Effects of exogenous crosslinking on in vitro tensile and compressive moduli of lumbar intervertebral discs

1. Introduction 

Mechanisms for load transfer within the intervertebral disc are inherently complex. For instance, fibers in the annulus fibrosus (AF) can sustain tensile stresses generated by hydrostatic pressure in the nucleus, while at the same time the annulus is carrying a substantial amount of compressive stress (Adams et al., 2002). The structure and composition of the AF intervertebral disc (IVD) is equally intricate with an inner region (capsular AF) and an outer region (ligamentous AF). The dense outer AF is highly organized, composed of numerous (15–20) concentric rings of fibrocartilageous tissue that inserts in the bony vertebra, while the inner AF is less organized and attaches to the cartilaginous endplate. During the process of degeneration, there is loss of the annular–nuclear demarcation as the inner annulus becomes more mucinous, infiltrated with nucleus-like material, while the nucleus displays changes in collagen and proteoglycan content. These complex and transforming structural properties make the intervertebral disc, and in particular the annulus difficult to measure and model. Because of these complexities several different methods have been employed to investigate the mechanical properties of the annulus. Stokes (1987) used cadaveric, physical, and mathematical models to demonstrate surface strains of the intervertebral disc. Other biomechanical studies have contributed to increased understanding of annular mechanics and the fidelity of analytical models (Holzapfel et al., 2005, Iatridis et al., 2005, Iatridis et al., 1998). In this biomechanical study the effect of crosslinking treatment on annulus mechanical tissues was determined using tensile and unconfined compressive loading of four types of tissue samples. The annular stresses included in this present analysis include: circumferential tension (CT), radial compression (RC), axial tension (AT), and axial compression (AC).

Collagen crosslinks play a major role in providing the mechanical strength for load-supporting tissues, with human intervertebral disc’s having a higher concentration of mature crosslinks than other tissues (Eyre et al., 1988, Pokharna and Phillips, 1998). This signals demanding mechanical requirements. Duance (Duance et al., 1998) found significantly higher quantities of reducible crosslinks on the convex sides rather than on the concave sides of human scoliotic discs. This suggests that a natural, cell-mediated crosslink augmentation mechanism exists in response to the elevated tensile environment on the convex side of scoliotic discs. Crosslinks have recently been described as providing “sacrificial bonds” that protect tissue and dissipate energy (Thompson et al., 2001).

Together these studies suggest that an increase in crosslinks may be advantageous for maintaining tissue integrity. However, natural crosslink augmentation, in the form of age-increasing glycation endproduct crosslinks, has frequently been implicated as a prominent contributor to load-supporting soft-tissue degradation. Age-increasing crosslinks have been described as degenerative factors in many tissues including arteries, lenses, skin, tendons and articular cartilage (Franke et al., 2003, Stein et al., 2003, Chen et al., 2002, Monnier et al., 1986, Wolffenbuttel et al., 1998, Bailey et al., 1993, Verzijl et al., 2002, Bank et al., 1998). Several of these authors have postulated that crosslinks may be responsible for brittleness and reduced fatigue resistance in load supporting tissues. The role of age-increasing crosslinks is not simple or fully understood. Endogenous non-enzymatic crosslinks increase with age in collagenous tissues, while collagen content declines with age, suggesting that even highly crosslinked collagen can be biodegraded. Though unable to prevent the loss of collagen, excessive crosslinking may prevent the shedding of degraded matrix molecules that are of no benefit to the matrix. A recent study by Degroot et al. (2004) demonstrated the capacity for non-enzymatic crosslinking to adversely affect repair of an iatrogenically transected anterior cruciate ligament. Another consideration, severely degenerated intervertebral discs have been shown to have lower quantities of age-related crosslinks (Duance et al., 1998). Their absence in more degenerated discs may again signal their importance in maintaining tissue integrity. An aim of this study is to investigate whether collagen crosslink augmentation may provide mechanical advantages towards preventing the ongoing process of degenerative disc disease.

