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Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Objective

To investigate the in vivo effects of dehydroepiandrosterone (DHEA) on knee joints during the development of experimentally induced osteoarthritis (OA).

Methods

Twenty-two mature NZW rabbits underwent bilateral anterior cruciate ligament transection (ACLT) and received 0.3-ml intraarticular injections of DHEA (at a concentration of 100 μM in phosphate buffered saline) and control solution in the right and left knees, respectively, beginning 4 weeks after ACLT and continuing once weekly for 5 weeks. All animals were killed 9 weeks after surgery, and the knee joints were assessed by gross morphologic, histologic, histomorphometric, and biochemical methods.

Results

Gross morphologic inspection following India ink application showed that the right femoral condyles, which received DHEA, demonstrated less severe cartilage damage than did the contralateral condyles. The thickness, area, and roughness of the DHEA-treated femoral condyles provided evidence of a cartilage-protecting effect of DHEA following ACLT. These results were supported by gene expression analysis. Messenger RNA expression of a proinflammatory cytokine, interleukin-1β, and catabolic enzymes, matrix metalloproteinases 1 and 3, was reduced in the cartilage of the DHEA-treated knee joints, and expression of tissue inhibitor of metalloproteinase 1 was increased.

Conclusion

Results of the present study demonstrate a cartilage-protecting effect of DHEA during the development of OA following ACLT in a rabbit model.

Osteoarthritis (OA) is a degenerative joint disease characterized by loss of articular cartilage, subchondral bone remodeling, joint space narrowing, and bone spur formation, as well as synovial inflammation. There are several treatment modalities, including administration of nonsteroidal antiinflammatory drugs (1, 2) or steroids (3, 4), physical therapy (5), and surgery such as osteotomy and joint replacement as a last resort.

Although intraarticular corticosteroid injection has been recommended for the relief of pain and swelling, adverse effects (including possible infection) and concerns about the possible development of progressive cartilage damage through repeated injections, together with the short duration of action, have limited wider usage (6–9). Meanwhile, intraarticular injection of hyaluronan, which is one of the principal components of cartilage matrix, is now frequently performed for the palliation of joint pain and has been reported to have some positive effects on the maintenance of cartilage matrix integrity during the development of OA (10, 11). However, the mechanism of action of hyaluronan remains unclear, because the duration of benefit reported exceeds its synovial half-life.

Dehydroepiandrosterone (DHEA) is a 19-carbon steroid hormone classified as an adrenal androgen. DHEA is synthesized from pregnenolone (derived from cholesterol) and is rapidly sulfated to its ester form, DHEA-S, the predominant form found in circulating plasma (12). Because the plasma level of DHEA declines with age, numerous studies of DHEA in various disease conditions, such as atherosclerosis (13), cancer (14), diabetes (15), obesity (16), aging (17), and inflammatory arthritis, including rheumatoid arthritis (RA) (18–23), have been performed.

Although RA shares some aspects with OA, there is little information about the effects of DHEA on OA, as far as we know. In a previous in vitro study (24), we demonstrated that in OA, DHEA has an ability to modulate the imbalance between matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases 1 (TIMP-1) at the transcription level, which suggests that DHEA has a protective role against articular cartilage loss.

The aim of the present study was to investigate the in vivo effects of intraarticular injections of DHEA on the maintenance of the cartilage matrix and on the gene expression of various inflammatory mediators during the development of OA. The anterior cruciate ligament transection (ACLT) model of rabbit knee joints was used for this study.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Animals.

Twenty-two mature NZW rabbits underwent bilateral ACLT, using a medial arthrotomy method. Briefly, all animals were anesthetized with an intramuscular injection of ketamine (100 mg/kg) and xylazine (8 mg/kg). Both knees were shaved and disinfected with Betadine solution. A medial parapatellar incision was made on the skin, followed by a medial arthrotomy. After lateral dislocation of the patella, the knee was fully flexed. The anterior cruciate ligament was visualized and transected with a #15 scalpel blade. The knee joint was irrigated with sterile saline and closed with a running suture of 4-0 nylon. The skin incision was then closed with a running suture of 4-0 nylon supplemented with some interrupted sutures. Postoperatively, the animals were permitted activity in the cage (60 cm × 60 cm × 40 cm), without immobilization.

