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.Aim: 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. Materials & 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. Conclusion: 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.
Bibliography
- 1 Francois RJ, Braun J, Khan MA: Entheses and enthesitis: a histopathologic review and relevance to spondyloarthritides. Curr. Opin. Rheumatol.13,255–264 (2001).
- 2 Gao J, Rasanen T, Persliden J, Messner K: The morphology of ligament insertions after failure at low strain velocity: an evaluation of ligament entheses in the rabbit knee. J. Anat.189(Pt 1),127–133 (1996).
- 3 Chu D, LeBlanc R, D’Ambrosia P, D’Ambrosia R, Baratta RV, Solomonow M: Neuromuscular disorder in response to anterior cruciate ligament creep. Clin. Biomech.18,222–230 (2003).
- 4 Wei X, Messner K: The postnatal development of the insertions of the medial collateral ligament in the rat knee. Anat. Embryol.193,53–59 (1996).
- 5 Benjamin M, Ralphs JR: Fibrocartilage in tendons and ligaments: an adaptation to compressive load. J. Anat.193(Pt 4),481–494 (1998).
- 6 Benjamin M, Toumi H, Ralphs JR, Bydder G, Best TM, Milz S: Where tendons and ligaments meet bone: attachment sites (‘entheses’) in relation to exercise and/or mechanical load. J. Anat.208,471–490 (2006).
- 7 Loty S, Foll C, Forest N, Sautier JM: Association of enhanced expression of gap junctions with in vitro chondrogenic differentiation of rat nasal septal cartilage-released cells following their dedifferentiation and redifferentiation. Arch. Oral. Biol.45,843–856 (2000).
- 8 Waggett AD, Benjamin M, Ralphs JR: Connexin 32 and 43 gap junctions differentially modulate tenocyte response to cyclic mechanical load. Eur. J. Cell Biol.85,1145–1154 (2006).
- 9 Nagineni CN, Amiel D, Green MH, Berchuck M, Akeson WH: Characterization of the intrinsic properties of the anterior cruciate and medial collateral ligament cells: an in vitro cell culture study. J. Orthop. Res.10,465–475 (1992).
- 10 Turhani D, Weissenböck M, Watzinger E et al.: In vitro study of adherent mandibular osteoblast-like cells on carrier materials. Int. J. Oral Maxillofac. Surg.34,543–550 (2005).
- 11 Bani-Yaghoub M, Bechberger JF, Underhill TM, Naus CC: The effects of gap junction blockage on neuronal differentiation of human NTera2/clone D1 cells. Exp. Neurol.156,16–32 (1999).
- 12 Czyz J, Irmer U, Schulz G, Mindermann A, Hulser DF: Gap-junctional coupling measured by flow cytometry. Exp. Cell Res.255,40–46 (2000).
- 13 Noreen NJ, Hickok AR, Tuan RS: Regulation of chondrocyte differentiation and maturation. Microsc. Res. Tech.43,174–190 (1998).
- 14 Heng BC, Cao T, Lee EH: Directing stem cell differentiation into the chondrogenic lineage in vitro.Stem Cells22,1152–1167 (2004).