Effects of Diurnal Loading on Intervertebral Disc using a Whole Organ in Vitro Culture Model
At a Glance
Section titled âAt a Glanceâ| Metadata | Details |
|---|---|
| Publication Date | 2015-05-01 |
| Journal | Global Spine Journal |
| Authors | James P. Stannard, Aaron M. Stoker, Ferris M. Pfeiffer, Keiichi Kuroki, James L. Cook |
| Institutions | University of Missouri |
| Analysis | Full AI Review Included |
Technical Documentation & Analysis: MPCVD Diamond in Advanced Biomechanics Research
Section titled âTechnical Documentation & Analysis: MPCVD Diamond in Advanced Biomechanics ResearchâExecutive Summary
Section titled âExecutive SummaryâThis study demonstrates the critical role of mechanical loading type (static vs. diurnal) and magnitude (physiological vs. supraphysiological) in preserving the viability and integrity of whole-organ Intervertebral Disc (IVD) explants in vitro.
- Core Finding: Diurnal, physiological loading (0.1 MPa, 12-hour cycle) significantly outperformed static loading and supraphysiological loading (1 MPa) in maintaining viable cell density and extracellular matrix composition (GAG/HP).
- Methodological Requirement: The creation of precise IVD explants necessitated the use of ultra-hard tooling, specifically a diamond band saw, highlighting the need for high-durability materials in preparation phases.
- Material Relevance: Replication and extension of this researchâparticularly the evaluation of new cyclic regimens and injury modelsârequires extremely robust, non-degrading materials for mechanical testing apparatuses and high-precision cutting tools.
- 6CCVD Value Proposition: 6CCVD provides the necessary MPCVD diamond materials (PCD plates for tooling, SCD/PCD substrates for non-reactive testing platforms, and BDD for potential electrochemical sensing) to advance complex biomechanical culture models.
- Future Application: The need for precise, long-term mechanical stimulation rigs suggests an ideal application for large-area, highly polished PCD substrates from 6CCVD, ensuring mechanical stability and chemical inertness.
Technical Specifications
Section titled âTechnical SpecificationsâThe following parameters were extracted from the study detailing the mechanical and biological conditions applied to the IVD explants.
| Parameter | Value | Unit | Context |
|---|---|---|---|
| Sample Source | Canine L1-L5 Discs | n=7 | Whole organ IVD explants |
| Physiological Load Level | 0.1 | MPa | Optimal load for cell viability and matrix preservation |
| Supraphysiological Load Level | 1 | MPa | Associated with significant loss of cell viability |
| Cyclic Loading Regimen | 12-hour cycle | Duration | Applied to Diurnal groups |
| Culture Duration | 3 | Days | Time point for assessment |
| Viability Result (0.1 MPa Diurnal) | Significantly Higher | p < 0.05 | Compared to static counterpart |
| Matrix Integrity Result (0.1 MPa) | Significantly Higher GAG/HP | p < 0.05 | Compared to 1 MPa groups |
| Biomechanical Assessment | Unconfined Compression | N/A | Used to calculate explant stiffness |
Key Methodologies
Section titled âKey MethodologiesâThe experimental design focused on precise mechanical manipulation and subsequent biochemical analysis, requiring high-tolerance preparation tools.
- Explant Preparation: Lumbar spine segments were harvested, and explants were created using a diamond band saw to ensure clean, precise sectioning of the hard and soft tissues.
- Randomization and Loading: Explants were divided into four groups based on load magnitude (0.1 MPa or 1 MPa) and load type (static or diurnal).
- Diurnal Cycling: Diurnal groups were subjected to their respective loads using a defined 12-hour cyclic regimen.
- Assessment: After 3 days, IVDs were bisected for quantification of viable cell density using an in-house program.
- Biochemical Analysis: Glycosaminoglycan (GAG) and collagen/hydroxyproline (HP) contents were calculated to assess extracellular matrix integrity.
- Biomechanical Analysis: Explant stiffness was determined by calculating the slope of the force-displacement curve derived from unconfined compression testing.
6CCVD Solutions & Capabilities
Section titled â6CCVD Solutions & CapabilitiesâThis research, particularly its reliance on precision cutting and the need for stable, non-reactive mechanical testing environments, presents several opportunities for integration of MPCVD diamond materials supplied by 6CCVD.
