with the baseline %DHI values before the annular puncture, p!.0001). Then, the intradiscal injection of OP-1 significantly affected the disc height in the postinjection time course (p!.0001). When the DHI at each time point was assessed, at 4 weeks postinjection, the %DHI of the OP-1-injected discs showed an increase compared with the lactoseinjected discs with statistical significance (p!.001).
called the phase angle d of between 0 (purely elastic) and 90 (purely viscous) [38]. The complex modulus E* is vectorially separated into an elastic modulus E0 and a viscous 00 0 00 modulus pffiffiffiffiffiffiffi E in the complex plane by E*5E þiE , where i5 1 [38] (Fig. 3B). The magnitude of the complex modulus |E*| is determined by |E*|5s/e, where stress s5F (force)/A (cross-sectional area), strain e5DDH (change in disc height)/DH (disc height) [39,40]. The elastic modulus E0 , viscous modulus E00 , and a ratio of viscousto-elastic moduli tan d are determined by E0 5|E*| cos d, E00 5|E*| sin d, and tan d5E00 / E0 , respectively. In order to determine the biomechanical effects of the intradiscal injection of rhOP-1, the elastic and viscous
moduli and the ratio of viscous-to-elastic moduli were compared among the three IVD groups (nonpunctured control discs, OP-1-injected punctured discs, lactose-injected punctured discs). Additionally, a correlation between the elastic and viscous moduli was assessed. Biochemical analyses After completion of the biomechanical testing, the gelatinous NP tissue was sharply dissected out from each disc. A white fibrous tissue found in the punctured disc, which was observed in the area corresponding to the anatomical location of the normal NP, was also collected as NP. The remaining fibrous AF tissue was removed from the end plates and adjacent vertebral bodies. Wet and dry weights were obtained for all specimens before digestion with papain at 60 C for 48 hours [41]. Contents of deoxyribonucleic acid (DNA), PG, and collagen were analyzed as previously described [41]. To minimize the size differences in the IVDs found among the levels within a single animal and among the individual animals, all biochemical parameters were standardized by the cross-sectional area of the IVD (mm2), as determined by radiographs which had been digitized and measured. Data and statistical analyses The significance of differences among means was analyzed by two-way repeated measurement analysis of variance (ANOVA) or one-way ANOVA and Fisher’s
698
K. Miyamoto et al. / The Spine Journal 6 (2006) 692–703
protected least significant difference test as a post hoc test on data obtained from X-ray measurements, biomechanical parameters, and biochemical parameters. All data are expressed as the mean6standard deviation (SD) or the mean6standard error (SE). The Kruskal-Wallis test and Mann-Whitney U test were used to analyze nonparametric data (MRI grading) for the effect of treatment.
Correlations between biochemical properties and dynamic viscoelastic parameters and between biochemical and biomechanical properties were examined using the Spearman signed rank test. The elastic modulus and the viscous modulus at typical physiological loading frequency of 0.5 Hz (1 s loading and 1 s unloading) were correlated with the PG and collagen contents of NP and AF tissues, standardized by the cross-sectional area. All statistical analyses were performed using Statview, Version 5.0 (Windows version XP, Abacus Concepts, Berkeley, CA). A level of p!.05 was used to determine statistical significance.
