J. Microbiol. Biotechnol. (2012), 22(2), 274–282http://dx.doi.org/10.4014/jmb.1110.10078First published online January 9, 2012

Efficacy Test of Polycan, a Beta-Glucan Originated from Aureobasidiumpullulans SM-2001, on Anterior Cruciate Ligament Transection and PartialMedial Meniscectomy-Induced-Osteoarthritis Rats

Kim, Joo-Wan1, Hyung-Rae Cho

1, and Sae-Kwang Ku

2*

1Glucan Corp. Research Institute, Marine Biotechnology Center, Busan 617-763, Korea2Department of Anatomy and Histology, College of Oriental Medicine, Daegu Haany University, Gyeongsan-si, Gyeongsanbuk-do712-715, Korea

Received: October 26, 2011 / Accepted: November 21, 2011

The object of this study was to assess the efficacy of

Polycan from Aureobasidium pullulans SM-2001, which is

composed mostly of beta-1,3-1,6-glucan, on osteoarthritis

(OA)-induced by anterior cruciate ligament transection

and partial medial meniscectomy (ACLT&PMM). Three

different dosages of Polycan (85, 42.5, and 21.25 mg/kg)

were orally administered once a day for 84 days to male

rats a week after ACLT&PMM surgery. Changes in the

circumference and maximum extension angle of each

knee, and in cartilage histopathology were assessed using

Mankin scores 12 weeks after Polycan administration. In

addition, cartilage proliferation was evaluated using

bromodeoxyuridine (BrdU). As the result of ACLT&PMM,

classic OA was induced with increases in maximum

extension angles, edematous knees changes, and capsule

thickness, as well as decreases in chondrocyte proliferation,

cartilages degenerative changes, and loss of articular

cartilage. However, these changes (except for capsule

thickness) were markedly inhibited in all Polycan- and

diclofenac sodium-treated groups compared with OA control.

Although diclofenac sodium did not influence BrdU uptake,

BrdU-immunoreactive cells were increased with all dosages

of Polycan, which means that Polycan treatment induced

proliferation of chondrocytes in the surface articular

cartilage of the tibia and femur. The results obtained in

this study suggest that 84 days of continuous oral treatment

of three different dosages of Polycan led to lesser degrees

of articular stiffness and histological cartilage damage

compared with OA controls 91 days after OA inducement,

suggesting that the optimal Polycan dosage to treat OA is

42.5 mg/kg based on the present study.

Keywords: Anterior cruciate ligament transaction, Aureobasidium

pullulans, β-glucan, osteoarthritis

Osteoarthritis (OA) is the most prevalent articular disease

in the elderly [10]. The process is characterized by changes

in the structure and function of the articulation, mainly due

to a degenerative process that takes place in the articular

cartilage [42]. An understanding of OA etiopathology,

however, has proven to be elusive [13]. Owing to the fact that

OA affects nearly 70% of all people at some point in their

lives, it has a major economic and social impact on patients

and health care systems [4, 5, 12]. Consequently, there is a

pressing need to develop disease-modifying OA drugs [1].

Before a disease-modifying OA drug can reach clinical

trials, it must first be successful in preclinical trials. This

requires animal models of OA in which specific aspects of

drug efficacy in articular cartilage, subchondral bone, and

other affected tissues can be examined, as well as potential

side effects in other organs [18].

Rodent models of OA were first developed in the late

1970s in mice and rats [11, 38, 45, 49]. Initially, experiments

employed models in which OA was induced in the

temporomandibular joint [9, 35, 50], but subsequently

models were developed in synovial joints, including the

knee [38], using either a chemical method (intra-articular

injection of, for instance, papain [33] or sodium iodoacetate

[28]) or a surgical method (structural alteration to the

tendons, muscle, or ligaments [3, 34, 37]). A review by

Schwartz [46] in 1987 summarized these early developments.

Other models developed since then rely on genetic

predisposition or engineering to stimulate OA pathology.

However, genetic models may require a long time for OA

to develop, and there is often considerable variability

between animals (e.g., in the temporal dynamics of OA

progression). Disease progression in surgical models is

faster and more consistent. Moreover, these models reflect

post-traumatic (secondary) OA, because they rely on

changes in weight bearing and unnatural joint articulation

for OA etiopathology [6, 55].

