Human Intervertebral Disc Cell Attachment, Proliferation and Matrix Production In Vitro: Experimental Studies With Selected Cell Carriers for Tissue Engineering
H.E. Gruber, PhD; E.N. Hanley, Jr., MD
Little is known about how disc cells attach, proliferate and form extracellular matrix (ECM) within carrier materials. Such information is needed to help formulate criteria for successful cell-carrier interactions in tissue engineering. The objective of this research was to compare proliferation, ECM production and gene expression in annulus cells cultured in a variety of cell carrier materials with potential application in tissue engineering of the disc. Annulus cells from discs of 29 individuals were tested in collagen sponge, collagen gel, agarose and alginate or fibrin gel formulations. In situ hybridization assessed ECM gene expression of Types I and II collagen, aggrecan and chondroitin-6 sulfotransferase. Cell proliferation, cell shape, attachment and ECM production were evaluated. Collagen sponges provided the best microenvironment for disc cell ECM production and gene expression. Although collagen gels often could support good cell growth, such constructs did not result in either abundant ECM production or ECM gene expression as shown by in situ hybridization. Growth and ECM production and gene expression in alginate, agarose and fibrin microenvironments were inferior. In conclusion, tissue engineering techniques open new therapeutic possibilities for use of autologous disc cells, but fundamental questions on how these cells interact with cell carriers are unexplored. Results provide novel data on disc cell gene expression within diverse microenvironments. The collagen sponge proved to be a superior microenvironment.
Intratransverse Process Arthrodesis: Efficacy of DC Stimulation in Nicotine Exposure Model
J.C. France, MD; T.L. Norman, PhD
Nicotine has been shown to have an inhibitory effect on the vascular ingrowth of autogenous bone graft leading to prolonged or inhibited bone healing. This effect of nicotine has been suggested as the reason for decreased effectiveness in spinal fusion procedures in smoking patients. In our previous work, direct current electrical stimulation was shown to enhance intertransverse process (ITP) fusion in a rabbit model In this study, the effect of DC current stimulation was investigated in rabbits exposed to nicotine. It was hypothesized that direct current stimulation would ameliorate the debilitating effect of nicotine on bone healing.
Five studies were conducted during the course of this grant due to problems we had administering correct and level nicotine. These problems were corrected using a nicotine patch rather than a miniosmotic pump for nicotine administration. In the final study, 30 adult (one year) 4.0 kg New Zealand white rabbits were obtained and randomly divided into three test groups (n=10). Each group received a single level posterolateral, intertransverse process fusion with autologous iliac crest bone. A control group (Control) received an autologous bone graft fusion. A nicotine group (Nic) received the autologous bone graft fusion and a 10.5 mg nicotine patch. A nicotine/stimulator group (Nic/Stim) received spine fusion with the addition of a 100-microamp direct current stimulator (EBI SpF-2T) (EBI Medical Systems, Inc., Parsippany, NJ) and the 10.5 mg nicotine patch. The fusion masses (L5-6) and the adjacent unfused control segment (L4-5) were harvested from each animal at five weeks. Radiographs of the segments were viewed and graded by orthopedic surgeons. The lumbar spine segments were also manually palpated and evaluated for fusion based on motion and graded as fused or not fused. Mechanical tests in tension (studies 1-4) and bending (study 5) were also performed on the fused segments.
The report contains results from all studies. For the final study, the fusion rates of the Nic/Stim group determined by manual palpation was significantly (p<0.05) higher than controls. The fusion rate of the Nic group was also significantly (p<0.05) greater than the Control group. The average fusion rates were: Nic/Stim (85% ± 12.7%), Nic (66.0% ± 7.2%) and Control (37.5% ± 12.5%). Radiographic assessment revealed significantly (p<0.0151) better trabeculation and bone incorporation in the Nic/Stim specimens (2.17 ± 0.38) compared to the Control group (1.56 ± 0.46). The Nic specimens also exhibited a higher mean (1.91 ± 0.33) than the Control group, although the difference was not significant. There were no significant differences in normalized bending stiffness determined via mechanical testing; we attributed this result to a problem with the test fixture binding. We developed a new bending test for future studies.
DC stimulation significantly improved healing in rabbits exposed to nicotine compared to those exposed to nicotine alone and compared to the control group as hypothesized. Surprisingly, the Nic group showed improved healing compared to the Control group. Nicotine has been shown to have an inhibitory effect on the vascular ingrowth of autogenous bone graft leading to prolonged or inhibited bone healing and this effect has been suggested as the reason for decreased effectiveness in spinal fusion procedures in patients who smoke. However, results of this study indicated improved fusion rates with nicotine delivered via patch. Because tobacco smoke is a complex mixture of over 4,000 chemicals, it is difficult to predict the effect of nicotine delivered via the use of tobacco. Results of the current study are supported by recent studies.
*The Effect of Anterior Wedge Fracture and Subsequent Augmentation on the Mechanical Stability of the Osteopenic Thoraco-Lumbar Spine
R.N. Alkalay, PhD; B.D. Snyder, MD, PhD
*Abstracts/permission forms not received at the time of publication
**Current and ongoing research