M. Ono et al.

lagens. Moreover, the nuclei of these fibroblastic-like cells were positive for PCNA (Fig.
1E). Type-II collagen was not noted in either
fibroblastic-like cells or fibrous connective tissue (Fig. 1C). IGF-1 (Fig. 1F) and IGF-1
receptor (Fig. 1G) showed almost the same distribution, being identified in the cytoplasm andareas around fibroblastic-like cells in the fibrous connective tissue. The results describedabove were obtained in all 5 animals. Chondrocytes noted in a layer deeper than fibroblastic-like cells were negative for PCNA in all
but 1 animal, and the cartilage matrix surrounding the chondrocytes was positive for type-IIcollagen. These chondrocytes were positive for type-I collagen in 3 of 5 animals
and for type-III collagen in 1 animal, but A
no animals were positive for PCNA andtype-I and -III collagens at the same time.
In the weight-bearing regions of thecartilage, the cartilage matrix was almost
homogeneously positive for type-II collagen. Chondrocytes in the tangential zone
were positive for type-I collagen (2/5 animals), IGF-1 (5/5 animals) and IGF-1
receptor (5/5 animals), almost continuously from the articular margins. Chondrocytes in the tangential zone were positivefor PCNA in only 1 animal.
B
Moderate group
Fibroblastic-like cells in the fibrousconnective tissue were positive for type-I(Fig. 2B) and -III (Fig. 2D) collagens andPCNA (Fig. 2E) in all animals, as well as in
the mild group. In chondrocytes clusteringin the layer deeper than fibroblastic-likecells in the fibrous connective tissue, typeI and -III collagens and PCNA were identified in 4 animals. The extracellular matrix
surrounding these chondrocytes was positive for type-II collagen (Fig. 2C). IGF-1(Fig. 2F) and IGF-1 receptor (Fig. 2G)
were identified in fibroblastic-like cells in
the fibrous connective tissue and chondrocytes in the layer deeper than fibroblasticlike cells in all animals.
In the superficial layer of the weightbearing regions of the cartilage, type-I (6/7animals) and -III (5/7 animals) collagens werenoted in the degenerated cartilage matrix.
Staining for type-II collagen was not heterogeneous, and the degenerated cartilage matrix
in the superficial layer of the weight-bearing
regions showed stronger staining. Neither IGF1 nor IGF-1 receptor was observed in the tangential zone or the deeper cartilage layer.
Severe group
At the articular margins, the bone component in the osteophytes was positive for type-I
collagen (Fig. 4B) (5/5 animals). Fibroblasticlike cells in the fibrous connective tissue aroundJSMTFig. 3. Safranin-O staining (A) reveals a hump-likeprocess (arrowheads) with a thick calcified zone from themedial articular margins of the tibial plateau in the severegroup of 12-month-old animals with end-stage
osteoarthritis. Chondrocytes are noted in the process, but
not stained by safranin O. A distinctive tide mark isobserved in a layer deeper than the cartilage layer (A). The
calcified zone below the tide mark is positive for type-II
collagen (B), suggesting that the hump-like process ischondrophyte. JS, joint space; M, medial meniscus; T,tibia. Original magnification, × 200.

136

Processes of osteophyte formation

the osteophyte were positive for type-I and -IIIcollagens (Fig. 4D) and slightly positive for
PCNA (Fig.4E) (5/5 animals). Type-II collagenwas not observed in the fibrous connective tissue (Fig. 4C). IGF-1 (Fig. 4F) and IGF-1 receptor (Fig.4G) were noted in the fibrous connective tissue of all animals.
In the weight-bearing regions, the staining
intensity for type-II collagen in the cartilage
matrix tended to decrease with osteoarthritis
progression. The staining intensities for type-I
and -III collagens were increased mainly in thesuperficial layer of the degenerated cartilage
compared with those in the mild and moderategroups. No PCNA-positive cells were detectedin the weight-bearing regions of the cartilage.
The superficial matrix of the degenerated cartilage was positive for IGF-1 and IGF-1 receptor(5/5 animals).
Synovial membrane
IGF-1 and IGF-1 receptor showed the samestaining pattern, a comparatively homogeneous
distribution in the cytoplasm of lining cells of
the synovial membrane. There was no intergroup difference in this staining pattern.

Discussion
A study in human knee joints by Allard et al.
(1990) has suggested the presence of fibrousconnective tissue that is connected to the synovial membrane, is not stained with safranin O,
and differs from articular cartilage at the margins. Using a rabbit model for experimental
osteoarthritis, Moskowitz and Goldberg (1987)
have demonstrated that cells with a very highregenerative capacity are present in the junction
between the synovial membrane and perichondrium or between the synovial membraneand periosteum at the margin of the articularcartilage, and these cells are involved in osteophyte formation. Telhag and Lindberg (1972)
also reported similar results. Moreover, an immunohistological study has demonstrated thatfibroblastic-like cells in the fibrous connective
tissue produce a connective tissue matrix
mainly composed of type-I and -III collagens(Aigner et al., 1995). The presence of type-I
and -III collagens was confirmed in totipotent
immature mesenchymal cells of the cartilagenous anlage during the embryonic period(Linsenmayer et al., 1973; von der Mark et al.,
1976; von der Mark and von der Mark, 1977).
In this study, we confirmed the presence offibroblastic-like cells that were embedded in thefibrous connective tissue and were positive for
type-I and -III collagens, but were negative for
type-II collagen at the cartilagenous margins of
the knee joint in guinea pigs. It was also demonstrated that these cells were positive for
PCNA as well, indicating that they have a highmitotic activity. Consequently, fibroblasticlike cells at the margins of the articular cartilage
seemed to be totipotent immature mesenchymal
cells based on the features of the staining pattern for collagens and on having differentiating
potential.
Fibrous connective tissue was present at thearticular margins, and fibroblastic-like cells
presumed to be totipotent immature mesenchymal cells were identified in this connective
tissue in all animals irrespective of the degree ofosteoarthritis. Mitotic activity of chondrocytes
observed in the layer deeper than these fibroblastic-like cells varied according to the degreeof osteoarthritis. In the mild group, no mitotic
activity of the chondrocytes at the margins wasenhanced. In the moderate group, however,chondrocytes with an enhanced mitotic activity
had proliferated and clustered. Around thesechondrocytes, a cartilage matrix positive for
type-II collagen was identified. In the severegroup, in which osteoarthritis had progressed
further, a hump-like process with a thick calcified zone, which was considered to be the chondrophyte, was observed. Furthermore, this area
had been substituted by bone tissue positive for
type-I collagen. These findings suggest that themechanism of osteophyte formation is mediated by endochondral ossification.
IGF-1 and IGF-1 receptor showed almost
the same distribution in all animals. This result
demonstrates that chondrocytes with an IGF-1
secretory activity and those with IGF-1 receptor
exist in the joints. In particular, totipotent immature mesenchymal cells at the margins of the137