Tissue engineering in an in vitro model of human cartilage repair

Abstract

Purpose: The purpose was to study the cartilage repair of in vitro lesion models using human bone marrow mesenchymal stromal cells (BMSCs) and different types of collagen scaffolds. Methods: 3 mm lesions were made in human cartilage biopsies. 2x105 BMSCs were seeded on type I collagen (C1), C1 and heparan sulfate (C1HS) and type I and II collagen and HS (C1C2HS) biomaterials. They were introduced inside the lesion, and cultured during 60 days in chondrogenic medium with 10 ng/ml TGFβ-3. Neotissue was evaluated by histochemistry and immunohistochemistry, and macroscopically using a modification of ICRS II scale. Results: In the in vitro model, histochemistry showed that after 60 days, the cells seeded on the three biomaterials filled almost the 100% of the lesion and we found integration of the neotissue with the border of the lesion. The cellular density in the grafts was higher than the native cartilage (Fig. 1, H-E) and spindle- and round- morphology cells were found. The biomaterial was fully degraded in the model except in C1HS. Regarding the extracellular matrix (ECM), Masson’s trichrome showed presence of collagens in all the constructs (Fig. 1, MT). Immunohistochemically we found that type II collagen (Fig. 1, COL II) was present in all the models, being more homogeneus in C1C2HS. The presence of proteoglycans was tested by Safranin O (Fig. 1, SO) that was more metachromatic in C1C2HS than in C1 and C1HS. However, aggrecan was neither present in C1HS nor C1C2HS, being weakly stained in C1 (Fig. 1, AGG). In the macroscopic ICRS II assessment, the repair score was 42.5% or fibrocartilage for C1, 39.16% or mixed fibrous/fibrocartilage for C1HS and 73.3% or mixed firbrocartilage/hyaline cartilage for C1C2HS. Conclusions: Data showed that hBMSCs seeded on collagen biomaterials in a repair model are able to fill the lesion and to form pre-chondrogenic ECM. Considering the modified ICRS II macroscopic evaluation, the better criteria for the repair of an in vitro human cartilage lesion model was obtained for the C1C2HS biomaterial. Acknowledgements: Opocrin S.P.A.; CAM (S2009/MAT-1472); CIBER-BBN; Red Gallega de Terapia Celular, Xunta de Galicia (R2014/050); GPC, Xunta de Galicia (GPC2014/048); Universidade da Coruña; Fundación Española de Reumatología (2014 grant); Fundación Profesor Novoa Santos.