The focus of this study was to determine effects of a single organic crosslinking agent on AF mechanical properties. As a first investigation of its type, methods of reagent application and contrasting effects of different types of crosslinking reagents were beyond the scope of this investigation.

2. Methods 

Twenty-four intervertebral discs from six 4-month-old bovine lumbar spines were included in the study. Gross inspection of the bovine intervertebral discs confirmed the cross-sectional area proportions of the nucleus, inner annulus and outer annulus were similar to those of human intervertebral discs, while disc height was less than half that of human discs. Four separate types of annulus fibrosus specimens were tested: CT (12), RC (12), AT (10), and AC (14). Specimens pairs were taken from symmetrical right and left portions of the disc.

2.1. Mechanical testing 

Simple tensile and unconfined compression tests matching specimen geometries were designed and appropriate fixtures were constructed for these experiments. The AT and AC test specimens consisted of (superior) bone – disc (outer AF) – bone (inferior) potted in polyurethane. The nominal cross-section of the specimens was selected on the basis of ensuring inclusion of more than two annular layers and inclusion of fibers spanning the intervertebral space with vertebral attachments on both ends. During the dissection and removal of the soft tissue around the disc, care was taken to keep the disc wrapped with a moist paper to prevent dehydration due to evaporation. In addition, irrigation of the specimen with a saline spray was performed at regular intervals to maintain a hydrated state. The annulus portion of the AT specimens was trimmed into a dumb-bell shape with circular cross-section. While the necked-down cross-section still included two or more annular layers, the existence of individual fibers that spanned the intervertebral space from endplate to endplate was not verified.

CT test specimen pairs were taken bilaterally (Fig. 1) from the annulus fibrosus with the region to be loaded coming from the anterolateral portion of outer annulus fibrosus including several annular layers. The specimens were then clamped at both ends with custom made metal clamps. For the RC tests, the specimens were taken from the same anterolateral portion of outer annulus fibrosus with a rectangular arc shape, again being careful to include multiple layers of the outer annulus (Fig. 2). A custom-made contoured compression testing apparatus was constructed (Fig. 2). The outer radius of the compressing cylinder was 17.5mm and the inner radius of the cylindrical depression was 22.5mm functioning to seat the specimens. The design fitted the shape of specimens so that the specimen was tested in compression with its original physiological orientation.

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Fig. 1. Location and shape of circumferential tensile test specimens.

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Fig. 2. A custom apparatus was constructed to apply uniform compressive strain to a curved specimen that followed the natural curvature of the posterolateral disc annulus. The specimen is shown here between the outer radius of the compressing cylinder and the inner radius of the cylindrical depression. The outer radius of the compressing cylinder was 17.5mm and the inner radius of the cylindrical depression was 22.5mm. These radii were computed from measurements of specimen curvatures in situ. The location and shape of radial compressive test specimens is showed at left upper corner in the figure.

Genipin, a gardenia fruit extract, thought to link amino acid groups in intramolecular, intermolecular and intermicrofibrillar bonds (Sung et al., 1993), was used as the crosslinker in this study. Each pair of specimens was separated into a control group that was soaked in a phosphate buffered solution (PBS) for 2 days under room temperature before the test was preformed, and a crosslinked group in which the specimens were soaked in a 0.33% genipin solution (98% pure, Challenge Bioproducts Co., Taichung, Taiwan) under the same conditions and duration as controls.

To ensure accurate cross-sectional area measurements, the external contour of the testing specimens, except for the RC specimen group, were measured by a custom-made non-contact measurement machine which contained a rotating laser displacement sensor (LK-081, Keyence, NJ, USA) and a fixed specimen platform. The rate of data acquisition was 20Hz with 800 data points for a 360° complete circle. The minimum cross-sectional area calculation was realized using custom-designed software based on the Matlab (Mathworks, Natik, MA, USA) programming environment. The sizes of specimens in the RC group were measured by an electric digital caliper (Mitutoyo; sensitivity: 0.01mm).

