title: Effect of growth and differentiation factor 6 on the tenogenic differentiation of bone marrow-derived mesenchymal stem cells.
abstract: BACKGROUND
  Recent studies showed that bone marrow-derived mesenchymal stem cells (BMSCs) had risk of ectopic bone formation. In this study, we aimed to investigate the effect of growth and differentiation factor 6 (GDF-6) on the tenogenic differentiation of BMSCs in vitro, and then combined with small intestine submucous (SIS) to promote tendon regeneration in vivo.
  
  
  METHODS
  The BMSCs were isolated from the green fluorescent protein (GFP) rats, and were characterized by multi-differentiation assays following our previous study protocol. BMSCs cultured with different concentrations of GDF-6, without growth factors served as control. After 2 weeks, mRNA expression and protein expression of tendon specific markers were examined by qRT-PCR and Western blotting to define an optimal concentration of GDF-6. Mann-Whitney U-test was used to compare the difference in relative mRNA expression among all groups; P ≤ 0.05 was regarded as statistically significant. The GDF-6 treated BMSCs combined with SIS were implanted in nude mice and SD rat acute patellar tendon injury model, the BMSCs combined with SIS served as control. After 12 and 4 weeks in nude mice and tendon injury model, the samples were collected for histology.
  
  
  RESULTS
  After the BMSCs were treated with different concentration of GDF-6 for 2 weeks, the fold changes of the specific markers (Tenomodulin and Scleraxis) mRNA expression were significantly higher in GDF-6 (20 ng/ml) group (P ≤ 0.05), which was also confirmed by Western blotting result. The BMSCs became parallel in orientation after GDF-6 (20 ng/ml) treatment, but the BMSCs in control group were randomly oriented. The GDF-6 (20 ng/ml) treated BMSCs were combined with SIS, and were implanted in nude mice for 12 weeks, the histology showed neo-tendon formation. In the SD rat patellar tendon window injury model, the histology also indicated the GDF-6 (20 ng/ml) treated BMSCs combined with SIS could promote tendon regeneration.
  
  
  CONCLUSIONS
  GDF-6 has tenogenic effect on the tenogenic differentiation of BMSCs, and GDF-6 (20 ng/ml) has better tenogenic effect compared to other concentrations. The GDF-6 (20 ng/ml) treated BMSCs combined with SIS can form neo-tendons and promote tendon regeneration.
authors.affiliation: Department of Orthopaedics, the General Hospital of Chinese People's Liberation Army, Beijing 100853, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Chai
authors.forename: Ming
authors.initials: M
authors.lastname: Ni
authors.forename: Yun-feng
authors.initials: YF
authors.lastname: Rui
authors.forename: Kai-yi
authors.initials: KY
authors.lastname: Zhang
authors.forename: Qiang
authors.initials: Q
authors.lastname: Zhang
authors.forename: Liang-liang
authors.initials: LL
authors.lastname: Xu
authors.forename: Kai-ming
authors.initials: KM
authors.lastname: Chan
authors.forename: Gang
authors.initials: G
authors.lastname: Li
authors.forename: Yan
authors.initials: Y
authors.lastname: Wang
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects*; D055431:Growth Differentiation Factor 6 / Q000494:pharmacology*; D008297:Male; D008565:Membrane Proteins / Q000235:genetics; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000187:drug effects*; D051379:Mice; D008819:Mice, Nude; D051381:Rats; D017207:Rats, Sprague-Dawley; D012038:Regeneration / Q000187:drug effects; D013710:Tendons / Q000187:drug effects* / Q000502:physiology
chemical_list: D055431:Growth Differentiation Factor 6; D008565:Membrane Proteins; C486027:Tnmd protein, rat