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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Spine (Phila Pa 1976). Author manuscript; available in PMC Nov 25, 2008.
Published in final edited form as:
PMCID: PMC2587060
NIHMSID: NIHMS71375

Needle Puncture Injury Affects Intervertebral Disc Mechanics and Biology in an Organ Culture Model

Abstract

Study Design

A bovine intervertebral disc organ culture model was used to study the effect of needle puncture injury on short-term disc mechanics and biology.

Objective

To test the hypothesis that significant changes in intervertebral disc structure, mechanics, and cellular response would be present within 1 week of needle puncture injury with a large-gauge needle but not with a small-gauge needle.

Summary of Background Data

Defects in anulus fibrosus induced by needle puncture injury can compromise mechanical integrity of the disc and lead to degeneration in animal models. The immediate and short-term mechanical and biologic response to anulus injury through needle puncture in a large animal model is not known.

Methods

Bovine caudal intervertebral discs were harvested, punctured posterolaterally using 25G and 14G needles, and placed in organ culture for 6 days. Discs underwent a daily dynamic compression loading protocol for 5 days from 0.2 to 1 MPa at 1 Hz for 1 hour. Disc structure and function were assessed with measurements of dynamic modulus, creep, height loss, water content, proteoglycan loss to the culture medium, cell viability, and histology.

Results

Needle puncture injury caused a rapid decrease in dynamic modulus and increase in creep during 1-hour loading, although no changes were detected in water content, disc height, or proteoglycan lost to the media. Cell viability was maintained except for localized cell death at the needle insertion site. An increase in cell number and possible remodeling response was seen in the insertion site in the nucleus pulposus.

Conclusion

Relatively minor disruption in the disc from needle puncture injury had immediate and progressive mechanical and biologic consequences with important implications for the use of discography, and repair-regeneration techniques. Results also suggest diagnostic techniques sensitive to mechanical changes in the disc may be important for early detection of degenerative changes in response to anulus injury.

Keywords: spine, intervertebral disc, organ culture, needle puncture, bovine, discography, mechanics, dynamic compression loading

Low back pain is a common and costly affliction leading to around 19 million physician visits and approximately $20 billion in costs in the United States per year.1 The causes of low back pain are multifactorial and complex, yet disc degeneration is often a contributor, particularly in its early unstable stage.2,3

Current and future procedures for intervertebral disc (IVD) diagnosis, repair, and regeneration often require needle injection to the nucleus pulposus (NP) through the anulus fibrosus (AF). For example, discography, which requires injection of a radio opaque dye into the NP, has a best-case positive predictive value of 50% to 60%, and results in potential AF damage through needle puncture.4 Intradiscal electrothermal treatment also requires puncture of the AF and additional anular disruption using a catheter.5 Future treatments including growth factor therapy,6 tissue engineering,7 and gene and cell therapy8,9 may also require puncture of the AF using a needle.

There is evidence in animal models that defects in the AF structure, such as those induced by needle insertion, can compromise disc and motion segment mechanical integrity,10-13 and lead to mild and moderate degeneration over time.13-17 It is generally believed that needle puncture injury with small-gauge needles is not expected to cause damage, whereas needle puncture injury with large-gauge needles leads to degenerative changes. AF needle puncture injury to the disc in rabbits using needles of different gauges has been demonstrated to result in slow progressive degeneration as measured using magnetic resonance imaging (MRI), radiograph, histology, and polymerase chain reaction.6,15,16,18 Differences in scaling and in biology between small and large animal models leave unanswered questions regarding extrapolation of needle puncture injury studies to the human condition. The creation of peripheral AF tears in sheep and pig models also demonstrated evidence of degeneration when evaluated longitudinally for morphologic, biochemical, and biomechanical changes.19-22 These studies focused on the medium to long-term effects of disc injury on degeneration, and there is very limited information on the immediate and short-term mechanical and biologic response to anulus injury through needle puncture in a large animal model.

