BACKGROUND:LMP-1 is known to increase proteoglycan production through the upregulating the BMPs and it is also known that BMP-2 acts on anulus fibrosus cells and chondrocytes to increase proteoglycan production.METHOD:We carried out an experiment, the effect of AdLMP-1 transfection on AF cells and chondrocytes in the production of sulfated-glycosaminoglycans, mRNA expression (aggrecan, type I, II collagen, LMP-1, BMP-2, and BMP-7), and immunofluorescence staining. AF cells and chondrocytes were grown in monolayer and treated for 6 days with AdLMP1-green fluorescence protein (GFP) (10, 20, and 30 multiplicity of infection [MOI]). After 6 days, the sGAG content in the media was quantified using 1,9-dimethylmethylene blue staining. The mRNA expression was measured with real-time PCR after 20 MOI infection of AdLMP1-GFP. The each cells treated with 20 MOI infection of AdGFP was used as a control group for the mRNA expression. The each cell group was immunofluorescence stained with each antibodies in the chamber slide at 3 x 10(4) cells/chamber.FINDINGS:1) The sGAG production was maximum in 20 MOI AdLMP1-GFP infection on the AdLMP-1 treatment for both of AF cells and chondrocytes. 2) The mRNA expression of aggrecan, type I collagen, type II collagen, LMP-1, BMP-2, and BMP-7 is increased in both AF cells and chondrocytes in 20 MOI AdLMP1-GFP infection. 3) On the immunofluorescence staining results, the positive immunofluorescence stained cell numbers are increased after 20 MOI AdLMP1-GFP infection concordant with upregulation of mRNA expression.CONCLUSIONS:The AdLMP-1 treatments in AF cells and chondrocytes may be useful for cell transplantation therapy in disc degeneration.

STUDY DESIGN:A biochemical and radiologic comparison of 4 disc injury models to produce disc degeneration in the rabbit was carried out in 2 experiments.OBJECTIVES:To develop a reliable animal model of intervertebral disc degeneration.SUMMARY OF BACKGROUND DATA:In order to study various interventions for retarding or preventing disc degeneration, a reliable animal model of disc degeneration is needed.METHODS:First experiment: 7 New Zealand white rabbits (1 year old, 3.5-4.5 kg body weight) were used to test 4 different disc injury models; intradiscal injection of Camptothecin (an apoptotic agent) using a 23-gauge needle at L2-L3, nucleus aspiration using a 21-gauge needle at L3-L4, 3 anulus punctures using a 21-gauge needle at L4-L5, and 1 anulus puncture using a 18-gauge needle at L5-L6. The L1-L2 level was used as a control. Rabbits were killed 12 weeks later. Lumbar spinal magnetic resonance images were assessed using 4 grades of disc degeneration. The water content of the nucleus was measured. Dimethylmethylene blue (DMMB) assay was used to measure the sulfated-glycosaminoglycan content. Second experiment: the 21-gauge 3-puncture and the 18-gauge 1-puncture models, thought most effective in producing disc degeneration in the first experiment, were again used in a second study. Six rabbits were killed 8 weeks later, the water and sulfated-glycosaminoglycan contents being measured as in the first experiment.RESULTS:In the first experiment, the water content in the aspiration and puncture models was significantly decreased. Only the sulfated-glycosaminoglycan content in the aspiration model showed a significant decrease as compared to the control. Disc heights and magnetic resonance grades documented significant degeneration occurring in the aspiration and puncture models. In the second experiment, the water content showed a significant decrease in the 21-gauge 3-puncture model, whereas neither of the results for the sulfated-glycosaminoglycancontent showed a significant difference as compared to the control data.CONCLUSION:In the first experiment, the 21-gauge 3-puncture and the 18-gauge 1-puncture models produced the most consistent disc degeneration in the rabbit lumbar spine. When these 2 models were again studied in the second experiment, the 21-gauge 3-puncture technique was superior in producing disc degeneration over a shorter period of time.