Results Radiographic analysis of the in vivo disc height In both lactose and rhOP-1-injected discs, which had received an identical puncture with an 18-gauge needle, the %DHI at the 4-week time point after the AF puncture showed a similar decrease (lactose vs. OP-1, not significant [NS]) of approximately 32% (compared with the baseline %DHI values before the AF puncture; p!.0001, Fig. 4), whereas the %DHI of the control level (L3–L4) remained constant. Repeated ANOVA showed that the intradiscal injection of rhOP-1 significantly affected the disc height in the postinjection time course (p!.0001). Although no significant difference among the groups was observed at the 2-week postinjection time point, at 4 weeks postinjection, the %DHI of the rhOP-1-injected discs showed an increase compared with the lactose-injected discs with statistical significance (4 weeks, p!.001, 6 weeks, 8 weeks, p!.0001) (Fig. 4). At 8 weeks postinjection, the disc height of the OP-1-injected discs achieved approximately 88% of the DHI in the nonpunctured controls, which was not significantly different (OP-1 vs. nonpunctured control, NS)
= Fig. 6. Effects of osteogenic protein-1 (OP-1) on the elastic modulus (E0 ), the viscous modulus (E00 ) and the loss tangent (E00 /E0 ) of rabbit intervertebral discs after annular puncture (mean6standard error). In the OP-1injected discs (closed circles), at all loading frequencies the elastic modulus (E0 ) was significantly higher than that in the lactose-injected discs (triangles, mean: þ43%, p!.05) and approached that of the nonpunctured control discs (open circle) (OP-1 vs. nonpunctured control, not significant, upper graph). The viscous modulus was significantly higher in the OP-1injected discs (closed circle) than that in the lactose-injected discs (reverse triangle) at loading frequencies of 0.05, 0.2, 0.5, and 1 Hz (average: þ55%, p!.001), while showing a strong tendency to be higher at 0.1 (p5.077), and 2 Hz (p5.093) (middle graph). In both the elastic and the viscous moduli, no significant difference was observed between the OP1-injected discs and nonpunctured discs at all loading frequencies (upper and middle graphs). No significant difference was observed in the loss tangent among the nonpunctured control and the recombinant human osteogenic protein-1-injected and lactose-injected discs.
K. Miyamoto et al. / The Spine Journal 6 (2006) 692–703
699
intensity, with an average grade of 3.1, than that of the lactose-injected discs, with an average grade 3.6 (p!.05, Mann-Whitney test, Fig. 5). Gross observation of the retrieved discs It is important to note that there was a significant formation of osteophytes in both lactose-injected and OP-1-injected punctured discs; these were located mostly in the right anterolateral surface of the IVDs where the puncture was performed. When the cross-sectional areas of the osteophytes were compared between the lactose- and the OP-1-injected discs, no significant differences were observed (OP-1: 17.9610.1% of the disc area; lactose: 19.6612.5% of the disc area). Because these osteophytes might affect the biomechanical properties of the disc by bridging the vertebral bodies, all biomechanical analyses were performed with and without removing osteophytes to confirm the accuracy of assessment. Because the biomechanical results of a disc with or without osteophytes showed essentially the same trend, the results after removal of osteophytes are reported in the following section. Biomechanical properties
Fig. 7. Effects of the treatment with osteogenic protein-1 (OP-1) or lactose on wet weight, DNA, proteoglycan (PG) and collagen contents of the nucleus pulposus (NP) and annulus fibrosus (AF) of rabbit intervertebral discs (mean6standard deviation). In the NP, as can be observed in data from the lactose-injected discs, the annular needle puncture induced dramatic decreases in wet weight, DNA content, and PG content, whereas the collagen content was increased (left panel). In the AF, the annular needle puncture did not cause significant changes in any of the four parameters (right panel). The intradiscal injection of OP-1 significantly increased the wet weight and contents of DNA, PG, and collagen in the NP, when compared with the lactose-injected discs. OP-1 also increased the wet weight and PG content of the AF.
(Fig. 4). On the other hand, in the lactose-injected discs, the disc height loss was sustained at a level of approximately 69% of the DHI in the nonpunctured control for up to 8 weeks postinjection (Fig. 4).