*Corresponding authorPhone: +82-53-819-1549; Fax: +82-53-819-1269;E-mail: gucci200@hanmail.net

275 Kim et al.

It is advantageous to develop surgical models in rats or

mice because genetic studies are also possible in these

animals [21, 26, 29]. Rat models are of interest because

their larger size compared with mice provides more tissue

for biochemical and gene expression analysis, and permits

cross-disciplinary studies (e.g., genomics, cell biology,

electrophysiology, and in vivo small animal imaging) [22].

Models developed in the rat include anterior cruciate

ligament transection (ACLT) [15, 20, 52] and partial medial

meniscectomy (PMM) [14, 40], or a combination of both

[44].

Although disease progression in surgical models is faster

and more consistent [1], peak symptoms (mainly stiffness)

generally develop within 3 months post-surgery owing to

fibrosis [43]. Management of OA has changed as the result

of current knowledge based on the physiology and

pharmacology of articular cartilage, as well as the use of

specific new drugs [43]. Polycan is purified β-glucan from

Aureobasidium pullulans SM-2001 composed mostly of

β-1,3-1,6-glucan and other organic materials, such as

amino acids, mono- or di-unsaturated fatty acids (linoleic

and linolenic acids), and fibrous polysaccharide [47].

Recently, we found that Polycan has anti-osteoporotic

effects [48, 51], inhibiting bone loss and accelerating bone

formation, which led us to hypothesize that Polycan would

show favorable effects on OA. Therefore, in the present

study, the efficacy of Polycan on OA induced by

ACLT&PMM was evaluated.

MATERIALS AND METHODS

Animals

Male, six-week-old Sprague-Dawley rats (N=120; SLC, Japan)

were used after seven days of acclimatization. Animals were housed

four or five per polycarbonate cage in a temperature (20-25oC) and

humidity (40-45%) controlled room with a 12 h:12 h light:dark cycle.

Feed (Samyang, Korea) and water were supplied ad libitum. Prior to

euthanasia, all animals were fasted overnight, and treatment was in

accordance with the Guide for the Care and Use of Laboratory

Animals (Institute of Laboratory Animal Resources, Commission on

Life Science, National Research Council, Washington DC, USA,

1996). Eight rats per group (total 48 rats) were selected based on

body weights and knee thicken seven days post-surgery.

Preparation and Administration of Polycan or Diclofenac Sodium

Three dosages of Polycan (85, 42.5, and 21.25 mg/kg; Glucan Co.

Korea) were dissolved in distilled water and orally administered,

once a day for 84 days in a volume of 1 ml/kg. In sham and OA

control groups, only 1 ml/kg of distilled water was orally administered.

As a reference control, 2 mg/kg of diclofenac sodium (Sigma, MO,

USA) dissolved in saline in a volume of 1 ml/kg was subcutaneously

injected once a day for 84 days from one week after OA inducement

by ACLT&PMM surgery (Table 1). In the present study, diclofenac

sodium, a well-known cyclooxygenase inhibitor nonsteroidal anti-

inflammatory drug (NSAID), was used as a comparable reference

drug for OA [14, 17, 23].

Induction of OA

Rats were anesthetized with a 25 mg/kg intraperitoneal injection of

Zoletile (Zoletile 50; Virbac Lab., France). The OA treatment group

underwent surgery involving ACLT&PMM via an incision on the

medial aspect of the joint capsule of the left knee, anterior to the

medial collateral ligament. Following surgery, the incision was

closed in two layers. The joint capsule was sutured independently

from peripheral tissues using dissolvable 5-0 Vicryl suture, and the

skin was closed with interrupted silk sutures. This procedure was

used to induce OA pathogenesis and referred to as the operated-

induced side. Conversely, the right knee joint (non-operated, intact

side) was used for contralateral treatment. The sham group of rats

underwent an operation in which a similar incision in the joint capsule

was made but ACLT&PMM were not performed. Only the left knees

of sham animals were used as controls for disease progression.

Body Weight Changes

Body weights were measured weekly from the start of treatment to

euthanasia, using an automatic electronic balance (Precisa Instruments

AG, Switzerland). Body weight gains during the 12 weeks of

observation were calculated as follows to reduce the individual

differences in body weights at the start of the study: Body weight

gain (g) = body weight 1 day before euthanasia - body weight at

treatment initiation.

Knee Thickness Measurement

The thickness of OA operated right knees was measured using an

electronic digital caliper (Mytutoyo, Japan) and recorded weekly. In

addition, knee thickness was also measured after joint capsule

Table 1. Experimental design.

Group OA Dose (mg/kg/day) Animal No.

