Jeffrey C. Lotz

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.

Intervertebral Disc Cell Therapy for Regeneration: Mesenchymal Stem Cell Implantation in Rat Intervertebral Discs

This study explores the use of mesenchymal stem cells (MSCs) for intervertebral disc regeneration. We used an in vivo model to investigate the feasibility of exogenous cell delivery, retention, and survival in the pressurized disc space. MSC injection into rat coccygeal discs was performed using 15% hyaluronan gel as a carrier. Injections of gel with or without MSCs were performed. Immediately after injection, fluorescently labeled stem cells were visible on sections of cell-injected discs. Seven and 14 days after injection, stem cells were still present within the disc, but their numbers were significantly decreased. At 28 days, a return to the initial number of injected cells was observed, and viability was 100%. A trend of increased disc height compared to blank gel suggests an increase in matrix synthesis. The results indicate that MSCs can maintain viability and proliferate within the rat intervertebral disc.

Intervertebral disc cell death is dependent on the magnitude and duration of spinal loading.

Study Design. An in vivo study of the toxic consequences of static compressive stress on the intervertebral disc. Objectives. To determine whether disc cell death is correlated with the magnitude and duration of spinal compressive loading. Summary of Background Data. Static compression in vivo has been demonstrated to induce cell death. Cell death, in turn, has been associated with disc degeneration in humans. There are currently no tolerance criteria for the intervertebral disc that combine both biomechanical and biologic factors, although both have been implicated in cases of accelerated degeneration. Methods. Mouse tail discs were loaded in vivo with an external compression device. Compressive stress was applied at one of two magnitudes (0.4 and 0.8 MPa) for 7 days, and at one additional magnitude (1.3 MPa) for 1, 3, and 7 days. Midsagittal sections of the discs were stained for apoptosis using the TdT-dUTP terminal nick-end labeling (TUNEL) reaction. Quantal analysis was used to correlate the extent of cell death to the magnitude and duration of loading. Results The probit transformation of the percentage of dying cells was proportional to the sum of the logarithmic transformations of the compressive stress and the time of loading. Conclusions. The results of this study demonstrate the feasibility of developing a quantitative correlation between spinal loading and disc degeneration. Such a correlation may be coupled in the future to existing engineering models that predict spinal loading in response to physical exposures and lead to improved definition of the bounds of healthy and unhealthy spinal loading, and ultimately, refined guidelines for low back safety.

In vivo growth factor treatment of degenerated intervertebral discs.

Study Design. An in vivo model was used to investigate the response of degenerated discs to various exogenous growth factors. Objectives. To study growth factor-induced alterations of the spatial and temporal patterns of disc cellularity and matrix gene expression. Summary of Background Data. Cell proliferation and proteoglycan synthesis have been stimulated by growth factors in normal disc cells, suggesting that growth factors may play a therapeutic role for degeneration. However, the response in situ in degenerated discs has not been characterized. Methods. Degeneration was induced in murine caudal discs by static compression. Degenerated discs were given single or multiple injections of growth and differentiation factor-5, transforming growth factor-&bgr;, insulin-like growth factor-1, basic fibroblast growth factor, or saline as control. Comparisons of disc morphology, anular cell density, proliferating cells, disc height, and aggrecan and type II collagen gene expression were made either 1 week or 4 weeks after treatment. Results. In some growth and differentiation factor-5 and transforming growth factor-&bgr; treated discs, expansion of inner anular fibrochondrocyte populations into the nucleus was observed. The cells actively expressed aggrecan and type II collagen mRNA. A lesser effect was observed for insulin-like growth factor-1 and little or no effect for basic fibroblast growth factor. Differences in cell density and proliferating cells were not significant between treatments but suggested a trend of increased cellularity and proliferation following growth factor treatment. A statistically significant increase in disc height 4 weeks after growth and differentiation factor-5 treatment was measured. Conclusions. Anular fibrochondrocytes in degenerated discs are responsive to some growth factors in vivo. The results have implications in the early intervention of disc degeneration to arrest or slow the degenerative process.