MRI analyses At 8 weeks postinjection, the MRI of the NP in the OP-1-injected discs showed a slightly stronger T2 signal
The elastic modulus (E0 ) A two-way ANOVA demonstrated that the treatment of the IVD (punctured vs. nonpunctured; OP-1 injection vs. lactose injection) affected the elastic modulus significantly (p!.0001), whereas the loading frequency had no significant effect. The elastic modulus (E0 ) in the lactose-injected discs (triangle) was significantly lower than that in the nonpunctured control discs (open circle) at all loading frequencies (mean: 64% of the control, p!.01) (Fig. 6, upper graph). On the other hand, at all loading frequencies, the elastic modulus in the OP-1-injected discs (closed circle) was significantly higher than that in the lactose-injected discs (mean: þ43%, p!.05) and approached that of the nonpunctured control levels (OP-1 vs. nonpunctured control, NS) (Fig. 6, upper graph). The viscous modulus (E00 ) Two-way ANOVA demonstrated that both the treatment of the IVD (punctured vs. nonpunctured; OP-1 injection vs. lactose injection) and loading frequency affected the viscous modulus significantly (p!.0001), with no interactive effects. The viscous modulus had a trend to be low in the higher frequencies (Fig. 6, middle graph). When the effect of the treatment was assessed, the viscous modulus E00 ) in the lactose-injected discs (triangle) was significantly lower than that in the nonpunctured control discs (open circle) only at 0.05, and 0.2 Hz (mean: 65% of the control, p!.05; Fig. 6, middle graph). The viscous modulus in the OP-1injected discs (closed circle) was significantly higher than that in the lactose-injected discs at 0.05, 0.2, 0.5, and 1 Hz (average: þ55%, p!.001) and with a strong tendency
700
K. Miyamoto et al. / The Spine Journal 6 (2006) 692–703
Fig. 8. Correlations between the biomechanical properties at a typical physiological loading frequency of 0.5 Hz and the biochemical properties of rabbit intervertebral discs from lactose-injected and osteogenic protein-1 (OP-1)-injected rabbit intervertebral discs using the Spearman signed rank test. The elastic modulus (E0 ) was shown to have a significant positive correlation with the PG and collagen contents in the NP and the collagen content in the AF tissues. Similarly, the viscous modulus was also shown to have a significant correlation with the PG and collagen content in the NP and the collagen content in the AF.
to be higher at 0.1 (p5.077), and 2 Hz (p5.093) (Fig. 6, middle graph). In the viscous modulus, no significant differences were observed between the OP-1-injected discs and nonpunctured control discs at any loading frequencies. The ratio of viscous-to-elastic moduli (E00 / E0 ; loss tangent) A two-way ANOVA demonstrated that the loading frequency had a significant effect on the loss tangent (p!.0001) with smaller values at higher frequencies, whereas the treatment of the IVD (punctured vs. nonpunctured; OP-1 injection vs. lactose injection) had no significant effect (Fig. 6, lower graph).
In the NP, the intradiscal injection of OP-1 resulted in significant increases in wet weight (þ57%, p!.0001), DNA content (þ48%, p!.0001), PG content (þ83%, p!.0001), and collagen content (þ76%, p!.001), compared with the lactose-injected discs (Fig. 7, NP: left column). In the AF, significant increases in wet weight (þ20%, p!.01) and PG content (þ35%, p!.05) were also observed (Fig. 7, AF: right column). It is important to note that, after the intradiscal injection of OP-1, the PG content in the AF and the collagen content of the NP were significantly higher than those in the nonpunctured control IVDs (PG in the AF: þ28%, p!.05; collagen in the NP: þ234%, p!.001).
Biochemical properties In the NP, when comparing data between the nonpunctured control discs and the lactose-injected discs, the needle puncture induced dramatic decreases in wet weight (47%), DNA content (49%), and PG content (68%), whereas the collagen content was increased by the puncture (þ90%) (Fig. 7, NP: left column). In the AF, when comparing data between the nonpunctured control discs and the lactose-injected discs, the needle puncture did not produce significant changes in any of the four parameters (Fig. 7, AF: right column).
Correlations between biomechanical and biochemical properties using the Spearman rank correlation test When discs from all treatment groups were evaluated together, the elastic modulus (E0 ) was shown to have a significant positive correlation with the PG content in the NP (p5.014) and the PG and collagen content in the AF (PG: p5.01, collagen: p5.019) (Fig. 8, left panels). Similarly, the viscous modulus was shown to have a significant positive correlation with the PG content in the NP (p5.020)
K. Miyamoto et al. / The Spine Journal 6 (2006) 692–703
and the PG and collagen content in the AF (p5.007) (PG: p5.028, collagen: p5.047) (Fig. 8, right panels).
