Background/Purpose: The aim of this study was to evaluate the chondrogenic potential of bone marrow mesenchymal stem cells (BM-MSCs) grown on type I collagen and different concentrations of heparan sulfate (HS) scaffolds and the quality of the neosynthesized cartilaginous tissue.

Methods: BM-MSCs were cultured on scaffolds for 16 and 30 days. BM-MSCs were cultured in chondrogenic differentiation medium or DMEM with 20% FBS (Fetal Bovine Serum), in both cases plus 100 nM PTHrP (Parathyroid hormone-related protein). Chondrogenic differentiation and neosythesized cartilage quality were evaluated by histochemical and immunohistochemical analysis, transmission and scanning electron microscopy and molecular biology techniques. Culture supernatants were collected every 3-4 days to determine collagen presence by Elisa assays.

Results: Isolated cells were able to proliferate on type I collagen and various concentrations of HS scaffolds, showing high percentages of positivity for PCNA proliferation marker. Hematoxylin-eosin and Massonxs trichrome stainings showed that BM-MSCs proliferated better when cultured in chondrogenic medium than in growth medium (DMEM 20%). Stimulated cells spread throughout the biomaterials in high percentage, showing a good morphology at both times as well as a wide distribution of ECM. They showed high percentages of positivity for safranin O, Toluidine Blue, aggrecan and type II collagen. Degradation of biomaterials was gradual, as fibers were replaced with ECM. Molecular analysis indicated the expression of cartilage-characteristic genes, such as Col II and Sox9. Scanning and transmission electron microscopy confirmed cell presence and ECM synthesis after 16 and 30 days of culture. Cells showed a high number of distended rough endoplasmic reticulum cisternae, electrodense vesicles and mitochondria. Finally, culture supernatants analysis showed the release of collagen in most of the time periods studied, confirming their differentiation and cartilage ECM characteristic compounds.

Conclusion: Our data demonstrated that type I collagen and HS scaffolds were optimal for BM-MSCs growth and differentiation towards chondrocytes-like cells after both 16 and 30 days in chondrogenic medium. Our scaffolds favour phenotypic maintenance of the differentiated cells and synthesis of cartilage-like tissue. Acknowledgements: Opocrin, S.P.A; CIBER BBN CB06-01-0040; SAI-UDC; P. Esbrit (Fundación Jiménez Díaz).

Figure 1. Analysis of BM-MSCs cultured over type I collagen and HS scaffolds on chondrogenic medium: Histochemical and immunohistochemical staining after 16 days in culture (A-D); transmission electron microscopy analysis after 16 days (E, F) and 30 days (G, H) in culture; scanning electron microscopy  after 30 days in culture (I-L). White arrows: cells. Black arrows: ECM.