Carol Muehleman

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

Study Design. An in vivo study to radiographically and histologically assess a new method of induction of disc degeneration. Objective. To establish a reproducible rabbit model of disc degeneration by puncturing the anulus with needles of defined gauges and to compare it to the classic stab model. Summary of Background Data. New treatment approaches to disc degeneration are of great interest. Although animal models for disc degenerative disease exist, the quantitative measurement of disease progression remains difficult. A reproducible, progressive disc degeneration model, which can be induced in a reasonable time frame, is essential for development of new therapeutic interventions. Methods. The classic anular stab model and the new needle puncture model were used in the rabbit. For the needle puncture model, 3 different gauges of needle (16G, 18G, and 21G) were used to induce an injury to the disc to a depth of 5 mm. Radiographic and histologic analyses were performed; magnetic resonance images were also assessed in the needle puncture model. Results. Significant disc space narrowing was observed as early as 2 weeks after stabbing in the classic stab model; there was no further narrowing of the disc space. In the needle puncture model, all needle sizes tested induced a slower and more progressive decrease in disc height than in the classic stab model. The magnetic resonance imaging supported the results of disc height data. Conclusions. The needle puncture approach, using 16G to 21G needles, resulted in a reproducible decrease of disc height and magnetic resonance imaging grade. The ease of the procedure and transfer of the methodology will benefit researchers studying disc degeneration.

Osteogenic Protein-1 Injection Into a Degenerated Disc Induces the Restoration of Disc Height and Structural Changes in the Rabbit Anular Puncture Model

Study Design. In vivo study of the effect of injection of osteogenic protein-1 (OP-1) on a rabbit anular needle puncture model of intervertebral disc (IVD) degeneration. Objective. To study radiographic, magnetic resonance imaging (MRI), biochemical, and histologic changes in the rabbit IVD after injection of OP-1 into the nucleus pulposus in a needle puncture disc degeneration model. Summary of the Background Data. Growth factors, such as OP-1, have the ability to stimulate synthesis of proteoglycans and collagen in vitro. The in vivo injection of OP-1 into the normal rabbit IVD has increased disc height and proteoglycan content in the anulus fibrosus and nucleus pulposus. However, to our knowledge, no attempts have yet been made to determine the effects of these growth factors in an in vivo model of disc degeneration. Methods. New Zealand adolescent white rabbits (n = 90, 8 for baseline evaluation, 82 at 8 times) received an anular puncture in 2 noncontiguous discs with an 18-gauge needle to induce disc degeneration. Four weeks later, either 5% lactose (10 &mgr;L) or OP-1 (100 &mgr;g in 10 &mgr;L 5% lactose) was injected into the center of the nucleus pulposus. The disc height was followed radiographically for up to 24 weeks after the injections. At the 2, 4, 8, 12, and 24-week times after the injection, rabbits were euthanized, and MRI of the harvested spinal columns was obtained to grade the degeneration. The discs injected with OP-1 or lactose and noninjected discs were subjected to biochemical and histologic analysis. The specimens at the 24-week time were limited to histologic evaluation. Results. The anular puncture with a needle induced a consistent disc narrowing within 4 weeks. The injection of OP-1 induced a restoration of disc height at 6 weeks, which was sustained for the entire experimental period, up to 24 weeks after the injection. The injection of lactose alone did not change the course of disc narrowing over the same time. MRI grading score showed significant differences between the OP-1 and lactose groups at the 8, 12, and 24-week times, suggesting an increase in water content in the nucleus pulposus of the OP-1 group. The proteoglycan content of the nucleus pulposus and anulus fibrosus was significantly higher in the OP-1 group than in the control group. The degeneration grades of the punctured discs in the OP-1 group were significantly lower than those in the lactose group. Conclusion. The results of this study show the feasibility of restoring degenerative rabbit discs by a single injection of OP-1 into the nucleus pulposus. Importantly, the effects of the OP-1 injection on disc height were sustained for up to 24 weeks. The metabolic changes in the cells, following a single injection, might be sustained and, thus, induce long-term changes in disc structure. An efficacy study in large animals is required to show further that the intradiscal injection of OP-1, or bone morphogenetic proteins or growth factors with similar properties would be useful for the structural restoration of the IVD in humans.