Objective An in vitro experiment study using rat disc cells was carried out to determine the effect of bone morphogenetic protein-2 (BMP-2) on extracellular matrix production, other BMPs, and BMP receptors (BMPRs). Methods Cells from the anulus fibrosus and transition zone were harvested and cultured. When the cells reached 80% confluence, BMP-2 was added to reach a final concentration of 200 ng/mL. Three days later, the culture media were collected for the assay of sulfated glycosaminoglycans (sGAG) and collagen types I and II. The cells were harvested for RNA extraction to determine the genes expressed. All experiments were performed at least three times to ensure repeatability. Results BMP-2 significantly increased aggrecan and collagen type II mRNA expression 8.30 and 4.61 times, respectively, and decreased versican mRNA expression 0.54 times as compared with control. Collagen type I production and mRNA level were not changed. BMP-2 significantly increased transforming growth factor-β1 (TGFβ1) and BMP-7 mRNA expression 2.32 and 2.45 times, respectively, compared with control. There was no significant change in BMP-6 mRNA expression. BMPR type IB and II mRNA expressions were increased and BMPR type 1A mRNA expression was decreased, but none of these differences was significant. Conclusions The results of this study show that in rat intervertebral disc cells, BMP-2 increases aggrecan and collagen type II mRNA expression and decreases versican gene expression. BMP-2 also up-regulates mRNA expression for BMP-7 and TGFβ but has no significant effect on the BMPRs.

Background: The hypothesis that injecting bone cement adjacent to one or both endplates would bring about degeneration in the intervening disc was tested. nucleus and the annulus and clear histologic changes in some of the discs.Methods: In 11 dogs, bone cement was injected just below the superior endplates of L1, L2, and L3 to block the nutritional supply through these endplates to the three intervertebral discs T13-L1, L1-L2, and L2-L3. In one other dog, both the superior and the inferior endplates of the same discs (T13-L1, L1-L2, and L2-L3) were blocked with bone cement. All 12 dogs were euthanized between 31 and 70 weeks after the Surgery. The three experimental discs (T13-L1, L1-L2,and L2-L3) and two control discs (T12-T13 and L4-L5) were excised and assessed using enzyme-linked immunosorbent assay (ELISA) and histology.Results: Radiographs of the lumbar spine at the time of death did not show any signs of disc bulging, disc space narrowing, or peripheral osteophyte formation in any of the 12 dogs. The experimental discs as well as the control discs appeared normal in every dog. After the discs were bisected, they were carefully inspected for any visible signs of degeneration. The experimental discs showed no clear signs of disc degeneration and were not distinguishable from the control discs on a gross level. The numerical results from the ELISA showed that in the experimental discs as opposed to the control discs, there were significant increases in proteoglycan content in both the nucleus (P = 0.033) and annulus (P = 0.01) and clear histologic changes in some of the discs.Conclusion: The results show that injecting bone cement adjacent to one or both endplates for up to 70 weeks does not produce degeneration in any visible form in the intervening disc. There were no disc bulging, no apparent annular fissures, and no disc spacing narrowing. There were, however, increases in protoglycan content in both the nucleus and the annulus and clear histologic changes in some of the discs.

Study Design. Experiments using both in vitro tissue culture and in vivo rabbit methods were used to study the effect of Lim Mineralization Protein-1 (LMP-1) on intervertebral disc (IVD) cell production of proteoglycans and bone morphogenetic proteins (BMPs).Objectives. To determine the effect of LMP-1 overexpression in IVD cells on the production of proteoglycans and BMPs both in vitro and in vivo and to show that LMP-1 mediates the control of proteoglycan production through its action on BMPs.Summary of Background Data. Because BMPs are known to increase proteoglycan synthesis and LMP-1 is known to upregulate BMPs in certain cells, it was hypothesized that LMP-1 may increase proteoglycan production in IVD cells.Methods. DMMB, real-time polymerase chain reaction, and ELISA methods were used to quantitate proteoglycan, mRNA, and protein levels, respectively. Noggin was used to block the effect of the adenovirus carrying LMP-1 (AdLMP-1) on proteoglycan synthesis. In vivo experiments using intradiscal AdLMP-1 injection were performed with New Zealand White rabbits. Three weeks later, the mRNA levels of LMP-1, aggrecan, BMP-2, and BMP-7 were measured.Results. In vitro experiments revealed that the sulfated glycosaminoglycan (sGAG) and aggrecan mRNA levels were significantly increased with AdLMP-1 treatment. Similarly, BMP-2 and BMP-7 mRNA and protein levels increased significantly, but BMP-4 and BMP-6 levels were unchanged. Noggin blocked the upregulation of proteoglycan by AdLMP-1. In vivo discs injected with AdLMP-1 had significantly elevated levels of LMP-1, BMP-2, and BMP-7 mRNA.Conclusions. LMP-1 overexpression increases disc cellproduction of proteoglycan, BMP-2, and BMP-7. LMP-1mediates the control of proteoglycan production through its action on BMP.

