Casey L. Korecki

Needle puncture injury affects intervertebral disc mechanics and biology in an organ culture model

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.

Complex loading affects intervertebral disc mechanics and biology

BACKGROUND Complex loading develops in multiple spinal motions and in the case of hyperflexion is known to cause intervertebral disc (IVD) injury. Few studies have examined the interacting biologic and structural alterations associated with potentially injurious complex loading, which may be an important contributor to chronic progressive degeneration. OBJECTIVE This study tested the hypothesis that low magnitudes of axial compression loading applied asymmetrically can induce IVD injury affecting cellular and structural responses in a large animal IVD ex-vivo model. METHODS Bovine caudal IVDs were assigned to either a control or wedge group (15°) and placed in organ culture for 7 days under static 0.2MPa load. IVD tissue and cellular responses were assessed through confined compression, qRT-PCR, histology and structural and compositional measurements, including Western blot for aggrecan degradation products. RESULTS Complex loading via asymmetric compression induced cell death, an increase in caspase-3 staining (apoptosis), a loss of aggrecan and an increase in aggregate modulus in the concave annulus fibrosis. While an up-regulation of MMP-1, ADAMTS4, IL-1β, and IL-6 mRNA, and a reduced aggregate modulus were induced in the convex annulus. CONCLUSION Asymmetric compression had direct deleterious effects on both tissue and cells, suggesting an injurious loading regime that could lead to a degenerative cascade, including cell death, the production of inflammatory mediators, and a shift towards catabolism. This explant model is useful to assess how injurious mechanical loading affects the cellular response which may contribute to the progression of degenerative changes in large animal IVDs, and results suggest that interventions should address inflammation, apoptosis, and lamellar integrity.

Notochordal cell conditioned medium stimulates mesenchymal stem cell differentiation toward a young nucleus pulposus phenotype

IntroductionMesenchymal stem cells (MSCs) offer promise for intervertebral disc (IVD) repair and regeneration because they are easily isolated and expanded, and can differentiate into several mesenchymal tissues. Notochordal (NC) cells contribute to IVD development, incorporate into the nucleus pulposus (NP), and stimulate mature disc cells. However, there have been no studies investigating the effects of NC cells on adult stem cell differentiation. The premise of this study is that IVD regeneration is more similar to IVD development than to IVD maintenance, and we hypothesize that soluble factors from NC cells differentiate MSCs to a phenotype characteristic of nucleus pulposus (NP) cells during development. The eventual clinical goal would be to isolate or chemically/recombinantly produce the active agent to induce the therapeutic effects, and to use it as either an injectable therapy for early intervention on disc disease, or in developing appropriately pre-differentiated MSC cells in a tissue engineered NP construct.MethodsHuman MSCs from bone marrow were expanded and pelleted to form high-density cultures. MSC pellets were exposed to either control medium (CM), chondrogenic medium (CM with dexamethasone and transforming growth factor, (TGF)-β3) or notochordal cell conditioned medium (NCCM). NCCM was prepared from NC cells maintained in serum free medium for four days. After seven days culture, MSC pellets were analyzed for appearance, biochemical composition (glycosaminoglycans and DNA), and gene expression profile (sox-9, collagen types-II and III, laminin-β1 and TIMP1(tissue inhibitor of metalloproteinases-1)).ResultsSignificantly higher glycosaminoglycan accumulation was seen in NCCM treated pellets than in CM or TGFβ groups. With NCCM treatment, increased gene expression of collagen III, and a trend of increasing expression of laminin-β1 and decreased expression of sox-9 and collagen II relative to the TGFβ group was observed.ConclusionsTogether, results suggest NCCM stimulates mesenchymal stem cell differentiation toward a potentially NP-like phenotype with some characteristics of the developing IVD.

