Jeffrey J. MacLean

In Vivo Remodeling of Intervertebral Discs in Response to Short- and Long-Term Dynamic Compression

This study evaluated how dynamic compression induced changes in gene expression, tissue composition, and structural properties of the intervertebral disc using a rat tail model. We hypothesized that daily exposure to dynamic compression for short durations would result in anabolic remodeling with increased matrix protein expression and proteoglycan content, and that increased daily load exposure time and experiment duration would retain these changes but also accumulate changes representative of mild degeneration. Sprague‐Dawley rats (n = 100) were instrumented with an Ilizarov‐type device and divided into three dynamic compression (2 week–1.5 h/day, 2 week–8 h/day, 8 week–8 h/day at 1 MPa and 1 Hz) and two sham (2 week, 8 week) groups. Dynamic compression resulted in anabolic remodeling with increased matrix mRNA expression, minimal changes in catabolic genes or disc structure and stiffness, and increased glysosaminoglycans (GAG) content in the nucleus pulposus. Some accumulation of mild degeneration with 8 week–8 h included loss of annulus fibrosus GAG and disc height although 8‐week shams also had loss of disc height, water content, and minor structural alterations. We conclude that dynamic compression is consistent with a notion of “healthy” loading that is able to maintain or promote matrix biosynthesis without substantially disrupting disc structural integrity. A slow accumulation of changes similar to human disc degeneration occurred when dynamic compression was applied for excessive durations, but this degenerative shift was mild when compared to static compression, bending, or other interventions that create greater structural disruption.

Effects of Mechanical Loading on Intervertebral Disc Metabolism In Vivo

The overall goal of this work is to define more clearly which mechanical loading conditions are associated with accelerated disc degeneration. This article briefly reviews recent studies describing the effects of mechanical loading on the metabolism of intervertebral disc cells and defines hypothetical models that provide a framework for quantitative relationships between mechanical loading and disc-cell metabolism. Disc cells respond to mechanical loading in a manner that depends on loading magnitude, frequency, and duration. On the basis of the current data, four models have been proposed to describe the effects of continuous loading on cellular metabolism: (1) on/off response, in which messenger ribonucleic acid (mRNA) transcription remains altered for the duration of loading; (2) maintenance, characterized by an initial change in mRNA levels with return to baseline levels; (3) adaptation, in which mRNA transcription is altered and remains at a new steady state; and (4) no response. In addition, five hypothetical mechanisms that describe the long-term consequences of these metabolic changes on disc-remodeling are presented. The transient nature of gene expression along with enzyme activation/inhibition is associated with changes at the protein level. The hypothetical models presented provide a framework for obtaining quantitative relationships between mechanical loading, gene expression, and changes at the compositional level; however, additional factors, such as regulatory mechanisms, must also be considered when describing disc-remodeling. A more quantitative relationship between mechanical loading effects and the metabolic response of the disc will contribute to injury prevention, facilitate more effective rehabilitation treatments, and help realize the potential of biologic and tissue engineering approaches toward disc repair.

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.

Measurements of proteoglycan and water content distribution in human lumbar intervertebral discs.

Study Design. Study of regional variations in composition in a sample of 9 mildly to moderately degenerated human intervertebral discs. Objective. The aim of this study was to obtain proteoglycan distribution in human lumbar discs with high position resolution in the: 1) sagittal, 2) coronal, and 3) axial directions. Summary of Background Data. Regional variation in disc proteoglycan content has only been reported in coronal sections in a small number of discs and with low spatial resolution in the sagittal direction, and has not been reported in the axial direction. Methods. Each of 9 human L2–L3 or L3–L4 lumbar discs (age, 53–56 years) were dissected into 36 to 41 specimens using a rectangular cutting die, measured for water content and analyzed for glycosaminoglycan content using the dimethylmethylene blue dye binding assay. Results. The intervertebral discs were mildly to moderately degenerated. They had glycosaminoglycan content ranging ∼40 to 600 &mgr;g/mg dry tissue, with largest values in the nucleus and lowest in the outer anulus. In general, posterior regions had greater glycosaminoglycan content than anterior regions, although values were not as high as in the nucleus. Small variations in glycosaminoglycan content in the axial direction were observed with the largest values in the center, although this variation was small compared with radial variations. Water content results followed similar trends as glycosaminoglycan content with average values ranging from ∼66% to 86%. Conclusions. A refined map of proteoglycan content is presented with 3 important findings. First, sagittal variations were distinct from coronal variations. Second, the proteoglycan content was not uniform across the nucleus regions. Third, some specimens had localized variations in proteoglycan and water contents suggestive of focal damage and degeneration.

Different effects of static versus cyclic compressive loading on rat intervertebral disc height and water loss in vitro.

