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 0.2 MPa 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.

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.

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, leading to activation of Rho-dependent signaling. Here we sought to determine whether the altered ECM produced by leiomyoma was accompanied by an altered state of mechanical homeostasis.Study Design-Measurement of the mechanical response in paired leiomyoma and myometrium, immunogold, confocal microscopy, and immunohistochemical analyses.Results-Leiomyoma were significantly stiffer than matched myometrium. The increased stiffness was associated with a moderate increase in total sulfated glycosaminoglycan content and a slight increase in hydroxyproline. Levels of the Rho-GEF, AKAP13, were increased and subcellular localization was altered in leiomyoma. Phosphorylation of p38MAPK was greater in leiomyoma extracts. Conclusions-Leiomyoma

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. Keywordsspine; intervertebral disc; organ culture; needle puncture; bovine; discography; mechanics; dynamic compression loading ©2008, Lippincott Williams & WilkinsAddress correspondence and reprint requests to James C. Iatridis, PhD, 201 Votey Building, 33 Colchester Avenue, Burlington, VT, 05405; E-mail: E-mail: james.iatridis@uvm.edu. NIH Public AccessAuthor Manuscript Spine (Phila Pa 1976 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 therapy 8,9 may also require puncture of t...