Prevalence of degenerative morphological changes in the joints of the lower extremity

Information on the prevalence and extent of degenerative morphological changes (DMC) in the joints of the lower extremity, including foot joints is sparse. In the present study, the first and fifth metatarsalphalangeal (MTP), transverse tarsal, subtalar, talocrural, knee and hip joints of 50 cadavers were examined grossly and graded on a five-point scale for signs of DMC. Selected samples were examined histologically. Our results confirm clinical findings that severe DMC in foot joints are uncommon except in the first MTP joint where the plantar aspect is most affected. The knee joint displayed the most numerous and severe signs of DMC followed by the first MTP joint. The hip, talocrural, subtalar and transverse tarsal joints displayed comparatively moderate levels of DMC while the fifth MTP was rarely affected. The only joint to display significantly greater levels of DMC on the distal side of the joint as compared with the proximal side, when a difference was present, was the hip. There were significantly greater levels of DMC on the medial aspect of two or more joints within an extremity than on the lateral aspect. Radiographs often showed few or no signs of DMC even when erosion down to subchondral bone was observed upon gross examination.

Multiple-image radiography

Conventional radiography produces a single image of an object by measuring the attenuation of an x-ray beam passing through it. When imaging weakly absorbing tissues, x-ray attenuation may be a suboptimal signature of disease-related information. In this paper we describe a new phase-sensitive imaging method, called multiple-image radiography (MIR), which is an improvement on a prior technique called diffraction-enhanced imaging (DEI). This paper elaborates on our initial presentation of the idea in Wernick et al (2002 Proc. Int. Symp. Biomed. Imaging pp 129-32). MIR simultaneously produces several images from a set of measurements made with a single x-ray beam. Specifically, MIR yields three images depicting separately the effects of refraction, ultra-small-angle scatter and attenuation by the object. All three images have good contrast, in part because they are virtually immune from degradation due to scatter at higher angles. MIR also yields a very comprehensive object description, consisting of the angular intensity spectrum of a transmitted x-ray beam at every image pixel, within a narrow angular range. Our experiments are based on data acquired using a synchrotron light source; however, in preparation for more practical implementations using conventional x-ray sources, we develop and evaluate algorithms designed for Poisson noise, which is characteristic of photon-limited imaging. The results suggest that MIR is capable of operating at low photon count levels, therefore the method shows promise for use with conventional x-ray sources. The results also show that, in addition to producing new types of object descriptions, MIR produces substantially more accurate images than its predecessor, DEI. MIR results are shown in the form of planar images of a phantom and a biological specimen. A preliminary demonstration of the use of MIR for computed tomography is also presented.

DIFFRACTION ENHANCED X-RAY IMAGING OF ARTICULAR CARTILAGE

OBJECTIVE To introduce a novel X-ray technology, diffraction-enhanced X-ray imaging (DEI), in its early stages of development, for the imaging of articular cartilage. DESIGN Disarticulated and/or intact human knee and talocrural joints displaying both undegenerated and degenerated articular cartilage were imaged with DEI. A series of three silicon crystals were used to produce a highly collimated monochromatic X-ray beam to achieve scatter-rejection at the microradian level. The third crystal (analyser) was set at different angles resulting in images displaying different characteristics. Once the diffraction enhanced (DE) images were obtained, they were compared to gross and histological examination. RESULTS Articular cartilage in both disarticulated and intact joints could be visualized through DEI. For each specimen, DE images were reflective of their gross and histological appearance. For each different angle of the analyser crystal, there was a slight difference in appearance in the specimen image, with certain characteristics changing in their contrast intensity as the analyser angle changed. CONCLUSIONS DEI is capable of imaging articular cartilage in disarticulated, as well as in intact joints. Gross cartilage defects, even at early stages of development, can be visualized due to a combination of high spatial resolution and detection of X-ray refraction, extinction and absorption patterns. Furthermore, DE images displaying contrast heterogeneities indicative of cartilage degeneration correspond to the degeneration detected by gross and histological examination.

