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Molecular and histological characterization of nasal chondrocyte-based cartilage grafts for the treatment of kissing lesions in the knee

Introduction: Articular cartilage "kissing" lesions in the knee are untreatable lesions that lead to osteoarthritis. They are characterized by two defects in direct contact to each other. The aim of this study was to characterize and evaluate the quality of engineered cartilage grafts prior to implantation in the defect induced in the sheep animal model. Grafts were produced using sheep nasal chondrocytes obrained from the nasal biopsy and cultured on the collagen-based scaffold for two days (N-CAM) or two weeks (N-TEC). Assessment of the N-CAM and N- TEC quality was performed using histological and molecular methods. Methods: Sheep nasal septum biopsy was dissected into small pieces and digested in collagenase solution. Chondrocytes were expanded in monolayer for 13 days, seeded on a collagen scaffold (Chondro-Gide membrane) and cultured in chondrogenic medium for 2 days for N- CAM grafts or 2 weeks for N-TEC grafts. Manufactured grafts were fixed and embedded in paraffin for histological analysis. Grafts were stained using HE, safranin O, picrosirius red and immunostained against collagen type I, II and aggrecan. Total RNA from cartilage grafts was isolated using TRIzol reagent and reverse transcribed to cDNA to perform RT-qPCR and quantify gene expression of collagen I, II and aggrecan. Results: Histologically, the N-TEC grafts contained higher amount of glycosaminoglycans (GAG) accompanied with higher content of collagen type II and aggrecan, compared to N-CAM grafts. Collagen type I level was low in both N-CAM and N-TEC grafts. qPCR analysis confirmed higher collagen type II and aggrecan expression in N-TEC grafts. Conclusion: Two types of engineered grafts shows different expression of GAG and extracellular matrix cartilage specific proteins which can impact the treatment of articular cartilage kissing lesions. N-CAM is immature graft with little or no extracellular matrix while N-TEC is mature graft with extracellular matrix containing cartilage specific proteins. The regenerative potential of these grafts will be evaluated after implantation. Acknowledgments: Funding for this research has been received from the European Union's Horizon 2020 research and innovation programme under grant agreement No. 681103, BIO- CHIP. References: 1. Langer R, Vacanti JP. Tissue Engineering. Science 260, 920, 1993.

nasal chondrocytes, tissue engineering, cartilage, kissing lesions

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