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.

Aims: The maintenance of the original phenotype by isolated chondrocytes grown in vitro is an important requisite for their use in repairing damaged articular cartilage. The methods to verify the expression of cartilage specific molecules usually involve destructive procedures to recover the cells from the scaffolds for tests. The aim of this study was to find a soluble marker able to attest the occurrence of a differentiation process by chondrocytes grown onto a biomaterial used for cell transplantation. We turned our attention to cathepsin B which is known to be abnormally synthesized in de-differentiated chondrocytes and scarcely produced in the differentiated ones.

Methods: The production of cathepsin B by human articular chondrocytes expanded in vitro and then grown onto a hyaluronan-based polymer derivative (Hyaff“-11) three-dimensional scaffold was evaluated with a specific ELISA and by immunohistochemical analysis at different experimental times (1hour, 1 day, 7, 14, 21 days) together with the expression of mRNA by Real Time PCR.

Results: Cathepsin B is always secreted by the cells grown onto the biomaterial but the protein levels increased from the first day after seeding up to 7 days (p< 0.01), then decreased progressively and significantly until day 21 (p< 0.01). The immunohistological data confirmed those obtained by the ELISA test. Cathepsin B staining was particularly evident at day 7 after cells were seeded onto the biomaterial, and then progressively decreased up to 21 days; at this experimental time point, the totality of cells were negative. Real-time PCR monitoring with the LightCycler using fluorescent dye allowed rapid and sensitive detection of cathepsin B mRNAs from the patient samples. The mRNA levels increased for up to 7 days of culture and slightly decreased until day 21. However, no significant differences were observed.

Conclusions: We can identify in cathepsin B a soluble marker of differentiated chondrocytes phenotype useful in the monitoring of autologous chondrocyte transplantation performed by means of different carriers. Its low concentration in the constructs culture medium could be indicative of a phenotypic stability. The introduction of mature cells inside the chondral defects could help to regenerate damaged hyaline articular cartilage better and faster.