Defining the Abnormal Kinematics of Lumbar Spine Instability as a Cause of Degenerative Low Back Pain-A Biomechanical Study of the Kinematics of Cadaver Lumbar Spine
Dilip K. Sengupta, MD
Purpose: Chronic low back pain (LBP) is a common musculoskeletal disorder that significantly impacts public health. However the mechanism of chronic LBP is still not fully understood. From a biomechanical point of view, it is believed that chronic LBP is related to spinal instability (abnormal motion), abnormal load transmission, or both in the degenerated motion segment. But the relationship between these factors and back pain is again not clear. There remains a need for more effective biomechanical measures that are able to characterize spine motion and load transmission, and clarify how these factors relate to pain generation. The long term goal of this study is to find effective biomechanical measures for characterizing spinal motion and load transmission, and use these measures to elucidate the biomechanical mechanism of chronic low back pain. The objective of this research is to validate in vitro that instantaneous axis of rotation (IAR) is an effective measure to define abnormal spinal motion (spinal instability), and intra-discal pressure profile (IDPP) an effective measure to define abnormal load-transmission; and further to understand the relationship between IAR and IDPP.
Hypotheses:1) Instantaneous axis of rotation (IAR) trace patters are different between normal and degenerated discs. 2) The load transmission across the disc space, as measured using intra-discal pressure profile (IDPP), is different between normal and degenerated discs. 3) There is a relationship between IAR and the load-transmission across the disc space.
Methods: An in vitro study of lumbar spine kinematics and load transmission is proposed to achieve our study aims. Ten human cadaver lumbar spine specimens will be procured. MRI scans of these spines will be performed to determine the degree of disc degeneration. Lateral radiographs of the specimens will be obtained to measure disc height, lordosis, listhesis, endplate sclerosis, etc. The specimens will then be potted with two motion segments in each potting for load-deformation tests on a six-degrees-of-freedom (6-DOF) spine tester. IAR will be determined from motion tracking data and hundreds of instances of IAR locations (continuous tracking of the locations of the IAR) will be calculated during one flexion-extension cycle. Disc pressures will be determined using two methods, with IDPP as the primary approach, to identify any abnormal load-transmission across the disc space. The two methods for measuring disc pressures are: 1)IDPP, as a continuous pressure profile across the disc space with the specimens held statically in flexion, extension and neutral posture under load-bearing; and 2)disc pressure graph, which is determined during dynamic flexion-extension motion from the center of the disc space. Finally, both qualitative and statistical comparison of IAR and IDPP will be performed based on degree of disc degeneration to understand their relationships.
1. Findings
1.1. Project summary
We have conducted tests on 40 discs from 14 spine specimens. Before tests, MRI was conducted on specimens and degenerated discs were graded using a clinical disc degeneration grading system as described by Pfirrmann et al (2001). Discs were divided into five groups: normal (grade I), minor (grade II), mild (grade III), moderate (grade IV), and severe degeneration (grade V). Table 1 summarizes degeneration grade distribution of tested discs.
The majority of tested discs were normal (grade I), and only one grade V disc was available. It was found obtaining higher degeneration grade discs (Grade V) was difficult, because of their low availability and the frequent spontaneous fusion of such motion segments. However, we obtained sufficient numbers of discs for Grade I~IV. Clinically, symptomatic discs with degeneration equal or greater than grade III are surgically treated, therefore our tested Grade III and IV discs should be sufficient to answer our research question.
In the following sections, IAR, ROM, NZ, IDPP, and disc pressure graph (IDP) data are presented.
1.2. Instantaneous Axis of Rotation (IAR)
Potted specimens were loaded onto a 6-DOF spine tester for load-deformation tests in flexion-extension direction. During tests, motion was captured with a motion tracking system and IAR was calculated from the continuous motion tracking data. After tests, lateral X-ray radiograph of the specimen was obtained to record the reference positions of motion tracking markers. IAR was then superimposed to the radiographs to reveal anatomic location of the IAR relative to the motion segment.
Figure 1 and Figure 2 are IAR for two typical normal discs (Grade I). Figure 3 and Figure 4 are IAR for two typical Grade II disc. Figure 5 and Figure 6 are IAR for two typical Grade III discs. Figure 7 and Figure 8 are IAR for two typical Grade IV disc. Figure 9 is the IAR result for one Grade V disc.
In summary, extensive tests have been conducted and IAR of 40 motion segments with various degrees of degeneration have been obtained. Baseline of IAR patterns of normal discs has been established. The IAR results of degenerated discs will be analyzed and compared with normal IAR. Further results will be submitted in the final report this project.
1.3. Intra-Discal Pressure Profile (IDPP)
To measure IDPP, the specimens were held statically in flexion, extension and neutral posture under load-bearing. A needle sensor was inserted through the disc until the tip of the needle sensor is beyond the posterior annulus. Disc pressure was then recorded when the needle sensor was drawn back through the disc. Figure 10 through Figure 13 are representative IDPP results of discs with degeneration grades I though IV. For each grade, IPDD of two discs are presented.
