Abstract
Autologous chondrocyte transplantation is a widely used technique for the treatment of cartilage lesions. This therapeutic strategy has been recently improved by the use of biocompatible scaffolds which allow a better fixation of the cells inside the defect together with the maintenance of their original phenotype. We have recently reported that human chondrocytes can efficiently grow on a hyaluronan acid derivative biomaterial (Hyaff-11, Fidia Advanced Biopolymers, Abano Terme, Italy) and are able to express and produce collagen type II and proteoglycans, molecules expressed by differentiated cells (Grigolo et al. Biomaterials 2002). However, from the histological evaluations of the grafted tissues there is not always evidence of hyaline cartilage neo-formation even in presence of good clinical symptoms. Only few studies deals with cellular, and biochemical processes that occur during the remodeling of the graft tissue after transplantation in humans. Biopsy samples harvested from the graft have been examined using a panel of specific antibodies. It was found that cell transplantation is followed not only by a process of cartilage repair but in some cases also by a regeneration achieved through the turnover of the initial fibrocartilagineous tissue via enzymatic degradation and synthesis of newly formed collagen type II. Therefore, we examined the expression of genes encoding extracellular matrix proteins and regulatory factors essential for cell differentiation in human cartilage biopsies of patients who underwent autologous chondrocyte transplantation.
Human cartilage biopsies of patients treated by autologous chondrocyte transplantation and from a multi-organ donor were used. A Real-Time RT-PCR analysis was performed in isolated chondrocytes to evaluate the expression of collagen type I, II, X, aggrecan, cathepsin B, early growth response protein-1 (Egr-1) and Sry-type high-mobility-group box transcription factor-9 (Sox-9) mRNAs. Immunohistochemical analysis for ECM proteins and regulatory proteins was carried out on paraffin embedded sections.
Real-time RT-PCR analysis showed that collagen type I mRNA was expressed in all the samples evaluated while collagen type II was present even if at lower levels compared to control. Collagen type X messenger was undetectable. Aggrecan mRNA was present in all the samples at lower levels compared to donor. Cathepsin B messenger was higher in the samples compared to control. Egr-1 and Sox-9 mRNAs were expressed at lower levels compared to donor. The immunohistochemical analysis showed a slight positivity for collagen type I in all the sections. Collagen type II was found in all the samples evaluated with a positivity confined inside the cells, while the control displayed a positivity which was diffuse in the ECM. Cathepsin B was slightly positive in all the samples while the control was negative. Egr-1 protein was particularly evident in the areas negative for collagen type II. Sox-9 was positive in all the samples, with evident localization in the superficial layer.
Our results provide evidence that the remodelling of the graft tissue after autologous chondrocyte transplantation is regulated by a sophisticated gene expression machinery control addressed to new cartilage formation.
The abstracts were prepared by Ms Grazia Gliozzi. Correspondence should be addressed to her at the Italian Orthopaedic Research Society, Laboratory for Pathophysiology, Instituti Ortopedici Rizzoli, University of Bologna, Bologna, Italy.