All tests were performed by a servo-hydraulic MTS 858 materials testing system. The AT and CT specimens were loaded to failure at a rate of 1mm/s after 10 cycles of preconditioning (5mm maximal displacement at 1mm/s). The axial and radial compressive specimens were loaded at 1mm/s to 75% of deformation after 10 cycles of preconditioning (0.5mm maximal displacement at 1mm/s).

3. Results 

3.2. Comparison between control and crosslinked specimens 

Table 1 shows a comparison between control and crosslinked specimens. The AC low stiffness modulus was 126% higher after crosslinking, P=0.031 (Fig. 3). The axial compressive high stiffness modulus and the radial compressive high and low stiffness moduli were not statistically different with crosslink augmentation (Fig. 3, Fig. 4).

Table 1. A comparison of mechanical properties between control and crosslinked specimens

		Control group	Crosslinked group	Remarks: increase from crosslinking (Crosslinked−control)/control (%)
	AC
	Low stiffness modulus	3.43MPa (2.83)	8.62MPa (2.44)	151.3
	High stiffness modulus	8.99MPa (2.45)	10.3MPa (6.52)	14.6
	RC
	Low stiffness modulus	1.23MPa (1.25)	1.38MPa (1.15)	12.2
	High stiffness modulus	10.2MPa (6.83)	17.1MPa (9.21)	67.6
	CT
	YS	6.75MPa (1.21)	12.0MPa (4.73)	77.8
	UTS	6.96MPa (1.04)	12.4MPa (4.85)	78.2
	Low stiffness modulus	19.7MPa (4.32)	24.1MPa (8.28)	22.3
	High stiffness modulus	35.6MPa (5.87)	56.8MPa (21.2)	59.6
	Toughness	0.83 MJ/m3 (0.30)	1.52 MJ/m3 (0.71)	83.1
	AT
	YS	1.26MPa (0.66)	1.82MPa (0.71)	44.8
	UTS	1.50MPa (0.81)	2.03MPa (0.71)	35.3
	Low stiffness modulus	1.83MPa (2.12)	2.17MPa (1.91)	18.6
	High stiffness modulus	5.75MPa (4.01)	5.42MPa (3.08)	−5.7
	Toughness	0.49 MJ/m3 (0.32)	0.86 MJ/m3 (0.41)	75.5

The value in the table is the mean value of the test. In the parentheses is the standard deviation. AC: axial compression, RC: radial compression, CT: circumferential tension, AT: axial tension, YS: yield stress, UTS: ultimate tensile stress, MPa: megapascal, MJ/m3: megajoules/cubic meters.

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Fig. 3. The comparison of low stiffness modulus (at 25% strain: deformation=25% of original length) between control and crosslinked groups (AT: axial tensile modulus, AC: axial compressive modulus, RC: radial compressive modulus, CT: circumferential tensile modulus).

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Fig. 4. The comparison of high stiffness (linear elastic region) modulus between control and genipin crosslinked groups (AT: axial tensile modulus, AC: axial compressive modulus, RC: radial compressive modulus, CT: circumferential tensile modulus).

The mean circumferential yield stress demonstrated a 78.0% (P=0.024) increase after crosslinking (Fig. 5). The ultimate tensile strength had a 78.7% increase in crosslinked groups compared to the controls, P=0.033. The circumferential low stiffness modulus was not statistically different after the crosslinking treatment, while the high stiffness modulus was increased 59.3% after a crosslinking treatment, P=0.045 (Fig. 4). The toughness was 83.1% increased after a crosslinking treatment (P=0.05).

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Fig. 5. The comparison of circumferential tensile mechanical properties between control and genipin crosslinked groups [UTS: ultimate tensile strength, the unit of yield stress and UTS is megapascal (MPa), unit of toughness is megajoules/cubic meters (MJ/m3)].

The AT yield stress was 44.8% higher with the crosslinking treatment (P=0.008) (Fig. 6). The ultimate tensile strength was 20.7% higher with crosslinking (P=0.036), while toughness was not significantly increased with the crosslink treatment (P=0.170). The AT low stiffness and high stiffness moduli were not statistically affected by the crosslinking treatment (P=0.843) (Fig. 3, Fig. 4).

