NEO-INTERVERTEBRAL DISC FORMATION BY SUBCUTANEOUS INJECTION OF BOVINE DISC CELLS IN MICE

Abstract

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.