Biological response of the intervertebral disc to dynamic loading

Disc degeneration is a chronic remodeling process that results in alterations of matrix composition and decreased cellularity. This study tested the hypothesis that dynamic mechanical forces are important regulators in vivo of disc cellularity and matrix synthesis. A murine model of dynamic loading was developed that used an external loading device to cyclically compress a single disc in the tail. Loads alternated at a 50% duty cycle between 0MPa and one of two peak stresses (0.9 or 1.3MPa) at one of two frequencies (0.1 or 0.01Hz) for 6h per day for 7 days. An additional group received static compression at 1.3MPa for 3h/day for 7 days. A control group wore the device with no loading. Sections of treated discs were analyzed for morphology, proteoglycan content, apoptosis, cell areal density, and aggrecan and collagen II gene expression. Dynamic loading induced differential effects that depended on frequency and stress. No significant changes to morphology, proteoglycan content or cell death were found after loading at 0.9MPa, 0.1Hz. Loading at lower frequency and/or higher stress increased proteoglycan content, matrix gene expression and cell death. The results have implications in the prevention of intervertebral disc degeneration, suggesting that loading conditions may be optimized to promote maintenance of normal structure and function.

Tenascin-x deficiency mimics ehlers-danlos syndrome in mice through alteration of collagen deposition

Tenascin-X is a large extracellular matrix protein of unknown function. Tenascin-X deficiency in humans is associated with Ehlers–Danlos syndrome, a generalized connective tissue disorder resulting from altered metabolism of the fibrillar collagens. Because TNXB is the first Ehlers–Danlos syndrome gene that does not encode a fibrillar collagen or collagen-modifying enzyme, we suggested that tenascin-X might regulate collagen synthesis or deposition. To test this hypothesis, we inactivated Tnxb in mice. Tnxb−/− mice showed progressive skin hyperextensibility, similar to individuals with Ehlers–Danlos syndrome. Biomechanical testing confirmed increased deformability and reduced tensile strength of their skin. The skin of Tnxb−/− mice was histologically normal, but its collagen content was significantly reduced. At the ultrastructural level, collagen fibrils of Tnxb−/− mice were of normal size and shape, but the density of fibrils in their skin was reduced, commensurate with the reduction in collagen content. Studies of cultured dermal fibroblasts showed that although synthesis of collagen I by Tnxb−/− and wildtype cells was similar, Tnxb−/− fibroblasts failed to deposit collagen I into cell-associated matrix. This study confirms a causative role for TNXB in human Ehlers–Danlos syndrome and suggests that tenascin-X is an essential regulator of collagen deposition by dermal fibroblasts.

Magnetic resonance imaging of trabecular bone structure in the distal radius: Relationship with X-ray tomographic microscopy and biomechanics

The contribution of trabecular bone structure to bone strength is of considerable interest in the study of osteoporosis and other disorders characterized by changes in the skeletal system. Magnetic resonance (MR) imaging of trabecular bone has emerged as a promising technique for assessing trabecular bone structure. In this in vitro study we compare the measures of trabecular structure obtained using MR imaging and higher-resolution X-ray tomographic microscopy (XTM) imaging of cubes from human distal radii. The XTM image resolution is similar to that obtained from histomorphometric sections (18 µm isotropic), while the MR images are obtained at a resolution comparable to that achievable in vivo (156×156×300 µm). Standard histomorphometric measures, such as trabecular bone area fraction (synonymous with BV/TV), trabecular width, trabecular spacing and trabecular number, texture-related measures and three-dimensional connectivity (first Betti number/volume) of the trabecular network have been derived from these images. The variation in these parameters as a function of resolution, and the relationship between the structural parameters, bone mineral density and the elastic modulus are also examined. In MR images, because the resolution is comparable to the trabecular dimensions, partial volume effects occur, which complicate the segmentation of the image into bone and marrow phases. Using a standardized thresholding criterion for all images we find that there is an overestimation of trabecular bone area fraction (∼3 times), trabecular width (∼3 times), fractal dimension (∼1.4 times) and first Betti number/ volume (∼10 times), and an underestimation of trabecular spacing (∼1.6 times) in the MR images compared with the 18-µm XTM images. However, even for a factor of 9 difference in spatial resolution, the differences in the morphological trabecular structure measures ranged from a factor of 1.4 to 3.0. We have found that trabecular width, area fraction, number, fractal dimension and Betti number/volume measured from the XTM and MR images increases, while trabecular spacing decreases, as the bone mineral density and elastic modulus increase. A preliminary bivariate analysis showed that in addition to bone mineral density alone, the Betti number, trabecular number and spacing contributed to the prediction of the elastic modulus. This preliminary study indicates that measures of trabecular bone structure using MR imaging may play a role in the study of osteoporosis.