Discussion The results of this study demonstrated that a single injection of OP-1 into the NP significantly ameliorated the dynamic viscoelastic properties of the degenerated disc in the rabbit annular-puncture disc degeneration model. The biochemical analysis of repaired tissues revealed that the OP-1 injection induced an increase in wet weight, DNA content, PG content, and collagen content of the NP, compared with the lactose-injected discs, whereas an increase was observed only in the wet weight and PG content of the AF. As has been primarily hypothesized, an increase in content of ECM components of the IVD tissues positively correlated with viscous and elastic moduli of the IVD, suggesting that biochemical changes induced by an injection of OP-1 may have resulted in the biomechanical restoration of the IVD. This is the first report providing evidence that a biological approach for disc repair is effective in restoring the biomechanical properties of degenerated IVDs. The biomechanical testing method used in this study enabled the measurement of the dynamic viscoelastic properties of the IVD over a broad range of loading frequencies (0.05–2 Hz), encompassing those occurring physiologically [36]. The analyses of the viscous and elastic response components of compressive stiffness have allowed detection of subtle variations in the biomechanical properties of IVDs from different levels (eg, L1–L2, L3–L4, L5–L6) [36]. Our previous study showed that annular puncture into the NP with an 18-gauge needle resulted, 4 weeks subsequently, in a marked decrease in both the elastic and viscous properties of the rabbit IVD [42]. Because our previous attempt, using static compression, failed to detect biomechanical changes in the degenerated disc in the rabbit stab model (data not shown), the method reported here, which tests dynamic viscoelastic properties, has proven useful to detect changes in biomechanical properties in a therapeutic modality. At the 12-week postpuncture time point analyzed in the current study, viscous and elastic moduli (Fig. 6) were similar to the corresponding values at 4 weeks postpuncture [42]; this suggests that the punctured and lactose-injected IVD does not have the capability of being restored in the short period used in this study, as seen radiographically (Fig. 4). The biomechanical analyses also provide insight into the therapeutic effects of the intradiscal administration of OP1. Such an injection, using a 26-gauge needle, restored those once lost viscoelastic properties by 8 weeks postinjection to levels that did not differ significantly from the nonpunctured control disc levels (Fig. 6). This indicates a functional biomechanical healing of the IVD damage during the 8-week time period. Interestingly, the loss tangent
701
(E00 /E0 ) was highest at the lowest frequency (0.05 Hz) tested in the present study, indicating that the viscous behavior of the rabbit IVD is most prominent at the lower frequency and that the frequency-dependence of compressive behavior in the radially unconfined configuration reflects decreased fluid exudation at the higher frequencies [43,44]. Similar loss tangents found among the nonpunctured control discs and both the OP-1-injected and lactose-injected punctured discs in this study may reflect an injury-associated decrease in the IVD compressive modulus and an increase in hydraulic permeability that are counterbalanced by a decrease in hydration with, thus, no overall effect on the characteristic response frequency. Also, similar loss tangents found between punctured OP-1-treated discs and lactose-injected discs provide further evidence for a full functional recovery associated with OP-1 injection. PGs and collagen both contribute to the mechanical properties of the NP and AF. PGs endow disc tissues with a fixed negative charge, which results in hydration and swelling of the tissue and the consequent ability to resist compressive loading [7]. The collagen network, which imparts tensile strength to the disc, entraps PG molecules in the tissues. In this study, a significant increase in wet weight and PG content was observed in both NP and AF tissues of the OP-1-injected discs, compared with the lactoseinjected discs, whereas an increase in collagen content was observed only in the NP. These results suggested that an increased