Nutrient supply and nucleus pulposus cell function: effects of the transport properties of the cartilage endplate and potential implications for intradiscal biologic therapy.

OBJECTIVE: Intradiscal biologic therapy is a promising strategy for managing intervertebral disc degeneration. However, these therapies require a rich nutrient supply, which may be limited by the transport properties of the cartilage endplate (CEP). This study investigated how fluctuations in CEP transport properties impact nutrient diffusion and disc cell survival and function. DESIGN: Human CEP tissues harvested from six fresh cadaveric lumbar spines (38-66 years old) were placed at the open sides of diffusion chambers. Bovine nucleus pulposus (NP) cells cultured inside the chambers were nourished exclusively by nutrients diffusing through the CEP tissues. After 72 h in culture, depth-dependent NP cell viability and gene expression were measured, and related to CEP transport properties and biochemical composition determined using fluorescence recovery after photobleaching and Fourier transform infrared (FTIR) spectroscopy. RESULTS: Solute diffusivity varied nearly 4-fold amongst the CEPs studied, and chambers with the least permeable CEPs appeared to have lower aggrecan, collagen-2, and matrix metalloproteinase-2 gene expression, as well as a significantly shorter viable distance from the CEP/nutrient interface. Increasing chamber cell density shortened the viable distance; however, this effect was lost for low-diffusivity CEPs, which suggests that these CEPs may not provide enough nutrient diffusion to satisfy cell demands. Solute diffusivity in the CEP was associated with biochemical composition: low-diffusivity CEPs had greater amounts of collagen and aggrecan, more mineral, and lower cross-link maturity. CONCLUSIONS: CEP transport properties dramatically affect NP cell survival/function. Degeneration-related CEP matrix changes could hinder the success of biologic therapies that require increased nutrient supply.

Inflammatory response of disc cells against Propionibacterium acnes depends on the presence of lumbar Modic changes.

PURPOSE: Intervertebral disc with Propionibacterium acnes (P. acnes) is suggested to be an etiology of Modic type I changes in the adjacent bone marrow. However it is unknown if disc cells can respond to P. acnes and if bone marrow cells respond to bacterial and disc metabolites draining from infected discs. METHODS: Human disc cells (n = 10) were co-cultured with 10- and 100-fold excess of P. acnes over disc cells for 3 h and 24 h. Lipopolysaccharide was used as positive control. Expression of IL1, IL6, IL8, and CCL2 by disc cells was quantified by quantitative PCR. Lipase activity was measured in culture supernatants (n = 6). Human vertebral bone marrow mononuclear cells (BMNCs) (n = 2) were cultured in conditioned media from disc cell/P. acnes co-cultures and expression of IL1, IL6, IL8, and CCL2 was measured after 24 h. RESULTS: All disc cells responded to lipopolysaccharide but only 6/10 responded to P. acnes with increased cytokine expression. Cytokine increase was time- but not P. acnes concentration-dependent. Disc cell responsiveness was associated with the presence of lumbar Modic changes in the donor. Lipase activity was increased independent of disc cell responsiveness. BMNCs responded with inflammatory activity only when cultured in supernatants from responsive disc cell lines. CONCLUSION: Disc cell responsiveness to P. acnes associates with the presence of lumbar Modic changes. Furthermore, bone marrow cells had an inflammatory response to the cocktail of disc cytokines and P. acnes metabolites. These data indicate that low virulent P. acnes infection of the disc is a potential exacerbating factor to Modic changes.

Comparison of vertebral and intervertebral disc lesions in aging humans and rhesus monkeys.