Applicable Materials
Section titled âApplicable Materialsâ| Research Requirement | 6CCVD Material Recommendation | Rationale & Benefit |
|---|---|---|
| Precision Cutting Tooling | Polycrystalline Diamond (PCD) Plates | Used for high-wear, durable diamond band saw blades. PCD offers superior abrasion resistance and longevity over traditional materials, ensuring consistent explant geometry. |
| Mechanical Testing Platforms | Optical Grade Single Crystal Diamond (SCD) | Ideal for non-reactive, ultra-stiff compression platens or windows in advanced bioreactors. SCD offers extreme hardness and chemical inertness, preventing contamination or material degradation during long-term cyclic loading. |
| Advanced Sensing/Monitoring | Heavy Boron-Doped Diamond (BDD) | BDD electrodes can be integrated into the culture system to perform in situ electrochemical monitoring of metabolic activity (e.g., oxygen consumption, pH changes) or nutrient diffusion under load. |
| Large-Scale Culture Rigs | Large-Area PCD Wafers | For scaling up the in vitro model, 6CCVD offers PCD plates up to 125mm in diameter, providing a stable, durable base for multi-sample mechanical testing rigs. |
Customization Potential
Section titled âCustomization Potentialâ6CCVD specializes in providing materials tailored to the exact needs of complex scientific and engineering applications:
- Custom Dimensions: We can supply large-area PCD plates up to 125mm for building robust, multi-well mechanical loading platforms, or SCD substrates up to 500”m thick for high-stiffness components.
- Surface Finish: For components requiring minimal friction or high optical clarity (e.g., viewing windows for cell viability assessment), 6CCVD provides ultra-smooth polishing: Ra < 1nm for SCD and Ra < 5nm for inch-size PCD.
- Integrated Metalization: If future research involves integrating strain gauges or BDD electrodes onto the diamond substrate for real-time load monitoring, 6CCVD offers internal metalization capabilities (Au, Pt, Pd, Ti, W, Cu) to create custom contact pads or circuit traces.
Engineering Support
Section titled âEngineering Supportâ6CCVDâs in-house PhD team can assist researchers in material selection for similar Biomechanical IVD Culture and Cyclic Loading projects. We provide expert consultation on:
- Selecting the optimal diamond grade (SCD vs. PCD) based on required stiffness, thermal management, and optical transparency.
- Designing custom diamond components for high-pressure, cyclic loading environments where material fatigue is a concern.
- Integrating BDD electrochemical sensors for non-invasive, real-time monitoring of cellular health and nutrient transport within the culture system.
For custom specifications or material consultation, visit 6ccvd.com or contact our engineering team directly.
View Original Abstract
Introduction Intervertebral disc (IVD) disorders associated with pain and disability are extremely prevalent and current treatment options do not result in restoration of tissue integrity or function. The exact mechanisms of IVD degeneration are not fully understood at this time although aging, injury, nutrition, metabolism, and mechanical stress are suspected to contribute. To more fully understand IVD degeneration, an in vitro whole organ model of IVD is desirable. It has been theorized that preservation of large animal IVDs in vitro is difficult due to poor diffusion of nutrients. To assess the effects of nutrient diffusion and more closely mimic in vivo loading of the human spine, we hypothesized that a diurnal loading regimen would be associated with significantly higher cell viability in IVDs compared with static loading. We further hypothesized that a supraphysiological load of 1 MPa would be associated with significant loss of cell viability and tissue morphology of IVD explants compared with a physiological load of 0.1 MPa. Material and Methods Under ACUC approval, lumbar spine segments containing L1-L5 discs were harvested from dogs ( n = 7) euthanized for reasons unrelated to this study. Soft tissue was aseptically dissected and explants were created using a diamond band saw. Explants were randomly divided into either diurnal or static load and were further divided into either 0.1 MPa load or 1 MPa load. Explants in the diurnal group were subjected to the respective load using a 12-hour cycle. After 3 days, IVDs were bisected and assessed for cell viability and viable cell density was quantified using an in-house cell counting program. Glycosaminoglycan (GAG) content and collagen content were calculated. Biomechanical analysis was determined using unconfined compression and calculated using the slope of the force is placement curve. All samples were evaluating statistically with significance set at p < 0.05 using Student t-test. Results For both static and diurnal loading, 0.1 MPa groups had significantly ( p < 0.05) higher viable cell density than 1 MPa groups after 3 days of loading. Viable cell density was significantly ( p < 0.05) higher in each diurnally loaded group compared with its statically loaded counterpart. GAG and HP contents in annulus fibrosus were significantly ( p < 0.05) higher in 0.1 MPa groups compared with 1 MPa groups. Explant stiffness was significantly ( p < 0.05) higher in the 0.1 MPa group at day 3 as compared with the day 0 controls. Conclusion To our knowledge, this is the first study to report the effects of physiological and supraphysiological static and diurnal loads on whole organ canine IVDs in culture. There were significant differences in viable cell density and extracellular matrix composition associated with level (0.1 and 1 MPa) and type (static vs. diurnal) of loading. Taken together, these data suggest that diurnal, physiological load best preserves IVD viability and integrity. Ongoing research is aimed at evaluating additional loading levels and cyclic regimens, as well as assessing the effects of insult and injury on loaded whole-organ IVD explants.