Comparative analysis in continuous expansion of bovine and human primary nucleus pulposus cells for tissue repair applications

Abstract

Autologous NP cell implantation is a potential therapeutic avenue for intervertebral disc (IVD) degeneration. However, monolayer expansion of cells isolated from surgical samples may negatively impact matrix production by way of dedifferentiation. Previously, we have used a continuous expansion culture system to successfully preserve a chondrocyte phenotype. In this work, we hypothesised that continuous expansion culture could also preserve nucleus pulposus (NP) phenotype. We confirmed that serial passaging drove NP dedifferentiation by significantly decreasing collagen type II, aggrecan and chondroadherin (CHAD) gene expression, compared to freshly isolated cells. Proliferation, gene expression profile and matrix production in both culture conditions were compared using primary bovine NP cells. Both standard culture and continuous culture produced clinically relevant cell populations. However, continuous culture cells maintained significantly higher collagen type II, aggrecan and CHAD transcript expression levels. Also, continuous expansion cells generated greater amounts of proteoglycan, collagen type II and aggrecan protein deposition in pellet cultures. To our surprise, continuous expansion of human intervertebral disc cells -isolated from acute herniation tissue -produced less collagen type II, aggrecan and CHAD genes and proteins, compared to standard culture. Also, continuous culture of cells isolated from young non-degenerate tissue did not preserve gene and protein expression, compared to standard culture. These data indicated that primary bovine and human NP cells responded differently to continuous culture, where the positive effects observed for bovine cells did not translate to human cells. Therefore, caution must be exercised when choosing animal models and cell sources for pre-clinical studies.

Transforming Growth Factor β Enhances Tissue Formation by Passaged Nucleus Pulposus Cells In Vitro

“…However, there are several possible explanations, such as the difference in cell source, human versus bovine, or seeding cell density. Culture environment can also affect NP cells as has been shown in a study by Rosenzweig et al who demonstrated that NP phenotype was better maintained in cells grown in continuous monolayer culture as opposed to those which undergo serial passage.…”

“…Others have also shown that NP cells dedifferentiate with passaging in monolayer culture . This could be a result of the stiffness of the polystyrene culture dishes, cell density, or trypsinization …”

“…Chondrocytes grown under such conditions undergo de‐differentiation and lose their ability to form cartilage . There is some evidence that NP cells will also undergo loss of phenotype under similar culture conditions . Additionally these cells can have a limited replicative capability .…”

Leaping the hurdles in developing regenerative treatments for the intervertebral disc from preclinical to clinical

“…The choice of cell source is an important question. It is well known that species‐specific responses can be observed and as such, caution must be applied when using nonhuman cells in initial proof of principle testing. These differences could arise from differential species‐dependent expression of proteins and receptors, cell types isolated from tissues (eg, notochordal cells), or the disease status of the tissues .…”

Do we damage nucleus pulposus tissue while treating cerebrovascular ischemic neurological deficits with nimodipine?

“…At a pharmaco-molecular level, the study aimed to analyze the effects of nimodipine on chondroadherin (CHAD) (19,20); NP-specific marker proteins; type II collagen (COL2A1) (21), which is responsible for extracellular matrix development; and hypoxia-inducible factor 1 alpha (HIF 1α) (22), which is induced by hypoxia and is a continuous expression of the NP-specific marker.…”