title: In vitro cartilage production using an extracellular matrix-derived scaffold and bone marrow-derived mesenchymal stem cells.
abstract: BACKGROUND
  Cartilage repair is a challenging research area because of the limited healing capacity of adult articular cartilage. We had previously developed a natural, human cartilage extracellular matrix (ECM)-derived scaffold for in vivo cartilage tissue engineering in nude mice. However, before these scaffolds can be used in clinical applications in vivo, the in vitro effects should be further explored.
  
  
  METHODS
  We produced cartilage in vitro using a natural cartilage ECM-derived scaffold. The scaffolds were fabricated by combining a decellularization procedure with a freeze-drying technique and were characterized by scanning electron microscopy (SEM), micro-computed tomography (micro-CT), histological staining, cytotoxicity assay, biochemical and biomechanical analysis. After being chondrogenically induced, the induction results of BMSCs were analyzed by histology and Immunohisto-chemistry. The attachment and viability assessment of the cells on scaffolds were analyzed using SEM and LIVE/DEAD staining. Cell-scaffold constructs cultured in vitro for 1 week and 3 weeks were analyzed using histological and immunohistochemical methods.
  
  
  RESULTS
  SEM and micro-CT revealed a 3-D interconnected porous structure. The majority of the cartilage ECM was found in the scaffold following the removal of cellular debris, and stained positive for safranin O and collagen II. Viability staining indicated no cytotoxic effects of the scaffold. Biochemical analysis showed that collagen content was (708.2-44.7) µg/mg, with GAG (254.7 ± 25.9) µg/mg. Mechanical testing showed the compression moduli (E) were (1.226 ± 0.288) and (0.052 ± 0.007) MPa in dry and wet conditions, respectively. Isolated canine bone marrow-derived stem cells (BMSCs) were induced down a chondrogenic pathway, labeled with PKH26, and seeded onto the scaffold. Immunofluorescent staining of the cell-scaffold constructs indicated that chondrocyte-like cells were derived from seeded BMSCs and excreted ECM. The cell-scaffold constructs contained pink, smooth and translucent cartilage-like tissue after 3 weeks of culture. We observed evenly distributed cartilage ECM proteoglycans and collagen type II around seeded BMSCs on the surface and inside the pores throughout the scaffold.
  
  
  CONCLUSIONS
  This study suggests that a cartilage ECM scaffold holds much promise for in vitro cartilage tissue engineering.
authors: Chinese People's Liberation Army General Hospital, Beijing, China.
authors: Yan-hong
authors: YH
authors: Zhao
authors: Qiang
authors: Q
authors: Yang
authors: Qun
authors: Q
authors: Xia
authors: Jiang
authors: J
authors: Peng
authors: Shi-bi
authors: SB
authors: Lu
authors: Quan-yi
authors: QY
authors: Guo
authors: Xin-long
authors: XL
authors: Ma
authors: Bao-shan
authors: BS
authors: Xu
authors: Yong-cheng
authors: YC
authors: Hu
authors: Bin
authors: B
authors: Zhao
authors: Li
authors: L
authors: Zhang
authors: Ai-yuan
authors: AY
authors: Wang
authors: Weng-jing
authors: WJ
authors: Xu
authors: Jun
authors: J
authors: Miao
authors: Yue
authors: Y
authors: Liu
mesh_terms: D000818:Animals; D001696:Biomechanical Phenomena; D002356:Cartilage / Q000166:cytology*; D002470:Cell Survival; D002478:Cells, Cultured; D004285:Dogs; D005109:Extracellular Matrix / Q000502:physiology*; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D023822:Tissue Engineering / Q000379:methods*; D054457:Tissue Scaffolds