Four weeks after ACLT (and after intravascular administration of anesthesia), each animal received a 0.3-ml intraarticular injection of DHEA (Sigma, St. Louis, MO) at a concentration of 100 μM in phosphate buffered saline (PBS) in the right knee and an injection of control solution (PBS) in the left knee, once a week for 5 weeks. All animals were killed 9 weeks after ACLT for assessment of knee joints by gross morphologic, histologic, histomorphometric, and gene expression analysis.

Gross morphology.

After the rabbits were killed, both knees from all rabbits were harvested. The femoral condyles were photographed using a high-resolution digital camera (3.3 million pixels, with a close-up lens [Coolpix995; Nikon, Tokyo, Japan]). After application of India ink, gross morphologic changes of the femoral condyles were evaluated by 3 independent researchers who reevaluated the specimens several times and were not aware of any prior data concerning each specimen. The criteria used (10) were as follows: for grade 1 (intact surface), surface appears normal and does not retain any ink; for grade 2 (minimal fibrillation), surface appears normal before staining but retains the India ink as elongated specks or light gray patches; for grade 3 (overt fibrillation), surface is velvety in appearance and retains ink as intense black patches; for grade 4 (erosion), loss of cartilage is evident, with exposure of the underlying bone. Grade 4 was further divided into 3 subgrades according to the length of the erosion, as follows: for grade 4a, erosion ≤2 mm; for grade 4b, erosion >2 mm and ≤5 mm; for grade 4c, erosion >5 mm. After morphologic grading, the knees were irrigated with saline solution to remove India ink and divided into 2 groups. The first group (n = 11) underwent histologic and histomorphometric evaluation, and the second group (n = 11) underwent gene expression analysis.

Histologic preparation.

The medial femoral condyles of both knees were divided sagittally into 2 equal parts and fixed in 10% neutral buffered formalin with 1% cetylpyridinium chloride (CPC) for 72 hours and then decalcified with 14% EDTA. CPC was used to prevent loss of glycosaminoglycan from the tissues during processing (25). When decalcification was completed, the 2 halves of the condyles were embedded in paraffin, and 5μ sections were cut, followed by staining with hematoxylin and eosin and Safranin O/fast green.

Histomorphometry.

Six Safranin O/fast green–stained sections per knee (3 sections from each half of the medial femoral condyle) were evaluated using an image analysis system (ImagePro Plus; Media Cybernetics, Silver Spring, MD) by 3 independent researchers who reevaluated the knee sections several times and were not aware of any prior data concerning each specimen. In each half, the first section was obtained from the region of most damaged articular cartilage, and the other 2 sections were obtained from regions that were equally distant from the first section point. Histologic sections were visualized with a microscope attached to a high-resolution color video camera. The live video images were captured by the image analysis system and viewed on a high-resolution color monitor (SyncMaster; Samsung, Suwon, Korea).

We developed customized software to measure the following 3 histomorphometric parameters: cartilage thickness, area, and surface roughness, as described by Yoshioka et al (26). The geometric parameters of the specimens were defined (at 12.5× magnification) on 3 different areas of 5-mm length on a femoral condyle (anterior [the patellofemoral joint surface], distal [weight-bearing area], and distal–posterior [the transition area from the weight-bearing area to the posterior articular surface]) (Figure 1) and were averaged. The thickness of the cartilage was defined as the distance from the surface to the tidemark and was calculated from the mean of 640 measurements made perpendicular to the surface of each section at 640 equally spaced points along the 5-mm width. The possible variability in the placement of the tidemark was kept to a minimum, with efforts to obtain clear staining of the specimens and to reach consensus among the evaluators on placement, using some pilot samples before real measuring. The area of the cartilage along the 5-mm width was likewise calculated. The thickness and area of the cartilage were computed with the coordinates of the articular cartilage and the tidemark (Figures 2A–C).

