title: [Fabrication and analysis of a novel tissue engineered composite biphasic scaffold for annulus fibrosus and nucleus pulposus].
abstract: OBJECTIVE
  To fabricate a novel composite scaffold with acellular demineralized bone matrix/acellular nucleus pulposus matrix and to verify the feasibility of using it as a scaffold for intervertebral disc tissue engineering through detecting physical and chemical properties.
  
  
  METHODS
  Pig proximal femoral cancellous bone rings (10 mm in external diameter, 5 mm in internal diameter, and 3 mm in thickness) were fabricated, and were dealed with degreasing, decalcification, and decellularization to prepare the annulus fibrosus phase of scaffold. Nucleus pulposus was taken from pig tails, decellularized with Triton X-100 and deoxycholic acid, crushed and centrifugalized to prepare nucleus pulposus extracellular mtrtix which was injected into the center of annulus fibrosus phase. Then the composite scaffold was freeze-dryed, cross-linked with ultraviolet radiation/carbodiimide and disinfected for use. The scaffold was investigated by general observation, HE staining, and scanning electron microscopy, as well as porosity measurement, water absorption rate, and compressive elastic modulus. Adipose-derived stem cells (ADSCs) were cultured with different concentrations of scaffold extract (25%, 50%, and 100%) to assess cytotoxicity of the scaffold. The cell viability of ADSCs seeded on the scaffold was detected by Live/Dead staining.
  
  
  RESULTS
  The scaffold was white by general observation. The HE staining revealed that there was no cell fragments on the scaffold, and the dye homogeneously distributed. The scanning electron microscopy showed that the pore of the annulus fibrosus phase interconnected and the pore size was uniform; acellular nucleus pulposus matrix microfilament interconnected forming a uniform network structure, and the junction of the scaffold was closely connected. The novel porous scaffold had a good pore interconnectivity with (343.00 +/- 88.25) microm pore diameter of the annulus fibrosus phase, 82.98% +/- 7.02% porosity and 621.53% +/- 53.31% water absorption rate. The biomechanical test showed that the compressive modulus of elasticity was (89.07 +/- 8.73) kPa. The MTT test indicated that scaffold extract had no influence on cell proliferation. Live/Dead staining showed that ADSCs had a good proliferation on the scaffold and there was no dead cell.
  
  
  CONCLUSIONS
  Novel composite scaffold made of acellular demineralized bone matrix/acellular nucleus pulposus matrix has good pore diameter and porosity, biomechanical properties close to natural intervertebral disc, non-toxicity, and good biocompatibility, so it is a suitable scaffold for intervertebral disc tissue engineering.
authors.affiliation: Department of Spinal Surgery, Cell Engineering Laboratory of Orthopaedic Institute, Tianjin Hospital, Tianjin, 300211, P.R.China.
authors.forename: Haiwei
authors.initials: H
authors.lastname: Xu
authors.forename: Baoshan
authors.initials: B
authors.lastname: Xu
authors.forename: Qiang
authors.initials: Q
authors.lastname: Yang
authors.forename: Xiulan
authors.initials: X
authors.lastname: Li
authors.forename: Xinlong
authors.initials: X
authors.lastname: Ma
authors.forename: Qun
authors.initials: Q
authors.lastname: Xia
authors.forename: Chunqiu
authors.initials: C
authors.lastname: Zhang
authors.forename: Yaohong
authors.initials: Y
authors.lastname: Wu
mesh_terms: D000273:Adipose Tissue / Q000166:cytology; D000818:Animals; D001696:Biomechanical Phenomena; D049109:Cell Proliferation; D002478:Cells, Cultured; D005109:Extracellular Matrix / Q000737:chemistry* / Q000378:metabolism; D005240:Feasibility Studies; D005260:Female; D005269:Femur / Q000166:cytology; D007403:Intervertebral Disc / Q000737:chemistry* / Q000166:cytology* / Q000378:metabolism; D055959:Intervertebral Disc Degeneration / Q000628:therapy; D008297:Male; D008422:Materials Testing; D008855:Microscopy, Electron, Scanning; D016062:Porosity; D011817:Rabbits; D013234:Stem Cells / Q000166:cytology / Q000648:ultrastructure; D013552:Swine; D023822:Tissue Engineering / Q000379:methods; D054457:Tissue Scaffolds / Q000737:chemistry*