The purpose of this study was to test the hypothesis that significant changes in disc structure, mechanics, and cellular response would be present within 1 week after needle puncture injury with a large-gauge needle but not with a small-gauge needle. To study the effect of the needle puncture injury under reproducible conditions in a large animal model, we used a bovine caudal IVD organ culture model. This ex vivo system allows precise control over the mechanical and chemical boundaries of the disc, the ability to obtain mechanical parameters over time for the same disc, and the ability to study these interactions in a large animal system where the effects of tissue disruption may be evaluated in the absence of substantial inflammatory response as found in vivo.

Materials and Methods

Bovine tails were obtained from a local abattoir within 4 hours postmortem and randomly assigned to an unpunctured control group (N = 10), and 1 of 2 needle puncture groups (small = 25G syringe, N = 11; large = 14G syringe, N = 12). Musculature surrounding the IVD was removed. Caudal discs were punctured using a posterolateral approach through the AF taking care to only puncture as far as the NP. Discs were removed from vertebral endplates, and initial disc heights, diameters, and wet weights were measured before culturing. Specimens were then placed in an organ culture chamber and incubated in standard culture conditions at 37°C and 5% CO2 under a 0.2 MPa static load as previously described.23 Media consisting of Dulbecco's Modified Eagle's medium (DMEM) (4.5 g/L glucose, 110 mg/L sodium pyruvate, with L-glutamine), supplemented with 10% fetal bovine serum, 100 U/mL of penicillin/streptomycin, 0.1 mg/mL gentamicin, 0.75 mg/L fungizone, 0.02 M HEPES buffer, and 50 μg/mL ascorbic acid, was continuously circulated through the chamber (1.1 mL/min) and changed every 2 days.

The loading protocol for all IVDs consisted of 4 conditions: Baseline, Test 1, Dynamic Loading, and Test 2 (Figure 1). IVDs were initially loaded under a baseline static load of 0.2 MPa for 12 hours.23 Chambers were then individually attached to an incubator-housed loading device for 3 cyclic tests lasting slightly more than 1 hour: Test 1 consisted of a 1-minute test (0.2–0.4 MPa, 1 Hz) that was sinusoidally applied to obtain a preloading dynamic nominal modulus; Dynamic Loading consisted of 1 hour of sinusoidal loading from 0.2–1.0 MPa at 1 Hz; and Test 2 consisted of a repeat of the 1-minute test to obtain a postloading nominal dynamic modulus. Creep during 1 hour of dynamic loading was calculated from displacements that were recorded from the loading device at points corresponding to 0.2 MPa load for the first and last cycles of the 1-hour dynamic loading test, and the initial height at the first cycle was compared between days to compare height lost over the culture duration. Dynamic stiffnesses were calculated using custom written MATLAB code (The MathWorks, Natick, MA), and for ease of comparison across animals, all stiffness measurements were normalized by initial IVD cross-sectional area and presented as a “nominal modulus.” After the 3 test cycles, the baseline 0.2 MPa static load was again applied to each chamber, and at least 12 hours of recovery was allowed between dynamic load cycles. Each chamber experienced loading once per day, adding to 5 total times during the 6-day culture period.

Figure 1
Timeline for mechanical intervention protocol. Daily loading consisted of Test 1, Dynamic Loading, and Test 2. A baseline static load (0.2 MPa) was applied for the remaining culture duration. The intervention protocol was repeated each day for 5 days ...

Glycosaminoglycan (GAG) content released to the media was assayed using the dimethylmethylene blue assay24 using DMEM (4.5 g/L glucose, 110 mg/L sodium pyruvate, with L-glutamine) and chondroitin-4 sulfate to create a standard curve. Media aliquots were collected before every loading experiment and frozen at −20°C before analysis. Regional water contents for the outer and inner anulus (OA, IA), and NP were determined for each group by comparing the wet weights and dry weights (after lyophilization) of tissue samples isolated from the disc.

