Anti-Apoptotic Effects of IGF-1 and PDGF on Human Intervertebral Disc Cells In Vitro
H.E. Gruber, PhD; H.J. Norton, PhD; E.N. Hanley, Jr., MD
Previous studies have shown that there is a small cell population in the human aging intervertebral disc. Earlier work from our laboratory suggested that apoptosis (programmed cell death) may be a major contributing factor to the decline in cell number. A wide variety of inhibitors of apoptosis have now been identified; the present report presents our findings on the actions of IGF-1 and PDGF in retarding or preventing apoptosis. The objective of this study was to determine whether two selected cytokines, insulin-like growth factor-1 (IGF-1) and platelet derived growth factor (PDGF), were effective in decreasing apoptosis in human cells from the annulus grown in culture for ten days. Human cells from the annulus were grown in tissue culture in an experimental design to study the anti-apoptotic effect of two selected cytokines. Positive and negative controls for apoptosis were included in the experimental tests. Experimental studies were approved by the authors’ human subjects institutional review board. Results from this work demonstrated a significant reduction in the percentage of apoptotic disc cells following exposure to 50 and 500 ng/ml IGF-1 or exposure 100 ng/ml PDGF. These findings expand our understanding of the cell biology of the disc cell and show that selected cytokines can retard or prevent programmed cell death in vitro. This type of research may have future therapeutic potential in the treatment of disc degeneration.
This technical report presented methods for in situ detection of apoptotic cells in vitro. Technical tips on procedures and useful laboratory techniques were presented. These methods were developed during our studies of programmed cell death in disc cells.
Gap junctions mediate cell-cell communication by allowing passage of small molecules (<1 kD in size) from one cell to another. Findings in this research provide evidence for gap junction formation and connexin (Cx) 43 and 45 gene expression in human intervertebral disc cells in vivo and in vitro. These findings in cells from the annulus are important in conjunction with the well recognized loss of disc cells during aging and disc degeneration. As a result of this loss of cells, cell-cell communication, which we propose is an important, but as yet poorly understood mechanism which links and coordinates cellular function throughout the entire population of disc cells, is also disrupted. These studies provide additional information on the fundamental cell biology of the disc cell and provide an additional framework for understanding aging, degeneration and potential repair of the human disc.
*Molecular and Cellular Effects of Nicotine on Posterior Lumbar Spine Fusion
M.A. Morone, MD, PhD; S.D. Boden, MD; E.C. Benzel, MD
Five Year Outcomes of Lumbar Spinal Stenosis Surgery
J.N. Katz, MD, MS; S.J. Lipson, MD; L.J. Grobler, MD; M.E. Husni, MD; A.H. Fossel; G.W. Brick, MD
Objective. Lumbar spinal stenosis surgery (LSSS) rates vary widely, suggesting there is considerable uncertainty among physicians regarding the indications for LSSS. The success rate for laminectomies also varies. More than 25% of patients were found to be dissatisfied with their surgery. It is well established that 3/4 of patients with LSS benefit from decompressive surgery in the first two years, but the condition can deteriorate with longer follow up. There is a very little research on long term outcomes of LSSS. The purpose of this prospective, multicenter observational study is to determine five year outcomes of laminectomy (in terms of pain, function, satisfaction, reoperations and death) and to identify preoperative factors associated with particularly favorable or poor outcomes.
Methods. Two hundred sixty-seven patients who underwent LSSS were evaluated at baseline and sixty months postoperatively in four academic centers. The principal outcomes, including walking capacity, back and leg pain and satisfaction with surgery, were assessed with validated measures. The association between preoperative variables and outcomes were examined with univariate and multivariate techniques.
Results. One hundred seventy-nine (78%) patients completed follow up questionnaires at five years (39 died and 49 refused or were lost to follow up). Mean age was 69 years and 60% were female. At five years, 72% were satisfied with their surgery, 61% had improvement in back and leg pain severity, 74% had improved walking capacity and 12.6 % required a reoperation. In multivariate models, the predictors of persistent pain, worse function and dissatisfaction included greater number of comorbidities and lower baseline functional status (p<.006).
Conclusion. Three quarters of patients were satisfied with their surgery five years postoperatively. Patients with better baseline function and fewer comorbidities had better outcomes. Advanced age, presence of neurologic compromise and greater number of levels decompressed were not associated with outcome. These findings will help patients and their doctors make informed decisions of whether to proceed with surgery for spinal stenosis.
