Proceeding of Veterinary and Animal Science Days 2015, 15th- 17th July, Milan, Italy 

riviste.unimi.it/index.php/haf 

   

 

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CORRESPONDING AUTHOR 

Francesco Ferrucci 

Francesco.ferrucci@unimi.it 

 

 

ABSTRACT  

Ghrelin, the natural ligand for GH secretagogue receptors, influences the activity of both human and rat joint 

chondrocytes, modulating the synthesis of eicosanoids in the cartilage1. This project aims to assess the possible 

protective effects of Ghrelin on equine cultured chondrocytes exposed to lipopolysaccharide (LPS), as a model of 

osteoarthritic joint. In the preliminary step of our study, we have characterized equine chondrocyte primary cell 

cultures and standardized the detrimental effects LPS on chondrocytes health. Equine articular cartilage was collected 

with aseptic procedure and equine chondrocytes were isolated by overnight incubation. Cells were cultured in 

monolayer culture to obtain third-passage (P3) chondrocytes and specific cartilaginous genes were identified by 

qualitative PCR. P3 chondrocytes were treated with increasing LPS concentrations (1-100 ng/ml) and the effects of LPS 

were assessed with the apoptosis-induction test and fluorescence microscopy after 1, 3, 6, 12, 24, 48 h of treatment. 

PCR evidenced type II collagenases and aggrecan expression in isolated cells confirming the isolation of chondrocytes. 

Healthy chondrocytes (HC) showed no fluorescence, necrotic chondrocytes (NC) red fluorescence, early apoptotic 

chondrocytes (EAC) green fluorescence and advanced apoptotic chondrocytes (AAC) mixed green-red fluorescence. In 

LPS-stimulated chondrocytes, there was a trend throughout an increased percentage of AAC that became equal to or 

overwhelmed the percentage of HC after 3 hours for all LPS treatments and lasted for 6 hours after the highest LPS 

concentration used. On the basis of these results, the concentration of LPS 100 ng/ml for 6 h will be used for 

subsequent experiments. 

 
REFERENCES  

            Caminos J.E., Gualillo O., Lago F. et al. 2005. Endocrinology 146(3): 1285-92.  

Effects of Ghrelin on metabolic activity of 

equine cartilage affected with inflammatory 

degeneration: preliminary isolation, 

characterization of equine cultured 

chondrocytes and assessment of LPS-

induced inflammatory stress. 

S. Ceriotti
1
, A. Lange Consiglio

2
, L. Casati

3
, F. Cremonesi

2
, E. Zucca

1
, V. 

Sibilia
3
, F. Ferrucci

1 

1 
Department of Health, Animal Science and Food Safety, School of Veterinary 

Medicine, Università degli Studi di Milano ῀ Milano 

2
Reproduction Unit, Large Animal Veterinary Hospital (Lodi), Università degli 

Studi di Milano ῀ Milano 
 
3
 Department of Medical Biotechnology and Translational Medicine, Università 

degli Studi di Milano ῀ Milano