In vitro study of stem cell communication via gap junctions for fibrocartilage regeneration at entheses.
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.
Nayak
Bibhukalyan Prasad
BP
Department of Biotechnology & Medical Engineering, National Institute of Technology, Rourkela, India. crl.bpnayak@gmail.com
Goh
James Cho Hong
JC
Toh
Siew Lok
SL
Satpathy
Gyan Ranjan
GR
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
D024043:Collagen Type II; D004396:Coloring Agents