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Study Design: Basic science study of the relationship between spine pathology and the contractile ability of the surrounding muscles., Objective: The aim of this study was to investigate single muscle fiber contractile function in a model of progressive spine mineralization (ENT1-/- mice)., Summary of Background Data: Altered muscle structure and function have been associated with various spine pathologies; however, studies to date have provided limited insight into the fundamental ability of spine muscles to actively contract and generate force, and how this may change in response to spine pathology., Methods: Experiments were performed on two groups (ENT1-/- [KO] and ENT1+/+ [WT]) of mice at 8 months of age (n = 12 mice/group). Single muscle fibers were isolated from lumbar multifidus and erector spinae, as well as tibialis anterior (a non-spine-related control) and tested to determine their active contractile characteristics., Results: The multifidus demonstrated decreases in specific force (type IIax fibers: 36% decrease; type IIb fibers: 29% decrease), active modulus (type IIax: 35% decrease; type IIb: 30% decrease), and unloaded shortening velocity (Vo) (type IIax: 31% decrease) in the ENT1-/- group when compared to WT controls. The erector spinae specific force was reduced in the ENT1-/- mice when compared to WT (type IIax: 29% decrease), but active modulus and Vo were unchanged. There were no differences in any of the active contractile properties of the lower limb TA muscle, validating that impairments observed in the spine muscles were specific to the underlying spine pathology and not the global loss of ENT1., Conclusion: These results provide the first direct evidence of cellular level impairments in the active contractile force generating properties of spine muscles in response to chronic spine pathology.Level of Evidence: N/A., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Study Design: Basic science study of the relationship between the structural properties of the spine and its surrounding musculature., Objective: To determine whether an increase in spine stiffness causes an inverse compensatory change in the passive stiffness of the adjacent paraspinal muscles., Summary of Background Data: Intervertebral disc degeneration causes an increase in multifidus passive stiffness; this was hypothesized to compensate for a decrease in spine stiffness associated with disc degeneration. Mice lacking equilibrative nucleoside transporter 1 (ENT1) develop progressive ectopic calcification of the fibrous connective tissues of the spine, which affects the lumbar spine by 6 months of age and likely creates a mechanically stiffer spine., Methods: Experiments were conducted on four groups of mice (n = 8 mice/group): wild-type (WT) and ENT1 knockout (KO) at 2 or 8 months of age. Lumbar spines were removed and tested in cyclic axial compression to determine neutral zone length and stiffness. Single muscle fibers and bundles of fibers were isolated from lumbar multifidus and erector spinae, as well as tibialis anterior (a non-spine-related control) and tested to determine elastic modulus (passive stiffness)., Results: At 2 months of age, neither spine nor muscle stiffness was different between KO and WT. At 8 months of age, compared with WT the lumbar spines of ENT1 KO mice had a stiffer and shorter neutral zone, and the paraspinal muscle fibers were less stiff; however, fiber bundles were not different. In addition, tibialis anterior was not different between KO and WT., Conclusion: This work has confirmed that calcification of spinal connective tissues in the ENT1 KO mouse results in a stiffened spine, whereas the concurrent decrease in muscle fiber elastic modulus in the adjacent paraspinal muscles suggests a direct compensatory relationship between the stiffness of the spine and the muscles that are attached to it., Level of Evidence: N/A.

