Restoration of disc height loss by recombinant human osteogenic protein-1 injection into intervertebral discs undergoing degeneration induced by an intradiscal injection of chondroitinase ABC.

STUDY DESIGN: In vivo study of the effect of an injection of recombinant human osteogenic protein-1 into degenerated discs induced by chondroitinase ABC. OBJECTIVE: To investigate the efficacy of an injection of recombinant human osteogenic protein-1 to induce the recovery of disc height, and biochemical and histologic repair, in discs degenerated through enzymatic digestion by chondroitinase ABC. SUMMARY OF THE BACKGROUND DATA: Chondroitinase ABC is currently proposed as a chemonucleolysis agent; however, postchemonucleolysis degeneration is currently unavoidable. Recombinant human OP-1 has been shown to promote extracellular matrix repair in vitro and in vivo. METHODS: Fifty-four adolescent New Zealand white rabbits were used. Four weeks after an initial injection of chondroitinase ABC (10 mU/disc), 5% lactose (10 microL/disc) or recombinant human osteogenic protein-1 (100 microg in 10 microL lactose/disc) was injected. Disc heights were monitored radiographically at 2-week intervals, and rabbits were killed at 6, 8, 12, and 16 weeks after the initial chondroitinase ABC injections. The intervertebral discs were subjected to histologic and biochemical analyses. RESULTS: Significant disc space narrowing was observed in both groups 2 weeks after the injection of chondroitinase ABC. In the chondroitinase ABC/lactose group, this narrowing progressed after the vehicle injection and was sustained for up to 16 weeks. In the chondroitinase ABC/recombinant human osteogenic protein-1 group, the disc height index showed a significant increase at 6 weeks (lactose vs. recombinant human osteogenic protein-1; P < 0.01); this recovery was sustained for up to 16 weeks. The proteoglycan content was higher in the chondroitinase ABC/recombinant human osteogenic protein-1 group than in the chondroitinase ABC/lactose group. However, histologic changes, after the recombinant human osteogenic protein-1 injection, were not observed. CONCLUSIONS: A single injection of recombinant human osteogenic protein-1 into a rabbit disc dramatically reversed the decrease in disc height induced by chondroitinase ABC chemonucleolysis. The recovery was significant and sustained over the next 12 weeks. The therapeutic effects of both chondroitinase ABC chemonucleolysis and recombinant human osteogenic protein-1 injections should be further explored in higher animals before it is applied to humans.

Osteogenic protein-1 injection into a degenerated disc induces the restoration of disc height and structural changes in the rabbit anular puncture model.

STUDY DESIGN: In vivo study of the effect of injection of osteogenic protein-1 (OP-1) on a rabbit anular needle puncture model of intervertebral disc (IVD) degeneration. OBJECTIVE: To study radiographic, magnetic resonance imaging (MRI), biochemical, and histologic changes in the rabbit IVD after injection of OP-1 into the nucleus pulposus in a needle puncture disc degeneration model. SUMMARY OF THE BACKGROUND DATA: Growth factors, such as OP-1, have the ability to stimulate synthesis of proteoglycans and collagen in vitro. The in vivo injection of OP-1 into the normal rabbit IVD has increased disc height and proteoglycan content in the anulus fibrosus and nucleus pulposus. However, to our knowledge, no attempts have yet been made to determine the effects of these growth factors in an in vivo model of disc degeneration. METHODS: New Zealand adolescent white rabbits (n = 90, 8 for baseline evaluation, 82 at 8 times) received an anular puncture in 2 noncontiguous discs with an 18-gauge needle to induce disc degeneration. Four weeks later, either 5% lactose (10 microL) or OP-1 (100 microg in 10 microL 5% lactose) was injected into the center of the nucleus pulposus. The disc height was followed radiographically for up to 24 weeks after the injections. At the 2, 4, 8, 12, and 24-week times after the injection, rabbits were euthanized, and MRI of the harvested spinal columns was obtained to grade the degeneration. The discs injected with OP-1 or lactose and noninjected discs were subjected to biochemical and histologic analysis. The specimens at the 24-week time were limited to histologic evaluation. RESULTS: The anular puncture with a needle induced a consistent disc narrowing within 4 weeks. The injection of OP-1 induced a restoration of disc height at 6 weeks, which was sustained for the entire experimental period, up to 24 weeks after the injection. The injection of lactose alone did not change the course of disc narrowing over the same time. MRI grading score showed significant differences between the OP-1 and lactose groups at the 8, 12, and 24-week times, suggesting an increase in water content in the nucleus pulposus of the OP-1 group. The proteoglycan content of the nucleus pulposus and anulus fibrosus was significantly higher in the OP-1 group than in the control group. The degeneration grades of the punctured discs in the OP-1 group were significantly lower than those in the lactose group. CONCLUSION: The results of this study show the feasibility of restoring degenerative rabbit discs by a single injection of OP-1 into the nucleus pulposus. Importantly, the effects of the OP-1 injection on disc height were sustained for up to 24 weeks. The metabolic changes in the cells, following a single injection, might be sustained and, thus, induce long-term changes in disc structure. An efficacy study in large animals is required to show further that the intradiscal injection of OP-1, or bone morphogenetic proteins or growth factors with similar properties would be useful for the structural restoration of the IVD in humans.

