Bone graft substitutes have been used routinely for spine fusion for decades, yet clinical evidence establishing comparative data remains sparse. With recent scrutiny paid to the outcomes, complications, and costs associated with osteobiologics, a need to improve available data guiding efficacious use exists. We review the currently available clinical literature, studying the outcomes of various biologics in posterolateral lumbar spine fusion, and establish the need for a multicenter, independent osteobiologics registry.

STUDY DESIGN:A rabbit model of disc degeneration adjacent to a lumbar spinal fusion.OBJECTIVE:To use a rabbit model to determine the long-term changes in the intervertebral discs at the levels above (cephalad) and below (caudad) 2 fused lumbar levels.SUMMARY OF BACKGROUND DATA:Lumbar spinal fusion is generally carried out to eliminate motion at a specific lumbar level. However, it is commonly thought that by eliminating motion at a level, one increases the motion at the adjacent levels cephalad and caudad the fused levels. There have been studies that have reported on degeneration occurring at the cephalad and caudad levels adjacent to the fused levels.METHODS:A total of 9 New Zealand white, female rabbits: 4 rabbits in the control group and 5 rabbits in the experimental group. The 5 rabbits in the experimental group underwent a posterolateral 2-level lumbar spinal fusion from L3 to L5. The changes in the lumbar discs were assessed using radiographs, magnetic resonance (MR) images, and histology at 6 months and 12 months.RESULTS:The results at 6 months are less clear than those at 12 months. The results at 12 months for the experimental group are (1) the intervertebral disc height decreased at the caudad adjacent level and to a lesser extent at the cephalad adjacent level; (2) the MRI scores for the discs at the caudad and cephalad adjacent levels showed severe loss of signal intensity as compared to the discs at the same levels in the control group. This loss was more pronounced at the caudad level where the loss of signal intensity was similar to that seen at the fused levels; (3) the histologic analysis showed severe degenerative changes with a lack of live cells in the nucleus pulposus and in the endplate at the caudad adjacent level. At the cephalad level, live cells were apparent (albeit few) in the nucleus pulposus, and there was a more normal looking endplate with live cells.CONCLUSION:The intervertebral discs at both the cephalad and the caudad levels adjacent to the 2 fused lumbar levels in this rabbit-model experiment carried out over 12 months after surgery showed degenerative changes asassessed using disc-height measurements, MR images, and histology, and the effect was more severe at the caudad adjacent level.

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.

Osteoporotic vertebral compression fractures are a significant source of morbidity. Recently described techniques of vertebroplasty and kyphoplasty have provided consistent successful outcomes in the short term. However, techniques to address the failure of vertebroplasty have not been addressed. We present two cases of failed vertebroplasty that were revised with a kyphoplasty technique. Both patients had near complete resolution of preoperative symptoms and had a significant vertebral height restoration more than 10 months after revision with kyphoplasty. In conclusion, kyphoplasty technique can be utilized to salvage failed vertebroplasty.

Osteoporotic burst fractures with neurologic symptoms are typically treated with neural decompression and multilevel instrumented fusion. These large surgical interventions are challenging because of patients' advanced ages, medical co-morbidities, and poor fixation secondary to osteoporosis. The purpose of this retrospective clinical study was to describe a novel technique for the treatment of osteoporotic burst fractures and symptomatic spinal stenosis via a limited thoracolumbar decompression with open cement augmentation [vertebroplasty (VP) or kyphoplasty (KP)]. Indications for decompression and cement augmentation were intractable pain at the level of a known osteoporotic burst fracture with symptoms of spinal stenosis. As such, 25 patients (mean age, 76.1 years) with low-energy, osteoporotic, thoracolumbar burst fractures (7 males, 18 females; 39 fractures) were included. In all cases, laminectomy of the stenotic level(s) was followed by vertebral cement augmentation (9 VP; 16 KP). When a spondylolisthesis at the decompressed level was present, instrumentation was applied across the listhetic level (n = 9). Clinical outcome (1 = poor to 4 = excellent) was assessed on last clinical follow-up (mean, 44.8 wks). In addition, a modified MacNab's grading criteria was used to objectively assess patient outcomes postoperatively. Radiographic analysis of sagittal contour was assessed preoperatively, immediately postoperatively, and at final follow-up. The average time from onset of symptoms to intervention was 19 weeks (range, 0.3-94 wks). A mean of 1.6 fractures/patient was augmented (range, 1-3 fractures) and 2.8 levels were decompressed (range, 1-6 levels). No statistical difference in anatomic distribution or number of fractures between the VP and KP groups or in the instrumented versus noninstrumented patients was noted (P > 0.05). An overall subjective outcome score of 3.4 was noted. Twenty of 25 patients were graded as excellent/good according to the modified MacNab's criteria. The choice of augmentation procedure or use of instrumentation did not predict outcome (P = 0.08). Overall, 1.7 degrees of sagittal correction was obtained at final follow-up. One patient was noted to have progressive kyphosis after KP. The use of a limited-posterior decompression and open cement augmentation via VP or KP is a safe treatment option for patients who have osteoporotic burst fractures and who are incapacitated from fracture pain and concomitant stenosis. After thoracolumbar decompression, open VP/KP provides direct visualization of the posterior vertebral body wall, allowing for safe cement augmentation of burst fractures, stabilizing the spine, and obviating the need for extensive spinal reconstruction. Although clinically successful, this technique warrants careful patient selection.

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.

Over 250000 patients each year undergo a spine fusion procedure in the US. This constitutes 50% of all bone graft procedures. Despite best efforts, a large percentage of spine fusions (up to 35%) fail to form a solid bony arthrodesis. This is a significant clinical problem and has led to research in bone formation biology to augment spine fusion rates. Both recombinant and purified osteoinductive cytokines have been studied in pilot and pivotal studies in humans. At this point, recombinant human bone morphogenetic protein-2 has received FDA approved for lumbar interbody application with titanium cages. Despite these successes, limitations of directly applying osteoinductive proteins related to cost and carriers remain to be overcome. Gene therapy for spine fusion and other bone healing applications are being pursued as an alternative strategy. This article will review the state of the art of local gene therapy for bone formation and to highlight specific issues, which must be addressed when pursuing such a program. A critical step in using gene therapy for bone formation is choosing an appropriate osteoinductive gene. In choosing the gene, one must consider the differences in efficacy of the gene as well as the gene availability due to proprietary constraints. The choice of delivery vector is important. Factors such as the potency of the gene and the specific application intended play a role in this decision. Next, the effective dose, transduction time, and gene transfer method must be established. The choice of carrier material to form the scaffold for the new bone formation is another critical step that must be optimized for successful bone formation. Finally, a strategy for in vitro and in vivo testing must be developed to maximize the chances of success in human trials.

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.

Research in biologic methods of treating disc degeneration is still in its infancy. Many different strategies are being evaluated, but the gene therapy strategy stands out because of its potential for long-term efficacy. Choosing the correct gene for use in gene therapy is critically important. Of the many different classes of potentially therapeutic genes, the regulatory genes hold the most promise. Of the different gene therapy delivery methods, the most work has been performed with viral vectors, either ex vivo or in vivo. Current research now is turning toward in vivo experiments in rabbits. Efficacy and safety will be demonstrated first with smaller animal models. Beyond that, nonhuman primate experiments demonstrating efficacy and safety will be the penultimate step before initiation of human studies.

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.