Aim of study: The development of tissue engineering techniques evidenced that the healing of injured ligaments require the interactions of different cell types, local cellular environment and the use of devices. In order to gain new information on the complex interactions between mesenchymal stem cells (MSCs) and biodegradable scaffold, we analysed in vitro the proliferation, vitality and phenotype of MSCs grown onto a multilayered-woven-cylindric-array of Hyaff-11A8 fiber configured as ligament scaffold.

Methods: Sheep MSCs were isolated from bone marrow aspirates and grown at two different density (7,5x106/cm and 15x106/cm) in the scaffold. At different time points (2, 4, 6 days) cellular proliferation was analysed by MTT test and cellular viability by calcein-AM immunofluorescence dye and confocal microscopy analysis. Moreover, hyaluronic acid receptor (CD44) and typical matrix ligament proteins (collagen type I, III, laminin, fibronectin, actin) were evaluated by immunohistochemistry.

Results: MSCs growth was cell density-dependent and cells were uniformly distributed inside and along the scaffold. Confocal analysis showed that MSCs completely wrap the fibers at both cell concentrations analysed and were all viable both outside and inside the scaffolds only using the lower cell concentration. Moreover, MSCs expressed CD44, collagen type I, III, laminin, fibronectin and actin.

Conclusion: These data demonstrate that MSCs well survive in a hyaluronic acid-configured ligament scaffold expressing a protein important for scaffold interaction, like CD44, and proteins responsible of the functional characteristic of the ligaments.