Effects of growth differentiation factor-5 on the intervertebral disc--in vitro bovine study and in vivo rabbit disc degeneration model study.

Study Design. In vitro studies on the effects of recombinant human growth and differentiation factor-5 (rhGDF-5) on matrix metabolism of bovine intervertebral disc cells and an in vivo study on the effect of rhGDF-5 in the rabbit anular puncture model. Objective. To determine the reparative capacity of rhGDF-5 on the intervertebral disc. Summary of Background Data. The in vitro and in vivo effects of rhGDF-5, a crucial protein in the developing musculoskeletal system, on repair of the degenerated intervertebral disc remain unidentified. Methods. In vitro, bovine nucleus pulposus and anulus fibrosus cells were cultured with or without rhGDF-5 (100 or 200 ng/mL). On days 7, 14, and 21, the contents of deoxyribonucleic acid and proteoglycan, and the synthesis of proteoglycan and collagen were assessed. In vivo, 16 adolescent New Zealand white rabbits received anular punctures in 2 lumbar discs. Four weeks later, phosphate buffered saline or rhGDF-5 (10 ng, 1 and 100 &mgr;g) was injected into the nucleus pulposus. The rabbits were followed up for 16 weeks for disc height, magnetic resonance imaging, and histologic grading. Results. In vitro, rhGDF-5 increased the deoxyribonucleic acid and proteoglycan contents of both cell types significantly after day 14. rhGDF-5 at 200 ng/mL significantly stimulated proteoglycan synthesis (nucleus pulposus: +138%, anulus fibrosus: +24%) and collagen synthesis (nucleus pulposus: +95%, anulus fibrosus: +23%) at day 21. In vivo, the injection of rhGDF-5 resulted in a restoration of disc height, improvement of magnetic resonance imaging scores, and histologic grading scores with statistical significance (P < 0.05−0.001). Conclusion. A single injection of rhGDF-5 has a reparative capacity on intervertebral discs, presumably based on its effects to enhance extracellular matrix production in vitro.

Proinflammatory cytokines stimulate the expression of nerve growth factor by human intervertebral disc cells.

Study Design. In vitro studies of the effects of proinflammatory cytokines on the production of nerve growth factor (NGF) by human intervertebral disc (IVD) cells. Objective. To determine the constitutive expression and production of NGF and the effect of cytokines on the expression of NGF by human IVD cells. Summary of the Background Data. NGF may play a role in the collateral sprouting of sensory axons, neural survival, and regulation of nociceptive sensory neurons. NGF is known to be up-regulated by proinflammatory cytokines. Methods. The presence of NGF protein was analyzed by immunohistochemistry using human IVD cells obtained from cadaveric human spines with no known disc disease (MRI Thompson grades 2–4). The effects of interleukin-1&bgr; (IL-1&bgr;) and tumor necrosis factor-&agr; (TNF-&agr;) on NGF production and mRNA expression of NGF by IVD cells were examined. The expression of NGF receptors, trkA and p75NGFR, was also assessed immunohistochemically. Results. Cadaveric anulus fibrosus (AF) and nucleus pulposus (NP) cells cultured in vitro in monolayer and in alginate beads positively stained with an anti-NGF antibody. The constitutive production of NGF protein in IVD cells was low (NP) or not detectable (AF). The expression of NGF mRNA was detectable in both cell types. IL-1&bgr; and TNF-&agr; up-regulated the NGF mRNA expression and the secretion of NGF protein into the media. TrkA was immunolocalized in AF and NP cells. Conclusion. Our results demonstrate, for the first time, that human AF and NP cells constitutively express NGF protein and mRNA, and that the proinflammatory cytokines IL-1&bgr; and TNF-&agr; stimulate the production of NGF. The precise role of NGF produced by IVD cells in the generation of discogenic pain or on the metabolism of IVD cells, especially under certain physiologic conditions in which cytokines are up-regulated, needs to be clarified in future experimentation.

Restoration of disc height loss by recombinant human osteogenic protein-1 injection into intervertebral discs undergoing degeneration induced by an intradiscal injection of chondroitinase ABC.

