title: In vitro study of stem cell communication via gap junctions for fibrocartilage regeneration at entheses.
abstract: BACKGROUND
  Entheses are fibrocartilaginous organs that bridge ligament with bone at their interface and add significant insertional strength. To replace a severely damaged ligament, a tissue-engineered graft preinstalled with interfacial fibrocartilage, which is being regenerated from stem cells, appears to be more promising than ligament-alone graft. Such a concept can be realized by a biomimetic approach of establishing a dynamic communication of stem cells with bone cells and/or ligament fibroblasts in vitro.
  
  
  OBJECTIVE
  The current study has two objectives. The first objective is to demonstrate functional coculture of bone marrow-derived stem cells (BMSCs) with mature bone cells/ligament fibroblasts as evidenced by gap-junctional communication in vitro. The second objective is to investigate the role of BMSCs in the regeneration of fibrocartilage within the coculture.
  
  
  METHODS
  Rabbit bone/ligament fibroblasts were dual-stained with DiI-Red and calcein (gap-junction permeable dye), and cocultured with unlabeled BMSCs at fixed ratio (1:10). The functional gap junction was demonstrated by the transfer of calcein from donor to recipient cells that was confirmed and quantified by flow cytometry. Type 2 collagen (cartilage extracellular matrix-specific protein) expressed by the mixed cell lines in the cocultures were estimated by real-time reverse transcription PCR and compared with that of the ligament-bone coculture (control).
  
  
  RESULTS
  Significant transfer of calcein into BMSCs was observed and flow cytometry analyses showed a gradual increase in the percentage of BMSCs acquiring calcein with time. Cocultures that included BMSCs expressed significantly more type 2 collagen compared with the control.
  
  
  CONCLUSIONS
  The current study, for the first time, reported the expression of gap-junctional communication of BMSCs with two adherent cell lines of musculoskeletal system in vitro and also confirmed that incorporation of stem cells augments fibrocartilage regeneration. The results open up a path to envisage a composite graft preinstalled with enthesial fibrocartilage using a stem cell-based coculture system.
authors.affiliation: Department of Biotechnology & Medical Engineering, National Institute of Technology, Rourkela, India. crl.bpnayak@gmail.com
authors.forename: Bibhukalyan Prasad
authors.initials: BP
authors.lastname: Nayak
authors.forename: James Cho Hong
authors.initials: JC
authors.lastname: Goh
authors.forename: Siew Lok
authors.initials: SL
authors.lastname: Toh
authors.forename: Gyan Ranjan
authors.initials: GR
authors.lastname: Satpathy
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology / Q000378:metabolism; D002450:Cell Communication; D019070:Cell Lineage; D018920:Coculture Techniques; D024043:Collagen Type II / Q000235:genetics / Q000378:metabolism; D004396:Coloring Agents / Q000378:metabolism; D051445:Fibrocartilage / Q000166:cytology / Q000502:physiology*; D005434:Flow Cytometry; D017629:Gap Junctions / Q000378:metabolism*; D005786:Gene Expression Regulation; D008022:Ligaments / Q000166:cytology / Q000502:physiology*; D008297:Male; D008856:Microscopy, Fluorescence; D011817:Rabbits; D012038:Regeneration / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism
chemical_list: D024043:Collagen Type II; D004396:Coloring Agents