Dynamic Compression Effects on Intervertebral Disc Mechanics and Biology

Study Design. A bovine intervertebral disc organ culture model was used to study the effect of dynamic compression magnitude on mechanical behavior and measurement of biosynthesis rate, cell viability, and mRNA expression. Objective. The objective of this study was to examine the effect of loading magnitude on intervertebral disc mechanics and biology in an organ culture model. Summary of Background Data. The in vivo and cell culture response of intervertebral disc cells to dynamic mechanical loading provides evidence the disc responds in a magnitude dependant manner. However, the ability to link mechanical behavior of the disc with biologic phenomena has been limited. A large animal organ culture system facilitates measurements of tissue mechanics and biologic response parameters on the same sample allowing a broader understanding of disc mechanobiology. Methods. Bovine caudal intervertebral discs were placed in organ culture for 6 days and assigned to a static control or 1 of 2 dynamic compression loading protocols (0.2–1 MPa or 0.2–2.5 MPa) at 1 Hz for 1 hour for 5 days. Disc structure was assessed with measurements of dynamic modulus, creep, height loss, water content, and proteoglycan loss to the culture medium. Cellular responses were assessed through changes in cell viability, metabolism, and qRT-PCR analyses. Results. Increasing magnitudes of compression increased disc modulus and creep; however, all mechanical parameters recovered each day. In the anulus, significant increases in gene expression for collagen I and a trend of increasing sulfate incorporation were observed. In the nucleus, increasing gene expression for collagen I and MMP3 was observed between magnitudes and between static controls and the lowest magnitude of loading. Conclusion. Results support the hypothesis that biologic remodeling precedes damage to the intervertebral disc structure, that compression is a healthy loading condition for the disc, and further support the link between applied loading and biologic remodeling.

Mechanical homeostasis is altered in uterine leiomyoma

OBJECTIVE Uterine leiomyoma produce an extracellular matrix (ECM) that is abnormal in its volume, content, and structure. Alterations in ECM can modify mechanical stress on cells and lead to activation of Rho-dependent signaling and cell growth. Here we sought to determine whether the altered ECM that is produced by leiomyoma was accompanied by an altered state of mechanical homeostasis. STUDY DESIGN We measured the mechanical response of paired leiomyoma and myometrial samples and performed immunogold, confocal microscopy, and immunohistochemical analyses. RESULTS Leiomyoma were significantly stiffer than matched myometrium. The increased stiffness was accompanied by alteration of the ECM, cell shape, and cytoskeleton in leiomyoma, compared with myometrial samples from the same uterus. Levels of AKAP13, a protein that is known to activate Rho, were increased in leiomyoma compared to myometrium. AKAP13 was associated with cytoskeletal filaments of immortalized leiomyoma cells. CONCLUSION Leiomyoma cells are exposed to increased mechanical loading and show structural and biochemical features that are consistent with the activation of solid-state signaling.

Intervertebral disc cell response to dynamic compression is age and frequency dependent.

The maintenance of the intervertebral disc extracellular matrix is regulated by mechanical loading, nutrition, and the accumulation of matrix proteins and cytokines that are affected by both aging and degeneration. Evidence suggests that cellular aging may lead to alterations in the quantity and quality of extracellular matrix produced. The aims of this study were to examine the role of loading and maturation (a subset of aging), and the interaction between these two factors in intervertebral disc cell gene expression and biosynthesis in a controlled 3D culture environment. Cells were isolated from young (4–6 months) and mature (18–24 months) bovine caudal annulus fibrosus and nucleus pulposus tissue. Isolated cells were seeded into alginate and dynamically compressed for 7 days at either 0.1, 1, or 3 Hz or maintained as a free‐swelling control. After 7 days, DNA and sulfated glycosaminoglycan contents were analyzed along with real time, quantitative reverse transcription‐polymerase chain reaction analysis for collagen types I and II, aggrecan, and matrix metalloproteinase‐3 gene expression. Results suggest that maturation plays an important role in intervertebral disc homeostasis and influences the cell response to mechanical loading. While isolated intervertebral disc cells responded to mechanical compression in 3D culture, the effect of loading frequency was minimal. Altered cellular phenotype and biosynthesis rates appear to be an attribute of the cell maturation process, potentially independent of changes in cellular microenvironment associated with lost nutrition and disc degeneration. Mature cells may have a decreased capacity to create or retain extracellular matrix components in response to mechanical loading compared to young cells.

