Cytokine expression in synovial membrane of sheep knee treated with tissue engineered osteochondral graft

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1 Cytokine expression in synovial membrane of sheep knee treated with tissue engineered osteochondral graft Maja Pusic 1, Andreja Vukasovic 1, Petar Kostesic 2, Drazen Maticic 2, Damir Hudetz 3, Davor Jezek 1, Marko Pecina 4, Alan Ivkovic 3,5 1 University of Zagreb, School of Medicine, Department of Histology and Embryology, Zagreb, Croatia 2 University of Zagreb, Faculty of Veterinary Medicine, Clinic for Surgery, Orthopedics and Ophthalmology, Zagreb, Croatia 3 University Hospital Sveti Duh, Department of Orthopedics, Zagreb, Croatia 4 University of Zagreb, School of Medicine, Zagreb, Croatia 5 University of Rijeka, Department of Biotechnology, Rijeka, Croatia

2 Introduction Treating big osteochondral defects and restoration of damaged articular cartilage is still a major clinical challange in orthopaedics. Aim of this study was to use autologous bioreactor-engineered tissue grafts for the treatment of chronic osteochondral defects in sheep knee enabling regeneration of both subchondral bone and cartilage. Chronic osteochondral defects could be related to the effect of osteoarthritis on articular cartilage and subchondral bone. Although osteoarthritis is noninflammatory disease, it is believed inflammation is involved in its development and progression. As a result we wanted to examine pro-inflammatory cytokines expression in the joint after treatment of chronic osteochondral defects with bioengineered tissue.

3 Materials and Methods 8 sheep were assigned to 4 treatment groups. All sheep underwent two surgical procedures. During the first procedure, chondral defects 4 mm in diameter were created on the weight bearing surfaces of both condyles of the right femur. After six weeks, all chondral defects were converted to osteochondral defects 6 mm in diameter and 5 mm deep. In groups NC (n=2) and AC (n=2) autologous three-dimensional osteochondral grafts engineered in perfusion bioreactor from scaffolds with nasal septum and articular chondrocytes respectively were implanted in the defect. In the CFS group (n=2) only scaffolds were implanted while CTR group (n=2) underwent defect conversion only (negative control). 6 weeks after treatment surgery, all sheep were sacrificed and tissue blocks were obtained for histology analysis. The samples were fixed in 4% buffered paraformaldehyde and decalcified in 15% EDTA. Paraffin blocks were sectioned at 5 µm and stained with hematoxylin-eosin and against TNF-alpha. To determine relative expression of IL-1β, IL-6 and TNFα in synovial membrane of treated and contralateral knee qrt-pcr was used.

4 contralateral knee treated knee Results TNF-α immunohistochemistry Immunohistochemistry of osteochondral explants against TNF-α showed positive chondrocytes in cartilage of all groups. However, there was no significant difference in TNF-α expression between operated knee and contralateral (non operated) knee in all groups. CTR CFS AC NC Figure 1. Chondrocytes positive for TNF alpha in osteochondral explants of treated and contralateral knee

5 Results TNF-α immunohistochemistry TNF alpha immunohistochemistry revealed positive giant cells, osteoblasts and fibroblasts in the defects of treated knees. In contralateral knee samples giant cells could not be found, while osteoblasts and fibroblasts were less positive for TNF-α. a) b) c) Figure 2. TNF alpha immunohistochemistry staining of osteochondral explants of operated and contralateral knee: a) giant cell, b) osteoblasts, c) fibroblasts

6 Results - qrt-pcr qrt- PCR results showed moderate expression of all three cytokines in samples from both, operated and contralateral knee with no significant correlation among groups. a) b) Figure 3. Relative gene expression of a) IL1β and b) IL-6 in synovial membrane from the operated knees. Each gene expression was compared to its relative gene expression in synovial membrane from the contralateral knee.

7 Results - qrt-pcr Figure 4. Relative gene expression of TNFα in synovial membrane from the operated knee. Each gene expression was compared to its relative gene expression in synovial membrane from the contralateral knee. Higher mrna levels of IL1β, IL 6 and TNFα were present in synovial membrane of one sheep from the CTR group and in one sheep from the NC group. They are not considered significant since mrna extraction from synovial membrane samples was not optimal. mrna was isolated only from some smaller percentage of cells in the tissue.

8 Take-home message Cytokine expression in synovial membrane of sheep knee treated with tissue engineered osteochondral graft was similar to cytokine expression in synovial membrane from non operated, contralateral knee. There was no significant difference among study groups. Immunochistochemistry staining of osteochondral explants against TNF alpha confirmed this.

9 References 1. Kapoor, M. et al. (2011) Role of proinflammatory cytokines in the pathophysiology o-f osteoarthritis.nat. Rev. ERheumatol. 7, Smith, MD. et al.(2003) Microarchitecture and protective mechanisms in synovial tissue from clinically and arthroscopically normal knee joints. Ann Rheum Dis 62: Ando W. et al. (2012) Ovine synovial membrane-derived mesenchymal progenitor cells retain the phenotype of the original tissue that was exposed to in-vivo inflammation: evidence for a suppressed chondrogenic differentiation potential of the cells. Inflamm. Res. 61: Acknowledgment The research leading to these results has received funding from the European Union's Seventh Framework Programme (FP7/ ) under grant agreement n