Control group

Sham-operated Vehicle 1 mg/kg (0.2 ml/head) Rat-01 ~ Rat-08

OA Vehicle 1 mg/kg (0.2 ml/head) Rat-09 ~ Rat-16

OA Diclofenac sodium 2 mg/kg Rat-17 ~ Rat-24

Experimental group

OA Polycan 85 mg/kg (orally) Rat-25 ~ Rat-32

OA Polycan 42.5 mg/kg (orally) Rat-33 ~ Rat-40

OA Polycan 21.25 mg/kg (orally) Rat-41 ~ Rat-48

(n=8), OA, osteoarthritis; All three dosages of Polycan were orally administered once a day for 84 days, dissolved in distilled water, from 1 week after

ACLT&PMM surgery in a volume of 1 ml/kg; Diclofenac sodium was subcutaneously administered, once a day for 84 days from 1 week after

ACLT&PMM surgery.

ANTI-OSTEOARTHRITIC EFFECTS OF POLYCAN 276

exposure at euthanasia using the same method to reduce differences

due to surrounding tissues.

Maximum Extensor Angle Measurement

OA operated knees in all animals were dissected from the

coxofemoral region to the ankle, leaving the articular capsule intact.

After the dissection, the maximum extension angle of each knee

was measured according to previous methods [43], with 0 degrees

corresponding to the maximum possible extension. In order to minimize

any possible bias, all operations and extension measurements were

performed by the same veterinarian.

Histopathology

Knee joints were excised with the joint capsule intact and fixed in

10% neutral buffered formalin. After five days of fixation, samples

were decalcified using 24.4% formic acid, and 0.5 N sodium hydroxide

for five days (decalcifying solution was exchanged once a day for

five days), after which median joint parts were longitudinally

trimmed and embedded in paraffin, sectioned (3-4 µm), and stained

with hematoxylin and eosin (H&E) or Safranin O for cartilage as

previously described [7, 27, 54]. Histological profiles of the knee

joints were observed and compared with intact controls.

Mankin scoring: Articular cartilage injuries were evaluated using

the Mankin score [2,36] with H&E and Safranin O staining. In this

system, the higher the score, the higher the level of OA. The entire

histological evaluation was performed by the same pathologist.

Histomorphometry - The thickness of tibia and femur articular

cartilage was measured by histomorphometrical analyses of prepared

longitudinally trimmed samples at µm levels using a digital image

analyzer (DMI-300, DMI, Korea).

BrdU-Uptake Measurement

To assess the effects of Polycan on the proliferation of cells within

the rat knee joints, proliferating cells were labeled by an intraperitoneal

injection of BrdU. One hour prior to the injection of diclofenac

sodium or oral administration of Polycan (on day 82 of treatment),

rats were given intraperitoneal injections of BrdU (MP Biomedicals,

OH, USA) 50 mg/kg, in a volume of 2 ml/kg dissolved in saline,

and euthanized 72 h later as previously described [24]. BrdU uptake

was detected with an anti-BrdU antibody as described by Moore et

al. [39]. Fixed tissues were prepared, embedded in paraffin, and

sectioned as described previously. Tissues were de-paraffinized through

a series of washes with xylene and graded alcohol.

After epitope retrieval by pretreatment of 2 N HCl as previously

described [25, 53], sections were immunostained as follows:

1. Incubate sections with methanol and 0.3% H2O2 for 30 min for

blocking endogenous peroxidase activity at room temperature.

Rinse in 0.01 M phosphate-buffered saline (PBS; pH 7.2) three

times.

2. Incubate sections with normal horse serum blocking solution

(Vector Lab. Inc., CA, USA) at a 1:100 dilution for 1 h to block

nonspecific binding of immunoglobulin at room temperature in

a humidified chamber. Rinse in 0.01M PBS three times.

3. Incubate sections with primary antisera [Anti-BrdU (HRP)

polyclonal antibody; code Ab2285; Abcam, UK] overnight at 4oC

in a humidified chamber. Rinse in 0.01 M PBS three times.

4. Incubate sections with biotinylated universal secondary antibody

(Vector Lab. Inc.) at a dilution of 1:50 for 1 h at room

temperature in a humidified chamber. Rinse in 0.01 M PBS

three times.

5. Incubate sections with ABC reagents (Vectastain Elite ABC

Kit; code PK-6200; Vector Lab. Inc.) at a 1:50 dilution for 1 h

at room temperature in a humidified chamber. Rinse in 0.01 M

PBS three times.