Tissue samples were isolated both along the needle track and on the opposite side of the disc from the insertion site to assess cell viability both local to the needle track and in the overall tissue (Figure 2). Samples were immersed in TBSS (Tyrode's Buffered Saline Solution) with 1 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT thiazole blue, Sigma Aldrich, St. Louis, MO) for live cell staining and 1 μmol/L ethidium homodimer-1 (Molecular Probes, Eugene, OR) for dead cell staining and allowed to incubate for 2 hours. Excess dye was removed by placing the tissue samples in phosphate buffered saline on a shaker for 10 minutes and samples were frozen at −80°C. Frozen tissue was sectioned using a cryotome into 10-μm thick slices either perpendicular or parallel to the needle insertion track to obtain radial or sagittal sections (Figure 2) for evaluation of the disc and needle insertion path. Images of each section were obtained at 20× under fluorescent (ethidium) and brightfield (MTT) lighting conditions. This technique was shown to be effective in assessing cell viability in all regions of the bovine IVD.23

Figure 2
Sectioning orientations for histology and viability images. Tissue was oriented in 2 different planes to capture localized response to needle puncture injury (radial section) or a more global tissue response (sagittal section). Radial and sagittal sections ...

Tissue samples encompassing the needle track were fixed in formalin for 7 to 10 days, embedded in paraffin, and stained with alcian blue (proteoglycans), picosirius red (collagen), and Weigert's hematoxylin (cell nucleus) for histologic appearance.25 Tissue was again sectioned either perpendicular or parallel to the needle track for radial and sagittal evaluation of the disc and needle insertion path.

For all quantitative variables, analysis of variance with Bonferroni-adjusted post hoc comparisons was performed using P < 0.05 significance level. All values are reported as averages ± SEM.

Results

The nominal dynamic modulus was significantly affected by needle puncture injury (P = 0.009), with average preload and postload values for the large needle group being significantly lower than for control (Figure 3). No significant differences existed for pre- or postload modulus between small and large needle groups, nor between small needle and control groups (P > 0.19). Regardless of experimental group, the nominal dynamic modulus increased postload when compared with preload; however, no significant differences were noted between groups (P = 0.076). A small but significant increase in preload dynamic modulus was observed between day 2 and day 1 in the small needle puncture group (P = 0.0028) (Figure 3A), and in postload dynamic modulus for the large needle puncture group between day 1 with days 2 and 5 (P = 0.042) (Figure 3B). No significant increase in either the preload or postload dynamic modulus was seen over time in the control group (P > 0.062).

Figure 3
Average ± SEM nominal dynamic loading modulus for preload (top) and postload (bottom) tests. A significant difference between groups or time points is indicated by sharing of a common symbol.

Needle puncture injury also affected the creep during the 1-hour dynamic loading, with significantly more creep observed in needle puncture than control groups (Figure 4, P < 0.006). No significant differences were seen between large and small needle groups. Disc height recorded at 0.2 MPa during the first cycle of dynamic loading decreased over time for all groups, and tended to decrease more for needle puncture group; however, no significant differences were detected (Table 1).

Figure 4
Average ± SEM creep during the one hour dynamic loading protocol at each day. Significantly more creep was observed at all time points in the needle puncture groups than in the dynamic control group (star indicates a difference relative to all ...
Table 1
Height Lost Between Culture Days

Regional tissue water contents were not significantly affected by needle puncture injury (P > 0.125). Combining all groups, average OA water content was 57.21% ± 0.75%, IA water content was 71.92% ± 0.75% and NP water content was 80.24% ± 0.56%. The amount of GAG released to the media, reported as a percentage of initial disc wet weight, was small and not significantly affected by either large or small needle puncture (P > 0.125). Average GAG release to the media was 0.060% ± 0.005% of the initial disc weight (Table 2).

Table 2
Average GAG Loss to Culture Media (as a Percentage of Total Initial Disc Weight) and Percent Water Content of Intervertebral Disc Tissue for Outer Annulus (OA), Inner Annulus (IA), Nucleus Pulposus (NP) Regions

Localized cell death was observed in the area adjacent to the needle tracks. Cell viability was maintained elsewhere in the disc, with no observable differences between groups (Figure 5). Histology revealed anulus fiber disruption (Figure 6), and a localized area of increased cell number and possible remodeling in the NP of both needle groups (Figure 7).