Real Time In Vivo Loading in the Baboon Lumbar Spine Using an Interbody Implant Load Cell
B.L. Sachs, MD; E.H. Ledet, MS; J.B. Brunski, PhD; C.E. Gatto, MD
Background. Mechanical properties of the spine have been characterized from previous in vitro studies on the spine and spinal implants. Many techniques have been implemented to indirectly determine in vivo loading. However, the actual loading in the anterior aspect of the lumbar spine has not been experimentally determined in vivo or in real time. Because the loading of the spine that is associated with everyday activities and specific posture is currently unknown, the activities that cause peak loading are also unknown. Additionally, it is unknown whether synthetic intradiscal implants, bone grafts and intervertebral disc prostheses that are used clinically, are mechanically sufficient to withstand the compressive loading in the lumbar spine during normal activities. Under such conditions, the implant must maintain the preoperative disc height during load. An implant whose intrinsic compressive strength is not sufficient to withstand the load placed on the spine is not mechanically suitable as an intradiscal spacer.
No comprehensive in vivo studies have been performed to determine the real time loading on the spine to date. There is a need for spinal research to go beyond the limitations of in vitro studies such that the loading under normal in vivo conditions can be accurately described.
Purpose. The purpose of this study was to directly measure the loads imposed on the lumbar spine of a baboon, a semi-upright animal, during the healing time course of interbody fusion.
Methods. A miniature load cell was fabricated from carbon fiber and from titanium synthetic intradiscal spinal implants (DePuy Acromed, Cleveland, OH) and instrumented with strain gages. The instrumented implant was inserted into the lumbar spine of four skeletally mature baboons following anterior discectomy and arthrodesis. Loading on the anterior aspect of the lumbar spine during different activities was determined in real time in vivo via a telemetry device implanted in the baboon.
Dorsal-ventral and lateral plain radiographs were taken monthly to assess the progress of healing over a 24 week period. Strain data were collected from the instrumented implants approximately three times weekly. As data from the implants were being collected, the animal’s activities were recorded electronically in real time to correlate spinal loading to sitting, standing upright (on two legs), standing (on four legs), laying down, hanging, bending laterally and bending sagittally. Correlations between changes in load during specific activities were determined.
Following euthanasia, the fused segments were grossly inspected, radiographed and placed into a combined axial compression/sagittal plane bending jig and mounted into a mechanical testing machine. The fused segment was loaded nondestructively in combined axial compression and sagittal plane bending while load, disc compression and rotation were measured. The load (via testing machine load cell and implant), displacement (via extensometer) and rotation (via potentiometer) were measured and the stiffness of the fused segments was calculated.
Results. Results from this study indicated that the implant/load cell is sufficiently sensitive to monitor changes in strain during normal activities of the animal. Furthermore, the strain on the implant was highly dependent on the activity of the animal. During extreme activity, the strain values exceeded the range that the system was able to measure. Although the exact load on the implants during these extreme cases is unknown, the highest measurable strain values are indicative of loads in excess of 2.8 times body weight for the 40 kg animals.
Preliminary histologic analysis and gross analysis indicate that the uniquely designed implant/load cell did not prohibit bone formation in the intervertebral disc space. These are consistent with radiographic indications that bony formation had occurred.
Discussion. The purpose of this pilot study was to develop an implant with a known relationship between strain, which was measured on the implant, and load that was applied to the implant. The data was then transmitted via a telemetry system such that in vivo loads were measured in real time. The implant was designed to measure in vivo loads and strains. The magnitude of these loads has not previously been measured. The system was designed to measure a range of loads based on previously reported models. However, the actual loads were higher than those anticipated.
Results from this study will help predict loading on the lumbar spine during everyday activities. This may assist physicians in understanding causes of chronic degenerative disc disease and may facilitate prevention.
Based on this pilot study, we have shown that our technique and technology are sufficient for measuring real time in vivo loads in the spine. The range of loads is higher than expected; therefore the implant/load cell was modified to measure these higher loads. The high loads may also indicate that the performance demands on the intervertebral disc and interbody implants are higher than expected.
Conclusion. A means of measuring the load on an intradiscal implant over the course of healing, provides key information about the mechanics of this process. Because the implant/load cell is initially the only weight bearing entity, the loading on the implant immediately following discectomy describes the full loading on the anterior aspect of the spine. By measuring the load imposed on the implant over the full course of fusion, the “long time” load demands on implants can also be determined. This can be used for performance or design criteria of intradiscal implants and artificial discs.
The future design of spinal implants and disc prostheses can be improved if the mechanical environment in which they are to perform is better understood. Further investigations are necessary to determine the specific forces imposed on the spine during the entire time course of fusion.
*Abstracts/permission forms not received at the time of publication
**Current and ongoing research