Study Design. Disc degeneration and osteoarthritis are diseases of the matrix. Chondrocytes that have been removed from damaged cartilaginous tissues maintain a capacity to proliferate, produce, and secrete matrix components, and respond to physical stimuli such as dynamic loading. A dog model was used to investigate the hypothesis that autologous disc chondrocytes can be used to repair damaged intervertebral disc.Objectives. Given the capacity for the cells in vitro to produce matrix molecules that would be appropriate for disc chondrocytes, the focus of the experiment was to investigate whether the cells would continue to sustain metabolic function after transplantation.Summary of the Background Data. No evidence for long-term integration exists for cell transplantation in species other than rats and rabbits. Furthermore, no controlled studies of 1-year duration have been published.Materials and Methods. Disc chondrocytes were harvested and expanded in culture under controlled and defined conditions, returned to the same animals from which they had been sampled (autologous transplantation) via percutaneous delivery. The animals were analyzed at specific times after transplantation by several methods to examine whether disc chondrocytes integrated with the surrounding tissue, produced the appropriate intervertebral disc extracellular matrix, and might provide a formative solution to disc repair.Results. In the context of degenerative changes in an injury model: (1) autologous disc chondrocytes were expanded in culture and returned to the disc by a minimally invasive procedure after 12 weeks; (2) disc chondrocytes remained viable after transplantation as shown by Bromodeoxyuridine incorporation and maintained a capacity for proliferation after transplantation as depicted by histology; (3) transplanted disc chondrocytes produced an extracellular matrix that displayed composition similar to normal intervertebral disc tissue. Positive evidence of proteoglycan content was supported by accepted histochemical staining techniques such as Safranin O-Fast Green; (4) both type II and type I collagens were demonstrated in the regenerated intervertebral disc matrix by immunohistochemistry after chondrocyte transplantation; and (5) when the disc heights were analyzed for variance according to treatment, a statistically significant-correlation between transplanting cells and retention of disc height was achieved.Conclusions. Autologous chondrocyte transplantation is technically feasible and biologically relevant to repairing disc damage and retarding disc degeneration.

