**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. Research Progress - Summary

Progress summary: We have conducted tests on 4 spine specimens. In this report, test results and their qualitative analysis are presented in Section 3. Quantitative analysis will be conducted after all 10 specimens are tested.

Impeding factor: presently the major factor that influences the progress of the project is the availability of high quality human cadaver lumbar spine specimens. We received 6 specimens from NDRI (National Disease Research Institute) but only 2 of them are appropriate for testing. Our study design has considered the specimen availability issue, and proposed a total time of 18 months to complete this study, which should be sufficient time for specimen procurement. We are actively working with NDRI to improve quality of received specimen and to speed up procuring process.

2. Findings to Date
The cadaver lumbar spines were potted with two motion segments in each potting. Four spine specimens with a total of 16 discs were tested. 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 no grade V disc was tested. In the following sections we present data of IAR, ROM, IDPP, and disc pressure graphs.  Among these, IDPP data are available from 16 discs, IAR, ROM and disc pressure graphs are available from 12 discs (4 discs have been tested but data not analyzed).

2.1. 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-A through Figure 1-I are IAR results for normal discs (Grade I). Figure 2 is the IAR result for one Grade II disc. Figure 3-A and Figure 3-B are the IAR results for two Grade III discs. Figure 4 is the IAR result for one Grade IV disc. Except for Figure 1-H and Figure 1-I, the IAR for Grade I discs showed fairly consistent locations within the disc, or below the disc and within the inferior vertebra body. The IAR for Grade II disc showed a location close to the superior edge of disc. The IAR for Grade III discs was located within the disc or within the inferior vertebra body.

We do not have a definite explanation for the drastically different IAR patterns in Figure 1-H and Figure 1-I at this point. But these IAR patterns came from L4-L5, L5-S1 of the same specimen, and although the discs were graded as “normal” according to MRI images, we found osteophytes around the vertebra bodies and the donor’s age was 66.

In summary, these IAR results have established a baseline of IAR patterns of normal discs. Our initial results on discs with Grade II through IV appear to indicate that there is no drastic change of IAR locations from normal discs. However more degenerated discs need to be tested to verify such a point. Our future tests on more discs of Grade II~V will yield IAR results that we will use to compare with normal disc IAR pattern.

2.2. 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. Pressures was then recorded when the needle sensor is drawn back through the disc. Figure 5 through Figure 8 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, as represented by multiple pressure spikes in the annulus region in Figure 6, Figure 7 and Figure 8. Two of the 4 discs in Figure 6 and Figure 8 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 8 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.

 Our results indicate that with increasing disc degeneration grade, IDPP showed a trend of changes from the behavior of “water-filled fluid bag” as represented by uniform pressures in nucleus region, towards the behavior of dehydrated and disrupted disc structure as represented by pressure spikes and depressurized states. Future tests of more discs may allow us to link specific IAR characteristics of various grade of disc degeneration to these IDPP characteristics.

2.3. Range of Motion (ROM)
Rang of motion (ROM) was measured during load-deformation tests for each specimens. Figure 9 through Figure 12 are representative load-deformation curves for normal discs and discs with degeneration grades from II to IV.

For normal discs (data from 8 discs), ROM in flex-extension (combined) is in the range of 3~8.5° with median 5.8°, ROM in lateral bending (combined)  range 2.3~7.2° with median 6.2°, and ROM in axial rotation (combined)  range 1.4~2.9° with median 2.2°.

For discs of Grade II ~ Grade IV, data are available from 1~2 discs for each grade. However it was seen that flex-extension and lateral bending ROM of some of these degenerated discs are beyond the range of normal discs, which may be caused by increased laxity of annulus and ligaments from degeneration. Further tests of more degenerated discs may link IAR and IDPP changes to ROM characteristic associated with degeneration grades.

2.4. Disc Pressure Graphs
Disc pressure graphs were measured using a miniature pressure sensor in the center of each disc during load-deformation tests. Figure 13 through Figure 16 are representative disc pressure graphs for normal discs and discs with degeneration grades from II to IV.

For normal discs (data from 8 discs), Flexion disc pressure change from neutral position showed a range 60~163 KPa with median 99KPa,   Extension disc pressure change from neutral position showed a range -33~109 KPa with median 63KPa. For discs of Grade II ~ Grade IV, disc pressure graphs are available from 1~2 discs for each grade. However with increasing disc degeneration, more irregular pressure profiles, i.e. lost pressures peaks at maximum loads or negative pressure changes from neutral positions etc., are present with increased frequency, especially in lateral bending mode. Further tests of more degenerated discs may link IAR and IDPP changes to these disc pressure characteristics associated with degeneration grades.

  
**Young Investigator:  Novel Scaffold Using Human Adipose-derived Stromal Cells
Francis H. Shen, MD

Background
Currently 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 reported cases. A successful spinal fusion requires the differentiation of osteoprogenitor cells into osteocytes. Currently iliac crest bone graft is currently considered the gold standard. However, iliac crest graft harvest is not without significant morbidity. Reported complications include chronic pain, hypersensitivity, increased blood loss, increased operative time, deep infections, vascular and neurologic injury, and poor cosmetic results. Furthermore, in certain clinical situations the available autograft material may be inadequate or insufficient. As a result, extensive work has been directed towards developing suitable bone graft substitutes. The ideal bone graft substitute should therefore contain all three components necessary for osteogenesis, specifically the osteogenic, osteoinductive, and osteoconductive properties.

