Notochord Cells Regulate Intervertebral Disc Chondrocyte Proteoglycan Production and Cell Proliferation

Abstract

MD, FRCP(C) † Study Design. Non-chondrodystrophic dog notochord cell conditioned medium was used to evaluate chondrocyte proteoglycan production and cell proliferation.Objectives. To evaluate the responsiveness of bovine disc-derived chondrocytes to notochord-cell conditioned medium with respect to proteoglycan and cell proliferation. In addition, to examine phenotypic changes of notochord cells cultured in monolayered as compared to 3-dimensional culture.Summary of Background Data. Non-chondrodystrophic dogs maintain their intervertebral disc notochord cells into adulthood and are protected from having degenerative disc disease develop. The chondrodystrophic breeds such as beagles do not preserve these cells and have disc disease develop much earlier in life. The role of the notochord cell within the disc nucleus is poorly understood.Methods. Canine notochord cells were cultured within alginate beads in serum-deficient conditions using Dulbecco modified Eagle medium to produce notochord cell conditioned medium (NCCM). NCCM was used to stimulate bovine disc chondrocytes from which we evaluated proteoglycan production and cell proliferation as compared to chondrocytes grown in DMEM alone. In addition, parallel cultures of notochord cells were seeded within alginate beads as well as in monolayer and cultured in order to examine for differences in phenotype between the 2 culture conditions.Results. The morphologic aspects of the intervertebral disc between the species differed markedly. A dosedependent relationship was seen between proteoglycan production and NCCM concentration across various concentrations of NCCM in repeated experiments. Although there was a 4-fold increase in cell proliferation under all NCCM concentrations, this increase in cell proliferation was not dose dependent in the concentrations tested. Unlike chondrocytes, notochord cells do not adhere to tissue culture plate (monolayer) until at least day 4 -6, do not markedly alter their phenotype, and rapidly assume masses of cells while floating within tissue culture medium.Conclusions. The biology of the disc-derived chondrocyte is profoundly affected by NCCM in that various concentrations of NCCM activate proteoglycan production in a dose-dependent fashion. However, in the doses tested in our study, cell proliferation was increased but in a nondose-dependent fashion. Notochord cells retain their phenotype even in monolayer and through the development of floating intimately associated masses of cells suggest the development and maintenance of cell-cell interaction. These masses of cells are retained even after 6 days in culture when they do attach to the tissue plate surface. The persistence of notochord cells in non-chondrodystrophic dog species suggests that these in vitro studies may mirror the milieu of the disc in vivo, in which the notochord cell may play a key role in disc homeostasis.

Notochordal intervertebral disc cells: Sensitivity to nutrient deprivation

“…The use of NCs for disc repair is attractive in view of the suggestion that they are stem cells or organizer cells for the NP (13). Implanted NCs improve repair in animal models of acute disc degeneration either directly by producing matrix (13) or indirectly by secreting soluble factors (17) that stimulate the matrix production of MNPCs (17,(47)(48)(49)(50). However, in the light of current findings, both of nutritional requirements and of sensitivity to nutrient deprivation, NCs at concentrations found in vivo may fail to survive in large degenerated human discs with limited nutrient availability ( Figure 4C); those contemplating their prospective use in disc repair should at least consider these possible limitations.…”

The enigma that is the nucleus pulposus cell: the search goes on

“…[5]. Is it a dose-response issue whereby the discs that are relatively deficient in notochordal cells are therefore lacking in the necessary and sufficient molecules synthesized by these cells that may act upon the NP cell [6,7]? It is thought that the notochordal cell-rich disc NP phenotype confers superior biomechanical properties [5,8].…”

Physical Limitations to Tissue Engineering of Intervertabral Disc Cells

“…In fact, transformation of the matrix by cells in human discs is very slow, and studies have shown that proteoglycan turnover takes about 20 years (Roughley, 2004) and collagen turnover takes more than 100 years (Verzijl et al, 2000). However, autologous transplantation of cells with a high proteoglycan production capacity such as notochordal cells and mesenchymal stem cells may lead to successful repair of degenerated discs , Aguiar et al,1999, Cappello et al, 2006, Erwin & Inman, 2006. Even if the transplantation of cells with a high proteoglycan production capacity into the nucleus pulposus is feasible, it is also essential for preventing the progression of disc degeneration that the transplanted cells survive in the degenerated disc and continue to produce appropriate macromolecules for maintenance of disc mechanical strength throughout life.…”

“…Aguiar et al have suggested that the notochordal cells found in the human nucleus pulposus up to approximately 10 years of age play an active role in nucleus pulposus development and in the maintenance of disc integrity through the production of soluble factors that induce nucleus pulposus cells to increase proteoglycan synthesis (Aguiar et al, 1999). Interestingly, it has been found that co-culture of immature cells such as mesenchymal stem cells with notochordal cells and adult nucleus pulposus cells (chondrocyte-like cells) stimulates proteoglycan synthesis more rapidly, and this method has been extensively used in studies on disc regeneration , Cappello et al, 2006, Erwin & Inman, 2006. However, previous studies on proteoglycan production by notochordal cells and chondrocyte-like nucleus pulposus cells have only compared proteoglycan synthesis on the basis of sulphate synthesis, and no studies have compared glycosaminoglycan accumulated by these cells.…”