STUDY DESIGN:An in vitro experiment to determine the molecular and cellular effect of recombinant human bone morphogenetic protein-2 on cultured rat intervertebral disc cells was performed.OBJECTIVES:To determine the effect of recombinant human bone morphogenetic protein-2 on cell proliferation, production of sulfated-glycosaminoglycan, and the expression of genes specific for chondrocytes (Type II collagen, aggrecan, and Sox9) in cultured rat intervertebral disc cells.SUMMARY OF BACKGROUND DATA:Intervertebral disc degeneration is associated with cellular and biochemical changes, which include decreased synthesis of cartilage specific gene products such as Type II collagen and aggrecan. Although bone morphogenetic protein-2 is known to induce chondrogenesis during new bone formation, the effects on intervertebral disc cells have not been characterized.METHOD:Cells were isolated from the anulus fibrosus and transition zones of lumbar discs from Sprague-Dawley rats. The cells were grown in monolayer and treated with recombinant human bone morphogenetic protein-2 (0, 10, 100, 1000 ng/mL) in Dulbecco's Modified Eagle Medium/F-12 with 1% fetal bovine serum (day 0). On days 2, 4, and 7 after recombinant human bone morphogenetic protein-2 treatment, sulfated-glycosaminoglycan content in the media was quantified using 1,9-dimethylmethylene blue staining. The results were normalized according to culture duration and cell number. On day 7, mRNA was extracted for reverse transcriptase-polymerase chain reaction and real-time polymerase chain reaction to quantitate mRNAs of Type I collagen, Type II collagen, aggrecan, Sox9, osteocalcin, and glyceraldehyde phosphate dehydrogenase. Cell number was determined with a hemocytometer.RESULTS:Recombinant human bone morphogenetic protein-2 at 100 and 1000 ng/mL yielded a 17% and 42% increase in cell number on day 4, and a 59% and 79% on day 7, respectively. Recombinant human bone morphogenetic protein-2 at 10 ng/mL had no effect on cell number. Sulfated-glycosaminoglycan increase was greatest at day 7, increasing by 1.3-, 2.1-, and 3.6-fold with recombinant human bone morphogenetic protein-2 treatments of 10, 100, and 1000 ng/mL, respectively. Increases in mRNA levels of Type II collagen, aggrecan, Sox9, and osteocalcin were observed with recombinant human bone morphogenetic protein-2 concentrations of 100 and 1000 ng/mL on day 7 as determined by reverse transcriptase-polymerase chain reaction. No detectable increase in mRNA level of Type I collagen was observed with any levels of recombinant human bone morphogenetic protein-2. Real-time polymerase chain reaction showed the greatest effect at 1000 ng/mL recombinant human bone morphogenetic protein-2, leading to an 11.5-fold increase in aggrecan, a 4.6-fold increase in Type II collagen, a 5.3-fold increase in Sox9, and a 1.9-fold increase in osteocalcin mRNA above untreated controls at day 7.CONCLUSION:The results of this study show that recombinant human bone morphogenetic protein-2 enhances disc matrix production and chondrocytic phenotype of intervertebral disc cells. Recombinant human bone morphogenetic protein-2 increases cell proliferation and sulfated-glycosaminoglycan (proteoglycan) synthesis. It increases mRNA of Type II collagen, aggrecan, and Sox9 genes (chondrocyte specific genes), and osteocalcin, but not Type I collagen or glyceraldehyde phosphate dehydrogenase.

OBJECT:Systemic nicotine has been hypothesized to cause degeneration of the intervertebral disc which in turn decreases vascular supply to the disc through a cholinergic receptor-mediated process. Another possible mechanism may be through direct regulatory effects on disc cells. In this study, the authors tested the hypothesis that nicotine adversely affects nucleus pulposus cells by directly inhibiting proteoglycan synthesis and gene expression of type II collagen (Phase I study). They also assessed the hypothesis that nicotine inhibits the bone morphogenetic protein (BMP)-2-induced upregulation of extracellular matrix (Phase II study).METHODS:Cells were isolated from nucleus pulposus obtained in rat lumbar discs and cultured on a monolayer. Media were treated with nicotine and/or recombinant human (rh)BMP-2 for 7 days. Sulfated glycosaminoglycan (SO4-GAG) in media was quantified using 1,9-dimethylmethylene blue (DMMB) assay. Gene assay of types I and II collagen, Sox9, and glyceraldehyde-3-phosphate dehydrogenase were quantified using reverse transcriptase-polymerase chain reaction (RT-PCR) and real time PCR. In the Phase I study, nicotine-treated (100 microg/ml) and non-treated cells were compared. The s-GAG production and messenger RNA (mRNA) of type II collagen and Sox9 decreased significantly in the nicotine-treated group. In the Phase II study, five groups were compared: 1) non-treatment; 2) rhBMP-2 only (100 ng/ml); and 3-5) with rhBMP-2 (100 ng/ml) and increasing doses of nicotine (1 [third group], 10, [fourth group], 100 [fifth group] microg/ml). The SO4-GAG production and mRNA of type II collagen and Sox9 decreased significantly in the groups treated with rhBMP-2 combined with 10 and 100 microg/ml of nicotine compared with the group treated with rhBMP-2.CONCLUSIONS:The results of this study raise the possibility that nicotine may contribute to the process of disc degeneration by a direct effect on the nucleus pulposus cells, possibly by antagonizing the effect of BMP-2.

