Cell polarity in the anulus of the human intervertebral disc: morphologic, immunocytochemical, and molecular evidence.
METHODS
Human intervertebral disc tissue was obtained in a prospective study of cell morphology and gene expression. Experimental studies were approved by the authors' Human Subjects Institutional Review Board. Discs were obtained from surgical specimens or control donors.


OBJECTIVE
To determine if there is morphologic and molecular evidence for polarity in cells of the human anulus.


BACKGROUND
In many tissues, cells become polarized as they develop functional specializations, which involve cell-cell and cell-extracellular matrix interactions and polarized targeting mechanisms. The highly specialized lamellar organization of the anulus is well recognized and suggests that this structure may be the result of directed secretion of extracellular matrix components by polarized disc cells.


METHODS
Human disc specimens from donor and surgical patients were examined with light and electron microscopy to assess morphology. Specimens were examined for immunocytochemical localization of PAR3 and claudin-1 and -11, recognized polarity proteins, and additional anulus specimens were examined for expression of polarity-related genes using microarray analysis. In vitro monolayer and 3-dimensional anulus cultures were also studied for gene expression, and additional surgical anulus specimens were used to obtain gene expression data using real time RT-PCR.


RESULTS
At the ultrastructural level, anulus cells showed localization of secretory organelles and directed deposition of extracellular matrix in one portion of the cell, with the nucleus positioned in the opposite side of the cell. Expression of the polarity-related genes claudin-11 and PAR3 and PARD6 was confirmed using RT-PCR and microarray studies and immunocytochemical analyses. The percentage of cells positive for PAR3 immunolocalization was significantly greater in the outer anulus (100%) than in either the inner anulus (43.8%) or nucleus pulposus (22.6%).


CONCLUSIONS
At the macroscopic level, the characteristic anular lamellar morphology implies a specialized architectural formation and organization, which is achieved by the tissue-specific function of polarized cells. Morphologic and molecular studies provided evidence for the presence of polarity in cells in the anulus. These findings advance our understanding of anulus disc cell function in production of highly aligned collagen fibrils and macroaggregates of these collagen fibrils into lamellar collagen bundles. Such disc cell activity is important in development and maintenance of the tissue-specific extracellular matrix of the disc.
Gruber
Helen E
HE
Department of Orthopaedic Surgery, Carolinas Medical Center, Charlotte, NC 28232, USA. helen.gruber@carolinashealthcare.org
Ingram
Jane
J
Hoelscher
Gretchen L
GL
Norton
H James
HJ
Hanley
Edward N
EN
D048868:Adaptor Proteins, Signal Transducing; D000328:Adult; D000368:Aged; D015415:Biomarkers / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D016764:Cell Polarity / Q000502:physiology*; D002478:Cells, Cultured; D062445:Claudin-1; D057167:Claudins; D003094:Collagen / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism; D005260:Female; D006801:Humans; D007120:Immunochemistry; D007403:Intervertebral Disc / Q000166:cytology* / Q000502:physiology* / Q000648:ultrastructure; D008297:Male; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008854:Microscopy, Electron; D008875:Middle Aged; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D022142:Secretory Vesicles / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D015415:Biomarkers; C568108:CLDN1 protein, human; C120453:CLDN11 protein, human; D018797:Cell Cycle Proteins; D062445:Claudin-1; D057167:Claudins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; C509689:PARD3 protein, human; D003094:Collagen