Characterization of tissue biomechanics and mechanical signaling in uterine leiomyoma.

Leiomyoma are common tumors arising within the uterus that feature excessive deposition of a stiff, disordered extracellular matrix (ECM). Mechanical stress is a critical determinant of excessive ECM deposition and increased mechanical stress has been shown to be involved in tumorigenesis. Here we tested the viscoelastic properties of leiomyoma and characterized dynamic and static mechanical signaling in leiomyoma cells using three approaches, including measurement of active RhoA. We found that the peak strain and pseudo-dynamic modulus of leiomyoma tissue was significantly increased relative to matched myometrium. In addition, leiomyoma cells demonstrated an attenuated response to applied cyclic uniaxial strain and to variation in substrate stiffness, relative to myometrial cells. However, on a flexible pronectin-coated silicone substrate, basal levels and lysophosphatidic acid-stimulated levels of activated RhoA were similar between leiomyoma and myometrial cells. In contrast, leiomyoma cells plated on a rigid polystyrene substrate had elevated levels of active RhoA, compared to myometrial cells. The results indicate that viscoelastic properties of the ECM of leiomyoma contribute significantly to the tumors inherent stiffness and that leiomyoma cells have an attenuated sensitivity to mechanical cues. The findings suggest there may be a fundamental alteration in the communication between the external mechanical environment (extracellular forces) and reorganization of the actin cytoskeleton mediated by RhoA in leiomyoma cells. Additional research will be needed to elucidate the mechanism(s) responsible for the attenuated mechanical signaling in leiomyoma cells.

Asymmetric Loading Promotes Early Signs of Intervertebral Disc Degeneration in Large Animal Organ Culture

Intervertebral disc (IVD) degeneration is a complex pathology, involving alterations in mechanical and biological function. Mechanical injury to IVDs may contribute to the development of IVD degeneration, and can arise following excessive loading or repeated exposure to loading levels which are not instantaneously damaging. Lateral bending and flexion produced the highest maximum shear strains in human IVDs and are considered the motions that place the IVD at greatest risk of injury (1). The biological response of the IVD to combined bending and compression has been examined in vivo in rat and mouse tail bending models demonstrating structural disruption, apoptosis and remodeling (2,4). However, there are practical limitations to current in vivo studies, as it can be difficult to apply repeated bending loads to the disc in vivo, and few large animal models exist capable of tracking the early biological, structural and compositional changes from asymmetrical loading. IVD organ culture allows control over mechanical boundary conditions and investigation of cellular responses to loading while the IVD remains largely intact, and allows the use of large animal models which more closely mimic the nutritional and compositional nature of human IVDs.Copyright