The current study is a biomechanical study using a cadaveric model of L5-S1 spondylolisthesis. The purpose of the current study was to compare, in a cadaveric model of simulated L5-S1 spondylolisthesis, the biomechanical stiffness of transdiscal fixation with traditional pedicle screw fixation, and transdiscal fixation with combined interbody/pedicle screw fixation. The surgical management of L5-S1 spondylolisthesis is a challenge because of the difficulties in achieving a reliable arthrodesis in the face of high mechanical forces. A method of lumbosacral fixation that has been used successfully in moderate grades of spondylolisthesis at our institution involves the use of transdiscal S1 pedicle screws. With this technique, S1 pedicle screws are placed through the S1 pedicle, through the superior endplate of S1, through the inferior endplate of L5, to terminate in the L5 body. Eighteen fresh human cadaveric (age 59-88 years) L5-S1 motion segments were obtained. The end of each intact motion segment was potted up to its midbody in a 10-cm-diameter polyvinylchloride end-cap using dental cement. The intact specimen was then biomechanically tested as follows: 1) axial compression (500 N), 2) flexion (10 Nm), 3) extension (10 Nm), 4) right lateral bending (10 Nm), and 5) left lateral bending (10 Nm). Stiffness values were calculated from the load-deflection curves obtained. Spondylolisthesis was then simulated by displacing L5 on S1 (% slip average = 41.3%) after performing a radical L5-S1 discectomy, L5 laminectomy, and bilateral L5-S1 facetectomies. The 18 motion segments were divided into two groups. Group I (n = 10) was biomechanically tested (as above) after pedicle screw fixation and again after replacing the S1 pedicle screws with transdiscal screws. Group II (n = 8) was biomechanically tested (as above) after combined interbody/pedicle screw fixation and again after fixation with transdiscal screws. Load-deflection curves were obtained each time, and stiffness values were calculated from the curves. Transdiscal fixation was 1.6-1.8 times stiffer than pedicle screw fixation (p < 0.05) in all loading modes tested. There were no differences in stiffness between transdiscal fixation and combined interbody/pedicle screw fixation. In a cadaveric model of simulated L5-S1 spondylolisthesis, transdiscal L5-S1 fixation produced a 1.6-1.8 times stiffer construct than traditional pedicle screw fixation. Further, the stiffness of the transdiscal fixation was equal to that of a combined interbody/pedicle screw fixation.

BACKGROUND:Nutrition to the cells in the disc is partly dependent on fluid flowing out during the day and flowing in during bed rest. In spaceflight there are little or no such diurnal changes, since the gravitational load is essentially zero.HYPOTHESIS:The questions we asked were: 1) How much fluid does the disc gain during the night and how quickly does the disc lose fluid during the following day? 2) Is it possible to carry out, in a reasonable amount of time, an exercise regimen on a spacecraft that would be rigorous enough to expel from the disc the equivalent amount of fluid lost during a normal day's activity?METHODS:in five normal subjects, magnetic resonance imaging was used to measure the volume changes (and the corresponding fluid changes) in the lumbar intervertebral discs (L1/L2, L2/L3, L3/L4, L4/L5) before and after a night's bed rest and again at specific intervals during the course of the day while carrying out three different protocols: walking, carrying a backpack, and exercising in a horizontal position.RESULTS:1) On average the disc gained 10.6% of its volume (or 0.90 cm3 of fluid) during an overnight bed rest; 2) on rising in the morning and after 8 h (using our walking protocol) the disc volume did not decrease to the volume measured at the end of the previous day; and 3) wearing a backpack that weighed 40% of body weight produced a volume decrease equivalent to the decrease in volume obtained at the end of the day, but it took 4 h to do it.CONCLUSIONS:The ability of the disc to retain water is substantial. It would be difficult to carry out an exercise regimen on a spacecraft that would be rigorous enough to expel from the disc the equivalent amount of fluid lost during a "normal" day's activity on Earth.