Study Design. In vivo study of the effect of an injection of recombinant human osteogenic protein-1 into degenerated discs induced by chondroitinase ABC. Objective. To investigate the efficacy of an injection of recombinant human osteogenic protein-1 to induce the recovery of disc height, and biochemical and histologic repair, in discs degenerated through enzymatic digestion by chondroitinase ABC. Summary of the Background Data. Chondroitinase ABC is currently proposed as a chemonucleolysis agent; however, postchemonucleolysis degeneration is currently unavoidable. Recombinant human OP-1 has been shown to promote extracellular matrix repair in vitro and in vivo. Methods. Fifty-four adolescent New Zealand white rabbits were used. Four weeks after an initial injection of chondroitinase ABC (10 mU/disc), 5% lactose (10 &mgr;L/disc) or recombinant human osteogenic protein-1 (100 &mgr;g in 10 &mgr;L lactose/disc) was injected. Disc heights were monitored radiographically at 2-week intervals, and rabbits were killed at 6, 8, 12, and 16 weeks after the initial chondroitinase ABC injections. The intervertebral discs were subjected to histologic and biochemical analyses. Results. Significant disc space narrowing was observed in both groups 2 weeks after the injection of chondroitinase ABC. In the chondroitinase ABC/lactose group, this narrowing progressed after the vehicle injection and was sustained for up to 16 weeks. In the chondroitinase ABC/recombinant human osteogenic protein-1 group, the disc height index showed a significant increase at 6 weeks (lactose vs. recombinant human osteogenic protein-1; P < 0.01); this recovery was sustained for up to 16 weeks. The proteoglycan content was higher in the chondroitinase ABC/recombinant human osteogenic protein-1 group than in the chondroitinase ABC/lactose group. However, histologic changes, after the recombinant human osteogenic protein-1 injection, were not observed. Conclusions. A single injection of recombinant human osteogenic protein-1 into a rabbit disc dramatically reversed the decrease in disc height induced by chondroitinase ABC chemonucleolysis. The recovery was significant and sustained over the next 12 weeks. The therapeutic effects of both chondroitinase ABC chemonucleolysis and recombinant human osteogenic protein-1 injections should be further explored in higher animals before it is applied to humans.

Radiography of soft tissue of the foot and ankle with diffraction enhanced imaging

Non‐calcified tissues, including tendons, ligaments, adipose tissue and cartilage, are not visible, for any practical purposes, with conventional X‐ray imaging. Therefore, any pathological changes in these tissues generally necessitate detection through magnetic resonance imaging or ultrasound technology. Until recently the development of an X‐ray imaging technique that could detect both bone and soft tissues seemed unrealistic. However, the introduction of diffraction enhanced X‐ray imaging (DEI) which is capable of rendering images with absorption, refraction and scatter rejection qualities has allowed detection of specific soft tissues based on small differences in tissue densities. Here we show for the first time that DEI allows high contrast imaging of soft tissues, including ligaments, tendons and adipose tissue, of the human foot and ankle.

A computed tomography implementation of multiple-image radiography

Conventional x-ray computed tomography (CT) produces a single volumetric image that represents the spatially variant linear x-ray attenuation coefficient of an object. However, in many situations, differences in the x-ray attenuation properties of soft tissues are very small and difficult to measure in conventional x-ray imaging. In this work, we investigate an analyzer-based imaging method, called computed tomography multiple-image radiography (CT-MIR), which is a tomographic implementation of the recently proposed multiple-image radiography method. The CT-MIR method reconstructs concurrently three physical properties of the object. In addition to x-ray attenuation, CT-MIR produces volumetric images that represent the refraction and ultrasmall-angle scattering properties of the object. These three images can provide a rich description of the objects physical properties that are revealed by the probing x-ray beam. An imaging model for CT-MIR that is based on the x-ray transform of the object properties is established. The CT-MIR method is demonstrated by use of experimental data acquired at a synchroton radiation imaging beamline, and is compared to the pre-existing diffraction-enhanced imaging CT method. We also investigate the merit of an iterative reconstruction method for use with future clinical implementations of CT-MIR, which we anticipate would be photon limited.