6. Incubate sections in peroxidase substrate (code SK-4100;

Vector Lab. Inc.) for 30 s at room temperature. Rinse in

0.01 M PBS three times.

7. Counterstain with Mayer’s hematoxylin solution. Rinse in tap

water for 30 min.

8. Dehydrate in 95% ethanol for 2 min and 100% ethanol three

times. Clear in xylene twice.

9. Add coverslip with permanent mounting medium and observe

under a light microscope (Nikon, Japan).

Among 100 chondrocytes, the number of cells occupied by over

10% BrdU immunoreactivity was detected in both the femur and

tibia surface articular cartilage with an automated digital image

analyzer (DMI-300, DMI, Korea) and calculated as percentages.

Statistical Analysis

All data are expressed as means±standard deviation (SD), and

statistical analysis was conducted using the Mann-Whitney U-

Wilcoxon Rank Sum W test with SPSS for Windows (Release 14;

SPSS Inc., USA).

RESULTS AND DISCUSSION

In the present study, to observe the long-term efficacy of

Polycan on OA, three different dosages were orally

administered once a day for 84 days to rats with OA

induced by ACLT&PMM surgery starting one week after

the operation. A vehicle control and a sham control group

were also included for comparison purposes, as well as a

group that received diclofenac sodium. Diclofenac sodium

is a well-known cyclooxygenase inhibitor NSAID used as

a reference drug for OA [14, 17, 23]. It has been shown to

preserve the joint cartilage relatively well at a subcutaneous

dose of 2 mg/kg in OA rats [23]. Therefore, 2 mg/kg of

diclofenac sodium was subcutaneously administered once

a day for 84 days from one week after OA inducement as a

reference control in the present study.

Body Weight Changes

In the present study, no meaningful changes in body weight

were detected in all Polycan-treated groups compared with

sham or OA control, and the body weights and gains of all

rats used in this study were within the range of normal age-

matched rats [32].

Changes on the Knee Thicknesses

OA, also known as a degenerative joint disease, is a

chronic inflammatory disease. The cartilage damage in OA

leads to edematous changes in the surrounding tissues, and

277 Kim et al.

the thicknesses of affected joints show marked increases

[19]. Significant (p<0.01) increases of OA operated knee

thickness were detected in OA controls compared with

sham controls from treatment initiation throughout 84 days

of experimental period. However, knee thickness of all

three doses of Polycan and diclofenac sodium was

significantly decreased (P<0.01 or P<0.05) compared with

OA controls from day 21 of treatment.

The thickness of OA-induced knees at euthanasia in

controls (12.96±0.38 mm) changed significantly (P<0.01)

compared with sham controls (10.92±0.56 mm). Diclofenac

sodium, and polycan 85, 42.5, and 21.25 mg/kg treatment

groups significantly changed (P<0.01 or 0.05) as 12.21±0.46,

12.23±0.45, 12.40±0.32, and 12.21±0.58 mm, respectively,

compared with OA controls (Table 2).

These favorable effects of Polycan are considered to be

the result of anti-inflammatory effects as previously

described [30, 31]. No marked changes were detected in

the thicknesses of knee joints after joint capsule exposure,

which means that no hyperplasia (overproliferation) of

chondrocytes was induced by treatment with Polycan or

diclofenac sodium.

Changes in Knee Thickness After Capsule Exposure

The fibrosis that occurs in OA from the chronic

inflammatory process limits joint motion, and the resultant

stiffness of joints is one of major symptoms of OA, which

reaches a peak approximately three months after OA

inducement. Stiffness of joints has been evaluated by the

maximum extension angle of the joint, considering 0

degree as maximum extension; therefore, the lower the

value, the better the knee function [43]. The thickness of

OA operated knees after joint capsule exposure was

significantly increased (p<0.01) in all OA-induced groups

compared with sham controls. Quite similar thicknesses

were detected with all three dosages of Polycan and

diclofenac sodium compared to OA controls.

The thickness of OA-induced control knees (9.24±0.30 mm)

after joint capsule exposure at euthanasia changed

significantly (P<0.01) compared with sham controls (8.00

±0.18 mm). Furthermore diclofenac sodium, and Polycan

85, 42.5, and 21.25 mg/kg treatment groups significantly

changed (P<0.01) as 9.14±0.23, 8.98±0.53, 9.14±0.43, and

9.04±0.48 mm, respectively, compared with sham controls.