OBJECTIVE: To compare gross and histologic patterns of age-related degeneration within the intervertebral disc and adjacent vertebra between rhesus monkeys and humans. MATERIALS AND METHODS: We examined age-related patterns of disc degeneration from mid-sagittal sections of the intervertebral disc and adjacent vertebral bodies (VB) among six rhesus monkey thoracolumbar and seven human lumbar spines. Gross morphology and histopathology were assessed via the Thompson grading scheme and other degenerative features of the disc and adjacent bone. RESULTS: Thompson grades ranged from 3 through 5 for rhesus monkey discs (T9-L1) and 2 through 5 for the human discs (T12-S1). In both rhesus monkey and human discs, presence of distinct lesions was positively associated with Thompson grade of the overall segment. Degenerative patterns differed for radial tears, which were more prevalent with advanced disc degeneration in humans only. Additionally, compared to the more uniform anteroposterior disc degeneration patterns of humans, rhesus monkeys showed more severe osteophytosis and degeneration on the anterior border of the vertebral column. CONCLUSIONS: Rhesus monkey spines evaluated in the present study appear to develop age-related patterns of disc degeneration similar to humans. One exception is the absence of an association between radial tears and disc degeneration, which could reflect species-specific differences in posture and spinal curvature. Considering rhesus monkeys demonstrate similar patterns of disc degeneration, and age at a faster rate than humans, these findings suggest longitudinal studies of rhesus monkeys may be a valuable model for better understanding the progression of human age-related spinal osteoarthritis (OA) and disc degeneration.

The role of the vertebral end plate in low back pain.

End plates serve as the interface between rigid vertebral bodies and pliant intervertebral disks. Because the lumbar spine carries significant forces and disks don't have a dedicated blood supply, end plates must balance conflicting requirements of being strong to prevent vertebral fracture and porous to facilitate transport between disk cells and vertebral capillaries. Consequently, end plates are particularly susceptible to damage, which can increase communication between proinflammatory disk constituents and vascularized vertebral bone marrow. Damaged end plate regions can be sites of reactive bone marrow lesions that include proliferating nerves, which are susceptible to chemical sensitization and mechanical stimulation. Although several lines of evidence indicate that innervated end plate damage can be a source of chronic low back pain, its role in patients is likely underappreciated because innervated damage is poorly visualized with diagnostic imaging. This literature review summarizes end plate biophysical function and aspects of pathologic degeneration that can lead to vertebrogenic pain. Areas of future research are identified in the context of unmet clinical needs for patients with chronic low back pain.

Structured three-dimensional co-culture of mesenchymal stem cells with chondrocytes promotes chondrogenic differentiation without hypertrophy.

OBJECTIVE: This study investigated a novel approach to induce chondrogenic differentiation of human mesenchymal stem cells (hMSC). We hypothesized that a structured three-dimensional co-culture using hMSC and chondrocytes would provide chondroinductive cues to hMSC without inducing hypertrophy. METHOD: In an effort to promote optimal chondrogenic differentiation of hMSC, we created bilaminar cell pellets (BCPs), which consist of a spherical population of hMSC encased within a layer of juvenile chondrocytes (JC). In addition to histologic analyses, we examined proteoglycan content and expression of chondrogenic and hypertrophic genes in BCPs, JC pellets, and hMSC pellets grown in the presence or absence of transforming growth factor-ß (TGFß) following 21 days of culture in either growth or chondrogenic media. RESULTS: In either growth or chondrogenic media, we observed that BCPs and JC pellets produced more proteoglycan than hMSC pellets treated with TGFß. BCPs and JC pellets also exhibited higher expression of the chondrogenic genes Sox9, aggrecan, and collagen 2A1, and lower expression of the hypertrophic genes matrix metalloproteinase-13, Runx2, collagen 1A1, and collagen 10A1 than hMSC pellets. Histologic analyses suggest that JC promote chondrogenic differentiation of cells in BCPs without hypertrophy. Furthermore, when cultured in hypoxic and inflammatory conditions intended to mimic the injured joint microenvironment, BCPs produced significantly more proteoglycan than either JC pellets or hMSC pellets. CONCLUSION: The BCP co-culture promotes a chondrogenic phenotype without hypertrophy and, relative to pellet cultures of hMSCs or JCs alone, is more resistant to the adverse conditions anticipated at the site of articular cartilage repair.

Laparoscopic surgical education--the experience of the first surgical unit Iasi.