For Grade I normal discs, IDPP profile showed relatively uniform disc pressures in the center of the discs in all loading conditions. With increasing degeneration, IDPP profiles become more and more irregular, showing multiple pressure spikes in the annulus region in Figure 11, Figure 12 and Figure 13. Two of the 4 discs in Figure 11 and Figure 13 showed a nearly “depressurized” state in some of the loading postures, and lost completely the uniform pressure plateau of normal discs. Such pressure profiles were consisted of regions of near-zero pressures and regions of pressure spikes, indicating significant disc structural changes such as nucleus dehydration and structure disruption. The upper profiles in Figure 13 showed higher pressures in annulus region than nucleus region, and also “broadened” annulus pressure region. Such profile was also observed in McNally et al’s IDPP studies.
These pressure profiles indicate that with increasing disc degeneration grade, IDPP showed a trend to shift from the behavior of “water-filled fluid bag” as represented by uniform pressures in nucleus region, to the behavior of dehydrated and disrupted disc structure as represented by pressure spikes and depressurized states.
Figure 14 shows overall magnitude changes of disc nucleus pressures, measured from IDPP. It is found the nucleus pressure continues to drop with increasing grade of disc degeneration. Despite of inherent large variation of disc pressure measurements, statistical significance were found in all modes of load in Grade I vs. Grade IV discs, confirming the trend of decrement.
Grade I (specimen 61785)
Grade II, (specimen 61960, 61317)
Grade III, (specimen 63160, 61960)
Grade IV, (specimen 63160)
1.4. Range of Motion (ROM) and Neutral Zone (NZ)
Rang of motion (ROM) was measured during load-deformation tests for each specimens. Figure 15 through Figure 18 are representative load-deformation curves for normal discs and discs with degeneration. The profiles of load-deformation curves, e.g. non-linearity, did not show significant changes in different degrees of degeneration.
Figure 19 shows the ROM comparison between different degeneration grades. There are some differences in ROM with increasing disc degeneration, but such differences did not demonstrate statistical significance.
Figure 20 shows the NZ comparison between different disc degeneration grades. NZ was calculated from Hysteresis width as defined by Wilke et al in 1998. In general, NZ increases with disc degeneration from grade I through grade III, in flex-ext, lateral bending and axial rotation, which is likely caused by increasing laxity of spinal ligaments and disc annulus, due to degeneration. Grade IV discs showed smaller NZ comparing to grade III disc in lateral bending and axial rotation, indicating stabilizing effect of advanced degeneration. Statistical significance was found in flexion of grade I vs. grade III, and grade I vs. grade IV discs.
It is interesting to note that there was tendency to reduce the ROM in Grade IV, but the NZ motion increased. It may be important that NZ in % of total ROM be studied to reveal the dramatic difference that degeneration may introduce.
1.5. Disc Pressure Graphs
Disc pressure graphs (IDP) were measured using a miniature pressure sensor in the center of each disc during load-deformation tests. Figure 22 through Figure 24 are representative disc pressure graphs for normal discs and discs with various degeneration grades. With increasing degeneration, the IDP showed a trend of change from “U” shape profiles of normal disc (pressure peaks at both full flexion and extension posture) to profiles that frequently lost their pressure peaks (Figure 23, Figure 24).
Figure 25 compares disc pressure graph rise magnitude among disc groups. Although statistical significance was only found in extension of grade I-IV due to large pressure variations, with increasing degeneration, a trend of decreasing disc pressure appears to be consistent, both in flexion and extension.
Grade III, (specimen 63160, 61960)
1.6. Summary of Findings to Date:
This study defines the normal parameters of the IAR, ROM, NZ, IDP rise, and IDPP profile across the disc space. Amongst the four parameters, the IDP and IDPP profile showed consistent changes with increasing disc degeneration. Results of IAR and ROM and NZ will be further analyzed to identify any additional linkages between these parameters and disc degeneration.
Young Investigator: Novel Scaffold Using Human Adipose-derived Stromal Cells
Francis H. Shen, MD
Abstract: Over 450,000 spinal fusions are performed in the United States a year. Despite improved techniques and instrumentation, nonunion rates continue to occur in up to 40% of cases. Iliac crest bone graft is considered the gold standard, however is not without significant morbidity. Recent studies suggest that adipose-derived stromal (ADS) cells are more than space fillers and, under appropriate conditions, can dedifferentiate and return to the status of an uncommitted stromal stem cell. The purpose for this research initiative is to determine the potential for hADS cells treated with recombinant human growth and differentiation factor-5 protein (rGDF-5) to undergo osteogenic differentiation on a novel bioengineered scaffold in vitro.
We studied the osteogenesis of hADS cells in vitro with the induction of rGDF-5. The osteogenesis differentiation was compared with von Kossa staining after treatment with osteogenic medium. The gene expressions were analyzed by real-time PCR. The cells cultured with different concentrations of GDF-5 were measured for an optimized application of the growth factor. Gene expression confirmed the osteogenic differentiation of hADS cells. Furthermore, real-time PCR revealed that the gene expression of VEGF groups demonstrated different reactions to the treatment of ODM with GDF-5 and confirmed the relationship between VEGF and BMP signaling pathways. This suggests that future studies investigating cell signal pathways during the stem cell differentiation may hold significant benefit for the investigation of spinal fusions and osteogenesis.
