Ellen Liebenberg

Compression-induced degeneration of the intervertebral disc : An in vivo mouse model and finite-element study

Study Design.An in vivo study of the biologic and biomechanical consequences of static compressive loading on the mouse tail intervertebral disc.Objectives.To determine whether static compression in vivo alters the biologic activity of the disc and leads to diminished biomechanical performance.SummaSTUDY DESIGN An in vivo study of the biologic and biomechanical consequences of static compressive loading on the mouse tail intervertebral disc. OBJECTIVES To determine whether static compression in vivo alters the biologic activity of the disc and leads to diminished biomechanical performance. SUMMARY OF BACKGROUND DATA Static compressive stress that exceeds the discs swelling pressure is known to change hydration and the intradiscal stress distribution. Alterations in hydration and stress have been associated with changes in disc cell activity in vitro and in other collagenous tissues in vivo. METHODS Mouse tail discs were loaded in vivo with an external compression device. After 1 week at one of three different stress levels, the discs were analyzed for their biomechanical performance, morphology, cell activity, and cell viability. A second group of mice were allowed to recuperate for 1 month after the 1-week loading protocol to assess the discs ability to recover. As an aid to interpreting the histologic and biologic data, finite-element analysis was used to predict region-specific changes in tissue stress caused by the static loading regimen. RESULTS With increasing compressive stress, the inner and middle anulus became progressively more disorganized, and the percentage of cells undergoing apoptosis increased. The expression of Type II collagen was suppressed at all levels of stress, whereas the expression of aggrecan decreased at the highest stress levels in apparent proportion to the decreased nuclear cellularity. Compression for 1 week did not affect the disc bending stiffness or strength but did increase the neutral zone by 33%. As suggested by the finite-element model, during sustained compression, tension is maintained in the outer anulus and lost in the inner and middle regions where the hydrostatic stress was predicted to increased nearly 10-fold. Discs loaded at the lowest stress recovered anular architecture but not cellularity after 1 month of recuperation. Discs loaded at the highest stress did not recover anular architecture, displaying islands of cartilage cells in the middle anulus at sites previously populated by fibroblasts. CONCLUSIONS The results of the current project demonstrate that static compressive loading initiates a number of harmful responses in a dose-dependent way: disorganization of the anulus fibrosus; an increase in apoptosis and associated loss of cellularity; and down regulation of collagen II and aggrecan gene expression. The finite element model used in this study predicts loss of collagen fiber tension and increased matrix hydrostatic stress in those anular regions observed to undergo programmed cell death after 1 week of loading and ultimately become populated by chondrocytes after one month of recuperation. This correspondence conforms with the suggestions of others that the cellular phenotype in collagenous tissues is sensitive to the dominant type of tissue stress. Although the specific mechanisms by which alterations in tissue stress lead to apoptosis and variation in cell phenotype remain to be identified, our results suggest that maintenance of appropriate stress within the disc may be an important basis for strategies to mitigate disc degeneration and initiate disc repair.

ISSLS prize winner: repeated disc injury causes persistent inflammation.

Study Design. An in vivo rat model of disc degeneration with emphasis on characterizing acute and chronic cytokine production. Objective. To compare the morphologic and proinflammatory response between a single and triple-stab injury in attempts to establish mechanisms of chronic disc inflammation. Summary of Background Data. The features that distinguish physiologic (asymptomatic) from pathologic (symptomatic) degeneration are unclear. Epidemiologic evidence suggests that cumulative damage and elevated disc cytokine levels may be linked to increased low back pain rates. Although acute injury stimulates a healing response that includes transient cytokine production, repetitive damage may be necessary to trigger the persistent inflammation suspected to underlie chronic pain. Methods. Tail discs were exposed surgically and stabbed with a number 11 blade. During the subsequent acute healing phase, triple-stab discs were percutaneously injured with a 23-gauge needle at day 3 and then again at day 6 after the initial blade incision. Cytokine (IL-1&bgr;, IL-6, IL-8, and TNF-&agr;) production was quantified using enzyme linked immunosorbent assay, and, in addition to MAPK signaling pathways (phosphorylated forms of ERK, JNK, and p38), was localized by immunohistochemistry. Disc architecture was evaluated using histology. Results. Both single-stab and triple-stab discs degenerated with time, yet degeneration was more severe with repeated injury where nuclear proteoglycan was replaced by disorganized collagen. Four days after single-stab, there was a transient peak in IL-1&bgr; and IL-8 production that was localized to the wound track and associated granulation tissue. By contrast, triple-stab induced an activated annular fibroblast phenotype (p38 positive) that caused a prolonged, diffuse inflammatory response with elevated levels of TNF-&agr;, IL-1&bgr;, and IL-8 up to 28 days after injury. Disc inflammation was accompanied by reactive changes in the adjacent vertebral marrow spaces that was initially lytic at day 4, becoming sclerotic by day 56. Conclusion. Our results demonstrate that repeated injury during active healing leads to persistent inflammation and enhanced disc degeneration. These data support the premise that damage accumulation and its associated inflammation may distinguish pathologic from physiologic disc degeneration. In the future, this triple-stab model may be useful to evaluate the efficacy of anti-inflammatory low back pain treatments.