The classic apprenticeship model for surgical training takes place into the operating theater under the strict coordination of a senior surgeon. During the time and especially after the introduction of minimally invasive techniques as gold standard treatment for many diseases, other methods were developed to successful fulfill the well known three stages of training: skill-based behavior, rule-based behavior and knowledge-based behavior. The skills needed for minimally invasive surgery aren't easily obtained using classical apprenticeship model due to ethical, medico-legal and economic considerations. In this way several types of simulators have been developed. Nowadays simulators are worldwide accepted for laparoscopic surgical training and provide formative feedback which allows an improvement of the performances of the young surgeons. The simulators currently used allow assimilating only skill based behavior and rule-based behavior. However, the training using animal models as well as new virtual reality simulators and augmented reality offer the possibility to achieve knowledge-based behavior. However it isn't a worldwide accepted laparoscopic training curriculum. We present our experience with different types of simulators and teaching methods used along the time in our surgical unit. We also performed a review of the literature data.

Minimizing errors associated with calculating the location of the helical axis for spinal motions.

One of the more common comparative tools used to quantify the motion of the vertebral joint is the orientation and position of the (finite) helical axis of motion as well as the amount of translation along, and rotation about, this axis. A survey of recent studies that utilize the helical axis of motion to compare motion before and after total disc replacement reveals a lack of concern for the relative errors associated with this metric. Indeed, intrinsic algorithmic and experimental errors that arise when interpreting motion tracking data can easily lead to a misinterpretation of the changes caused by replacement disc devices. While previous studies examining these errors exist, most have overlooked the errors associated with the determination of the location of the helical axis and its intersection with a chosen plane. The purpose of the study presented in this paper was to evaluate the sensitivity and reliability of the helical axis of motion as a comparative tool for kinematically evaluating spinal prostheses devices. To this end, we simulated a typical spine biomechanics testing experiment to investigate the accuracy of calculating the helical axis and its associated parameters using several popular algorithms. The resultant data motivated the development of a new algorithm that is a hybrid of two existing algorithms. The improved accuracy of this hybrid method made it possible to quantify some of the changes to the kinematics of a spinal unit that are induced by distinct placements of a total disc replacement.

Inguinal hernia--the practicalities of TAPP repair.

In a previous paper in this Journal, the case for laparoscopic inguinal hernia repair by the transabdominal preperitoneal (TAPP) approach was made, in terms of superiority of outcomes in both recurrence rate and long-term symptoms. This approach has been used by many people, but not always with such good outcomes. This may be due to inattention to detail. The technique used by the author is described, as if following the patient through his surgical pathway. Instrumentation, materials (including composition, configuration and placement) are delineated, suggesting that tailoring the mesh to the individual patient and his hernia are justified, after looking at the outcomes for different mesh sizes.

Laparoscopic hernia repair--the best option?

For 100 years the Bassini-type repair for inguinal hernia was the standard method. The Lichtenstein "tension free" mesh repair replaced it on the grounds of much lower recurrence rates, < 5% vs approximately 15%. However, open procedures all have significant long-term discomfort rates of up to 53%. Laparoscopic repair has become a genuine option in the last 15 years and offers low recurrence (< 1%) and minimal long-term discomfort. However, it has not been widely taken up. There is a common misconception that it takes longer to perform, has more complications and is much more expensive. None of these caveats stand up under objective scrutiny. It is time that laparoscopic repair became the method of choice for most elective inguinal hernia repairs.

Disc regeneration: why, when, and how.

There is increasing acknowledgment that patients with back pain who are candidates for surgery, will benefit over the long term from less invasive procedures that facilitate dynamic stabilization, rather than fusion. Dynamic stabilization can be addressed by providing assistance using mechanical devices, or relying on biologic processes such as tissue regeneration and repair. The concept of biologic disc repair has grown in recent years because of improved understanding of the cellular and molecular events of disc aging and degeneration. This article describes approaches to cell therapy, reviews relevant studies, and discusses ways to maximize clinical efficacy. Tissue engineering approaches for disc regeneration and healing have significant clinical potential.

Biaxial testing of human annulus fibrosus and its implications for a constitutive formulation.