Figure 5
Viability images of OA (left) and NP (right) in the large needle group. Needle puncture regions are on top oriented radially (perpendicular to needle track, with needle track centered in field of view), control images are on bottom oriented sagittally ...
Figure 6
Representative histology images of needle puncture discs (top) and control discs (bottom) of the OA (left), IA (middle), and NP (right) revealing annulus fiber disruption in the needle group. Collagen stains red, proteoglycans blue, and cell nuclei black. ...
Figure 7
Evidence of an increase in NP cell number and possible remodeling local to an insertion site in a large needle puncture disc. Image on left is at 2.5×, scale bar = 1 mm. Boxed area on left is magnified on the right, image is at 20×, scale ...

Discussion

The purpose of this study was to evaluate the immediate and short-term changes in disc structure, mechanics, and cellular response resulting from small- and large-gauge needle puncture injury. A bovine organ culture model was used to ensure homogeneous mechanical and chemical boundary conditions, to allow for multiple dependant variables to be examined on the same tissue, and to separate the inherent IVD tissue response from a more systemic inflammatory response. Localized disruption in the disc tissue from both small and large needle puncture injury was demonstrated to rapidly compromise local disc structure and elastic and viscoelastic mechanical properties. Evidence of a cellular response was also present in the NP region of both needle puncture injury groups, with increased cell death around the needle track, and regions of increased cell number and matrix remodeling along the needle track. Needle puncture injury did not affect GAG released from the disc or water content after recovery. Significant biologic and structural alterations in the disc in response to large-gauge needle puncture were anticipated and consistent with the hypothesis, but the significant alterations in response to small-gauge needle puncture were surprising and contrasted the hypothesis.

The results of this study indicate that needle puncture, with even a small needle, is sufficient to initiate immediate and progressive alterations in disc height, stiffness, and viscoelastic properties (i.e., creep during one hour of loading) that do not recover. In all groups, the largest changes in disc mechanics were observed between days 1 and 2, indicating the largest response to the mechanical loading occurred during the first day. Although some of the changes are probably due to a preconditioning type of response, disc mechanics in needle puncture groups were further altered, indicating that the majority of additional tissue damage probably occurred during the first day's loading. A previous in vitro study demonstrated that mechanical stiffness, viscoelastic relaxation, and water content all recovered within 18 hours after cyclic loading (although no measurements were taken at the 12-hour time point).26 Although discs were only allowed 12 hours of recovery between loading events in this study, the removal of vertebral endplates leads to significantly faster recovery times.27 Therefore, it can be concluded that mechanical changes reported in this study are associated with needle puncture injury and not due to the loading protocol. It is also noteworthy that water content in all discs in this study did recover within 12 hours after loading, consistent with MRI measurements of Johannessen et al.26 In this context, this study provides support to the concept that diagnostic techniques capable of evaluating biomechanical behaviors may be effective at evaluating early degenerative changes in the disc, whereas traditional MRI evaluations that focus on water content alone may miss important structural changes resulting from injury.

Degenerative changes of the disc may be induced as a result of pathologic loading and mechanical damage, biologic remodeling, response to injury and proinfloammatory cytokines, or a combination of all of these. In this study, no significant increase in GAG lost to the medium was detected for small and large needle puncture due to leaching of proteoglycans from the needle track, but we did find structural disruptions and altered mechanics. We infer that loss of GAG in anulus injury models of degeneration may be associated with 3 interactive pathways: damage accumulation from pathologic loading that might involve depressurization of the NP and larger, more ubiquitous structural defects than AF needle puncture injury alone, chronic biologic remodeling that includes proteolytic cleavage of aggrecan into smaller fragments; and biologic response to proinflammatory cytokines in response to injury in vivo.28

