Yonago Acta medica 2000;43:131–140

Processes of Osteophyte Formation in Guinea Pigs with SpontaneousOsteoarthritis
Masanori Ono, Yasutsugu Yamashita, Takeshi Minamizaki and Yasuo Morio
Department of Orthopedic Surgery, Faculty of Medicine, Tottori University, Yonago 6830826 Japan
In this study, we investigated osteophyte formation processes in guinea pigs withspontaneous osteoarthritis, histochemically and immunohistochemically. Serial thin
frontal sections of right knee joints were prepared from Hartley guinea pigs aged 1, 3,5, 8, 12 and 18 months. The severity of osteoarthritis was evaluated by safranin-O
staining, and the animals were classified into 3 groups: mild, moderate and severe. In
addition, immunostaining was performed by using primary antibodies against the proliferating cell nuclear antigen (PCNA), type-I, -II and -III collagens, insulin-like growth
factor 1 (IGF-1) and IGF-1 receptor. In the mild group, there was fibrous connective
tissue continuous with the synovial membrane and covering the margins of the articularcartilage of the medial tibial condyle. This tissue contained spindle-shaped fibroblasticlike cells. These cells were positive for PCNA, type-I and -III collagens, IGF-1 andIGF-1 receptor. In the moderate group, the chondrocytes beneath the fibroblastic-likecell layer had proliferated and were clustered together. These chondrocytes were also
positive for PCNA, type-I and -III collagens, IGF-1 and IGF-1 receptor. In the severegroup, this marginal area had been replaced by type-II collagen-positive chondrophytes,
which further changed to osteophytes due to the process of endochondral ossification.
In guinea pigs, fibroblastic-like cells at the margins of the articular cartilage of theknee joints seemed to be totipotent immature mesenchymal cells. These cells may be
the precursors of osteophytes, and IGF-1 appears to be involved in their formation.
Key words: guinea pig; insulin-like growth factor 1; osteoarthritis; osteophyte formation

The pathological changes in osteoarthritis include articular cartilage degeneration and proliferative changes, such as osteophyte formation and sclerosis of the subchondral bone.
Sclerosis of the subchondral bone is regarded asa reactive phenomenon to excessive loading,but little is known about the mechanism of osteophyte formation (Jewell et al., 1998). It has
been reported that acromegalic osteoarthropathy, which is categorized as secondary osteoarthritis, is characterized by hyperplasia of thearticular cartilage and marked osteophyte
formation (Bluestone et al., 1971; Jaffe, 1972;
Johanson et al., 1983; Resnick, 1988). Insulinlike growth factor 1 (IGF-1) is thought to induce the characteristic pathology of this disease(Lieberman et al., 1992). IGF-1 has been
demonstrated to promote mitosis and differentiation of articular chondrocytes, and to induce
matrix synthesis (Ash and Francis, 1975;
McQuillan et al., 1986; Luyten et al., 1988;
Trippel et al., 1989). These facts suggest thatIGF-1 may be generally involved in osteophyte
formation in cases of osteoarthritis other thanacromegalic osteoarthropathy, but this issue remains to be clarified.
Because osteophytes generally appear later
than cartilage degeneration, the early processesof osteophyte development cannot be examinedin human specimens, although observation ofmature osteophytes is possible. We examined
the processes of osteophyte formation in guinea

Abbreviations: IGF-1, insulin-like growth factor 1; PCNA, proliferating cell nuclear antigen

131M. Ono et al.

pigs, which show histological changes
very similar to those in human osteoarthritis (Bendele et al., 1989; Okada et al.,
1992; de Bri et al., 1996; Tokuda, 1997),
using histological, histochemical andimmunohistochemical techniques.