Study Design. An in vitro biomechanical study of adjacent segment motion (at L3–L4 and L5–S1) after a simulated lumbar interbody fusion of L4–L5 in different sagittal alignments was carried out. Objectives. To test the hypothesis that an L4–L5 fixation in different sagittal alignments causes increased angular motion at the adjacent levels (L3–L4 and L5–S1) in comparison with the intact spine. Summary of Background Data. Clinical experience has suggested that lumbar fusion in a nonanatomic sagittal alignment can increase degeneration of the adjacent levels. It has been hypothesized that this is the result of increased motion at these levels; however, to the authors’ knowledge no mechanical studies have demonstrated this. Methods. Eight fresh human cadaveric lumbar spines (L3–S1) were biomechanically tested. Total angular motion at L3–L4 and L5–S1 under flexion-extension load conditions (7-Nm flexion and 7-Nm extension) was measured. Each specimen was tested intact, and then again after each of three different sagittal fixation angles (at L4–L5): (1) in situ (21°lordosis), (2) hyperlordotic (31°lordosis), and (3) hypolordotic (7°lordosis). The simulated anterior/posterior fusion was performed at L4–L5 with pedicle screws posteriorly, vertebral body screws anteriorly, and an interbody dowel. Results. The averaged values for flexion-extension motion at L3–L4 were as follows: intact specimen 2.0°, in situ fixation 4.0°, hyperlordotic fixation 1.7°, hypolordotic fixation 6.5°. The averaged values for flexion-extension motions at L5–S1 were as follows: intact specimen 2.3°, in situ fixation 2.6°, hyperlordotic fixation 3.6°, hypolordotic fixation 2.9°. Conclusion. Hypolordotic alignment at L4–L5 caused the greatest amount of flexion-extension motion at L3–L4, and the differences were statistically significant in comparison with intact specimen, in situ fixation, and hyperlordotic fixation. Hyperlordotic alignment at L4–L5 caused the greatest amount of flexion-extension motion at L5–S1, and the difference was statistically significant in comparison with intact specimen but not in situ fixation or hypolordotic fixation.

Study Design. A rabbit and rhesus monkey model of posterolateral intertransverse process spine arthrodesis was used.Objective. To test two new soft tissue compression resistant ceramic/collagen sponge carriers for recombinant ant human bone morphogenetic protein-2.Summary of Background Data. After determining that plain collagen sponge was too compressible for large animals in a posterolateral fusion application, the authors demonstrated good bone induction using biphasic ceramic phosphate granules (60% hydroxyapatite/40% tri-calcium phosphate) as the carrier matrix for recombinant 6 V U man bone morphogenetic protein 2 in rhesus monkeys. A limitation of 60:40 biphasic ceramic phosphate its slow resorption time caused by the high hydroxy-apatite content, making radiographic detection of new formation very difficult.Methods. Adult New Zealand white rabbits (n = 14) underwent posterolateral spine arthrodesis at L5-L6 using 5:95 biphasic ceramic phosphate (5% hydroxyapatite/tricalcium phosphate) impregnated Type I collagen sponges (17 X 36 X 2.5 mm, two per side) loaded with 0.86 mg recombinant human bone morphogenetic protein 2. Additional rabbits (n = 14) received 60:40 hydroxyphosphate granules as the carrier for morphogenetic protein 2. Adult rhesus monkeys (n = 6) underwent posterolateral arthrodesis at L4-L5 with ceramic/collagen sponge carrier loaded with 9 mg human bone morphogenetic protein 2 per side. Two monkeys received ceramic/collagen sponges containing 15:85 biphasic ceramic phosphate (15% hydroxyapatite/85% hydroxyapatite/85% tricalcium phosphate) with two pieces per side; two received sponges containing 5:95 biphasic Phosphate with two pieces per side, and two received sponges containing 5:95 biphasic ceramic phosphate with four pieces per side. The rabbits were killed 5 weeks and the monkeys after 24 weeks; the spines were evaluated by manual palpation, radiographs, tensile testing (rabbits only), and histology.Results. The recombinant human bone morphogenetic protein 2 delivered in the 5:95 biphasic ceramic sponge achieved fusion in 100% of rabbits and had improved handling properties compared with the biphasic ceramic phosphate granules. Biomechanical results with 5:95 biphasic ceramic phosphate/collagen carrier were comparable to those obtained with the 60:40 biphasic ceramic phosphate granules and superior to those of autogenous bone graft (p < 0.05). The recombinant human bone morphogenetic protein 2 delivered in the 15:85 or the 5:95 biphasic ceramic phosphate/collagen sponge carrier (two pieces per side) induced fusion in nonhuman primates with normal bone histology, less residual ceramic, and more bone in the center of the carrier matrix in comparison with BCO granules alone. The 15:85 biphasic ceramic phosphate/collagen sponge resulted infusion mass sizes closer to the original size of the matrix implanted than did the 5:95 biphasic ceramic phosphate/collagen sponge, which was considered a desirable feature. The monkeys with 9 mg recombinant human bone morphogenetic protein 2 spread over four sponges per side instead of two had half the effective recombinant human bone morphogenetic protein 2 concentration per sponge and inferior results.Conclusions. The new compression-resistant biphasic ceramic phosphate/collagen sponge matrices were biologically compatible with recombinant human bone morphogenetic protein 2 bone formation, resulted in biomechanically stiffer fusion masses than autograft, better space maintenance than plain collagen sponges, and improved handling and radiographic resorption properties over the ceramic carriers previously tested.