Study Design. In vitro biomechanical study on rat caudal motion segments to evaluate association between compressive loading and water content under static and cyclic conditions. Objective. To test hypotheses: 1) there is no difference in height loss and fluid (volume) loss of discs loaded in compression under cyclic (0.15–1.0 MPa) and static conditions with the same root-mean-square (RMS) magnitudes (0.575 MPa); and 2) after initial disc bulge, tissue water loss is directly proportional to height loss under static loading. Summary of Background Data. Disc degeneration affects water content, elastic and viscoelastic behaviors. There is limited understanding of the association between transient water loss and viscoelastic creep in a controlled in vitro environment where inferences may be made regarding mechanisms of viscoelasticity. Methods. A total of 126 caudal motion segments from 21 Wistar rats were tested in compression using 1 of 6 protocols: Static loading at 1.0 MPa for 9, 90, and 900 minutes, Cyclic loading at 0.15 to 1.0 MPa/1 Hz for 90 minutes, Mid-Static loading at 0.575 MPa for 90 minutes, and control. Water content was then measured in anulus and nucleus regions. Results. Percent water loss was significantly greater in nucleus than anulus regions, suggesting some water redistribution, with average values under 1 MPa static loading of 23.0% and 14.9% after 90 minutes and 26.9% and 17.6% after 900 minutes, respectively. Cyclic loading resulted in significantly greater height loss (0.506 ± 0.108 mm) than static loading with the same RMS value (0.402 ± 0.096 mm), but not significantly less than static loading at peak value (0.539 ± 0.122 mm). Significant and strong correlations were found between percent water loss and disc height loss, suggesting water was lost through volume decrease. Conclusion. Peak magnitude of cyclic compression and not RMS value was most important in determining height change and water loss, likely due to differences between disc creep and recovery rates. Water redistribution from nucleus to anulus occurred under loading consistent with an initial elastic compression (and associated disc bulge) followed by a reduction in disc volume over time.

In vivo intervertebral disc remodeling: Kinetics of mRNA expression in response to a single loading event

Kinetics of mRNA expression following a single loading event was measured using an in vivo rat tail model. Animals were instrumented and loaded in compression for 1.5 h at 1 MPa and 1 Hz. Real‐time RT‐PCR was used to measure mRNA levels 0, 8, 24 and 72 h after mechanical stimulation for genes associated with matrix proteins (aggrecan, collagen‐I, collagen‐II), proteases (MMP‐2, MMP‐3, MMP‐13, ADAMTS‐4), and their inhibitors (TIMP‐1, TIMP‐3) in anulus fibrosus and nucleus pulposus regions. Baseline mRNA levels were of greatest abundance for matrix proteins and lowest for proteases. The mRNA levels reached maximum levels 24 h following mechanical stimulation for the majority of genes evaluated, but some had maximum levels 8 and 72 h following loading. The mRNA levels returned to baseline levels for all genes in the nucleus 72 h following loading, but the majority of genes in the anulus remained upregulated. Results support a coordinated strategy of relative mRNA expression that varied over time beginning with inhibition of tissue breakdown, followed by synthesis of aggrecan and matrix degrading enzymes, and eventually collagen metabolism days following loading. Consequently, optimal time for tissue harvest for mRNA measurements depends on genes of interest. Results suggest attempts at anabolic remodeling must be given adequate time for metabolic processes and protein synthesis to occur, and that changes in TIMP and MMP levels may have greater potency in affecting structural protein abundance than direct changes in the structural protein messages. Results have important implications for disc remodeling and tissue engineering.

Dynamics at Lys-553 of the acto-myosin interface in the weakly and strongly bound states.

Lys-553 of skeletal muscle myosin subfragment 1 (S1) was specifically labeled with the fluorescent probe FHS (6-[fluorescein-5(and 6)-carboxamido]hexanoic acid succinimidyl ester) and fluorescence quenching experiments were carried out to determine the accessibility of this probe at Lys-553 in both the strongly and weakly actin-bound states of the MgATPase cycle. Solvent quenchers of varying charge [nitromethane, (2,2,6, 6-tetramethyl-1-piperinyloxy) (TEMPO), iodide (I(-)), and thallium (Tl(+))] were used to assess both the steric and electrostatic accessibilities of the FHS probe at Lys-553. In the strongly bound rigor (nucleotide-free) and MgADP states, actin offered no protection from solvent quenching of FHS by nitromethane, TEMPO, or thallium, but did decrease the Stern-Volmer constant by almost a factor of two when iodide was used as the quencher. The protection from iodide quenching was almost fully reversed with the addition of 150 mM KCl, suggesting this effect is ionic in nature rather than steric. Conversely, actin offered no protection from iodide quenching at low ionic strength during steady-state ATP hydrolysis, even with a significant fraction of the myosin heads bound to actin. Thus, the lower 50 kD subdomain of myosin containing Lys-553 appears to interact differently with actin in the weakly and strongly bound states.

Development of a System for Application of Dynamic Mechanical Compression on Intervertebral Discs in Organ Culture and Investigation of Load Magnitude Effects

In vivo studies on the intervertebral disc (IVD) indicate that the magnitude, frequency, and duration of applied compression loading results in alterations in mRNA expression, composition, and annulus fibrosus structure [1]. In vivo models typically use small animal models or small sample sizes that make it difficult to evaluate multiple dependent variables on the same tissue. In this study, it was considered a priority to utilize a large animal model to investigate the effects of magnitude of compression loading on interacting dependent variable measurements of disc cell viability, biosynthesis, composition, structure, and biomechanics. A bovine IVD organ culture system was used because it provides control over mechanical and chemical boundary conditions while maintaining viable cells and normal cell-matrix interactions. To date, there are no studies investigating the response of the IVD in organ culture to dynamic mechanical loading.Copyright

Mechanical Damage to the Intervertebral Disc Annulus Fibrosus Subjected to Cyclic Tensile Loading

Damage progression in the circumferential direction of the disc annulus is likely to occur in vivo in response to cyclic loading with associated degradation in tensile material properties, yet this information is not available in the literature. We hypothesize that damage of the annulus will be increased by the number of cycles and magnitude of strain applied to the tissue. Therefore, the objective of this study is to obtain a quantitative relationship between number of cycles and magnitude of tensile strain and damage on the annulus fibrosus. Damage to the annulus is assessed through measurement of permanent deformation (% elongation) and peak stress in the tissue under cyclic loading conditions.Copyright