Study Design: In vitro-formed bovine nucleus pulposus (NP) tissues were used as a model for tumor necrosis factor-alpha (TNF-alpha) induced NP degeneration., Objective: To elucidate the signal transduction mechanisms regulating TNF-alpha induced matrix metalloproteinase (MMP) activity., Summary of Background Data: TNF-alpha is thought to contribute to the pathophysiology of intervertebral disc (IVD) degeneration by up-regulating MMPs, such as MMP-2. MMP-2 has been implicated in influencing disease progression and in the induction of neovascularization., Methods: In vitro-formed bovine NP tissues were treated with TNF-alpha to examine its effect on MMP-2 gene and protein levels and activity. The effect of TNF-alpha on membrane type (MT)1-MMP, an activator of MMP-2, was also assessed. MT1-MMP functional activation by TNF-alpha was confirmed using promoter-reporter luciferase constructs. Immunoblots and electrophoretic mobility shift assays were used to examine the expression and DNA binding activity of transcription factors known to regulate transcriptional activation of MT1-MMP., Results: TNF-alpha treatment induced MMP-2 gelatinase activity, which occurred in the absence of any change in MMP-2 gene or protein expression, but did correlate with increased MT1-MMP mRNA and protein levels. Up-regulation of MMP-2 activity was dependent on the ERK-MAPK pathway. ERK-1/2 activation up-regulated early growth factor (Egr-1) expression and its DNA binding activity to the MT1-MMP promoter. There was no effect on Sp-1 binding activity. Reporter constructs demonstrated that TNF-alpha induced MT1-MMP transcriptional activation and that this response was dependant on ERK MAPK and Egr-1., Conclusion: TNF-alpha induced MMP-2 gelatinase activity correlated with induction of MT1-MMP and not MMP-2 expression. MMP-2 activation was dependent on the ERK-MAPK pathway. As ERK also appeared to regulate MT1-MMP production, this suggests that TNF-alpha induction of MMP-2 gelatinase activity may be regulated by MT1-MMP. These findings elucidate the regulation of gelatinase activity and identify a mechanism whereby TNF-alpha may contribute to matrix degradation in NP tissue.

Study Design: This study examines changes in the production of extracellular matrix molecules as well as the induction of tissue degradation in in vitro formed nucleus pulposus (NP) tissues following incubation with tumor necrosis factor (TNF)alpha., Objective: To characterize the response of NP cells to TNF-alpha, a proinflammatory cytokine present in herniated NP tissues., Summary of Background Data: TNF-alpha is a proinflammatory cytokine expressed by NP cells of degenerate intervertebral discs. It is implicated in the pain associated with disc herniation, although its role in intervertebral disc degeneration remains poorly understood., Methods: In vitro formed NP tissues were treated with TNF-alpha (up to 50 ng/mL) over 48 hours. Tissues were assessed for histologic appearance, proteoglycan and collagen contents, as well as proteoglycan and collagen synthesis. Reverse transcriptase polymerase chain reaction was used to determine the effect of TNF-alpha on NP cell gene expression. Proteoglycan degradation was assessed by immunoblot analysis., Results: At doses of 1-5 ng/mL, TNF-alpha induced multiple cellular responses, including: decreased expression of both aggrecan and type II collagen genes; decreases in the accumulation and overall synthesis of aggrecan and collagen; increased expression of MMP-1, MMP-3, MMP-13, ADAM-TS4, and ADAM-TS5; and induction of ADAM-TS dependent proteoglycan degradation. Within 48 hours, these cellular responses resulted in NP tissue with only 25% of its original proteoglycan content., Conclusions: Because low levels of TNF-alpha, comparable to those present physiologically, induced NP tissue degradation, this suggests that TNF-alpha may contribute to the degenerative changes that occur in disc disease.

Study Design: This study describes the formation of nucleus pulposus tissue using a novel tissue engineering approach., Objectives: To determine if a construct composed of nucleus pulposus tissue on the surface of a calcium polyphosphate substrate could be formed in vitro with properties similar to native nucleus pulposus tissue., Summary of Background Data: There is no optimal treatment for the persistent pain associated with intervertebral disc degeneration. Disc replacement using artificial intervertebral discs has met with some success, and biologic transplantation is limited by the availability of donor tissues., Methods: Nucleus pulposus cells were isolated from bovine caudal intervertebral discs. Cells were seeded at high density on the upper surface of a porous bone substitute material (calcium polyphosphate) and maintained up to 6 weeks in culture. In vitro formed tissue was compared to native nucleus pulposus for histologic appearance, biochemical composition (tissue cellularity, proteoglycan and collagen accumulation), and compressive mechanical properties., Results: When maintained on the surface of a three-dimensional substrate, nucleus pulposus cells formed a continuous layer of tissue with a proteoglycan content equivalent to the native tissue. Although collagen accumulation attained only 26% than that of the native tissue, there was no difference in tissue stiffness, viscosity, or weight-bearing capacity of the in vitro formed tissue when compared with the native tissue., Conclusion: Nucleus pulposus-like tissue formed in vitro on the surface of a calcium polyphosphate substrate resembles the native tissue in terms of proteoglycan content and compressive mechanical properties. These studies are the first step toward developing a functional spinal unit in vitro.