These experiments also investigated the ability of hADS cells to adhere and proliferate on novel bioengineered scaffolds in vitro. The sintered microsphere matrix (SMM) were fabricated using the solvent evaporation technique. Using micron sieves, selected diameter size ranges of 500–600 mm were packed in a stainless steel mold and heated at 80 oC. The hADS cells demonstrated a good compatibility and similar level of osteogenesis on PLGA scaffold compared with monolayer culture. Next, nanofiber PLGA scaffold were developed utilizing a flexible nanofiber PLGA scaffold, which could be bent while maintaining its strength. The hADS cells were then co-cultured on the spun nanofibers (fiber sizes, 700-1000nm), while the cell density was 2000cells/scaffold. Using SEM, the results demonstrate both cell viability and growth between nanofibers.
Results of SMM and spun nanofibers suggest that fabrication of these novel bioengineered scaffolds are feasible using PLGA and currently available tissue engineering techniques in our laboratory. Furthermore, human ADS cells appear to be compatible on the bioengineered, biodegradable scaffold and that growth appears viable in an in vitro environment. Moreover, this nanofiber scaffold has the capability to be combined with the microsphere scaffold for use during in vivo bone formation in the spine. The ability for differentiation and migration of hADS cells on these novel scaffolds are still unclear and warrant further investigation. Furthermore, extensive in vivo experiments will be required in order to further confirm biocompatibility between scaffold and osteogenic cells and ability to undergo a spinal fusion.
Young Investigator: Intervertebral Disc Regeneration from Co-cultured Disc and Stem Cells in Biomimetic Engineered Extracellular Matrix Stimulated by Mechanically Active Bioreactor
Wan-Ju Li, Phd; Paul A. Anderson, MD, Tsung-Lin Tsai, M.D.*, Brenton Nelson, M.S.*
Purpose: To tissue-engineer functional intervertebral disc (IVD) for disc replacemen using extracellular matrix (ECM) structure-mimetic scaffolds cultured with mesenchymal stem cell (MSC)/disc cell mixtures, stimulated by compressive loading in a bioreactor.
Background: IVD failure resulted from tissue degeneration is a challenging orthopedic problem. Disc replacement using prostheses or vertebral body fusion has been used to treat IVD failure but both the treatments have their own limits on achieving satisfactory outcomes. Tissue engineering using cell, biomaterial scaffold, growth factor, and advanced culture technique holds a great promise to generate a functional, biological substitute for disc replacement. Previous studies using various types of cells cultured in different biomaterial scaffolds to fabricate tissue-engineered IVD have shown varying degrees of success. However, among these studies, few have used a scaffold that consists of three distinct structures for annulus fibrosus (AF), nucleus pulposus (NP), and endplate to structurally mimic ECM of native IVD for tissue regeneration. It is important to use an ECM structure-mimetic scaffold for IVD tissue engineering since scaffold structure affects biological response and biomechanical properties. Another challenge is associated with the choice of cultured cell types. AF and NP cells occupy about 1% of the total tissue volume. With such a low amount of cells in IVD, it is challenging to prepare a sufficient quantity of cells for culture. In contrast, multi-potential MSCs are easy to expand and can be a suitable cell source for IVD tissue engineering.
Study design: We fabricated an engineered IVD scaffold constructed of 1) the peripheral AF consisting of concentric multi-lamellae with oblique polymeric nanofibers alternatively oriented in successive layers, 2) the core NP filled with hydrogel, and 3) the endplate composed of non-woven nanofibers to structurally imitate the ECM structure of native IVD. These three structural components were seeded with MSCs/AF cells, MSCs/NP cells, and osteogenic MSCs, respectively, and induced by transforming growth factor beta-1 (TGF-β1) and compressive stimulation in a bioreactor for IVD regeneration.
Results: We first tested different ratios of MSC/disc cell mixtures for chondrogenesis. The results showed that after 28 days, the culture with the ratio of 1:2 (MSC:AF cell) or the ratio of 2:1 (MSC:NF cell) expressed the highest levels of cartilage-related ECM markers. We then used the optimal ratios of cell mixtures for cell seeding in the AF and NP components of an IVD construct. The cellular constructs cultured in a bioreactor and stimulated by compressive loading showed that mechanical stimulation upregulated the expression of collagen type II and aggrecan in NP but not in AF. Mechanical stimulation increased the ratio of collagen type II/I in the NP culture while decreased the ratio in the AF culture, suggesting that chondrogenesis and ECM production of MSCs/disc cells are regulated by the combinatory cues of scaffold structure and mechanical stimulation.
Conclusions: We demonstrate that our tissue engineering approach using MSCs and disc cells co-cultured in ECM structure-mimetic scaffolds is promising to regenerate IVD ex vivo for disc replacement.
* The authors contributed equally to this work
*Abstracts/permission forms not received at the time of publication
**Current and ongoing research