There were no significant changes between diclofenac,

Polycan, and OA controls (Table 3). Decreases of maximum

extension angles in OA-induced knees are considered as

direct evidence that Polycan ameliorated OA in this study.

Changes in Knee Maximum Extension Angles

The maximum extensor angle of OA-induced knees was

significantly increased (p<0.01) in OA controls compared

with sham controls. However, these angles were significantly

decreased (p<0.01) with diclofenac sodium and all three

doses of Polycan compared with OA controls.

The maximum extensor angles of OA-induced knees at

euthanasia in OA controls (74.25±4.13o) changed significantly

(P<0.01) compared with sham controls (28.63±3.20o).

Diclofenac sodium, and polycan 85, 42.5, and 21.25 mg/

kg treatment groups significantly changed (P<0.01) as

60.75±6.78o, 60.50±3.30o, 52.75±6.82o, and 56.38±4.31o,

respectively, compared with OA controls (Table 3).

Table 2. Changes on the OA-induced knee thickness during 84 days continuous oral treatment of Polycan or subcutaneous treatment ofdiclofenac sodium in OA rats.

Items D01) D7 D14 D21 D28 D35 D42 D49 D56 D63 D70 D77 D83 euthanasia

Control

Sham11.26±0.62

11.11±0.24

11.62±0.16

11.70±0.24

10.81±0.28

10.78±0.28

11.15±0.28

10.95±0.18

10.79±0.41

10.38±0.41

10.54±0.54

10.80±0.66

11.18±0.37

10.92±0.56

OA13.75±0.28a

13.64±0.37a

13.16±0.52a

13.13±0.40a

13.04±0.43a

13.30±0.52a

13.41±0.34a

12.82±0.43a

12.52±0.51a

12.74±0.44a

12.82±0.55a

13.00±0.48a

12.98±0.46a

12.96±0.38a

Diclofenac13.75±0.33

a13.26±0.51

a12.64±0.32

a12.43

±0.71ab

12.13±0.59

ab12.42

±0.80ab

12.39±0.28

ab12.05

±0.24ab

12.15±0.25

ac11.57

±0.59ab

12.04±0.60

ab12.26

±0.45ab

12.34±0.43

ab12.21

±0.46ab

Polycan treated

85 mg/kg13.73±0.20a

13.29±0.40a

12.79±0.26a

12.63±0.62ac

12.46±0.56ac

12.45±0.52ab

12.43±0.54ab

12.40±0.39a

12.25±0.34a

12.15±0.29ac

11.99±0.71ab

12.33±0.20ab

12.21±0.45ab

12.23±0.45ab

42.5 mg/kg13.87±0.14a

13.33±0.42a

12.83±0.39a

12.51±0.49ac

12.43±0.24ac

12.56±0.47ab

12.43±0.27ab

12.37±0.22ac

12.39±0.29a

12.10±0.57ab

12.23±0.38ac

12.32±0.59ac

12.50±0.28ac

12.40±0.32ac

21.25mg/kg13.62±0.35

a13.29±0.42

a12.95±0.57

a12.53

±0.27ac

12.26±0.35

ab12.71

±0.32ac

12.57±0.31

ab12.50±0.28

a12.36±0.26

a11.98

±0.37ab

12.18±0.37

ac12.41±0.80

a12.57±0.54

a12.21

±0.58ac

Values are expressed as Means ± SD (n = 8); unit, mm; OA, osteoarthritis; 1)day of Polycan administration;

ap<0.01 compared with sham control;

bp<0.01

cp<0.05 compared with OA control.

ANTI-OSTEOARTHRITIC EFFECTS OF POLYCAN 278

Changes in the Mankin Scores

The Mankin scoring system is generally used for

histopathological evaluation to detect articular cartilage

injuries. In this system, the higher the score, the higher the

level of OA [2, 43]. We found favorable decreases in

Mankin scores with diclofenac sodium treatment and all

three dosages of Polycan for both the tibia and femur. This

is also considered as direct evidence that Polycan ameliorated

OA. Marked decreases of articular cartilage thickness

occurr in OA [8, 39, 41]. In the present study, Polycan

effectively inhibited the decreases in articular cartilage

thickness.

Various degrees of articular cartilage surface damages,

hypocellularity, clones, and stain intensity for Safranin

O were detected in all OA-induced groups. The total

Mankin scores in tibia and femur of OA controls were

significantly increased (p<0.01) compared with sham

controls. Although individual scores varied in all groups,

Table 3. Knee thicknesses after joint capsule exposure and maximum extensor angles.