AJS
CL

Materials and Methods

T

Preparation of experimental materials
One-, 3-, 5-, 8-, 12- and 18-month-oldHartley guinea pigs (Charles River JapanInc., Tokyo, Japan) were sacrificed byintraperitoneal injection of pentobarbital
sodium (150 mg/kg). We examined two 1month-old (1 male, 1 female), three 3month-old (1 male, 2 females), four 5month-old (1 male, 3 females), four 8month-old (2 males, 2 females), two 12month-old (1 male, 1 female) and two 18month-old (1 male, 1 female) guinea pigs.
The right knee joints from these animals
were fixed in 10% neutral formalin in a 90˚flexed position for 3 days. Next, the knee
joints were decalcified with 10% EDTA
(pH 7.4) for 4 weeks at room temperature.
The specimens were confirmed by soft Xray that the specimens had been completely decalcified. After being dehydrated
with ascending grades of ethanol and xylene, the specimens were embedded inparaffin. These specimens were cut into
serial sections 7-µm thick each in thefrontal plane passing through the midline
between the medial and lateral menisci.
These sections were subjected to hematoxylin and eosin, safranin-O and immunohistological staining.

Evaluation of osteoarthritis
By using safranin O-stained specimensfrom each animal, the degree of degeneration of the medial condyle of the tibia, inwhich cartilage degeneration develops
earliest, was evaluated based on thehistological-histochemical grading system

BJSCLTCFig. 1. Safranin-O staining (A) and immunohistologicalstaining (B–G) show no chondrophyte formation in themedial margin of the tibial plateau in the mild group of 3month-old animals without degenerative changes due toosteoarthritis. Areas in the fibrous connective tissue (arrows) negative for safranin-O staining (A) and for type-II
collagen (C) are positive for type-I (B) and -III (D) collagens. The nuclei of fibroblastic-like cells in the fibrousconnective tissue are positive for PCNA (E), IGF-1 (F)and IGF-1 receptor (G). CL, medial colateral ligament;
JS, joint space; T, tibia. Original magnification, × 200.
[Figs. 1A–C on p. 132 and Figs. 1D–G on p. 133]132

Processes of osteophyte formation(Mankin et al., 1971). Because pannus formation was not observed in the degenerated
cartilage of any of the guinea pigs at anyage, this item was excluded. Thus, thedegree was graded on a 6-tiered scale instead of a 7-tiered scale, with 13 as the total
number of points. According to the cartilage degeneration score, animals were classified into the following 3 groups: the mildgroup (grades 0–2), the moderate group(grades 3–7) and the severe group (grades8–13).

DE
Immunohistological staining
Deparaffinized sections were reacted in solution containing bovine testicular hyaluronidase (Wako Kogyo Co. Ltd., Osaka,Japan; 2 mg/mL phosphate-buffered saline,
pH 5.3, for 30 min at room temperature).
For detection of type-II collagen, pronasetreatment (Dako, Kyoto, Japan; 1 mg/mL
phosphate-buffered saline, pH 7.3, for 30
min at room temperature) was added (Aigneret al., 1993). Protein treatment was not performed for detection of proliferating cell
nuclear antigen (PCNA). For the secondary antibody, enzyme reagent and mixtureof substrate and dye, Histofine streptavidinbiotin-PO (M) and (P) kits (Nichirei, Tokyo)
were used, and staining was performed withthe streptavidin-biotin method. Among primary antibodies, polyclonal anti-type-I
collagen antibody (LSL, Tokyo; diluted at1:500), monoclonal anti-PCNA antibody(Nichirei; non-diluted), monoclonal antiIGF-1 antibody (Upstate Biotechnology,Lake Placid, NY; 100 µg/mL) and polyclonal anti-IGF-1 receptor antibody (SantaCruz Biotechnology Inc., Santa Cruz, CA;
1 µg/mL) were reacted in a moist chamber
maintained at 4°C for about 24 h. Monoclonal anti-type-II and -type-III collagenantibodies (Fuji Chemical Industries,Takaoka, Japan; diluted at 1:1000 and
1:500, respectively) were reacted for 1 h atroom temperature. Counterstaining wasperformed with 3% methylene green foranti-PCNA antibody and hematoxylin forFGFigs. 1D–G. Continued from the previous page.

133