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Fig. 6. The comparison of axial tensile (AT) mechanical properties between control and genipin crosslinked groups [UTS: ultimate tensile strength, the unit of yield stress and UTS is megapascal (MPa), unit of toughness is megajoules/cubic meters (MJ/m3)].

4. Discussion 

This study involved the measurement of material property changes in the annulus fibrosus due to a genipin mediated increase in collagen crosslinking. The inherent complexity of form and function of the annular tissue necessitated a multi-directional analysis. The annulus fibrosus is inhomogeneous both in structure (Acaroglu et al., 1995, Iatridis et al., 1998) and composition (Best et al., 1994). Significant radial and circumferential variations in tensile properties in a single annular layer have been documented, with the anterior being stiffer than the posterolateral regions, and the outer being stiffer than the inner regions (Skaggs et al., 1994). The mechanical properties of annulus fibrosus depend not only on fiber and matrix but also fiber–matrix interactions (Adams and Green, 1993). Although it is important to understand the intrinsic mechanical properties of a single layer of annulus fibrosus, the mechanical behavior of annulus fibrosus in vivo depends on its composite structure (Ebara et al., 1996). This was the impetus for measuring material properties of multiple-layer annulus fibrosus specimens in this study.

In control specimen tissues, circumferential moduli, in both low and high stiffness regions, were higher than in other principal directions. If the principle of form following function holds, this would suggest that circumferential tension is the predominant type of stress experienced by non-degenerated discs. This may also indicate that circumferential tension in the annulus plays an important role in mechanical load transfer from the nucleus pulposus.

After a crosslinking treatment, the modulus increased more in CT as compared to AT. Also in the compression group specimens, the low stiffness modulus in the axial direction had a greater increase after crosslink augmentation than in the radial direction. The other relevant result was that the tensile properties (0.5% yield stress, ultimate tensile strength, and toughness) had higher increases due to crosslinking in the circumferential direction than in the axial direction. These results were compatible with the results in the control groups’ comparison which demonstrated that the discs had higher mechanical performance in CT than AT. Since the cellular response of intervertebral disc is closely related to its loading condition (Handa et al., 1997, Matsumoto et al., 1999, Ohshima et al., 1995), the above differences may be influenced by cellular modification of collagen tissue throughout the complex structure of the disc according to its corresponding loading environment. So the disc annulus may sustain higher mechanical stress in CT than in AT, and the disc may sustain higher stress in AC than RC.

AT 0.5% yield stress, ultimate tensile strength, and toughness all increased after crosslinking. These results were compatible with the results of parallel tests (Hedman et al., 2003) which investigated the durability/fatigue resistance of intervertebral discs after exogenous crosslink augmentation. CT yield stress, ultimate tensile strength, and toughness all increased after crosslinking.

In previous studies of multiple layer circumferential annulus fibrosus specimens, the tensile failure stress was found to be 1–3MPa (megapascal) at a strain of 10–25% and a strain rate of 0.01%/s (Ebara et al., 1996, Acaroglu et al., 1995). For comparison, the normal circumferential ultimate tensile strength of these bovine specimens was at least twice as high, 6.96MPa on average, and this circumferential strength increased 78% with crosslink augmentation. Concerning site variation, multiple layer annulus fibrosus specimens have been shown to be stronger in the outer than in the inner regions, and also stronger in the anterior than the posterior regions (Acaroglu et al., 1995). In the direction of lamella collagen fibers, the failure stress in human specimens can be up to 10MPa (Galante, 1967). It is assumed that differences between human and bovine disc composition, orientation and structure account for the differences in mechanical properties. It follows then that intrinsic differences between species may influence the degree to which crosslink augmentation affects mechanical properties. There are also differences based on age, the bovine specimens having been harvested from young animals, and degenerative disc disease most prevalent in the forth through sixth decade of life in humans. It should be noted, however, that one possible application for crosslink augmentation would be to prevent curve progression in adolescent scoliosis. Over all, the similarities in size, structure and composition, relative to other potential experimental models, combined with the minimal variation of these properties relative to human discs, and the difficulties and costs inherent in procuring sufficient numbers of human cadaveric specimens, make the calf intervertebral disc a preferred model for analyses of this type. A number of previous biomechanical experiments in our laboratory, utilizing both bovine and human discs, suggest to us that while there are differences, the general effects observed and quantified in bovine discs in this study would carry over to human tissues.