PG content, induced by the injection of OP-1, resulted in tissue hydration in both the NP and AF. However, the possibility cannot be excluded that changes in collagen content contribute to changes in biomechanical properties. Although the biomechanical tests performed on the ‘‘whole disc’’ bone–disc–bone complex made correlation analyses difficult, several biochemical properties of the IVD, including the contents of two key ECM components, PGs and collagen, have been shown to correlate significantly with biomechanical parameters. This correlation supports our contention that stimulation of ECM metabolism by the in vivo application of growth factors is beneficial to the repair of degenerated IVDs [13]. The positive correlation between the collagen content of the AF with both the elastic and the viscous moduli especially suggests that a treatment which aims to increase the collagen content of the IVD might be one possible therapeutic approach to disc degeneration. The IVD samples harvested from punctured discs in this study at the 8-week postinjection time point were frequently associated with osteophytes, as has previously been reported [22,45]. In most punctured disc specimens, osteophytes were located on the right side where the initial annular puncture was made; this suggests that the etiology of the development of the osteophytes was due to extruded discs induced by the needle puncture. Our quantitative comparison of the cross-sectional area of osteophytes between the lactose-injected and OP-1-injected discs did not show any
702
K. Miyamoto et al. / The Spine Journal 6 (2006) 692–703
statistical differences. Because of concerns about the influence of the presence of osteophytes on the biomechanical properties of the IVDs caused by partial bridging between two vertebral bodies, the biomechanical test measurements were repeated before and after removal of osteophytes. The results showed that measurements after the removal of osteophytes provided results essentially similar to those before osteophyte removal, but with less variation (data not shown). There are several limitations to this study. First, adolescent rabbits (5 months old) still have notochordal cells present in their IVDs. However, adolescent rabbits were used in the belief that this is an acceptable model in which to start preliminary in vivo experiments. The use of mature animals or larger animals, which have a cellular phenotype closer to that of human IVD cells, might provide more critical information on the efficacy of OP-1 injections into the degenerated IVD. Second, this experiment to determine if there was an initial recovery of biomechanical properties after a single injection of OP-1 protein was done to test the feasibility of achieving a biological effect by growth factors on the biomechanical properties of a degenerated disc. In future research, longer follow-up data are needed to prove the long-term efficacy of this therapy. Third, this study did not address the biodistribution of rhOP-1 that was injected into the center of the NP. Because the presence of endogenous OP-1 made an experimental design to address this question difficult without the use of radiolabeled rhOP-1, a decision was made to elucidate the half-life of injected rhOP-1 in a separate experiment that is currently under way. The results of this biodistribution study may provide us with the frequency of injections needed for effective clinical treatment. Fourth, the biomechanical tests used in this study were performed on a bone–disc–bone complex. To reveal the involvement of the AF and NP tissues in the repair process, further biomechanical studies on individual tissues may be required. Finally, the biomechanical testing was performed applying uniaxial compression only. Because the human spine has multidirectional flexibility, supported by the mechanical properties of the IVDs, several variations in the biomechanical testing on in vivo rabbit IVDs need to be performed in the future. In conclusion, the findings of the current study provide preliminary evidence that an injection of OP-1 restored biomechanical properties of degenerated discs in the rabbit annular-puncture disc degeneration model. The biomechanical and biochemical data in this study suggested that OP-1induced biochemical changes by anabolic stimulation may have resulted in the biomechanical restoration of the IVD. Although further efficacy studies with larger animals and studies using pain as a primary endpoint for a therapeutic approach for disc degeneration are needed, the preliminary evidence in this rabbit study established that an injection of growth factor may be clinically applicable as a therapeutic approach to repair the degenerated IVD.