The effect of static in vivo bending on the murine intervertebral disc

BACKGROUND CONTEXT Intervertebral disc cell function in vitro has been linked to features of the local environment that can be related to deformation of the extracellular matrix. Epidemiologic data suggest that certain regimens of spinal loading accelerate disc degeneration in vivo. Yet, the direct association between disc cell function, spinal loading and ultimately tissue degeneration is poorly characterized. PURPOSE To examine the relationships between tensile and compressive matrix strains, cell activity and annular degradation. STUDY DESIGN/SETTING An in vivo study of the biologic, morphologic and biomechanical consequences of static bending applied to the murine intervertebral disc. SUBJECT SAMPLE: Twenty-five skeletally mature Swiss Webster mice (12-week-old males) were used in this study. OUTCOME MEASURES Bending neutral zone, bending stiffness, yield point in bending, number of apoptotic cells, annular matrix organization, cell shape, aggrecan gene expression, and collagen II gene expression. METHODS Mouse tail discs were loaded for 1 week in vivo with an external device that applied bending stresses. Mid-sagittal sections of the discs were analyzed for cell death, collagen II and aggrecan gene expression, and tissue organization. Biomechanical testing was also performed to measure the bending stiffness and strength. RESULTS Forceful disc bending induced increased cell death, decreased aggrecan gene expression and decreased tissue organization preferentially on the concave side. By contrast, collagen II gene expression was symmetrically reduced. Asymmetric loading did not alter bending mechanical behavior of the discs. CONCLUSIONS In this model, annular cell death was related to excessive matrix compression (as opposed to tension). Collagen II gene expression was most negatively influenced by the static nature of the loading (immobilization), rather than the specific state of stress (tension or compression).

Structured coculture of stem cells and disc cells prevent disc degeneration in a rat model

BACKGROUND CONTEXT Harnessing the potential of stem cells is an important strategy for regenerative medicine. This study explores the use of bilaminar coculture pellets (BCPs) of mesenchymal stem cells (MSCs) and nucleus pulposus cells (NPCs) as a cell-based therapy for intervertebral disc regeneration. Prior in vitro experiments have shown that BCP can help differentiate MSCs and substantially improve new matrix deposition. PURPOSE To evaluate the clinical relevance of BCPs by testing the system in vivo. STUDY DESIGN/SETTING We have designed a novel spherical BCP where MSCs are enclosed in a shell of NPCs. The pellets were tested in vivo in a rat tail model of disc degeneration. METHODS Rat caudal intervertebral discs were denucleated and treated with BCP in a fibrin sealant (FS) carrier (controls were MSCs suspended in FS; NPCs suspended in FS; MSCs and NPCs suspended in FS; FS only; and surgery only). At 14 and 35 days after implantation, the animals were euthanized and discs were evaluated for proteoglycan content, enzyme-linked immunosorbent assay for inflammatory cytokines, cell retention using polymerase chain reaction, disc height, histology, and disc grade based on a blinded scoring system. RESULTS The proteoglycan and cytokine levels were not significantly different among groups. The BCP group had higher cell retention than controls. Disc height and disc grade increased over time only in the BCP group. Bilaminar coculture pellets were the only treatment to show proteoglycan staining in the nucleus space at 35 days. CONCLUSIONS This study shows that BCPs may prevent postnucleotomy disc degeneration in vivo. Larger animals and longer time points will be necessary to further judge potential clinical impact. As opposed to strategies that require growth factor supplements, predifferentiation, or genetic manipulations, BCPs are a self-sustaining and targeted method for tissue regeneration in situ.

Innervation of pathologies in the lumbar vertebral end plate and intervertebral disc.