Anti-adhesion properties of a thrombin-based hemostatic gelatin in a canine laminectomy model: a biomechanical, biochemical, and histologic study.

STUDY DESIGN: An in vivo study on the effects of an agent for scar prevention in a dog laminectomy model. OBJECTIVE: To examine the anti-adhesion properties of a thrombin-based hemostatic gelatin (FLOSEAL; Baxter International Inc., Deerfield, IL). SUMMARY OF BACKGROUND DATA: Postlaminectomy dural adhesion sometimes causes symptomatic problems. Although a new type of hemostatic agent, a thrombin-based hemostatic gelatin, has been developed, its effect on postlaminectomy scar prevention is unknown. METHODS: A 4-level lumbar laminectomy was performed on 11 adult mongrel dogs. There were 2 levels randomly chosen to receive the thrombin-based hemostatic gelatin treatment, while the remaining levels were untreated (control). Dogs were euthanized 8 weeks after surgery. To assess the tenacity of adhesion between the dura and scar, peel-off testing was performed. Gross, biochemical, and histologic analyses were then used to examine the samples. RESULTS: Peel-off stiffness was significantly decreased in the treatment group (64.8% of the control, P = 0.032; 2-tailed unpaired t test). Histologically, there was significantly less scar tissue formation in the treatment group (P = 0.04). The gross analyses showed a trend toward a smaller amount of scar tissue and tenacity of adhesion between the dura and scar in the treatment group. Biochemically, there was a trend toward a lower collagen cross-links content in the treatment group (P = 0.07). CONCLUSIONS: The thrombin-based hemostatic gelatin decreases the tenacity of adhesion between the dura and scar at laminectomy sites.

Exposure to pulsed low intensity ultrasound stimulates extracellular matrix metabolism of bovine intervertebral disc cells cultured in alginate beads.

STUDY DESIGN: In vitro study on the effects of pulsed low intensity ultrasound on the cellular metabolism of bovine intervertebral disc cells. OBJECTIVE: To determine whether pulsed low intensity ultrasound has effects on cell proliferation and extracellular matrix metabolism by bovine intervertebral disc cells. SUMMARY OF BACKGROUND DATA: The application of pulsed low intensity ultrasound is known to be effective in stimulating fracture and cartilage repair. However, the effects of pulsed low intensity ultrasound on intervertebral disc cells are not known. METHODS: Cells of the nucleus pulposus and inner and outer anulus fibrosus were enzymatically isolated from bovine coccygeal tissue and precultured in alginate beads for 14 days. In the ultrasound group, pulsed low intensity ultrasound was administered to the culture for 20 minutes daily for an additional 20 days. The control group was cultured in the same way but without administration of ultrasound. Cell viability, DNA content, proteoglycan and collagen synthesis, and proteoglycan content at days 10 and 20 after the initiation of treatment were evaluated. Characterization of newly synthesized collagen and proteoglycan was performed. RESULTS: No significant differences in cell viability and DNA content were observed between the two groups. On day 20, proteoglycan synthesis was increased by the application of pulsed low intensity ultrasound in nucleus pulposus and inner and outer anulus fibrosus cells (24%-26% increase, P < 0.001). The application of pulsed low intensity ultrasound increased proteoglycan content in alginate beads containing inner and outer anulus fibrosus cells (P < 0.05). Collagen synthesis by cells isolated from all three zones of the intervertebral disc was increased by the application of pulsed low intensity ultrasound (16%-19% increase, P < 0.05-0.0001). CONCLUSIONS: The application of pulsed low intensity ultrasound stimulated extracellular matrix metabolism in intervertebral disc cells. Pulsed low intensity ultrasound may prove useful for the physical stimulation of cell metabolism for tissue engineering of intervertebral disc tissue.

