title: [Effect of bone marrow mesenchymal stem cells-derived extracellular matrix scaffold on chondrogenic differentiation of marrow clot after microfracture of bone marrow stimulation in vitro].
abstract: OBJECTIVE
  To evaluate the feasibility and validity of chondrogenic differentiation of marrow clot after microfracture of bone marrow stimulation combined with bone marrow mesenchymal stem cells (BMSCs)-derived extracellular matrix (ECM) scaffold in vitro.
  
  
  METHODS
  BMSCs were obtained and isolated from 20 New Zealand white rabbits (5-6 months old). The 3rd passage cells were cultured and induced to osteoblasts, chondrocytes, and adipocytes in vitro, respectively. ECM scaffold was manufactured using the 3rd passage cells via a freeze-dying method. Microstructure was observed by scanning electron microscope (SEM). A full-thickness cartilage defect (6 mm in diameter) was established and 5 microholes (1 mm in diameter and 3 mm in depth) were created with a syringe needle in the trochlear groove of the femur of rabbits to get the marrow clots. Another 20 rabbits which were not punctured were randomly divided into groups A (n=10) and B (n=10): culture of the marrow clot alone (group A) and culture of the marrow clot with transforming growth factor beta3 (TGF-beta3) (group B). Twenty rabbits which were punctured were randomly divided into groups C (n=10) and D (n=10): culture of the ECM scaffold and marrow clot composite (group C) and culture of the ECM scaffold and marrow clot composite with TGF-beta3 (group D). The cultured tissues were observed and evaluated by gross morphology, histology, immunohistochemistry, and biochemical composition at 1, 2, 4, and 8 weeks after culture.
  
  
  RESULTS
  Cells were successfully induced into osteoblasts, chondrocytes, and adipocytes in vitro. Highly porous microstructure of the ECM scaffold was observed by SEM. The cultured tissue gradually reduced in size with time and disappeared at 8 weeks in group A. Soft and loose structure developed in group C during culturing. Chondroid tissue with smooth surface developed in groups B and D with time. The cultured tissue size of groups C and D were significantly larger than that of group B at 4 and 8 weeks (P < 0.05); group D was significantly larger than group C in size (P < 0.05). Few cells were seen, and no glycosaminoglycan (GAG) and collagen type II accumulated in groups A and C; many cartilage lacunas containing cells were observed and more GAG and collagen type II were synthesized in groups B and D. The contents of GAG and collagen increased gradually with time in groups B and D, especially in group D, and significant difference was found between groups B and D at 4 and 8 weeks (P < 0.05).
  
  
  CONCLUSIONS
  The BMSCs-derived ECM scaffold combined with the marrow clot after microfracture of bone marrow stimulation is effective in TGF-beta3-induced chondrogenic differentiation in vitro.
authors.affiliation: Department of Orthopaedics, Nanjing Hospital Affiliated to Nanjing Medical University, Nanjing First Hospital, Nanjing Iiangsu, 210006, P.R.China.
authors.forename: Bo
authors.initials: B
authors.lastname: Wei
authors.forename: Chengzhe
authors.initials: C
authors.lastname: Jin
authors.forename: Yan
authors.initials: Y
authors.lastname: Xu
authors.forename: Cheng
authors.initials: C
authors.lastname: Tang
authors.forename: Wenhao
authors.initials: W
authors.lastname: Hu
authors.forename: Liming
authors.initials: L
authors.lastname: Wang
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology; D002358:Cartilage, Articular / Q000473:pathology / Q000601:surgery*; D002454:Cell Differentiation; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology* / Q000378:metabolism; D020219:Chondrogenesis / Q000187:drug effects; D004195:Disease Models, Animal; D005109:Extracellular Matrix / Q000737:chemistry* / Q000378:metabolism; D005240:Feasibility Studies; D005260:Female; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D016062:Porosity; D011817:Rabbits; D023822:Tissue Engineering / Q000379:methods; D054457:Tissue Scaffolds / Q000737:chemistry*; D016212:Transforming Growth Factor beta / Q000378:metabolism / Q000494:pharmacology
chemical_list: D016212:Transforming Growth Factor beta