The necking-down of the axial tensile specimens, while necessary to ensure mid-tissue focused failure of the specimen, may have eliminated the existence of individual fibers spanning the intervertebral space from insertion site to insertion site. This may have reduced the tensile stiffness and strength and heightened the proportional effect of crosslink augmentation on these properties.

Another possible limitation of this study could be the presence of unmeasured electrochemical differences induced by crosslinking. Long-term soaking of disc tissues is known to lead to gross swelling and eventual loss of proteoglycans. These effects could potentially differ between the control and cross-linked specimens, subsequently producing an effect on mechanical properties. Previous hydration studies had demonstrated that these 2 day soakings did not result in statistically significant changes in disc hydration levels, with gross changes of less than 2%, and with non-significant average differences between the two treatment types of approximately 1% in the inner annulus and 0% in the outer annulus.

As mentioned above, age-increasing crosslinks have been frequently implicated as deleterious elements of the aging process. It has been suggested that age increasing crosslinks cause tissue brittleness and loss of fatigue resistance in load supporting tissues (Bank et al., 1998, Duance et al., 1998, Bailey et al., 1993, Chen et al., 2002, Pokharna and Phillips, 1998). However, in each of these instances, direct evidence to support this hypothesis was absent. To the contrary, biomechanical data has been presented which suggests that crosslink augmentation may in fact increase fatigue resistance, and modestly stiffen tissue without a loss of joint range of motion (Hedman et al., 2003).

The anisotropic mechanical properties of the annulus fibrosus are non-uniformly affected by crosslink augmentation. The dominant effects were in the directions with the greater inherent stiffnesses. These directions likely correspond with the orientation of principal physiological stresses in the tissue. These results suggest some beneficial effects of crosslink augmentation on the mechanical properties of the annulus fibrosus: increase in strength, yield stress, toughness, and increased modulus in the principal stress directions. Together with joint stabilizing effects and improvements in tissue durability of previous studies, these results suggest that crosslink augmentation should be further explored as a possible non-invasive injection therapeutic to resist mechanical disc degeneration.

This sort of treatment should not be confused with injection of sclerosing agents (prolotherapy) in biologically sufficient, vascularized connective tissues (Dagenais et al., 2005). The goal of prolotherapy is understood to be the creation of an acute inflammatory response in biologically active and nutritionally sufficient ligamentous tissue. The inflammatory response triggers neo-vascularization and the subsequent creation of new connective tissue. The treatment under investigation in this study, crosslink augmentation, while possibly eliciting a mild inflammatory response, is directed towards structural modification of the extracellular matrix of the largely avascularized and nutritionally insufficient intervertebral disc, not triggering, or for that matter, requiring a biological response.

5. Conclusions 

Assuming function influences form, circumferential tension would be the predominant type of stress experienced by non-degenerated discs according to this multi-directional study of annular mechanical properties. The anisotropic mechanical properties of the annulus fibrosus are non-uniformly affected by crosslink augmentation with the dominant effects occurring in the directions with the greater inherent stiffnesses. These results suggest some beneficial effects of crosslink augmentation on the mechanical properties of the annulus fibrosus: increase in strength, yield stress, toughness, and increased modulus in the principal stress directions. Together with joint stabilizing effects, improvements in tissue durability, and increased fluid flow to the nucleus as demonstrated by previous studies, crosslink augmentation should be further explored as a possible therapeutic to resist disc degeneration.