BACKGROUND CONTEXT Magnetic resonance imaging (MRI) has limited diagnostic value for chronic low back pain because of the unclear relationship between any anatomic abnormalities on MRI and pain reported by the patient. Assessing the innervation of end plate and disc pathologies-and determining the relationship between these pathologies and any abnormalities seen on MRI-could clarify the sources of back pain and help identify abnormalities with enhanced diagnostic value. PURPOSE To quantify innervation in the vertebral end plate and intervertebral disc and to relate variation in innervation to the presence of pathologic features observed by histology and conventional MRI. STUDY DESIGN/SETTING A cross-sectional histology and imaging study of vertebral end plates and intervertebral discs harvested from human cadaver spines. METHODS We collected 92 end plates and 46 intervertebral discs from seven cadaver spines (ages 51-67 years). Before dissection, the spines were scanned with MRI to grade for Modic changes and high-intensity zones (HIZ). Standard immunohistochemical techniques were used to localize the general nerve marker protein gene product 9.5. We quantified innervation in the following pathologies: fibrovascular end-plate marrow, fatty end-plate marrow, end-plate defects, and annular tears. RESULTS Nerves were present in the majority of end plates with fibrovascular marrow, fatty marrow, and defects. Nerve density was significantly higher in fibrovascular end-plate marrow than in normal end-plate marrow (p<.001). Of the end plates with fibrovascular and fatty marrow, less than 40% were Modic on MRI. Innervated marrow pathologies collocated with more than 75% of the end plate defects; hence, innervation was significantly higher in end plate defects than in normal end plates (p<.0001). In the disc, nerves were observed in only 35% of the annular tears; in particular, innervation in radial tears tended to be higher than in normal discs (p=.07). Of the discs with radial tears, less than 13% had HIZ on T2 MRI. Innervation was significantly less in radial tears than in fibrovascular end-plate marrow (p=.05) and end-plate defects (p=.02). CONCLUSIONS These findings indicate that vertebral end-plate pathologies are more innervated than intervertebral disc pathologies and that many innervated end-plate pathologies are not detectable on MRI. Taken together, these findings suggest that improved visualization of end-plate pathologies could enhance the diagnostic value of MRI for chronic low back pain.

Biological and biomechanical effects of fibrin injection into porcine intervertebral discs.

Study Design. Surgically denucleated porcine intervertebral discs (IVD) were injected with BIOSTAT BIOLOGX Fibrin Sealant (FS), and the in vivo effects were assessed over time by histological, biochemical, and mechanical criteria. Objective. The objectives were to test whether the intradiscal injection of FS stimulates disc healing. Summary of Background Data. Disc avascularity prevents the deposition of a provisional fibrin scaffold that typically facilitates soft tissue repair. Poor disc wound healing leads to disc damage accumulation and chronic inflammation characterized by overproduction of proinflammatory cytokines and proteolytic enzymes. Methods. Four lumbar IVDs from each of 31 Yucatan minipigs were randomized to untreated controls; degenerative injury (nucleotomy); and nucleotomy plus FS injection. Animals were killed at 1, 2, 3, 6, and 12 weeks postsurgery. IVDs were harvested to quantify (1) architecture using morphological and histological grading; (2) proteoglycan composition using DMMB assay; (3) cytokine content using ELISA; and (4) mechanical properties using quantitative pressure/volume testing. Results. There was progressive invasion of annular tissue into the nucleus of nucleotomy discs and concomitant reduction in proteoglycan content. By contrast, FS supplementation inhibited nuclear fibrosis and facilitated proteoglycan content recovery over time. FS discs synthesized significantly less TNF-&agr; than degenerate discs (66% vs. 226%, P < 0.05) and had upregulation of IL-4 (310% vs. 166%) and TGF-&bgr; (400% vs. 117%) at 2 to 3 weeks posttreatment. At the third week postsurgery, the denucleated discs were less stiff than controls (pressure modulus 779.9 psi vs. 2754.8 psi; P < 0.05) and failed at lower pressures (250.5 psi vs. 492.5 psi; P < 0.05). The stiffness and leakage pressure of the FS-treated discs recovered to control values after 6 and 12 weeks, respectively. Conclusion. FS facilitated structural, compositional, and mechanical repair of the surgically damaged IVD. These FS-derived benefits are likely due to its conductive scaffold properties and metabolically active constituents such as thrombin, factor XIII, and aprotinin acetate.

Innervation patterns of PGP 9.5‐positive nerve fibers within the human lumbar vertebra

Intervertebral disc injury or degeneration is a common cause of low back pain, and yet the specific source of pain remains ambiguous in many cases. Previous research indicates that the central vertebral endplate is highly innervated and can elicit pain responses to pressure. In effort to trace the origin of nerves located at the endplate, we used protein gene product 9.5 (PGP 9.5) to stain neurofibers and then quantified the spatial pattern of nerve distribution within a human L4 lumbar vertebra. The majority of nerves were adjacent to blood vessel walls, and consequently the nerve distribution closely resembled previously established vascularity patterns. We observed that the majority of nerves enter the vertebral body posteriorly, via the basivertebral foramen, and cluster in the vertebral center. These nerves follow the course of the nutrient artery, which enters the vertebral body through the basivertebral foramen, then branches toward the superior and inferior endplates. Our observations support the notion that nerves found at the central endplate could originate from sinuvertebral nerves accompanying the nutrient artery into the vertebral body. We also stained neighboring histological sections with calcitonin gene‐related protein and noted significant co‐localization with PGP 9.5, substantiating a nociceptive role for the nerves constituting our distribution pattern.