Objective. To test the hypothesis that hydrostatic pressure directly affects the synthesis of collagen and proteoglycan by intervertebral disc cells.Design. By the use of pressure vessels, hydrostatic pressure was applied to intervertebral disc cells cultured in alginate.Background. The influence of compression (both hydrostatic and axial) on chondrocyte metabolism was examined in a number of earlier studies. However, in most of these studies, articular cartilage, not intervertebral disc was used, and in none of these was hydrostatic pressure applied to intervertebral disc cells cultured in alginate.Methods. Fresh, cells were harvested from the lumbar intervertebral discs of dogs. Before their suspension in an alginate gel system, the cells were plated and expanded until they reached confluence. Then, by use of the alginate gel system, the cells were exposed (for up to 9 days) to specific values of hydrostatic pressure inside two stainless steel pressure vessels. One vessel was kept at 0.35 MPa and the other at atmospheric pressure (approximately 0.1 MPa). The effects of 0.35 MPa were compared against atmospheric pressure by measuring the incorporation of [H-3]-proline and [S-35]-sulfate into collagen and proteoglycans, respectively, for the anulus cells and nucleus cells separately, and by determining whether this incorporation was reflected by changes in the levels of mRNA for aggrecan and Types I and If collagen.Results. Proteoglycan synthesis was inhibited at 0.35 MPa as compared to atmospheric pressure for both the nucleus and anulus cells, whereas collagen synthesis was stimulated in the nucleus cells, but inhibited in the anulus cells. The mRNA levels of collagen IA and collagen 2A decreased in the anulus. but showed a differential response in the nucleus (collagen I A increased, while collagen 2A decreased). The mRNA levels for aggrecan core protein decreased in the anulus and increased in the nucleus.Conclusion. Hydrostatic pressure directly affects the synthesis of collagen and proteoglycan by the intervertebral disc cells.