Groups Knee thicknesses after joint capsule exposure at sacrifice (mm) Maximum extensor angles (degrees, o)

Controls

Sham 8.00±0.18 28.63±3.20

OA 9.24±0.30a 74.25±4.13a

Diclofenac 9.14±0.23a 60.75±6.78ab

Polycan treated

85 mg/kg 8.98±0.53a 60.50±3.30ab

42.5 mg/kg 9.14±0.43a 52.75±6.82ab

21.25 mg/kg 9.04±0.48a 56.38±4.31ab

Values are expressed as Means ± SD (n = 8); OA, osteoarthritis; ap<0.01 compared with sham control;

bp<0.01 compared with OA control.

Table 4. Mankin scores detected in femur at sacrifice.

Groups Surface Hypocellularity Clones Safranin O Totals1)

Controls

Sham 0.25±0.46 0.25±0.46 0.00±0.00 0.25±0.46 0.75±0.89

OA 2.75±0.46a

2.38±0.52a

2.25±1.16a

2.50±0.53a

9.88±1.55a

Diclofenac 2.13±0.35ad 1.38±0.52ab 2.38±0.92a 1.25±0.46ac 7.13±1.36ac

Polycan treated

85 mg/kg 2.13±0.64ad

1.13±0.83ac

1.88±0.83a

1.13±0.83bc

6.25±2.49ac

42.5 mg/kg 1.88±0.35ac 0.88±0.64c 1.38±0.92a 1.00±0.76c 5.13±1.46ac

21.25 mg/kg 2.13±0.64ad 1.63±0.74ad 1.88±0.99a 1.13±0.99d 6.75±2.66ad

Values are expressed as Means ± SD (n = 8); OA, osteoarthritis; 1)Totals mean Mankin score [Max = 12];

ap<0.01 and

bp<0.05 compared with sham control;

cp<0.01 and

d p<0.05 compared with OA control.

Table 5. Mankin scores detected in tibia.

Groups Surface Hypocellularity Clones Safranin O Totals1)

Controls

Sham 0.25±0.46 0.00±0.00 0.00±0.00 0.13±0.35 0.38±0.74

OA 2.63±0.52a

2.00±0.53a

2.38±0.52a

1.50±0.93a

8.50±1.93a

Diclofenac 2.00±0.53ab 1.63±0.52a 1.75±0.71a 1.38±0.74a 6.75±1.16ab

Polycan treated

85 mg/kg 2.00±0.76ab

1.13±0.83ab

1.75±0.89a

1.13±1.13 6.00±2.39ab

42.5 mg/kg 2.25±0.71a 1.38±0.74a 1.50±0.93a 1.00±0.93 6.13±2.42ab

21.25 mg/kg 2.13±0.64a 1.63±0.74a 1.88±0.99a 1.13±0.99 6.75±2.66a

Values are expressed as Means ± SD (n = 8); OA, osteoarthritis; 1)Totals mean Mankin score [Max = 12];

ap<0.01 compared with sham control;

bp<0.05

compared with OA control.

279 Kim et al.

the total Mankin scores in both tibia and femur with

diclofenac sodium and all three doses of Polycan were

dramatically decreased as compared with OA controls.

The total Menkin scores of femur articular cartilage in

OA controls (9.88±1.55) changed significantly compared with

sham controls (0.75±0.89). Diclofenac sodium, and polycan

85, 42.5, and 21.25 mg/kg treatment groups significantly

changed (P<0.01) as 7.13±1.36, 6.25±2.49, 5.13±1.46,

and 6.75±2.66, respectively compared with OA controls.

The total Menkin scores of tibia articular cartilage in

OA controls (8.50±1.93) changed significantly compared

with sham controls (0.38±0.74). Diclofenac sodium, and

polycan 85, 42.5, and 21.25 mg/kg treatment groups

significantly changed (P<0.01) as 6.75±1.16, 6.00±2.39,

6.13±2.42, and 6.75±2.66, respectively, compared with

OA controls (Tables 4-6 and Fig. 1).

Changes in the Articular Cartilage Thickness

Significant decreases (p<0.01) in articular cartilage thickness

were detected in OA controls compared with sham controls

in both the tibia and femur. However, these decreases in

cartilage thickness were significantly inhibited (p<0.01)

by treatment with diclofenac sodium and all three dosages

of Polycan in both the tibia and femur, except for the

Polycan 21.25 mg/kg treatment group in which the tibia

articular cartilage thickness was also significantly increased

(p<0.01) compared with OA controls, and femur articular

cartilage thickness was nonsignificantly increased.

