Although the etiology of low back pain is unclear, it is believed that intervertebral disc (IVD) degeneration plays a major role. In the present study, we sought to determine if bovine IVD cells maintain their phenotype in a mouse subcutaneous injection model, while embedded or not in biocompatible matrices.

Nucleus pulposus (NP) cells were isolated from adult bovine tails. Ten million cells were resuspended either in 500 ƒÝl of DMEM or in a negatively (alginate) or positively (chitosan) charged matrix. The mixtures were then injected subcutaneously in Balc/c nude mice. After two weeks, the mice were sacrificed and the implants harvested. The implants were examined histologically with a hematoxylin and eosin stain. The implant size was measured and the cells were counted. Proteoglycan was assessed by the GAG assay. The expression of type I and II collagens, aggrecan, and CD24 genes was analyzed by reverse transcription ¡V polymerase chain reaction (RT-PCR).

Histologic evaluation confirms the presence of cells in all NP implants. The presence of alginate increased the implant size, the number of cells in the implants, and to a lesser extent, the proteoglycan content, compared to implants formed with cells injected alone. However, chitosan had no effect on the implant size, the number of cells and the aggrecan content. NP implants expressed the same pattern of genes as the native NP tissue (i.e. type I and II collagens, aggrecan, and CD24). The presence of alginate did not affect this expression pattern whereas chitosan decreased slightly their expression.

After injection in mice, bovine NP cells appeared to retain their native phenotype. The RT-PCR analysis revealed that NP cells expressed aggrecan, type I and type II collagens as well as CD24, a specific marker for the NP phenotype. Also, NP cells can be embedded in matrices to produce NP-like features in vivo. In conclusion, we have developed a simple mouse subcutaneous injection model that recreates the features of the native IVD and avoids the need to use a disc degeneration model.

Purpose: To investigate the effect of amifostine and dexrazoxane on bone mass of the vertebrae and femurs of doxorubicin treated male rats.

Methods: Amifostine, Doxorubicin and Dexrazoxane were purchased from SMBD-Jewish General Hospital Pharmacy. Lactating Sprague Dawley dams with 14 male pups were purchased from Charles River Canada. At neonate day 10, rat pups were randomly divided into 4 groups of n=5. Pups were injected once intraperitoneally with either Phosphate Saline Buffer 1X (saline), or drugs, AMF (50 mg/kg), AMF + DOX (50 mg/kg +3 mg/ kg), or with AMF + DXR + DOX (50 mg/kg + 60 mg + 3 mg/kg, 20:1 DXR to DOX ratio). AMF and DXR were injected 30 minutes prior to the DOX injection. After injection, rat pups were returned to their mothers until weaning on neonate day 22. Rats were then sacrificed at day 38 (28 Post-Injection, PI). Bone mineral density (BMD) and micro computed tomography were analyzed.

Results: Dissection of male pups days 1, 5 or 9 post-injection did not reveal any intestinal or organ damage. AMF treatment alone led to a slight but not significant increase in the right femoral, left femoral and lumbar vertebral BMDs. Similarly, AMF + DOX or AMF + DXR + DOX treated rats had no significant change in either femoral and vertebrae BMD.

Conclusions: We recently showed that a single injection of DOX in young female rats is associated with low bone turnover resulting in vertebrae and femur bone growth deficits. However, no such a difference was detected when similarly treated males were examined. The role of sex steroid hormone at this age is unclear as sex hormones level are very low in neonates at the time of injection and the rats, male and female, were sacrificed prior to puberty. To define the role of sex hormone in the observed gender-specific drug susceptibility we plan on comparing the response of intact to ovariectomized female rats to the drug regimen.

Funding : Other Education Grant

Funding Parties : CIHR

Purpose: To develop an improved understanding of the in vivo behavior of intervertebral disc (IVD) cells for determining the phenotype of a differentiated stem cell in tissue engineering applications.

Methods: Nucleus pulposus (NP) and annulus fibrosus (AF) cells were isolated from adult bovine tails while notochordal cells were extracted from fetal bovine intervertebral disc. Ten million cells (of each cell type) in 500 & #61549;l of DMEM were then injected subcutaneously in C57Bl/6 mice. After 2 weeks, the mice were sacrificed and the specimens harvested. They were examined grossly, histologically and by scanning electronic microscopy (SEM) for the evidence of IVD-like structure formation. Proteoglycan was assessed by the GAG assay and PCR for analysis of gene expression. Control tissue (from bovine NP and AF) were directly fixed in glutaraldehyde, without any isolation technique and examined in SEM.

Results: After 2 weeks, SEM examination of specimens from AF and NP closely resembled normal bovine AF and NP. Of special interest here was the finding that some mice injected with cells from the AF developed an organized arrangement of parallel collagen fibres while NP cells injected mice had an amorphous structure with few collagen fibers. The GAG assay showed pro-teoglycan content for each samples, ranging from 3.8 microg to 26 microg. The morphology of the specimens retrieved from notochordal cells injected mice were also amorphous punctuated with thin collagen fibrils.

Conclusions: This study demonstrates that subcutaneous injection of bovine disc cells in mice can result in formation of disc structures similar to those of the bovine IVD. We believe that the cellular communication of the bovine disc cells is maintained in the mouse leading to architectural organization of the collagen fibers with the mouse as a source of nutrients. This technology may be useful in determining the phenotype of a differentiated stem cell for tissue engineering of IVD.

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