Inhibition of cartilage degradation: a combined tissue engineering and gene therapy approach.
OBJECTIVE
To determine if tissue-engineered cartilage can be protected from cytokine-induced degradation using a gene therapy approach.


METHODS
Chemical and pantropic retroviral gene transfer methodologies were compared for their ability to introduce a luciferase reporter gene into adult bovine cartilage chondrocytes grown in monolayer. Pantropic retrovirus was then used to transduce these cells with human tissue inhibitor of metalloproteinases 1 (TIMP-1), and the stability of expression in monolayer or pellet culture was monitored for 6 weeks. Untransduced and TIMP-1-transduced cells were also used to tissue engineer 3-dimensional cartilage constructs that were then challenged with interleukin-1 (IL-1) for 4 weeks. Conditioned media and residual cartilage were collected for analysis of matrix components, including type II collagen and proteoglycans, and for TIMP-1 production and matrix metalloproteinase (MMP) activity.


RESULTS
Chemical transfection of adult bovine chondrocytes gave rise to short-lived reporter expression that was virtually undetectable after 4 weeks of culture. In contrast, pantropic retroviral transduction gave rise to stable expression that persisted at a high level for at least 6 weeks. Pantropic transduction of the cells with TIMP-1 gave rise to similar long-term expression, both in monolayer and pellet cultures. TIMP-1-transduced tissue-engineered cartilage also retained TIMP-1 expression for an additional 4 weeks of culture in the presence of IL-1. Compared with control samples, TIMP-1-transgenic cartilage resisted the catabolic effects of IL-1, with MMP activity reduced to basal levels and a decreased loss of type II collagen.


CONCLUSIONS
Pantropic retroviral transduction permits long-term expression of potentially therapeutic transgenes in adult tissue-engineered cartilage. While TIMP-1 transduction could be used to prevent collagen breakdown, alternative transgenes may be necessary to protect cartilage proteoglycans.
Kafienah
Wael
W
University of Bristol, Bristol, UK.
Al-Fayez
Fayza
F
Hollander
Anthony P
AP
Barker
Michael D
MD
D000818:Animals; D019149:Bioreactors; D002356:Cartilage / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D002417:Cattle; D002449:Cell Aggregation; D002472:Cell Transformation, Viral; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005109:Extracellular Matrix / Q000737:chemistry / Q000378:metabolism; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D007375:Interleukin-1 / Q000494:pharmacology; D008979:Moloney murine leukemia virus / Q000235:genetics; D009940:Organoids; D010003:Osteoarthritis / Q000628:therapy*; D011100:Polyglycolic Acid; D023822:Tissue Engineering / Q000379:methods*; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000494:pharmacology
D007375:Interleukin-1; D019715:Tissue Inhibitor of Metalloproteinase-1; D011100:Polyglycolic Acid