Internal pressure in the healthy human annulus fibrosus leads to multiaxial stress in vivo, yet uniaxial tests have been used exclusively to characterize its in vitro mechanical response and to determine its elastic strain energy function (W). We expected that biaxial tension tests would provide unique and necessary data for characterizing the annular material response, and thereby, for determining W. We performed uniaxial and biaxial tests on specimens of annulus, then developed an objective methodology for defining an appropriate form for W that considers data from multiple experiments simultaneously and allows the data to dictate more directly the form and the number of parameters needed. We found that the stresses attained in the biaxial tests were higher, while the strains were considerably lower, than those observed in the uniaxial tests. A comparison of strain energy functions determined from the different data sets demonstrated that constitutive models derived from uniaxial data could not predict annulus behavior in biaxial tension and vice versa. Since the annulus is in a state of multaxial stress in vivo, we conclude that uniaxial tests alone are insufficient to prescribe a physiologically relevant W for this tissue.

Polarization-modulated second harmonic generation imaging: method for quantitative assessment of disorganization in anulus.

An experimental method for quantifying disorder within the anulus fibrosus is described based on polarization-modulated second harmonic generation imaging (PM-SHG-I). This method is demonstrated by imaging the anular lamellar architecture of a mouse model of compressive loading. Results were consistent with those obtained in an earlier study where organization was quantified directed secants image analysis on photomicrographs. In this study the orientation within individual lamellia is quantified by average orientation of the collagen molecules within a defined volume of a single lamellar as measured by the PM-SHG-I. Lamellar boundaries can be identified through the SHG intensity images, and confirmed through co-registration with photomicrographs of the same region. The orientation within the lamellar is quantified by the polarization angle of the maximum second harmonic intensity. PM-SHG-I offers several advantages as compared with the method of directed secants: first, it is nondestructive, allowing repeated measurements of the same tissue; second, images are captured on the order of seconds and capable of obtaining information up to a depth of 200-300 microns, thus allowing for real-time assessment of load damage; third, organization is measured at a much higher resolution, as it is based on disorder within the molecular arrays of a single lamella.

Mechanobiology in intervertebral disc degeneration and regeneration.

The intervertebral disc is an avascular, pliant, composite structure that separates spinal vertebrae and, in health, serves to support compression and facilitate movement. Its morphological organization is directed by fluid pressure and consists of a central swelling gel (nucleus), surrounded peripherally by a constraining ligament (annulus fibrosus), and separated from adjacent vertebrae by semi-permeable membranes (endplate). These three tissues serve differing structural roles, are subjected to differing mechanical environments, and are composed of unique matrices and cells. Viewing disc cells as mechanosensors, we use in vivo models of disc loading to identify spatial and temporal relationships between stress/strain and cell function that define normal morphology and drive the architectural changes attributed to normal aging and degeneration. Intra-discal stress patterns consistent with disc health can then be elucidated based on these relationships, and in turn, help us develop spine-loading criteria that parameterize injury tolerance. This same perspective is critical for tissue engineering approaches for disc repair. Cells and matrices meant to guide healing need to withstand the demanding mechanical forces in the acute phases, and differentiate/remodel along the appropriate trajectory in the long-term. Because of their unique potential for adaptation, we are exploring the mechanoplasticity of mesenchymal stem cells (MSCs) and their use in disc repair strategies. Our data demonstrate that these cells respond differentially to pressure and distortion, and can be delivered, retained, and survive in the disc's demanding mechanical/biochemical environment. Because of these features, MSCs are qualified as an intriguing autograft cell type for disc repair.

Mechanobiology of the intervertebral disc.

Intervertebral disc degeneration has been linked in humans to extreme spinal loading regimens. However, mechanisms by which spinal force influences disc cellularity, morphology and consequently biomechanical function are unclear. To gain insight into mechanobiological interactions within the disc, we developed an in vivo murine tail-compression model. Results from this model demonstrate how deviations in spinal stress induce a cycle of altered cell function and morphology as the disc remodels to a new homoeostatic configuration.