Background: The LIM mineralization protein-1 (LMP-1) gene encodes for an intracellular protein that induces the expression of several bone growth factors. The purpose of the present study was to determine the feasibility and the optimal dose of adenoviral delivery of the LMP-1 cDNA to promote spinal fusion. Methods: A replication-deficient human recombinant adenovirus was constructed with the LMP-1 cDNA driven by a cytomegalovirus promoter. In phase 1, an in vitro dose-response experiment was performed to determine the optimal adenovirus-LMP-1 (AdLMP-1) concentration and infection time. In phase 2, nine rabbits had a single-level posterolateral arthrodesis of the lumbar spine with implantation of a carrier matrix loaded with bone-marrow-derived buffy-coat cells that had been infected for ten minutes with adenovirus containing the cDNA for LMP-1 (AdLMP-1) or b-galactosidase (AdBgal). In phase 3, posterolateral arthrodesis of the spine was performed with implantation of cells infected with AdLMP-1 (ten rabbits) or cells infected with an empty adenovirus that did not contain LMP-1 cDNA (ten rabbits) and the results were compared. In this phase, peripheral-blood-derived buffy-coat cells were used instead of bone-marrow-derived cells and a collagen-ceramic-composite sponge was used as the carrier. Results: In phase 1, the in vitro dose-response experiment showed that a multiplicity of infection of 0.25 plaque-forming units per cell was the most efficient dose. In phase 2, the implants that had received cells infected with AdLMP-1 induced a solid, continuous spinal fusion mass at five weeks. In contrast, the implants that had received cells infected with AdBgal or a lower dose of AdLMP-1 induced little or no bone formation. In phase 3, a solid spinal fusion was observed at four weeks in all ten rabbits that had received cells infected with AdLMP-1 and in none of the ten rabbits that had received cells infected with the empty adenovirus. Biomechanical and histological testing of the AdLMP-1-treated specimens revealed findings that were consistent with a high-quality spinal fusion. Conclusions: Adenoviral delivery of LMP-1 cDNA promotes spinal fusion in immune-competent rabbits. Clinical Relevance: The use of delivery cells that are readily available from peripheral blood and the short infection time should allow this technique to be performed in any operating room. The use of an ex vivo gene-transfer protocol with a very low dose of virus should minimize the immune response and toxicity seen in association with other adenoviral applications.

There are drawbacks to using threaded cylindrical cages (e.g., limited area for bone ingrowth and metal precluding radiographic visualization of bone healing). To somewhat offset these drawbacks, a barbell-shaped cage has been designed. The central core of the barbell can be wrapped with collagen sheets infiltrated with bone morphogenetic protein. The obvious theoretical advantages of a barbell cage have to be weighed against potential biomechanical disadvantages. Our purpose was to compare the biomechanical properties of an anterior lumbar interbody reconstruction using 18-mm-diameter threaded cylindrical cages, with a reconstruction using barbell cages (18-mm diameter and 6 mm wide at both cylindrical ends, with a round 4-mm-diameter bar joining the two ends). Twelve cadaveric lumbar motion segments were tested. Three L5-S1 segments received two threaded cylindrical cages, and three L5-S1 segments received two barbell cages. Three L3-L4 segments received one threaded cylindrical cage, and three L3-L4 segments received one barbell cage. A series of biomechanical loading sequences were carried out on each motion segment, and stiffness curves were obtained. After the biomechanical testing, an axial compressive load was applied to the motion segments until failure. They were then radiographed and bisected through the disc, and the subsidence (or penetration) of the cage(s) in the cancellous bone of the vertebral bodies was measured. There was no difference in terms of stiffness between the motion segments with the threaded cylindrical cage(s) inserted and those with the barbell cage(s) inserted (p > 0.15). The average values of subsidence was 0.96 mm for the threaded cylindrical cage group and 0.80 mm for the barbell cage group (difference not significant: p = 0.38). The results suggest that a reconstruction using barbell cages is a biomechanically acceptable alternative to one using threaded cylindrical cages.

This study was designed to compare the tensile strength of ruptured Achilles tendons repaired using either the triple bundle technique or the Krakow locking loop technique. Eight pairs of fresh frozen cadaveric Achilles tendons were harvested. A simulated “Achilles tendon rupture” was created 4 cm from the calcaneal insertion in all sixteen tendons by transversely cutting the tendon with a scalpel. One Achilles tendon “rupture” of a pair was repaired using the triple bundle technique, while the other tendon of the pair was repaired using the Krakow locking loop technique. Then, using a servohydraulic testing machine, each tendon was tested to failure in tension at a displacement rate of 2.54 cm/sec. The average rupture load for the triple bundle technique was 453 N (range 397 ñ 549 N), while the average rupture load for the Krakow locking loop technique was 161 N (range 121 ñ 216 N). This difference in averages represents a statistically significant superiority of 2.8 to 1 (p < 0.001) in favor of the triple bundle technique.