title: Culture of human anulus fibrosus cells on polyamide nanofibers: extracellular matrix production.
abstract: METHODS
  Studies were approved by the authors' Human Subjects Institutional Review Board. Human anulus cells were tested for growth and extracellular matrix (ECM) production in vitro.
  
  
  OBJECTIVE
  To investigate cell attachment, cell proliferation, and ECM production of human intervertebral disc anulus cells seeded onto randomly oriented electrospun polyamide nanofibers.
  
  
  BACKGROUND
  Because nanofibrillar matrices have the potential to promote microenvironments, which may mimic in vivo conditions and resemble connective tissue, their utilization opens new avenues for cell-based tissue engineering applications for disc cells.
  
  
  METHODS
  Anulus cells were isolated from 4 cervical spine surgical disc specimens, expanded, and seeded into either routine plastic culture (control) or a nanofiber surface of randomly oriented electrospun polyamide nanofibers (Ultra-Web-coated culture dish, Corning) with a positive charge or without a charge. Cells were cultured for 9 days, digital images captured, cells harvested, embedded in paraffin, and examined for production of extracellular matrix (ECM). Additional anulus cultures were tested to quantitatively assess total proteoglycan production and cell proliferation under control or nanofiber cultures.
  
  
  RESULTS
  Cells attached well and exhibited cell extensions within the nanofiber layers; cells on the charged nanofiber surface deposited greater amounts of chondroitin sulfate than of type II collagen than cells cultured on the uncharged nanofiber surface. Results showed that culture of anulus cells on nanofibers was permissive for secretion and assembly of type II collagen and chondroitin sulfate. Significantly greater total proteoglycan formation was present after culture on the nanofiber with added charge conditions {control, 0.6116 microg/mL +/- 0.186 [4] [mean +/- sem(n)] vs. 1.201 +/- 0.2509 [4], P < 0.05}. Cell proliferation, however, did not differ among treatment groups.
  
  
  CONCLUSIONS
  Culture of anulus cells on nanofibers was found to be permissive for secretion and assembly of type II collagen and chondroitin sulfate, and culture on nanofibers with added charge significantly increased total proteoglycan production. These novel findings point to the need for further examination of nanofibrillar 3D culture of anulus cells for tissue engineering applications.
authors.affiliation: Department of Orthopaedic Surgery, Carolinas Medical Center, Charlotte, NC 28232, USA. helen.gruber@carolinashealthcare.org
authors.forename: Helen E
authors.initials: HE
authors.lastname: Gruber
authors.forename: Gretchen
authors.initials: G
authors.lastname: Hoelscher
authors.forename: Jane A
authors.initials: JA
authors.lastname: Ingram
authors.forename: Edward N
authors.initials: EN
authors.lastname: Hanley
mesh_terms: D000328:Adult; D000368:Aged; D049109:Cell Proliferation; D002478:Cells, Cultured; D002809:Chondroitin Sulfates / Q000378:metabolism; D024043:Collagen Type II / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism*; D005260:Female; D006801:Humans; D007403:Intervertebral Disc / Q000166:cytology* / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D009757:Nylons; D011509:Proteoglycans / Q000378:metabolism; D046509:Tissue Culture Techniques / Q000379:methods*; D023822:Tissue Engineering / Q000379:methods*
chemical_list: D024043:Collagen Type II; D009757:Nylons; D011509:Proteoglycans; D002809:Chondroitin Sulfates