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Figure 1. Geometric parameters of the histologic specimen. Three 5-mm–long images were obtained from the medial femoral condyle. Three geometric parameters (thickness, area, and roughness) were measured on each image, and the values were averaged.

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Figure 2. Quantitative histologic assessment using customized image analysis software. A, Original histologic image of the medial femoral condyle (grade 3). B, Thickness measurement. C, Area measurement. D, Roughness measurement. Each measurement was performed 640 times, and the values were averaged and are indicated as the colored portions in each figure.

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The surface profile was evaluated by the root mean square (RMS) surface roughness (26) and was expressed as the following equation:

  • equation image

where N is the number of digitized points, Y idealizedi is the theoretical coordinate of the idealized smooth surface of articular cartilage determined from the coordinates of the subchondral bone and mean thickness of the cartilage region, and Y reali is the actual coordinate of the articular cartilage surface (Figure 2D).

Surface roughness is, in part, dependent on thickness, which means a thicker cartilage surface is more susceptible to variations in wear, resulting in a larger surface roughness measurement. Conversely, a joint surface completely denuded of cartilage would yield a falsely low surface roughness measurement. Therefore, we used cartilage roughness normalized to the thickness (roughness/cartilage thickness). All roughness data are presented with normalization.

Extraction of RNA and synthesis of complementary DNA (cDNA).

Cartilage from the femoral condyle and the tibial plateau was harvested following gross morphologic assessment and stored frozen in liquid nitrogen until used. For extraction of total RNA from the cartilage, the frozen tissue was crushed and homogenized in 4M guanidinium thiocyanate containing 25 mM sodium citrate, 0.5% sodium sarkosyl, and 0.1M 2-mercaptoethanol. Total RNA was extracted using the acid guanidinium thiocyanate–phenol–chloroform method, as previously described (27), and an RNA extraction kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions. Complementary DNA was synthesized with 0.5 μg of RNA and random hexamers using a commercially available kit (First Strand cDNA Synthesis Kit; MBI Fermentas, Vilnius, Lithuania) according to the manufacturer's recommendation.

Polymerase chain reaction (PCR).

The resulting cDNA was then amplified by PCR using a commercially available kit (AccuPower; Bioneer, Taejon, Korea) in a volume of 20 μl using 3 thermocycler temperatures (Perkin-Elmer, Norwalk, CT). GAPDH reverse transcriptase–PCR (RT-PCR) products were used for normalization. All reactions were determined to be in a linear range of amplification within cyclic numbers of 14–30. The primers used for rabbit GAPDH, TIMP-1, MMP-1, MMP-3, and interleukin-1β (IL-1β) are shown in Figure 3. A cycle profile consisted of 30 seconds at 94°C for denaturation, 30 seconds at 60°C for annealing, and 30 seconds at 72°C for extension. Electrophoresis of 10 μl of the reaction mixture on a 1.5% agarose gel containing ethidium bromide was performed to evaluate the amplification and size of the generated fragments. A 100-basepair DNA ladder (Bioneer) was used as a standard size marker. A densitometric computer program (TINA; Raytest, Straubenhardt, Germany) was used to scan the RT-PCR agarose gel after photographic documentation. The program measured relative mean density corrected by background.

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Figure 3. Comparative mRNA expression in the dehydroepiandrosterone (DHEA) and control groups, and corresponding primer sequences. The expression of tissue inhibitor of metalloproteinases 1 (TIMP-1) was significantly increased in the DHEA-treated group, while that of matrix metalloproteinase 1 (MMP-1), MMP-3, and interleukin-1β (IL-1β) was decreased. Bars show the mean and SD. ∗ = P < 0.05.

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Statistical analysis.

All data are expressed as the mean ± SD. The data were verified using paired t-test, except for variability testing of gross morphologic grading. Interobserver and intraobserver variation in gross morphologic grading were verified by measuring agreement with the kappa statistic. P values less than 0.05 were considered significant.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Gross morphology.