Acknowledgments The authors would like to acknowledge Stryker Biotech for providing the osteogenic protein-1. References [1] Andersson GB. Epidemiological features of chronic low-back pain. Lancet 1999;354:581–5. [2] Aprill C, Bogduk N. High-intensity zone: a diagnostic sign of painful lumbar disc on magnetic resonance imaging. Br J Radiol 1992;65: 361–9. [3] Freemont AJ, Peacock TE, Goupille P, Hoyland JA, O’Brien J, Jayson MI. Nerve ingrowth into diseased intervertebral disc in chronic back pain. Lancet 1997;350:178–81. [4] O’Neill CW, Kurgansky ME, Derby R, Ryan DP. Disc stimulation and patterns of referred pain. Spine 2002;27:2776–81. [5] Pye SR, Reid DM, Smith R, et al. Radiographic features of lumbar disc degeneration and self-reported back pain. J Rheumatol 2004; 31:753–8. [6] Antoniou J, Steffen T, Nelson F, et al. The human lumbar intervertebral disc: evidence for changes in the biosynthesis and denaturation of the extracellular matrix with growth, maturation, ageing, and degeneration. J Clin Invest 1996;98:996–1003. [7] Urban JP, Roberts S. Degeneration of the intervertebral disc. Arthritis Res Ther 2003;5:120–30. [8] Urban JP, McMullin JF. Swelling pressure of the lumbar intervertebral discs: influence of age, spinal level, composition, and degeneration. Spine 1988;13:179–87. [9] Adams MA, McNally DS, Dolan P. ’Stress’ distributions inside intervertebral discs: the effects of age and degeneration. J Bone Joint Surg Br 1996;78:965–72. [10] Fujiwara A, Lim TH, An HS, et al. The effect of disc degeneration and facet joint osteoarthritis on the segmental flexibility of the lumbar spine. Spine 2000;25:3036–44. [11] Zhao F, Pollintine P, Hole BD, Dolan P, Adams MA. Discogenic origins of spinal instability. Spine 2005;30:2621–30. [12] Nishida K, Kang JD, Suh JK, Robbins PD, Evans CH, Gilbertson LG. Adenovirus-mediated gene transfer to nucleus pulposus cells. Implications for the treatment of intervertebral disc degeneration. Spine 1998;23:2437–42. discussion 43. [13] Masuda K, Oegema TR Jr, An HS. Growth factors and treatment of intervertebral disc degeneration. Spine 2004;29:2757–69. [14] An HS, Takegami K, Kamada H, et al. Intradiscal administration of osteogenic protein-1 increases intervertebral disc height and proteoglycan content in the nucleus pulposus in normal adolescent rabbits. Spine 2005;30:25–31. [15] Sakai D, Mochida J, Iwashina T, et al. Differentiation of mesenchymal stem cells transplanted to a rabbit degenerative disc model: potential and limitations for stem cell therapy in disc regeneration. Spine 2005;30:2379–87. [16] Osada R, Ohshima H, Ishihara H, et al. Autocrine/paracrine mechanism of insulin-like growth factor-1 secretion, and the effect of insulin-like growth factor-1 on proteoglycan synthesis in bovine intervertebral discs. J Orthop Res 1996;14:690–9. [17] Okuda S, Myoui A, Ariga K, Nakase T, Yonenobu K, Yoshikawa H. Mechanisms of age-related decline in insulin-like growth factor-I dependent proteoglycan synthesis in rat intervertebral disc cells. Spine 2001;26:2421–6. [18] Gruber HE, Leslie K, Ingram J, Hoelscher G, Norton HJ, Hanley EN Jr. Colony formation and matrix production by human anulus cells: modulation in three-dimensional culture. Spine 2004;29: E267–74. [19] Tim Yoon S, Su Kim K, Li J, et al. The effect of bone morphogenetic protein-2 on rat intervertebral disc cells in vitro. Spine 2003;28: 1773–80.
