Purpose: Knowledge of factors regulating the turnover, repair, and degeneration of the intervertebral disc (IVD) is lacking. Although type II collagen (CII) fragments accumulate in the degenerative IVD, little is known of how they affect the degenerative process. In this study, the effect of a CII fragment, CII-(245–270), known to be critical in arthritis was investigated on gene expression of proteinases, collagen, and proteoglycan by bovine disc cells to determine its role in matrix turnover.

Methods: Cells isolated from the nucleus pulposus (NP) and annulus fibrosus (AF) of adult bovine tails were cultured in the absence (control) or presence of the fragment. The fragment CII-(245–270) (US Biological, Massachusetts) was dissolved in culture medium to a final concentration of 1& #956;g/ml. PCR was performed and products were visualized by ethidium bromide staining.

Results: Addition of the CII-(245–270) peptide at 1& #956;g/ml to NP and AF cells enhanced expression of genes for MMP-1, cathepsin K, and aggrecan after 48 hours compared with the control. MMP-13 was also upregulated in the NP. In contrast, the effect in the AF was time dependent. Type II collagen was upregulated throughout the culture time in the NP as opposed to the AF where its expression was enhanced only on day 2.

Conclusions: We have shown that the CII-(245–270) peptide can alter gene expression of proteinases, collagen, and proteoglycan in bovine disc cells. The present study reveals the complex interrelationships of gene expression in the disc that accompany fragmentation of type II collagen. This new information suggests that increased levels of these fragments, in degenerated discs, may stimulate disc breakdown but may also attempt to protect the disc, by unknown mechanisms Funding: Other Education Grant Funding Parties: AO foundation, Switzerland

Low back pain is the primary cause of disability in individuals younger than 50 years. Potential sources of low back pain include the intervertebral disks, facet joints, vertebrae, neural structures, muscles, ligaments, and fascia. Increasing evidence is available as to the importance of cytokines in acute and particular chronic pain. Cytokines can influence transduction, conduction, and transmission of the nociceptive signal, resulting in prolonged or permanent signalling to the brain’s cognitive centres in the absence of a painful noxious or nonnoxius stimulus.

Several cytokines, including IL-1, TNFa, IL-6, and IL-10 are thought to influence nociception or pain.

To date, there have been no studies of the production of inflammatory mediators in blood from patients with low back pain. We have therefore analysed levels of the proinflammatory mediators IL-1ß, IL-6, TNF-α in sera from patients with sciatica and low back pain, and their possible relationship to pain dimensions.

In this prospective longitudinal study with a follow-up of six months, the course of serum concentration of IL-1ß, IL-6 and TNF-α was measured by Bio-Plex cytokine assay in 31 patients with acute sciatica and 41 patients with chronic low back pain. Blood samples were taken at ten fixed times during follow-up, and cytokine values were adjusted to possible influential factors and correlated to the course of pain and clinical function to evaluate the predictive role of cytokine regarding therapy outcome.

At admission of the study and 10 days later, the proportion of TNF-α positive subjects was significant elevated among patients with low back pain compared to patients with acute sciatica. Median (SD) of serum TNF-α concentrations were significant higher in patients with chronic low back pain (n=41) than in patients with acute sciatica (n=31). In the whole period the pain of patients reduced from time to time. Elevated TNF-α serum levels are associated with a significantly improved pain in patients with chronic low back pain but not with acute sciatica. A close coherence exists between the cytokines IL-1ß, IL-6 and TNF-α together in blood of patients as with acute sciatica as with chronic low back pain. But no connection of IL-1ß, IL-6 or TNF-α and CRP in blood was observed. Neither age, sex, BMI, nicotine and alcohol consumption are not related to the serum levels of cytokines.

As far as we know, this is the first analysis of parameters predicting a major clinical connection of cytokines in blood and low back pain. Our findings indicate that elevated serum levels of the proinflammatory cytokine TNF-α are associated with a significantly improved pain in patients with chronic low back pain but not with acute sciatica. We concluded that Detection of high level of TNF-α might be a marker for more pain in patients with chronic low back pain. and TNF-α probably play an important role in the chronic process of low back pain.

The thrombin-related peptide, TP508, is a synthetic 23 amino acid peptide, which represents the receptor binding domain of thrombin. TP508 mimics thrombin by interacting with receptors on cells involved in tissue repair. TP508 has been shown to enhance revascularization of injured tissue, and promote soft tissue wound healing, cartilage repair, and fracture repair. The aim of this study is to (1) test the effect of TP508 on bone regeneration during distraction osteogenesis; (2) study the chemotactic effect of TP508 on human osteoblasts.

Unilateral tibial osteoectomies were performed and stabilized with MX100 Orthofix lengthener in 5 male adult NZW rabbits. After 7 days, distraction was initiated at rates of 1.4 mm / day for 6 days. TP508 (100 μg/ml, n=2; 10 μg/ml, n=1) or saline (300 μl, n=2) was injected into the osteotomy / lengthening gap at days 1, 7 and 14 post surgery. Animals were sacrificed at 2 weeks after leg lengthening. Bone formation in the regenerate was assessed by radiography, quantitative computed tomography (pQCT) and histology. For chemotaxis studies, MG63 cells were cultured on glass cover slips for three days, and then inverted onto a Dunn chamber slide and sealed with dental wax. Gradients of TP508 (1, 10, 100 μg/ml) were added to the outer well and plain medium to the inner well. A sequence of images of the cells between the wells was taken via a CCD camera for 9 hours at interval of 10 minutes. Movements of individual cells were tracked and statistically analysed by a specially written Macro program. The Rayleigh test for unimodal clustering was used to determine the directional chemotactic movements.

The radiographic evaluation indicated a significant increase in new bone in the distraction regenerate in the TP508 treated groups at 1 and 2 weeks. pQCT images at 2 weeks demonstrated more advanced bone formation in the TP508 treated animals compared to the control. The mean total bone mineral density (BMD) of the regenerate, obtained from 3 slices was significantly greater (p = 0.019, t-test) in the TP508 treated group (BMD = 479.20 +/− 35.57 mg/ccm) than that in the saline control group (BMD = 355 +/− 2.83 mg/ccm). The histological evaluation supported the radiographic and the pQCT results. For chemotaxis study, no directional movements of the cells were found in the controls, whereas the MG63 cells were strongly chemotactic to TP508 at 1, 10 and 100 μg/ml concentrations.

This preliminary study shows that administration of TP508 enhances bone formation during distraction osteogenesis in the rabbit. The findings also show that TP508 has a chemotactic effect on osteoblasts, consistent with the effect of TP508 on fracture repair. A large animal study is in the process to confirm these findings and explore the underlying mechanisms.