All knee joints exhibited complete transection of the ACL at the time of evaluation. The most extreme area of degeneration usually occurred along the posteromedial aspect of the medial femoral condyle, which is consistent with a previous report (10). Generally, condyles in the DHEA group showed less severe cartilage damage than did those in the control group (Figure 4). Six (27.3%) of 22 condyles in the DHEA group showed grade 1 damage, compared with none of the specimens in the control group (Table 1). Seven specimens (31.8%) in the DHEA group and 6 (27.3%) in the control group showed grade 2 damage, while 3 specimens (13.6%) in the DHEA group and 8 (36.4%) in the control group showed grade 3 damage. Grade 4 cartilage damage occurred in 8 specimens (36.4%) in the control group (grade 4a, n = 1; grade 4b, n = 3; grade 4c, n = 4). Although 6 specimens (27.3%) in the DHEA group also showed grade 4 cartilage damage, the damage in the DHEA group was less severe than that in the control group. In the DHEA group, half of the specimens with grade 4 damage were categorized as 4a (grade 4a, n = 3; grade 4b, n = 2; grade 4c, n = 1), but in the control group the majority of grade 4 specimens were categorized as grades 4b and 4c. The interobserver and intraobserver variations for grading of cartilage damage in the DHEA and control groups were not statistically significant (κ = 0.45 and κ = 0.57, respectively [P > 0.05]).

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Figure 4. Gross morphologic and histologic findings after dehydroepiandrosterone (DHEA) (A and C) or control (B and D) treatment. The DHEA-treated femoral condyle showed minimal erosion, with some dotted India ink (grade 2) on its medial side, while cartilage in the control-treated femoral condyle is fully eroded, and the underlying subchondral bone is exposed (grade 4b). Color figure can be viewed in the online issue, which is available at http://www.arthritisrheum.org.

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Table 1. Gross morphologic assessment of the femoral condyles (n = 22)*
GradeDHEA groupControl group
  • *

    Values are the number of specimens. DHEA = dehydroepiandrosterone.

160
276
338
468
4a31
4b23
4c14

Qualitative histologic assessment.

Histologic analysis demonstrated a variety of degenerative changes in the knees that underwent ACLT. Usually the most damaged articular surface is in the distal weight-bearing area (the posteromedial aspect), followed by the transition area from the distal to the posterior articular surface, and then the anterior patellofemoral joint area. Six specimens in the DHEA group, but none in the control group, exhibited normal appearance of articular cartilage with normal Safranin O/fast green staining. Although 6 specimens in the DHEA group showed grade 4 damage, the extent of ulceration in the cartilage was less than that in the control group. Loss of Safranin O staining was observed in the fibrillated area of articular cartilage. There was no apparent difference in the staining pattern between the DHEA and control groups.

Quantitative histomorphometric assessment.

Because in most specimens the most severe area of degeneration occurred at the medial femoral condyles, we performed the histomorphometric assessment on them using the following 3 parameters: thickness, area, and roughness. Three images of 5-mm length from the patellofemoral joint surface, the weight-bearing area, and the transition area from the weight-bearing area to the posterior articular surface together covered nearly the total length of the sagittal contour of the medial femoral condyle of a rabbit. This suggests that our method of measurement adequately represents the whole articular surface. The interobserver and intraobserver variation among the measurements were not statistically significant using paired t-test (P > 0.05).

Cartilage thickness and area.

The mean ± SD cartilage thickness in the DHEA group was 0.45 ± 0.12 mm and was significantly greater compared with that in the control group (0.33 ± 0.09 mm) (Figure 5A). The mean ± SD cartilage area in the DHEA group was 2.24 ± 0.59 mm2, compared with 1.67 ± 0.46 mm2 in the control group (P < 0.05) (Figure 5B).

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Figure 5. Calculated cartilage thickness, area, and normalized root mean square (RMS) surface roughness of the articular cartilage from dehydroepiandrosterone (DHEA)–treated and control-treated knees. Articular cartilage obtained from DHEA-treated knees was significantly thicker (A), larger (B), and smoother (C) than that obtained from the control-treated knees. Bars show the mean and SD. ∗ = P < 0.05.

