Hypertrophy and physiological death of equine chondrocytes in vitro.
BACKGROUND
Equine osteochondrosis results from a failure of endochondral ossification during skeletal growth. Endochondral ossification involves chondrocyte proliferation, hypertrophy and death. Until recently no culture system was available to study these processes in equine chondrocytes.


OBJECTIVE
To optimise an in vitro model in which equine chondrocytes can be induced to undergo hypertrophy and physiological death as seen in vivo.


METHODS
Chondrocytes isolated from fetal or older (neonatal, growing and mature) horses were cultured as pellets in 10% fetal calf serum (FCS) or 10% horse serum (HS). The pellets were examined by light and electron microscopy. Total RNA was extracted from the pellets, and quantitative PCR carried out to investigate changes in expression of a number of genes regulating endochondral ossification.


RESULTS
Chondrocytes from fetal foals, grown as pellets, underwent hypertrophy and died by a process morphologically similar to that seen in vivo. Chondrocytes from horses age >5 months did not undergo hypertrophy in pellet culture. They formed intramembranous inclusion bodies and the cultures included cells of osteoblastic appearance. Pellets from neonatal foals cultured in FCS resembled pellets from older horses, however pellets grown in HS underwent hypertrophy but contained inclusion bodies. Chondrocytes from fetal foals formed a typical cartilage-like tissue grossly and histologically, and expressed the cartilage markers collagen type II and aggrecan mRNA. Expression of Sox9, collagen type II, Runx2, matrix metalloproteinase-13 and connective tissue growth factor mRNA increased at different times in culture. Expression of fibroblast growth factor receptor-3 and vascular endothelial growth factor mRNA decreased with time in culture.


CONCLUSIONS
Freshly isolated cells from fetal growth cartilage cultured as pellets provide optimal conditions for studying hypertrophy and death of equine chondrocytes.


CONCLUSIONS
This culture system should greatly assist laboratory studies aimed at elucidating the pathogenesis of osteochondrosis.
Ahmed
Y A
YA
School of Veterinary Science, University of Melbourne, Parkville, Victoria 3010, Australia.
Tatarczuch
L
L
Pagel
C N
CN
Davies
H M
HM
Mirams
M
M
Mackie
E J
EJ
D000367:Age Factors; D000375:Aging / Q000502:physiology; D000704:Analysis of Variance; D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis; D016923:Cell Death; D002478:Cells, Cultured; D019902:Chondrocytes / Q000502:physiology* / Q000648:ultrastructure*; D024043:Collagen Type II / Q000378:metabolism; D005786:Gene Expression Regulation; D006734:Horse Diseases / Q000473:pathology; D006736:Horses; D002479:Inclusion Bodies; D053509:Matrix Metalloproteinase 13 / Q000378:metabolism; D008854:Microscopy, Electron / Q000662:veterinary; D010007:Osteochondritis / Q000473:pathology / Q000662:veterinary; D010012:Osteogenesis / Q000502:physiology*; D016133:Polymerase Chain Reaction / Q000662:veterinary; D012333:RNA, Messenger / Q000378:metabolism*
D024043:Collagen Type II; D012333:RNA, Messenger; D053509:Matrix Metalloproteinase 13