Magnetic resonance imaging measurement of relaxation and water diffusion in the human lumbar intervertebral disc under compression in vitro.

STUDY DESIGN: Twelve lumbar intervertebral disc specimens were imaged with magnetic resonance imaging to estimate relaxation constants, T1 and T2, and tissue water diffusion, before and after applying compression. OBJECTIVES: The objectives of the study were to measure T1, T2, and water diffusion for differences with loading state, region of the disc (anulus fibrosus or nucleus pulposus), and grade of degeneration. SUMMARY OF BACKGROUND DATA: Magnetic resonance imaging can be used qualitatively to estimate water content and degeneration of the intervertebral disc. Beyond structural information of images, the relaxation times T1 and T2 may contain information on the changes occurring with degeneration. A modified spin-echo sequence can be used to estimate tissue water diffusion in cartilage and disc specimens with the ability to measure anisotropy. METHODS: Specimens were imaged in a 1.5-Tesla clinical scanner. T1, T2, and water diffusion were estimated from midsagittal images. Magnetic resonance imaging parameters were calculated before and after axial loading. The measured T1, T2, and D (diffusion coefficient) were compared before and after compression, and for the diffusion data, also by direction to consider anisotropy. RESULTS: For the T1 data, a significant difference was found by region, nucleus > anulus, and loading state, loaded > unloaded. For the T2 values, there was a significant difference by region, nucleus > anulus, and Thompson grade. For diffusion, significant differences were found by region, nucleus > anulus, Thompson grade, direction of diffusion, and state of compression, loaded > unloaded. CONCLUSIONS: This study demonstrated that magnetic resonance imaging can be used to measure significant changes in T1, T2, or diffusion in intervertebral disc specimens by region, loading condition, or Thompson grade.

Acute biomechanical and histological effects of intradiscal electrothermal therapy on human lumbar discs.

STUDY DESIGN: Human cadaver lumbar spines were used to assess the acute effects of intradiscal electrothermal therapy in vitro. OBJECTIVE: To determine whether intradiscal electrothermal therapy produces acute changes in disc histology and motion segment stability. SUMMARY OF BACKGROUND DATA: Intradiscal electrothermal therapy has been introduced as an alternative for the treatment of discogenic low back pain. Several hypothesized mechanisms for the effect of intradiscal electrothermal therapy have been suggested including shrinkage of the nucleus or sealing of the anulus fibrosus by contraction of collagen fibers, and thermal ablation of sensitive nerve fibers in the outer anulus. METHODS: Intradiscal electrothermal therapy was performed with the Spinecath by Oratec on 19 fresh, frozen human lumbar cadaver specimens. In a separate study, eight specimens were tested biomechanically and instrumented to map the thermal distribution, whereas five specimens were tested only biomechanically, both before and after intradiscal electrothermal therapy. Six additional specimens were heated with intradiscal electrothermal therapy, and the resulting canal was backfilled with a silicone rubber compound to allow colocalization of the catheter and anular architecture. RESULTS: A consistent pattern of increased motion and decreased stiffness was observed. For the specimens in which only biomechanical measurements were taken, a 10% increase in the motion, on the average, at 5 Nm torque was observed after intradiscal electrothermal therapy. No apparent alteration of the anular architecture was observed around the catheter site in the intradiscal electrothermal therapy-treated discs. CONCLUSION: The data from this study suggest that the temperatures developed during intradiscal electrothermal therapy are insufficient to alter collagen architecture or stiffen the treated motion segment acutely.

Parametric finite element analysis of vertebral bodies affected by tumors.