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Surface roughness.

The mean ± SD normalized surface roughness value for the DHEA group (0.17 ± 0.29 mm) was significantly less than that for the control group (0.98 ± 1.27 mm; P < 0.05). Figure 5C shows the results of normalized surface roughness.

Effects of DHEA on gene expression of IL-1β and inflammatory enzymes.

Each RT-PCR was performed in triplicate and yielded almost identical results. Representative results are shown in Figure 3. GAPDH messenger RNA was well expressed in all samples, which confirmed uniformity of the RNA preparation. Expression of TIMP-1 was significantly increased in the DHEA group compared with that in the control group (P < 0.05). Messenger RNA expression of MMP-1, MMP-3, and IL-1β was significantly decreased in the DHEA group compared with the control group (P < 0.05). Expression of MMP-1 was suppressed most prominently, decreasing to 17% of that in the control group.

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

This study is the first to demonstrate the in vivo effects of DHEA on articular cartilage during experimentally induced OA. The gross morphologic osteoarthritic changes of the femoral condyles revealed that the extent and severity of articular cartilage damage were less in the DHEA-treated knees than in the control knees. Furthermore, quantitative histomorphometric analysis showed that cartilage thickness, area, and roughness in the DHEA-treated knees were all statistically superior compared with controls. Results of the gene expression analysis of articular cartilage support those of the gross morphologic and histomorphometric analyses. Gene expression of TIMP-1, an antagonist to the MMPs, was increased in the DHEA-treated knees, while gene expression of MMP-1 and MMP-3, which are important catabolic enzymes, and IL-1β, a proinflammatory cytokine, was decreased in the DHEA-treated knees more than in the control-treated knees. It has been suggested that DHEA is protective against age-related illnesses (13, 15, 17, 28, 29). We concentrated on the relationship between DHEA and OA development caused by ACLT in the rabbit. The findings of the present study are in accordance with those of our previous in vitro study (24), in which application of DHEA treatment to chondrocytes cultured in alginate beads increased the gene expression of TIMP-1 and decreased that of MMP-1 and MMP-3.

In this study, we injected DHEA at a concentration of 100 μM into the rabbit knee joint. The choice of that concentration was based on results of a previous in vitro study, in which use of a 100-μM concentration yielded the best results, with no toxic effects observed. However, we arbitrarily selected the number and sequence of the injections, because no work with DHEA has been performed in this experimental model. Because results of the present study are encouraging, further studies focusing on the dose, number, and sequence of injections are warranted.

Although ACLT for inducing OA was first introduced in the dog model (30), it has now also been popularized in the rabbit model, as evidenced by gradual and progressive changes in the morphology, histopathology, biochemistry, and the gene expression pattern of the articular cartilage of the operated knee (10, 26, 31–38). In the rabbit model of ACLT, the most extreme area of degeneration is known to occur along the posteromedial aspect of the medial femoral condyle (26), which is consistent with the results of this study. This is the reason we performed the histomorphometric assessment on the medial femoral condyles.

In the present study, we revealed the effects of DHEA quantitatively using histomorphometric parameters such as surface roughness, cartilage area, and thickness, which were first described by Yoshioka et al (26). However, we slightly modified the previous method to remove the selection bias in obtaining images of the region of greatest cartilage damage. Instead, we divided the sagittal contour of the medial femoral condyle into 3 regions (anterior, distal, and distal–posterior) and obtained 3 images. Because the length of the sagittal contour of the rabbit femoral condyle is ∼15–20 mm, the 3 images can include nearly all of the region of the femoral condyle. We believe that we could avoid the selection bias through this modification and subsequently obtain more objective measurements.

This study represents the first attempt to examine the effects of DHEA during the development of OA following ACLT. Using histomorphometric and gene expression analyses, we demonstrated quantitatively that exogenously administered DHEA has positive effects on the maintenance of articular cartilage matrix integrity. Despite the lack of understanding of the exact mechanism of DHEA action, the present study demonstrated protective effects of DHEA on articular cartilage during experimentally induced OA.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES
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