A stable isotope method for the simultaneous measurement of matrix synthesis and cell proliferation in articular cartilage in vivo.
OBJECTIVE
Measurements of cell proliferation and matrix synthesis in cartilage explants have identified regulatory factors [e.g., interleukin-1 (IL-1)] that contribute to osteoarthritis and anabolic mediators [e.g., bone morphogenic protein-7 (BMP-7)] that may have therapeutic potential. The objective of this study was to develop a robust method for measuring cell proliferation and glycosaminoglycan synthesis in articular cartilage that could be applied in vivo.


METHODS
A stable isotope-mass spectrometry approach was validated by measuring the metabolic effects of IL-1 and BMP-7 in cultures of mature and immature bovine cartilage explants. The method was also applied in vivo to quantify physiologic turnover rates of matrix and cells in the articular cartilage of normal rats. Heavy water was administered to explants in the culture medium and to rats via drinking water, and cartilage was analyzed for labeling of chondroitin sulfate (CS), hyaluronic acid (HA) and DNA.


RESULTS
As expected, IL-1 inhibited the synthesis of DNA and CS in cartilage explants. However, IL-1 inhibited HA synthesis only in immature cartilage. Furthermore, BMP-7 was generally stimulatory, but immature cartilage was significantly more responsive than mature cartilage, particularly in terms of HA and DNA synthesis. In vivo, labeling of CS and DNA in normal rats for up to a year indicated half-lives of 22 and 862 days, respectively, in the joint.


CONCLUSIONS
We describe a method by which deuterium from heavy water is traced into multiple metabolites from a single cartilage specimen to profile its metabolic activity. This method was demonstrated in tissue culture and rodents but may have significant clinical applications.
Li
K W
KW
KineMed, Inc, Emeryville, CA 94608, USA. kli@kinemed.com
Siraj
S A
SA
Cheng
E W
EW
Awada
M
M
Hellerstein
M K
MK
Turner
S M
SM
D000818:Animals; D055419:Bone Morphogenetic Protein 7 / Q000494:pharmacology*; D002358:Cartilage, Articular / Q000166:cytology / Q000378:metabolism*; D002417:Cattle; D049109:Cell Proliferation / Q000187:drug effects*; D002809:Chondroitin Sulfates / Q000096:biosynthesis / Q000302:isolation & purification; D002849:Chromatography, Gas; D004247:DNA / Q000096:biosynthesis; D017666:Deuterium Oxide; D006025:Glycosaminoglycans / Q000096:biosynthesis; D006821:Hyaluronoglucosaminidase / Q000096:biosynthesis / Q000302:isolation & purification; D007375:Interleukin-1 / Q000494:pharmacology*; D053582:Interleukin-1alpha / Q000494:pharmacology; D007553:Isotope Labeling / Q000379:methods*; D008297:Male; D013058:Mass Spectrometry; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins; D013264:Stifle
D055419:Bone Morphogenetic Protein 7; D006025:Glycosaminoglycans; D007375:Interleukin-1; D053582:Interleukin-1alpha; D011994:Recombinant Proteins; D002809:Chondroitin Sulfates; D004247:DNA; D006821:Hyaluronoglucosaminidase; D017666:Deuterium Oxide