NEEDLE PUNCTURE AFFECTS INTERVERTEBRAL DISC MECHANICS AND BIOLOGY IN AN ORGAN CULTURE MODEL

INTRODUCTION Many potential treatments to regenerate or repair degenerated intervertebral discs require needle delivery systems. However, there is evidence that insertion of 27G and 28G needles into the annulus fibrosus or nucleus pulposus (NP), without herniation, leads to mild and moderate degeneration over time in rabbit and sheep models [1,2]. The early mechanical and biological response to needle puncture in a large animal model is not clear. We hypothesized that significant changes in disc structure, mechanics, and cellular response would be present within one week after small or large gage needle puncture in a bovine organ culture model. METHODS Bovine tails obtained from a local abattoir within 4 hours postmortem were randomly assigned to an unpunctured control group (N=10), and one of two needle puncture groups (small = 25G syringe, N=11; large = 14G syringe, N=12). Musculature surrounding the intervertebral disc was removed. Caudal discs c3-c5 were punctured using a posterolateral approach through the annulus 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 prior to culturing. Specimens were then placed in an organ culture chamber and incubated in standard culture conditions at 37C and 5% CO2 under a 0.2 MPa static load as previously described [3]. Media was continuously circulated through the chamber (1.1mL/min) and changed every 2 days. After an initial 12 hour equilibration period under 0.2 MPa [3], chambers were individually attached to an incubator-housed dynamic loading device. A one minute test (0.2-0.4MPa, 1Hz) was applied to obtain a pre-loading dynamic nominal modulus for all groups, followed by one hour of dynamic loading (0.2-1.0MPa, 1 Hz), and finally a repeat of the one minute test to obtain a post-loading nominal dynamic modulus. Change in disc height over the one hour loading period was assessed from displacement data obtained through the loading device. After the mechanical intervention, 0.2 MPa static load was again applied to allow at least 12 hours of recovery. Loading occurred once per day (5 times) during the 6 day culture period. Glycosaminoglycan (GAG) content released to the media was assayed using the dimethylmethylene blue (DMMB) assay and was normalized to initial wet weight of the intact disc. Regional water contents for tissue samples in the outer and inner annulus (OA, IA), and NP were determined for each group by comparing wet and dry weights. Cell viability was assessed as previously described [3]. Live and dead cells were stained with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT thiazole blue, Sigma Aldrich, St. Louis, MO) and ethidium homodimer-1 (Molecular Probes, Eugene, OR), respectively. Samples were sectioned into 10 μm thick slices and images of each section were obtained at 20x under fluorescent (ethidium) and brightfield (MTT) conditions. Tissue samples were also fixed in formalin, embedded in paraffin, and stained with alcian blue (proteoglycans), picosirius red (collagen), and Weigert’s hematoxylin (cell nucleis) for histologic appearance. For all quantitative variables, ANOVA with Bonferroni-adjusted post-hoc comparisons were performed using p<0.05 significance level. RESULTS The nominal dynamic modulus was significantly affected by needle puncture (P=0.009), with average values for the large needle group being significantly lower than for control (Figure 1, P=0.023). No significant differences existed between small and large needle groups, nor small needle and control groups (P>0.19). Needle puncture also affected the height lost during the one hour load cycle, with significant differences between needle puncture and control groups (Figure 1, P<0.006). The amount of GAG released to the media and regional tissue water contents were not significantly affected by needle puncture (P>0.125). Cell viability was maintained in discs although localized cell death was observed in the area adjacent to the needle tracks. Histology also revealed annulus fiber disruption, and increased cell number and remodeling in the NP of the large needle group (Figure 2).

Effect of Age and Frequency on Intervertebral Disc Cell Response to Dynamic Compression

The intervertebral disc (IVD) is a unique orthopaedic tissue consisting of at least two cell types: fibroblast-like annulus fibrosus (AF) cells and chondrocyte-like nucleus pulposus (NP) cells. Culture of cells in 3D gel matrices (such as alginate or agarose), maintains the normal morphology and ECM molecule production of chondrocytes for extended periods of time and also allows the application of various forms of mechanical stimulation, such as hydrostatic or compressive loading. In vivo studies have shown IVD cells to be responsive to frequency, duration, and amplitude of mechanical load [1]. IVD literature on mechanobiology uses varying methodologies to apply dynamic loads (compression, hydrostatic forces), with different times of mechanical stimulation, differences in model systems (in vivo, tissue culture, cell culture), species, and ages, and an optimal loading protocol to stimulate extracellular matrix protein accumulation is unknown. The overall goal of this work is to evaluate the potential, and perhaps even feasibility, of mechanical stimulation for extracellular matrix (ECM) regeneration using intervertebral disc cells. Also of interest is whether cells from mature tissue are capable of serving as a potential cell source for future IVD regeneration [2,3].