Peripheral nerve lengthening by controlled isolated distraction: a new animal model.

We have developed a simple and effective animal model to study the distraction neurogenesis utilizing the sciatic nervelengthening technique in rats. The model allows macroscopic, physiological, and histological evaluation of the distraction site. Fourteen adult Harlan Sprague Dawley rats (300–350 g) were used in this study. A 10 mm segment of the right sciatic nerve of each animal in the nerve‐lengthening group was resected. Gradual nerve lengthening was performed by advancing the proximal nerve stump at a rate of 1 mm/day. The proximal stump neuroma was then resected and a direct nerve anastomosis was performed. On the left side a standard autogenous nerve‐grafting procedure was performed with a 10 mm segment of sciatic nerve used as an in situ nerve graft. Three months after the second surgery, the sciatic nerves were exposed and investigated by gross observation and EMG followed by histological processing and tissue analysis. Neomicrovascularization was observed surrounding the sciatic nerve anastomosis in all five specimens of the nerve‐lengthening group as compared to the more white‐colored scar tissue that was observed in the nerve‐grafting group. The EMG results were similar for both groups. Histological studies of the lengthened nerves showed axon morphology equivalent to the grafted nerves. This study demonstrated a clear evidence of the successful nerve regeneration within a segmental nerve gap by nerve lengthening.

Propionibacterium acnes infected intervertebral discs cause vertebral bone marrow lesions consistent with Modic changes.

Modic type I change (MC1) are vertebral bone marrow lesions adjacent to degenerated discs that are specific for discogenic low back pain. The etiopathogenesis is unknown, but occult discitis, in particular with Propionibacteria acnes (P. acnes), has been suggested as a possible etiology. If true, antibiotic therapy should be considered for patients with MC1. However, this hypothesis is controversial. While some studies report up to 40% infection rate in herniated discs, others fail to detect infected discs and attribute reports of positive cultures to contamination during sampling procedure. Irrespective of the clinical controversy, whether it is biologically plausible for P. acnes to cause MC1 has never been investigated. Therefore, the objective of this study was to test if P. acnes can proliferate within discs and cause reactive changes in the adjacent bone marrow. P. acnes was aseptically isolated from a symptomatic human L4/5 disc with MC1 and injected into rat tail discs. We demonstrate proliferation of P. acnes and up‐regulation of IL‐1 and IL‐6 within three days of inoculation. At day‐7, disc degeneration was apparent along with fibrotic endplate erosion. TNF‐α immunoreactivity was enhanced within the effected endplates along with cellular infiltrates. The bone marrow appeared normal. At day‐14, endplates and trabecular bone close to the disc were almost completely resorbed and fibrotic tissue extended into the bone marrow. T‐cells and TNF‐α immunoreactivity were identified at the disc/marrow junction. On MRI, bone marrow showed MC1‐like changes. In conclusion, P. acnes proliferate within the disc, induce degeneration, and cause MC1‐like changes in the adjacent bone marrow.

Alterations in intervertebral disc composition, matrix homeostasis and biomechanical behavior in the UCD-T2DM rat model of type 2 diabetes.

Type 2 diabetes (T2D) adversely affects many tissues, and the greater incidence of discogenic low back pain among diabetic patients suggests that the intervertebral disc is affected too. Using a rat model of polygenic obese T2D, we demonstrate that diabetes compromises several aspects of disc composition, matrix homeostasis, and biomechanical behavior. Coccygeal motion segments were harvested from 6‐month‐old lean Sprague‐Dawley rats, obese Sprague‐Dawley rats, and diabetic obese UCD‐T2DM rats (diabetic for 69 ± 7 days). Findings indicated that diabetes but not obesity reduced disc glycosaminoglycan and water contents, and these degenerative changes correlated with increased vertebral endplate thickness and decreased endplate porosity, and with higher levels of the advanced glycation end‐product (AGE) pentosidine. Consistent with their diminished glycosaminoglycan and water contents and their higher AGE levels, discs from diabetic rats were stiffer and exhibited less creep when compressed. At the matrix level, elevated expression of hypoxia‐inducible genes and catabolic markers in the discs from diabetic rats coincided with increased oxidative stress and greater interactions between AGEs and one of their receptors (RAGE). Taken together, these findings indicate that endplate sclerosis, increased oxidative stress, and AGE/RAGE‐mediated interactions could be important factors for explaining the greater incidence of disc pathology in T2D.