INTRADISCAL INJECTION OF OSTEOGENIC PROTEIN-1 CAN RESTORE
VISCOELASTIC PROPERTIES OF DEGENERATED INTERVERTEBRAL DISCS
*Miyamoto, K; +*,**Masuda, K; #Kim, JG; *Inoue, N; *Akeda, K; *Andersson, GBJ; *An, HS
Depts. of +*Orthopedic Surgery and **Biochemistry, Rush University Medical Center, 1735 W. Harrison, Cohn 720, Chicago, IL 60612.
TEL: 312-942-4661, Fax: 312-942-8828, [email protected]
RESULTS: Disc Height: Annulus puncture induced a 32% decrease in
disc height four weeks after puncture (Fig. 1). At 4-weeks post-injection,
OP-1 injections significantly restored disc height; this was sustained for
up to eight weeks after the injection (OP-1 vs Lactose, p<0.001, Fig. 1).
Biomechanical Properties: The elastic modulus (K’) in the lactose group
(triangle) was significantly lower than that in the control group (closed
%DHI
100
80
Injection
*
**
**
OP-1 vs Lactose
* p<0.01, ** p<0.001
L3/4 non-punctured
OP-1 (100 µg)
60
5% Lactose (10 µl)
0
-4
-2
AF Puncture
0
2
4
6
8
Weeks after Injection
Fig. 1 Effects of OP-1 on disc height (mean ± S.E.)
Control
OP-1
10
8
*
6
##
4
0
*
*
##
##
##
*
*
*
##
##
Lactose
0.05 0.1
0.2
0.5
1
2
Loading frequency (Hz)
Viscous Modulus (MPa)
Elastic Modulus (MPa)
OP-1 vs Lactose: * p<0.05, Lactose vs Control : # p<0.05, ## p<0.01
Control
OP-1
Lactose
1.2
1.0
0.8
*
*
*
0.6
#
0.4
#
0.2
0.0
0.05 0.1
0.2
0.5
1
2
Loading frequency (Hz)
Fig. 2 Effects of OP-1 on elastic and viscous moduli (mean ± S.D.)
* p<0.05,**
** p<0.01,
p<0.001, ***
*** p<0.001
p<0.001
* p<0.05,
NP
AF
***
**
*
**
Fig. 3 Effect of OP-1 on biochemical
properties of rabbit IVDs.
Elastic Modulus (MPa)
Total PG Content (µg/disc)
circle) at all loading frequencies (mean: 55%, p<0.001). At all loading
frequencies, K’ in the OP-1 group was significantly higher than that in
the lactose group (mean: 41%, p<0.001) and approached that of the
control level (OP-1 vs Control, n.s.) (Fig. 2, left).
The viscous modulus (K”) in the OP-1 group was significantly higher
than that in the lactose group at 0.05, 0.2, and 1 Hz (average: 56%,
p<0.001) while showing a strong tendency to be higher at 0.1, 0.5, and 2
Hz (p = 0.06-0.10) (Fig. 2, right). In both the elastic and viscous
moduli, no significant difference was observed between the OP-1 and
control groups at all loading frequencies.
Biochemical Properties: The DNA contents of both the NP and AF in
the OP-1 group were slightly but significantly higher than those in the
lactose group (p<0.001). The PG contents of both the NP and AF in the
OP-1 group were significantly higher than that the lactose group (Fig.
3). The elastic modulus (K’) showed a positive correlation with total PG
content in the NP (p<0.05) (Fig. 4).
Co
nt
La
c
O
P1
Co
nt
La
c
O
P1
INTRODUCTION: Changes in the biomechanical properties of
degenerated intervertebral discs (IVDs) are associated with biochemical
changes in the extracellular matrix (ECM) of the IVD, such as loss of
proteoglycan (PG) and collagen, which lead to subsequent instability
and inflammation. Recently, as a minimally invasive therapeutic
approach for degenerated IVDs, biological repair using growth factors
has gained interest.
