Human Anulus Fibrosis and Nucleus Pulposus Cells of the Intervertebral Disc: Effect of Degeneration and Culture System on Cell Phenotype

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Abstract

Human intervertebral disc cells were harvested from patients with adolescent idiopathic scoliosis (AIS) and from donors with degenerative disc disease. Anulus fibrosis (AF) was separated from nucleus pulposus (NP), and cells were cultured separately in two different cell culture models. To investigate changes in gene expression of human disc cells during in vitro expansion and to determine whether cells from adolescent idiopathic scoliosis donors show different gene expression profiles compared with cells from patients operated for degenerative disc disease. During in vitro expansion, cells undergo a dedifferentiation process, which is characterized by a switch in gene expression. Markers for the differentiation and dedifferentiation status of human disc cells are not yet known. Moreover, it is not known whether changes in the gene expression pattern occur during the degeneration process. Cells from AF and NP tissues were expanded in monolayer and alginate cultures under controlled and defined conditions. Cells were then harvested, and analysis of phenotype was performed using quantitative real-time polymerase chain reaction (PCR). The mRNA expression of Type I, II, and X collagen, aggrecan, and interleukin-1beta in scoliosis and degenerative human intervertebral disc cells was analyzed. The gene expression of Type II and X collagen and of aggrecan significantly decreased for both cell types during monolayer expansion. Reexpression of all genes was observed when cells were cultured in alginate. Additionally, NP cells from degenerative tissues displayed significant lower levels of Type II collagen compared with NP cells from scoliosis donors. These results provide a better understanding of how the phenotype of human healthy and degenerative disc cells is influenced by in vitro expansion. This may be useful for future tissue engineering purposes.

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Inflammatory cytokines in the herniated disc of the lumbar spine.

M Motegi, Y Okada, Y Okazima … (1996)

Tissues in the area of herniated lumbar discs were examined for inflammatory cytokines to elucidate the causes of sciatic pain in lumbar disc herniation. To determine the role of inflammatory cytokines in the stimulation of sciatic pain in lumbar disc herniation. It is postulated that in addition to mechanical compression of lumbar nerve roots and sensory root ganglia by herniated discs, there is a chemical stimulus to the production of sciatic leg pain. The exact mechanisms of chemical stimulation are not clearly defined. During surgery, cases of lumbar disc herniation in 77 patients were classified macroscopically into protrusion, extrusion, and sequestration types. Tissues adjacent to nerve roots at the herniation were excised and analyzed biochemically and immunohistochemically for the presence of inflammatory cytokines and for the production of these cytokines and prostaglandin E2 in vitro. The homogenates of samples were analyzed for interleukin-1 alpha, interleukin-1 beta, interleukin-6, tumor necrosis factor-alpha, and granulocyte-macrophage colony stimulating factor, which were detectable. Most of the cytokine-producing cells were histiocytes, fibroblasts, or endothelial cells in extrusion and sequestration types, and chondrocytes in protrusion type. The secretion of these cytokines and prostaglandin E2 was decreased by the addition of betamethasone. The prostaglandin E2 production was dramatically enhanced by additional interleukin-1 alpha, but decreased by the addition of tumor necrosis factor-alpha. The results demonstrate that at the site of lumbar disc herniation, inflammatory cytokines such as interleukin-1 alpha are produced, which increases prostaglandin E2 production. Further studies are required to elucidate the role of inflammatory cytokines in causing sciatic pain.

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Reexpression of cartilage-specific genes by dedifferentiated human articular chondrocytes cultured in alginate beads.

P Freisinger, N Kadhom, J. Bonaventure … (1994)

We have used the three-dimensional culture system in alginate beads to redifferentiate human articular chondrocytes which were first expanded on a plastic support. After 15 days in alginate beads, electron microscopy showed that cells had synthesized an extracellular matrix containing collagen fibrils. Electrophoretic analysis of proline-labeled cells demonstrated that redifferentiated chondrocytes synthesized mainly type II collagen and its precursors (pro alpha 1II, pc alpha 1II, and pn alpha 1II). After pepsin digestion a small amount of collagen type XI was also detected. These results were confirmed by Northern blot analysis of total RNAs. Hybridization with collagen cDNA probes coding for the alpha 1(II) and alpha 1(I) chains of collagen types II and I showed that chondrocytes cultured in alginate expressed mainly alpha 1(II) mRNA, whereas alpha 1(I) mRNA transcripts were almost undetectable. Such a result was observed even after several passages on plastic flasks, suggesting that dedifferentiated cells were able to revert to a chondrocytic phenotype in this three-dimensional system. However, SV40-transformed chondrocytes were not able to redifferentiate in alginate as no alpha 1(II) mRNAs were detected. Total RNA was converted into cDNA by reverse transcription and amplified by polymerase chain reaction. This technique was employed to amplify mRNAs specific for collagen type II and type X and the large aggregating proteoglycan aggrecan. Two transcripts resulting from an alternative splicing of the complement regulatory protein (CRP)-like domain of aggrecan were originally identified in chondrocytes in monolayers. Like intact cartilage, chondrocytes in alginate expressed only the larger transcript with the CRP domain, whereas the two transcripts were equally expressed in SV40-transformed chondrocytes. Thus, the alginate system appears to represent a relevant model for the redifferentiation of human chondrocytes, especially when only a small cartilage biopsy is available, and could prove useful for pulse-chase studies of patients with skeletal chondrodysplasias. However it was unable to restore the chondrocytic phenotype in virally transformed cells.

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Molecular markers predictive of the capacity of expanded human articular chondrocytes to form stable cartilage in vivo.

F. Dell'Accio, C De Bari, F Luyten (2001)

To establish a model and associated molecular markers for monitoring the capacity of in vitro-expanded chondrocytes to generate stable cartilage in vivo. Adult human articular chondrocytes (AHAC) were prepared by collagenase digestion of samples obtained postmortem and were expanded in monolayer. Upon passaging, aliquots of chondrocyte suspensions were either injected intramuscularly into nude mice, cultured in agarose, or used for gene expression analysis. Cartilage formation in vivo was documented by histology, histochemistry, immunofluorescence for type II collagen, and proteoglycan analysis by 35S-sulfate incorporation and molecular sieve chromatography of the radiolabeled macromolecules. In situ hybridization for species-specific genomic repeats was used to discriminate human-derived from mouse-derived cells. Gene expression dynamics were analyzed by semiquantitative reverse transcription-polymerase chain reaction. Intramuscular injection of freshly isolated AHAC into nude mice resulted in stable cartilage implants that were resistant to mineralization, vascular invasion, and replacement by bone. In vitro expansion of AHAC resulted in the loss of in vivo cartilage formation. This capacity was positively associated with the expression of fibroblast growth factor receptor 3, bone morphogenetic protein 2, and alpha1(II) collagen (COL2A1), and its loss was marked by the up-regulation of activin receptor-like kinase 1 messenger RNA. Anchorage-independent growth and the reexpression of COL2A1 in agarose culture were insufficient to predict cartilage formation in vivo. AHAC have a finite capacity to form stable cartilage in vivo; this capacity is lost throughout passaging and can be monitored using a nude mouse model and associated molecular markers. This cartilage-forming ability in vivo may be pivotal for successful cell-based joint surface defect repair protocols.