The OA-induced femur articular cartilage thickness in

OA controls (303.07±91.40 µm) changed significantly

(P<0.01) compared with sham controls (569.43±95.77 µm).

Diclofenac sodium, and Polycan 85, 42.5, and 21.25 mg/kg

treatment groups significantly changed (P<0.01 or 0.05)

as 409.48±69.80, 520.81±173.05, 566.34±203.02, and

513.34±162.18 µm, respectively, compared with OA controls.

The OA-induced tibia articular cartilage thickness in

OA controls (296.51±73.69 µm) changed significantly

(P<0.01) compared with sham controls (780.16±145.83 µm).

Diclofenac sodium, and Polycan 85, 42.5, and 21.25 mg/kg

treatment groups significantly changed (P<0.01 or 0.05)

as 500.53±196.69, 571.85±137.17, 532.26±177.22, and

352.08±105.69 µm, respectively, compared with OA controls

(Table 6 and Fig. 1).

Changes in BrdU Uptake

Among the methods for the detection of cell proliferation

in histological sections, immunohistochemistry using BrdU

Table 6. Histomorphometrical scores.

GroupsThickness of articular cartilages (µm)

Femur Tibia

Controls

Sham 569.43±95.77 780.16±145.83

OA 303.07±91.40a 296.51±73.69a

Diclofenac 409.48±69.80b 500.53±196.69bd

Polycan treated

85 mg/kg 520.81±173.05c 571.85±137.17ac

42.5 mg/kg 566.34±203.02c 532.26±177.22ac

21.25 mg/kg 513.34±162.18c 352.08±105.69a

Values are expressed as Means ± SD (n=8); OA, osteoarthritis; ap<0.01 and

bp<0.05 compared with sham control;

cp<0.01 and

dp<0.05 compared with

OA control.

Fig. 1. Histopathological observation in tibia and femur articularsurface cartilages of the sham control (A, B), OA control (C, D),diclofenac sodium (E, F), and Polycan 85 (G, H), 42.5 (I, J), and21.25 (K, L) mg/kg treatment groups.Note that the articular surface of both the tibia and femur were markedly

damaged (as detected by the Mankin scoring system, showed marked

abnormal changes on surface, hypocellularity, multiple clones, and

decreases in Safranin O staining intensity) in the OA control with

decreases in both the cartilage thickness and the gap between these two

articular surface. However, these damages were markedly inhibited by

treatment of Polycan or diclofenac sodium, respectively; arrows indicate

the thickness of tibia or femur articular cartilages; OA, osteoarthritis; Gap

mean intra-joint space between articular surface of tibia and femur.

Safranin O stain; Scale bars = 80 µm.

ANTI-OSTEOARTHRITIC EFFECTS OF POLYCAN 280

is the most preferred [16, 39]. BrdU staining is easier to

read and reflects cell proliferations more specifically than

other staining methods [24]. In addition, BrdU uptake is

also prevalently used to detect chondrocyte proliferation

in OA cartilage [39]. Cells containing BrdU indicate

proliferated or proliferating cells.

Significant decreases (p<0.01) in BrdU-immunoreactive

cells were detected in both the tibia and femur articular

cartilage of OA controls compared with sham controls,

which indicates that proliferation of chondrocytes was

markedly inhibited. However, these decreases in BrdU-

immunoreactive cells were significantly inhibited (p<0.01)

by treatment with all three dosages of Polycan, except for

Polycan 21.25 mg/kg, which produced nonsignificant increases

in BrdU-immunoreactive cells in the tibia articular cartilage

but a similar number of immunoreactive cells in the femur

articular cartilage compared with OA controls. Similar

numbers of BrdU-immunoreactive cells were detected in

both the femur and tibia of diclofenac sodium-treated rats

compared with OA control.

BrdU-immunoreactive cell numbers in femur articular

cartilage OA controls (7.88±3.56/100 chondrocytes) changed

significantly (P<0.01) compared with sham controls (45.50

±9.49/100 chondrocytes). Diclofenac sodium, and Polycan

85 and 42.5 mg/kg treatment groups significantly changed

(P<0.01) as 8.63±4.03, 26.75±8.37, and 24.25±6.71/100

chondrocytes, respectively, compared with OA controls.