A novel rabbit model of mild, reproducible disc degeneration by an anulus needle puncture: correlation between the degree of disc injury and radiological and histological appearances of disc degeneration.

STUDY DESIGN: An in vivo study to radiographically and histologically assess a new method of induction of disc degeneration. OBJECTIVE.: To establish a reproducible rabbit model of disc degeneration by puncturing the anulus with needles of defined gauges and to compare it to the classic stab model. SUMMARY OF BACKGROUND DATA: New treatment approaches to disc degeneration are of great interest. Although animal models for disc degenerative disease exist, the quantitative measurement of disease progression remains difficult. A reproducible, progressive disc degeneration model, which can be induced in a reasonable time frame, is essential for development of new therapeutic interventions. METHODS: The classic anular stab model and the new needle puncture model were used in the rabbit. For the needle puncture model, 3 different gauges of needle (16G, 18G, and 21G) were used to induce an injury to the disc to a depth of 5 mm. Radiographic and histologic analyses were performed; magnetic resonance images were also assessed in the needle puncture model. RESULTS: Significant disc space narrowing was observed as early as 2 weeks after stabbing in the classic stab model; there was no further narrowing of the disc space. In the needle puncture model, all needle sizes tested induced a slower and more progressive decrease in disc height than in the classic stab model. The magnetic resonance imaging supported the results of disc height data. CONCLUSIONS: The needle puncture approach, using 16G to 21G needles, resulted in a reproducible decrease of disc height and magnetic resonance imaging grade. The ease of the procedure and transfer of the methodology will benefit researchers studying disc degeneration.

Transplantation of mesenchymal stem cells embedded in Atelocollagen gel to the intervertebral disc: a potential therapeutic model for disc degeneration.

Intervertebral disc degeneration is considered to be one of the major causes of low back pain. Despite this irreversible phenomenon, attempts to decelerate disc degeneration using various techniques have been reported. However, to date there has been no proven technique effective for broad clinical application. Based on previous studies, we hypothesize that maintenance of proteoglycan content in the disc is achieved by avoiding the depletion of nucleus pulposus and preserving the structure of the annulus is a primary factor in decelerating disc degeneration. One novel approach to solve the dilemma of intervertebral disc degeneration is found at the stem cell level. Mesenchymal stem cells (MSCs) are known to possess the ability to differentiate into various kinds of cells from mesenchymal origin. Although the majority of cells that contribute to disc formation are known to obtain chondrocyte-like phenotypes, no reported study has emphasized the correlation with mesenchymal stem cells. To evaluate the possible potential of MSCs in disc cell research and treatment of degenerative disc disease, autologous MSCs embedded in Atelocollagen gel were transplanted into the discs of rabbits which had undergone a procedure proven to induce degeneration. The results suggest that MSC transplantation is effective in decelerating disc degeneration in experimental models and provided new hopes for treatment of degenerative disc disease in humans. Atelocollagen gel served as an important carrier of MSCs in transplantation, permitting proliferation, matrix synthesis and differentiation of MSCs. This study strengthens the viable efficacy of practical application of MSCs in treatment of intervertebral disc disease.

Effect of reinsertion of activated nucleus pulposus on disc degeneration: an experimental study on various types of collagen in degenerative discs.

We examined the emergence and sequential changes in type I, II, and VI collagen production in an experimental rabbit model of disc degeneration. Type I collagen was minimally present initially and did not change over 24 weeks. Type I collagen seemed to have no effect on the degenerative process in this model. Staining for type II collagen was positive circumferentially in chondrocytelike cells and was mild in the early phase of disc degeneration, when the chondrocytelike cells began to appear in the inner layers of the annulus fibrosus. The stain became stronger during the middle phase when the chondrocytelike cells arranged themselves in cluster. Compared with type II collagen, the staining for type VI collagen was relatively strong early in the degenerative process. These findings led us to speculate that these chondrocytelike cells play an active role in the degenerative process. The reinsertion of nucleus pulposus cells cocultured with annulus fibrosus delayed disc degeneration and the emergence of chondrocytelike cells. Considering that the emergence of chondrocytelike cells which produce type II and type VI collagen is delayed in discs with the injection of cocultured nucleus pulposus cells by annulus fibrosus cells, we conclude that chondrocytelike cells that produce type VI collagen also seems to accelerate degeneration. Type VI collagen is produced at an earlier phase than type II collagen and may be both active agent and a marker for disc degeneration.