K. Miyamoto et al. / The Spine Journal 6 (2006) 692–703 [20] Li J, Yoon ST, Hutton WC. Effect of bone morphogenetic protein-2 (BMP-2) on matrix production, other BMPs, and BMP receptors in rat intervertebral disc cells. J Spinal Disord Tech 2004;17:423–8. [21] Masuda K, Takegami K, An H, et al. Recombinant osteogenic protein-1 upregulates extracellular matrix metabolism by rabbit annulus fibrosus and nucleus pulposus cells cultured in alginate beads. J Orthop Res 2003;21:922–30. [22] Masuda K, Imai Y, Okuma M, et al. Osteogenic protein-1 (OP-1) injection into a degenerated disc induces the restoration of disc height and structural changes in the rabbit annular puncture model. Spine 2006;31:742–54. [23] Takegami K, An HS, Kumano F, et al. Osteogenic protein-1 is most effective in stimulating nucleus pulposus and annulus fibrosus cells to repair their matrix after chondroitinase ABC-induced in vitro chemonucleolysis. Spine J 2005;5:231–8. [24] Masuda K, Aota Y, Muehleman C, et al. A novel rabbit model of mild, reproducible disc degeneration by an anulus needle puncture: correlation between the degree of disc injury and radiological and histological appearances of disc degeneration. Spine 2005;30:5–14. [25] Bradford DS, Oegema TR Jr, Cooper KM, Wakano K, Chao EY. Chymopapain, chemonucleolysis, and nucleus pulposus regeneration: a biochemical and biomechanical study. Spine 1984;9:135–47. [26] Best BA, Guilak F, Setton LA, et al. Compressive mechanical properties of the human anulus fibrosus and their relationship to biochemical composition. Spine 1994;19:212–21. [27] Leahy JC, Hukins DW. Viscoelastic properties of the nucleus pulposus of the intervertebral disk in compression. J Mater Sci Mater Med 2001;12:689–92. [28] Sarver JJ, Elliott DM. Altered disc mechanics in mice genetically engineered for reduced type I collagen. Spine 2004;29:1094–8. [29] Elliott DM, Sarver JJ. Young investigator award winner: validation of the mouse and rat disc as mechanical models of the human lumbar disc. Spine 2004;29:713–22. [30] Johannessen W, Elliott DM. Effects of degeneration on the biphasic material properties of human nucleus pulposus in confined compression. Spine 2005;30:E724–9. [31] Iatridis JC, Weidenbaum M, Setton LA, Mow VC. Is the nucleus pulposus a solid or a fluid? Mechanical behaviors of the nucleus pulposus of the human intervertebral disc. Spine 1996;21:1174–84. [32] Iatridis JC, Setton LA, Weidenbaum M, Mow VC. Alterations in the mechanical behavior of the human lumbar nucleus pulposus with degeneration and aging. J Orthop Res 1997;15:318–22.
703
[33] Beek M, Aarnts MP, Koolstra JH, Feilzer AJ, van Eijden TM. Dynamic properties of the human temporomandibular joint disc. J Dent Res 2001;80:876–80. [34] Zhu W, Mow VC, Koob TJ, Eyre DR. Viscoelastic shear properties of articular cartilage and the effects of glycosidase treatments. J Orthop Res 1993;11:771–81. [35] Tanaka E, Aoyama J, Tanaka M, Murata H, Hamada T, Tanne K. Dynamic properties of bovine temporomandibular joint disks change with age. J Dent Res 2002;81:618–22. [36] Kim JG, An HS, Masuda K, Inoue N. Dynamic viscoelastic properties of rabbit lumbar discs. Trans Orthop Res Soc 2005;3:1280. [37] Lu DS, Shono Y, Oda I, Abumi K, Kaneda K. Effects of chondroitinase ABC and chymopapain on spinal motion segment biomechanics: an in vivo biomechanical, radiologic, and histologic canine study. Spine 1997;22:1828–34. [38] Ferry JD. The nature of viscoelastic behavior. In: Ferry JD, editor. Viscoelastic properties of polymers. 1st ed. New York: John Wiley & Sons, 1961:1–20. [39] Ferry JD. The nature of viscoelastic behavior. In: Ferry JD, editor. Viscoelastic properties of polymers. 3rd ed. New York: John Wiley & Sons, 1980:1–32. [40] Tanaka E, Aoyama J, Tanaka M, et al. The proteoglycan contents of the temporomandibular joint disc influence its dynamic viscoelastic properties. J Biomed Mater Res A 2003;65:386–92. [41] Chiba K, Andersson GBJ, Masuda K, Momohara S, Williams JM, Thonar EJ-MA. A new culture system to study the metabolism of the intervertebral disc in vitro. Spine 1998;23:1821–8. [42] Kim JG, Miyamoto K, Masuda K, An HS, Andersson G, Inoue N. Correlations among biomechanical, biochemical and structural properties of the degenerated intervertebral disc. Trans Orthop Res Soc 2006;31:49. [43] Mow VC, Kuei SC, Lai WM, Armstrong CG. Biphasic creep and stress relaxation of articular cartilage in compression? Theory and experiments. J Biomech Eng 1980;102:73–84. [44] Kim YJ, Bonassar LJ, Grodzinsky AJ. The role of cartilage streaming potential, fluid flow and pressure in the stimulation of chondrocyte biosynthesis during dynamic compression. J Biomech 1995;28: 1055–66. [45] Sobajima S, Kompel JF, Kim JS, et al. A slowly progressive and reproducible animal model of intervertebral disc degeneration characterized by MRI, X-ray, and histology. Spine 2005;30: 15–24.