The BrdU-immunoreactive cell numbers in tibia articular

cartilage of OA controls (8.00±4.24/100 chondrocytes)

changed significantly (P<0.01) compared with sham controls

(40.88±8.43/100 chondrocytes). Diclofenac sodium, and Polycan

85 and 42.5 mg/kg treatment groups significantly changed

(P<0.01) as 8.13±3.52, 28.75±7.57, and 27.63±3.66/100

chondrocytes, respectively, compared with OA controls.

These results are considered as direct evidence that Polycan

induced proliferation of chondrocytes that differed from

diclofenac sodium (Table 7 and Fig. 2).

A period of 84 days after initiation of treatment was

assumed sufficient for the development of fibrotic stiffness

of OA joints based upon a previous study [43]. Changes

in the thickness and maximum extension angle of each

knee, thickness of capsular joint regions, and cartilage

histopathology were measured using the Mankin score

(histomorphometry included the thickness of surface

articular cartilages of femur and tibia in individual rats) at

Table 7. BrdU-immunoreactive cell numbers.

Groups

Number of BrdU-immunoreactive cells(/100 chondrocytes)

Femur Tibia

Controls

Sham 45.50±9.49 40.88±8.43

OA 7.88±3.56a

8.00±4.24a

Diclofenac 8.63±4.03a 8.13±3.52a

Polycan treated

85 mg/kg 26.75±8.37ab

28.75±7.57ab

42.5 mg/kg 24.25±6.71ab 27.63±3.66ab

21.25 mg/kg 8.38±4.03a 12.63±6.63a

Values are expressed as Means ± SD (n=8), percentages; OA, osteoarthritis;ap<0.01 compared with sham control;

bp<0.01 compared with OA control.

Fig. 2. BrdU-immunoreative cells observations in the tibia andfemur articular surface cartilages of the sham control (A, B), OAcontrol (C, D), diclofenac sodium (E, F), polycan 85 (G, H), 42.5(I, J) and 21.25 (K, L) mg/kg treated groups.Note that marked decreases of BrdU-immunoreactive cells were detected

in both the tibia and femur articular cartilages of OA control compared to

sham control, respectively. However, these decreases in BrdU-immunoreactive

cells were significantly inhibited by treatment of Polycan (except 21.25 mg/kg

treated group) (P<0.01), in which non-significant increases of BrdU-

immunoreactive cells in the tibia articular cartilage were also observed

compared to OA control but quite similar immunoreactive cells were detected

in femur articular cartilage. Similar numbers of BrdU-immunoreactive

cells were detected in both the femur and tibia of diclofenac sodium

treatment group compared to OA control, respectively; Gray-black stained

cells are indicated BrdU-immunoreactive cells; OA, osteoarthritis. ABC

methods; Scale bars = 80 µm.

281 Kim et al.

necropsy. In addition, the proliferation of cartilage was

also assessed using BrdU. As a result of ACLT&PMM,

classic OA was induced, identified by changes in maximum

extension angles, limited extension values, edematous

changes, capsule thickness increases, decreases in chondrocyte

proliferation (detected by BrdU uptake), cartilages degenerative

changes, Mankin score differences, and loss of articular

cartilage. However, these OA changes were markedly

inhibited by 84 days of diclofenac sodium treatment and

all three dosages of Polycan compared with OA controls,

with the excetption of capsule thickness in that similar

thickness was detected regardless of treatment. Whereas

diclofenac sodium did not influence BrdU uptake, BrdU-

immunoreactive cells were increased by treatment at all

three dosages of Polycan, which means that Polycan

induced proliferation of chondrocytes in tibia and femur

surface articular cartilage.

Similar or more favorable effects on stiffness and

cartilage losses induced by OA were detected with all three

oral dosages of Polycan compared with diclofenac sodium

2 mg/kg subcutaneously treated rats. These overall effects

of Polycan showed clear dosage dependencies between

42.5 and 21.25 mg/kg treatment groups, but slightly lower

or similar effects were detected between the 85 and

42.5 mg/kg treatment groups. Therefore, based upon these

results, we suggest that the optimal concentration to treat

OA is 42.5 mg/kg in the present study. This dosage can be

converted to 425 mg/head/day in a 60 kg human, based on the

body surface differences of rats being 1/6 that of humans.

Acknowledgment

This work was supported by the National Research

Foundation of Korea (NRF) grant funded by the Korea

government [MEST] (No.2011-0030124).

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