2002 - University of Washington Bone and Joint Sources

Evidence that FGF-18 is a Growth Factor for Mature Human
Articular Chondrocytes
RUSSELL J. FERNANDES, PH.D., JEFF L. ELLSWORTH, PH.D., EMMA E. MOORE, PH.D.,
STEVEN D. HUGHES, PH.D., AND DAVID R. EYRE, PH.D.
Articular cartilages are specialized
connective tissues that dissipate
loads as the bearing surfaces in
joints. Cartilage cells, the chondrocytes,
synthesize a complex extracellular
matrix containing collagens,
proteoglycans, and noncollagenous
proteins. Type II collagen, the
predominant collagen of cartilage is
polymerized into a network and is
responsible for the tensile strength of
cartilage.
Trauma and disease of the synovial
joint can cause structural damage to the
articular cartilage. Changes include
age-related fibrillation, cartilage
degeneration due to osteoarthritis, and
focal chondral and osteochondral
defects. In most other tissues, such
lesions would be rapidly repaired.
Adult articular cartilage, however, has
only a very limited capacity to heal.
Efforts to stimulate cartilage healing
have focussed on two general areas:
surgical procedures, designed to
stimulate the endogenous repair
response, and biological repair,
designed to stimulate chondrocyte
proliferation and/or matrix production
by the application of peptide growth
factors or by cell transplantation.
The fibroblast growth factors
(FGF’s) play roles in tissue repair,
embryonic development, and growth in
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FGF18
Figure 1: Effect of FGF-18 on chondrocyte proliferation.
animals. FGF-18, a novel member of
the FGF family has been shown to
stimulate hepatocytes and intestinal
cells to proliferate. As part of a screen
for factors that regulate angiogenesis,
we expressed Fgf18 by adenovirusmediated
gene transfer in the pinnae of
nude mice. Although an angiogenic
response was not observed, a surprising
phenotype developed: compared to
pinnae that received null adenovirus,
those that received adenovirusexpressing
Fgf18 became visibly thicker.
The increase in thickness was largely
due to an Fgf18-mediated increase in
chondrocyte proliferation, type-II
collagen synthesis, and extracellular
matrix production.
These interesting observations
prompted us to investigate if Fgf18 was
expressed by articular chondrocytes
and examine the effects of highly
purified FGF-18 protein on articular
chondrocytes in vivo and vitro. Our
results suggest that Fgf18 may play a
role in the biology of normal cartilage.
METHODS
Cell and tissue culture
Full thickness adult human
cartilage, and chondrocytes (high
density monolayer or micromass)
isolated from talus joints (Northwest
Tissue Center, Seattle) were maintained
+FGF18
Control
0 1 2 3 4 5 6
Week in culture
in serum free culture (DMEM) in the
presence or absence of 100ng/ml
recombinant FGF-18 protein. Cell
numbers from monolayer cultures were
determined weekly with the aid of a
Neubauer haemocytometer. Cell layer
collagen from monolayer or micromass
cultures was solubilized by pepsin
digestion and the a1(II)chains were
identified by western blotting after
SDS-PAGE.
Histochemistry
and
Immunohistochemistry
Micromass cultures were fixed,
embedded in paraffin, sectioned and
stained with saffranin O, H&E and
immunostained with type II collagen
antibody and proliferating cell nuclear
antigen (PCNA) antibody.
RESULTS AND DISCUSSION
In situ hybridizations revealed that
Fgf18 mRNA and mRNA for two of its
receptors, Fgfr3-(IIIc) and Fgfr2-(IIIc),
were localized within chondrocytes of
human talus articular cartilage.
Incubation of primary cultures of
adult human talus articular
chondrocytes with FGF-18 protein
increased the proliferation of these cells
(Figure 1).
Western blot analysis of collagen
deposited in the extracellular matrix
showed an increase in type II collagen
accumulation within one week after
culture in the presence of FGF18
(Figure 2).
In high-density micromass cultures,
(Figure 3) the thickness of the cell layer,
cell numbers, staining of chondrocyte
nuclei with antibodies to proliferating
cell nuclear antigen (PCNA) (Figure 3,
panels B, D) and type II collagen
accumulation in the extracellular
matrix (Figure 3, panels A, C) were
increased by incubation with media
containing 100ng FGF-18/ml for 4
weeks. Increased PCNA staining was
also seen in the nuclei of chondrocytes
from explant cultures treated with FGF-
18 (data not shown).
These data imply that FGF-18 can
act as a trophic factor for adult human
articular chondrocytes in primary cell
40 2002 ORTHOPAEDIC RESEARCH REPORT