The vertebral column is the most frequent site of metastatic involvement of the skeleton. Due to the proximity to the spinal cord, from 5% to 10% of all cancer patients develop neurologic manifestations. As a consequence, fracture risk prediction has significant clinical importance. In this study, we model the metastatically involved vertebra so as to parametrically investigate the effects of tumor size, material properties and compressive loading rate on vertebral strength. A two-dimensional axisymmetric finite element model of a spinal motion segment consisting of the first lumbar vertebral body (no posterior elements) and adjacent intervertebral disc was developed to allow the inclusion of a centrally located tumor in the vertebral body. After evaluating elastic, mixed, and poroelastic formulations, we concluded that the poroelastic representation was most suitable for modeling the metastatically involved vertebra's response to compressive load. Maximum principal strains were used to localize regions of potential vertebral trabecular bone failure. Radial and axial vertebral body displacements were used as relative indicators of spinal canal encroachment and endplate failure. Increased tumor size and loading rate, and reduced trabecular bone density all elevated axial and radial displacements and maximum tensile strains. The results of this parametric study suggest that vertebral tumor size and bone density contribute significantly to a patients risk for vertebral fracture and should be incorporated in clinical assessment paradigms.

Effect of frozen storage on the creep behavior of human intervertebral discs.

STUDY DESIGN: A biomechanical study of the compressive creep behavior of the human intervertebral disc before and after frozen storage. OBJECTIVES: To determine whether frozen storage alters the time-dependent response of the intact human intervertebral disc. SUMMARY OF BACKGROUND DATA: The biomechanical properties of the intervertebral disc are generally determined using specimens that have been previously frozen. Although it is well established that freezing does not alter the elastic response of the disc, recent data demonstrate that freezing permanently alters the time-dependent mechanical behavior of porcine discs. METHODS: Twenty lumbar motion segments from 10 human spines were harvested between 12 and 36 hours postmortem. The specimens were randomly assigned to one of two groups: Group 1 was tested promptly, stored frozen for 3 weeks, then thawed, and tested a second time; Group 2 was stored frozen for 3 weeks, thawed, and then tested. Each specimen was subjected to 5 cycles of compressive creep under 1 MPa for 20 minutes, followed by a 40-minute recovery under no load. After testing each specimen was graded on a degeneration scale. A fluid transport model was used to parameterize the creep data. RESULTS: There was no statistically significant effect of freezing on the elastic or creep response of the discs. The degree of pre-existing degeneration had a significant effect on the creep response, with the more degenerated discs appearing more permeable. CONCLUSIONS: Frozen storage for a reasonable time with a typical method does not significantly alter the creep response of human lumbar discs. Freezing may produce subtle effects, but these potential artifacts do not appear to alter the discs' time-dependent behavior in any consequential way. These results may not apply to tissue kept frozen for long durations and with poor packaging.

The effect of static in vivo bending on the murine intervertebral disc.

BACKGROUND CONTEXT: Intervertebral disc cell function in vitro has been linked to features of the local environment that can be related to deformation of the extracellular matrix. Epidemiologic data suggest that certain regimens of spinal loading accelerate disc degeneration in vivo. Yet, the direct association between disc cell function, spinal loading and ultimately tissue degeneration is poorly characterized. PURPOSE: To examine the relationships between tensile and compressive matrix strains, cell activity and annular degradation. STUDY DESIGN/SETTING: An in vivo study of the biologic, morphologic and biomechanical consequences of static bending applied to the murine intervertebral disc. SUBJECT SAMPLE: Twenty-five skeletally mature Swiss Webster mice (12-week-old males) were used in this study. OUTCOME MEASURES: Bending neutral zone, bending stiffness, yield point in bending, number of apoptotic cells, annular matrix organization, cell shape, aggrecan gene expression, and collagen II gene expression. METHODS: Mouse tail discs were loaded for 1 week in vivo with an external device that applied bending stresses. Mid-sagittal sections of the discs were analyzed for cell death, collagen II and aggrecan gene expression, and tissue organization. Biomechanical testing was also performed to measure the bending stiffness and strength. RESULTS: Forceful disc bending induced increased cell death, decreased aggrecan gene expression and decreased tissue organization preferentially on the concave side. By contrast, collagen II gene expression was symmetrically reduced. Asymmetric loading did not alter bending mechanical behavior of the discs. CONCLUSIONS: In this model, annular cell death was related to excessive matrix compression (as opposed to tension). Collagen II gene expression was most negatively influenced by the static nature of the loading (immobilization), rather than the specific state of stress (tension or compression).