Ex VivoObservation of Human Nucleus Pulposus Chondrocytes Isolated From Degenerated Intervertebral Discs

Abstract

Study DesignWe performed an ex vivo study to observe cell morphology and viability of human nucleus pulposus (NP) chondrocytes isolated from degenerated intervertebral discs (IVD).PurposeTo better understand the biological behavior of NP chondrocytes in monolayer cultures.Overview of LiteratureBiological repair of IVDs by cell-based therapy has been shown to be feasible in clinical trials. As one of the most promising transplanting seeds, how the isolated NP chondrocytes behavior ex vivo has not been fully understood.MethodsHuman NP chondrocytes were harvested from 20 degenerated IVDs and cultured in monolayers. Histological and immunochemistry staining was used to detect cell morphology change. Cell viability was studied by analyzing cell cycle distribution and apoptotic rate in the primary and subculuted cells.ResultsThe round or polygonal primary NP chondrocytes had an average adherence time of 7 days and took nearly 31 days to reach 95% confluence. The spindle-shaped P1 NP chondrocytes increased growth kinetics and took about 12 hours to adhere and 6.6 days to get 95% confluent. Immunochemistry staining of collagen II was positive in the cell cytoplasm. Nearly 90% of the confluent NP chondrocytes stayed in G1 phase while 16% underwent apoptosis. No significant difference of the collagen II expression, cell cycle distribution or the apoptosis indices were detected between the primary and subcultured NP chondrocytes.ConclusionsHuman NP chondrocytes undergo significant morphological change in monolayer cultures. Cell cycle distribution pattern and apoptosis index of the cutured NP chondrocytes potentially influence their clinical transplantation or laboratory use.

Reconstruction of an In Vitro Niche for the Transition from Intervertebral Disc Development to Nucleus Pulposus Regeneration

“…This issue is further complicated by the extensive passaging procedures required to obtain sufficient numbers of cells for in vivo implantation. When passaged in monolayer, NP cells (NPCs) are known to proliferate slowly [18] and undergo increasing dedifferentiation [19][20][21]. Cells from the degenerated NP also become increasingly senescent, which makes them minimally responsive to a growth factor or cytokine stimulation; the cells display an increased catabolic metabolism characterized by the increased production of matrix degrading enzymes [22][23][24][25].…”

Inhibition of microRNA-34a prevents IL-1β-induced extracellular matrix degradation in nucleus pulposus by increasing GDF5 expression

“…33,34 Four normal tissue specimens were obtained from four patients with idiopathic scoliosis (average age 17.8, range 16-20 years) and washed twice in phosphate-buffered saline (PBS) and cut into pieces (2-3 mm 3 ). The NP cells, which were chondrocyte-like cells, were isolated by enzymatic digestion for 8 h at 37 C in Dulbecco's modified Eagle medium (DMEM; Gibco, Grand Island, NY, USA) with 0.25 mg/mL type II collagenase (Invitrogen, Carlsbad, CA, USA).…”

Co-culture of annulus fibrosus cells and bone marrow mesenchymal stem cells

“…More than 20% of all low back pain (LBP) originates from degenerative changes in the intervertebral discs (IVDs), leading to progressive cell loss and degradation of the extracellular matrix (ECM) within the IVD (Wang et al, 2011). Disc degeneration due to age or injury is considered a major cause of LBP.…”