Osteogenic protein-1 (OP-1) is known for its efficacy in stimulating
ECM production and restoring disc height in the rabbit disc degeneration
model [1,2]. However, whether these effects of OP-1 result in restoration
of the biomechanical properties of IVDs has not been explored. The
present study was performed to investigate the effects of an intradiscal
injection of OP-1 on both biomechanical and biochemical properties
using the rabbit IVD annular puncture model [3] and a newly developed
dynamic viscoelastic property testing system [4] to reveal any
relationship between these two properties.
METHODS: Operations: Fourteen New Zealand White rabbits (3-3.5
kg) underwent annulus fibrosus (AF) puncture (18G) at both the L2/3
and L4/5 discs to induce disc degeneration [3]. The L3/4 discs served as
non-punctured controls. Four weeks later, both punctured discs in each
animal received the same injection of either 5% lactose (10 µl) or OP-1
(100 µg/10 µl) into the nucleus pulposus (NP). Eight weeks after the
injection, lumbar spines were harvested after sacrifice and bone-discbone complexes were removed.
Dynamic Viscoelastic Properties: When sinusoidal strain is applied to
viscoelastic materials, the resultant stress will be out-of-phase with a
time-lag called phase angle δ. A complex modulus K* has two
components: the elastic modulus K’ and the viscous modulus K”. K* =
K’ + iK”, where i = √-1. [5,6]. The magnitude is determined by |K*| =
σ(stress)/ε(strain). σ = ∆F(resultant force)/A(cross sectional area). ε =
∆L(length change)/DH(disc height). Finally, the properties are defined
as K’ = |K*| cos δ, and K” = |K*| sin δ [5].
Testing Protocol: Using a custom-made biomechanical testing system
[4], a cycle of sinusoidal strain, ε(t)=−εO+εA cos(ωt) was applied to each
IVD in an uniaxial unconfined compression. Strain amplitude of,
εO=εA=10% disc height was applied. The average resultant force of this
study was 95 N, which represents 249% of the rabbit’s body weight.
After pre-conditioning by 10 cycles at 1 Hz, six different physiological
loading frequency tests (0.05, 0.1, 0.2, 0.5, 1, and 2 Hz) were performed.
Radiological Analysis: Disc height was radiographically monitored
biweekly. Disc height was expressed as the disc height index [%DHI =
(Post-operative DHI/Pre–operative DHI)x100)] [3].
Biochemical Analyses: After the biomechanical testing, the NP and AF
were separated. Specimens were analyzed for DNA content using the
Hoechst 33258 dye method, PG content by the DMMB method [1].
Correlations between PG content and dynamic viscoelastic parameters
(K’ and K”) were examined using the Spearman’s signed rank test.
Statistical Analysis: Repeated ANOVA or one-way ANOVA with Fisher
PSLD test was used.
Rho=0.40
(P<0.05)
Total PG Content in NP (µg/disc)
Fig. 4 Relationship between biochemical
and biomechanical properties
DISCUSSION: We have shown for the first time that an injection of a
growth factor, OP-1, successfully restored the disc height and
biomechanical properties of the IVD in the rabbit IVD annular puncture
model. The results of the positive correlation between PG content in the
NP and the elastic modulus of the IVD suggest that biochemical changes
induced by an injection of OP-1 may result in structural and mechanical
restoration. Our data support the feasibility that a single injection of OP1 may serve as a therapeutic solution for a degenerative IVD to
regenerate into “a functioning shock absorber” in well-selected
indications. However, because the biomechanical function of human
IVDs includes multidirectional flexibility and complex motion, further
biomechanical testing of biologically-treated IVDs should be performed
in larger animals that more closely resemble the human IVD structure.
REFERENCES: [1] Masuda K+, J Orthop Res 21:922, 2003. [2] An
HS+, Spine 30:31, 2005. [3] Masuda K+, Spine 30:5, 2005. [4] Kim
J;ORS trans 2005;1280. [5] Ferry J, Viscoelastic Properties of Poly, NY,
Wiley, 1961. [6] Tanaka E; J Biomed Mater Res 65A:386, 2003.
ACKNOWLEDGMENTS: NIH grants P01-AR48152 and P50AR39329.
#
University of Illinois at Chicago, Chicago, IL.
52nd Annual Meeting of the Orthopaedic Research Society
Paper No: 0397