Currently, bone marrow is still considered the most reliable source for providing osteogenic mesenchymal stromal cells. Unfortunately, frequently cellular yield can be relatively low, with less then 1% of the nucleated cells in the marrow having multipotential characteristics. 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. As a result, a few select laboratories are now beginning to intensively investigate the use of human ADS cells as the primary source for osteogenic progenitors. The use of ADS cells for osteogenic differentiation however, will likely require sufficient levels of an osteoinductive stimulus. Several growth factors have been shown to promote osteogenesis to varying degrees. Careful selection for the most appropriate osteoinductive factors can greatly help assist in driving the process of osteogenic differentiation to completion. Lastly, the use of a three-dimensional structural scaffold will facilitate a bony fusion by providing a physical environment that protects and shields the ADS cells from the abnormal biomechanical shear stresses present during spinal fusions. Furthermore, they can provide scaffolding capable of cellular in-growth while placing both the osteogenic cells and osteoinductive factors within a single structural carrier.

Therefore the purpose for this research initiative is to determine the potential for hADS cells treated with rGDF-5 to undergo osteogenic differentiation on a novel bioengineered scaffold in vitro. This interim report will provide an update on the current status of the project and provide a summary of the ongoing and planned experiments for the second half of the funding period.

Current Status
Harvest human ADS cells:
A human protocol was approved by our HIC/IRB. The hADS were isolated according to an established procedure. Briefly, 10 cc of human adipose tissue were harvested from the groin/abdominal or gluteal/lumbodorsal region under sterile technique. The fat was then washed extensively using Dulbecco’s Modified Eagle’s Medium (DMEM; Invitrogen, Carlsbad, CA) with 2% penicillin/streptomycin.

Collagenase 0.01% (Sigma, St. Louis, MO) was added to the washed sample and the mixture agitated at 37°C for 1-2hr. The collagenase was inactive with an equal volume of DMEM/10% fetal bovine serum (FBS) and the infranatant centrifuged for 10 min at 60g. The pellet was resuspended in DMEM/10% FBS and transfer into T-75 flask containing 15 ml of control medium (DMEM, 10% FBS, 1% penicillin/streptomycin). Cell culture medium was changed every third day. Cells were maintained at subconfluent levels (80%) and passaged every 5 days to prevent spontaneous differentiation.

  
**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

Purpose:  To tissue-engineer a biologically and biomechanically functioning intervertebral disc (IVD) ex vivo using the designed, structurally mimic biomaterial scaffold and the mechanically active bioreactor for the IVD replacement.

Hypothesis:  The structurally mimic biomaterial IVD, co-cultured stem cells and disc cells, multiple growth factor combination, and mechanical loading will maintain disc cell activities and enhance chondrogenesis and IVD matrix production.

Methods:  We will fabricate and engineered IVD scaffold composed of the peripheral AF consisting of concentric multi-lamellae with oblique polymeric nanofibers alternatively oriented in successive layers, and the core NP filling with hydrogel, to imitate the native IVD structure.  The initial architecture will physically encourage the deposition of newly formed extracellular matrix (ECM) along the pre-built nanofibrous structure, expediting the formation of a neo-tissue with enhanced functional characteristics.  The IVD scaffolds will be seeded with co-cultured mesenchymal stem cells (MSCs) and annulus fibrosus (AF) cells in AF nanofibers and with co-cultured MSCs and nucleus pulposus (NP) cells in NP hydrogel.  We will also use the growth factor combination of transforming growth factor beta-1 and bone morphogenetic protein-7 to enhance cartilage-specific matrix production.  In addition, the cellular IVDs will be stimulated in the mechanically active bioreactor to increase matrix production and keep the discs healthy.

Findings to Date
In this research, we proposed to accomplish two specific aims, Specific Aim 1: Construction of Structurally Mimic IVD Scaffold, and Specific Aim 2: Regeneration of Biologically and Biomechanically Functioning IVD. We are currently in the stage of working toward to accomplishing Specific Aim 1 and starting the experiments for accomplishing Specific Aim 2. 

We have accomplished a few sub-aims of Specific Aims 1 and 2. These sub-aims accomplished in the past 4 months include 1) fabrication of intervertebral disc (IVD) scaffolds, 2) mesenchymal stem cell (MSC) isolation and culture, and 3) cell response to growth factors.

Fabrication of IVD scaffolds. We have constructed IVD scaffolds composed of a part of two layers of aligned electrospun nanofibers as annulus fibrosus (AF), and another part of hydrogel as nucleus pulposus at the core. The scaffold fabrication procedure has been optimized and standardized.   

Mesenchymal stem cell isolation and culture. We have successfully harvested and isolated MSCs from bovine bone marrow, and cultured, and maintained the cells for IVD regeneration.  

Cell response to growth factors. We tested four growth factors, TGF-1 (10 ng/mL), IGF-1 (50 ng/mL), BMP-7 (10 ng/mL), and bFGF (2 ng/mL), for the biological effect on IVD regeneration. In the 42 days of study, sole or various combinations of the four growth factors were used to stimulate bovine chondrocytes seeded in nanofibrous scaffolds to select the growth factor(s) for the effective IVD ECM production. The levels of mRNA of cartilage-specific markers, collagen type II and aggrecan, at Days 14, 28, and 42, showed that the combination of TGF-1 and BMP-7 were the most effective group enhancing cartilage-specific ECM production.          

  *Abstracts/permission forms not received at the time of publication
**Current and ongoing research