Several animal models of disc degeneration use needle puncture or other anulus injury to induce degenerative changes. Rabbit IVDs, when subjected to anular stab and needle puncture into the NP using 16 to 21G needles resulted in changes that were consistent with degeneration after 8 weeks.15,16 With a 23G needle, Kim et al reported nuclear herniation after needle puncture.17 In our study, no extrusion of NP material was observed, but immediate and significant mechanical and cellular changes were found. With smaller-gauge needle (28G), and saline injection into the rabbit NP, nonsignificant trends of decreased disc height and proteoglycan and collagen content in the AF and NP were observed at 2 weeks that persisted after 8 weeks.6 Moderate degeneration occurred after 12 months, and marked degeneration after 18 months in pig and sheep models that had peripheral AF tears induced surgically with clear loss of disc height and biochemical changes in the matrix.19-21 In an 18-month sheep study, AF delamination was produced by injecting saline using a 27G needle into the outer third of, and parallel to, the anterolateral AF fibers, and compared to a 27G needle puncture without saline injection.13 Of particular interest was the finding that both the saline injected and noninjected needle injuries showed morphologic evidence of mild to moderate degeneration, lamellar thickening in the region of the injury, and altered biomechanical behaviors. This study supports anulus injury as a potential pathway toward progressive disc degeneration and demonstrated that even small needle puncture resulted in immediate and progressive changes to the IVD biomechanics and cellular response.

Overall disc cell viability remained high, consistent with previous studies using this organ culture system,23,29 with the only exception in the area immediately adjacent to the needle track. The maintenance of cell viability in the disc away from the needle injury suggests that cell viability was not affected by altered disc mechanics associated with needle puncture injury. On the other hand, localized cell death was likely due to the severing of collagen fibers as the needle entered the disc, and it is possible that the observed changes in mechanical behavior could have induced more general apoptosis in response to altered stresses.30 In a rabbit needle puncture injury model, Sobajima et al reported an early upregulation of mRNA for IL-1β, MMP-3, and I-NOS in the nucleus that may have been associated with altered mechanics of the IVD because the needle puncture only penetrated the AF and not the NP.31 Catabolic remodeling of mRNA expression in response to altered mechanical loading is well documented32,33 and also supported by the results of this study that found early mechanical changes of the IVD in response to needle puncture injury.

Bovine discs were used in this study because they are a large animal model with IVDs that are reported to have composition and biosynthetic rates similar to human IVDs.34 Sparse notochordal cell populations in both adult bovine and sheep populations also mimic the situation seen in humans, where notochordal cells disappear during the second decade.35 The NP in bovine and other large animals also tends to be more fibrous, preventing an immediate prolapse of NP material through the experimental AF defect when the needle is pulled out, similar to an adult human.

We conclude that a relatively minor disruption in the disc from small- and large-gauge needle puncture had immediate and progressive mechanical and biologic consequences with important implications for the use of needle puncture in discography, and repair/regeneration techniques of degenerated discs. Results suggest that altered mechanics and subsequent changes in metabolism resulting from small and large needle puncture injury may be a possible mechanism for degenerative remodeling. Results also suggest early matrix disruption results in mechanical changes that would be difficult to detect from traditional imaging techniques that do not assess mechanical function. This study provides a greater basic science understanding of needle puncture models of degeneration in large animals and suggests that altered mechanics resulting from needle puncture injury may be a possible mechanism for degenerative changes. As with any model system, further studies on human tissue are warranted before any direct recommendations can be made on needle size for clinical applications.

Key Points

  • The influence of insertion of 25G and 14G needles on the short-term mechanical and biologic response of the disc was investigated using a bovine organ culture model.
  • Immediate and progressive changes in disc stiffness and viscoelastic behaviors were detected after needle puncture injury with both small- and large-gauge needles.
  • Needle puncture injury resulted in localized structural disruption, loss of cell viability, and matrix remodeling. Gross tissue water and proteoglycan contents and cell viability were maintained.
  • Results suggest that needle puncture injury results in important mechanical changes that may lead to subsequent degenerative remodeling, in a manner that would be difficult to detect with traditional imaging techniques that do not assess biomechanical function.
  • Anulus puncture injury by small- and large-gauge needles results in localized and generalized biologic and mechanical consequences with implications for discography and injection of biologic treatment agents.

Acknowledgments

The authors gratefully acknowledge Arthur Michalek for assistance with the creation of computer code for mechanical parameter analysis.

Supported by the Whitaker Foundation and National Institutes of Health (R01AR051146).

Federal funds were received in support of this work. No benefits in any form have been or will be received from a commercial party related directly or indirectly to the subject of this manuscript.

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