Intervertebral Disc Degeneration: The Role of the Mitochondrial Pathway in Annulus Fibrosus Cell Apoptosis Induced by Overload

Degeneration of the intervertebral disk (IVD) is a major pathological process implicated in low back pain and is a prerequisite to disk herniation. Although mechanical stress is an important modulator of the degeneration, the underlying molecular mechanism remains unclear. The association of human IVD degeneration, assessed by magnetic resonance imaging, with annulus fibrosus cell apoptosis and anti-cytochrome c staining revealed that the activation of the mitochondria-dependent apoptosome was a major event in the degeneration process. Mouse models of IVD degeneration were used to investigate the role of the mechanical stress in this process. The application of mechanical overload (1.3 MPa) for 24 hours induced annulus fibrosus cell apoptosis and led to severe degeneration of the mouse disks. Immunostaining revealed cytochrome c release but not Fas-L generation. The role of the caspase-9-dependent mitochondrial pathway in annulus fibrosus cell apoptosis induced by overload was investigated further with the use of cultured rabbit IVD cells in a stretch device. Mechanical overload (15% area change) induced apoptosis with increased caspase-9 activity and decreased mitochondrial membrane potential. Furthermore, Z-LEHD-FMK, a caspase-9 inhibitor, but not Z-IETD-FMK, a caspase-8 inhibitor, attenuated the overload-induced apoptosis. Our results from human samples, mouse models, and annulus fibrosus culture experiments demonstrate that the mechanical overload-induced IVD degeneration is mediated through the mitochondrial apoptotic pathway in IVD cells.

Multiple Differentiation Capacity of STRO-1+/CD146+ PDL Mesenchymal Progenitor Cells

Although mesenchymal progenitor cells can be isolated from periodontal ligament (PDL) tissues using stem cell markers STRO-1 and CD146, the proportion of these cells that have the capacity to differentiate into multiple cell lineages remains to be determined. This study was designed to quantify the proportions of primary human PDL cells that can undergo multilineage differentiation and to compare the magnitude of these capabilities relative to bone marrow-derived mesenchymal stem cells (MSCs) and parental PDL (PPDL) cells. PDL mesenchymal progenitor (PMP) cells were isolated from PPDL cells using the markers STRO-1 and CD146. The colony-forming efficiency and multilineage differentiation potential of PMP, PPDL, and MSCs under chondrogenic, osteogenic, and adipogenic conditions were determined. Flow cytometry revealed that on average 2.6% of PPDL cells were STRO-1(+)/CD146(+), whereas more than 63% were STRO-1(-)/CD146(-). Colony-forming efficiency of STRO-1(+)/CD146(+) PMP cells (19.3%) and MSCs (16.7%) was significantly higher than that of PPDL cells (6.8%). Cartilage-specific genes, early markers of osteoblastic differentiation, and adipogenic markers were significantly upregulated under appropriate conditions in PMP cells and MSCs compared to either their noninduced counterparts or induced PPDL cells. Consistent with these findings, immunohistochemistry revealed substantial accumulation of cartilaginous macromolecules, mineralized calcium nodules, and lipid vacuoles under chondrogenic, osteogenic, or adipogenic conditions in PMP and MSC cultures, respectively, compared to noninduced controls or induced PPDL cells. Thus STRO-1(+)/CD146(+) PMP cells demonstrate multilineage differentiation capacity comparable in magnitude to MSCs and could potentially be utilized for regeneration of the periodontium and other tissues.

Animal models of intervertebral disc degeneration: lessons learned.

Study Design. A literature review of intervertebral disc degeneration animal models. Objectives. Focus is placed on those models that suggest degeneration mechanisms relevant to human. Summary of Background Data. Medical knowledge from observational epidemiology and intervention studies suggest many etiologic causal factors in humans. Animal models can provide basic science data that support biologic plausibility as well as temporality, specificity, and dose-response relationships. Methods. Studies are classified as either experimentally induced or spontaneous, where experimentally induced models are subdivided as mechanical (alteration of the magnitude or distribution of forces on the normal joint) or structural (injury or chemical alteration). Spontaneous models include those animals that naturally develop degenerative disc disease. Results. Mechanobiologic relationships are apparent as stress redistribution secondary to nuclear depressurization (by injury or chemical means) can cause cellular metaplasia, tissue remodeling, and pro-inflammatory factor production. Moderate perturbations can be compensated for by cell proliferation and matrix synthesis, whereas severe perturbations cause architectural changes consistent with human disc degeneration. Conclusions. These models suggest that two stages of architectural remodeling exist in humans: early adaptation to gravity loading, followed by healing meant to reestablish biomechanical stability that is slowed by tissue avascularity. Current animal models are limited by an incomplete set of initiators and outcomes that are only indirectly related to important clinical factors (pain and disability).