Caloric restriction minimizes aging effects on the femoral medial condyle

Abstract

This study aimed to analyze the effects of caloric restriction on aged femoral articular cartilage of Wistar rats. Three groups of eight animals each were considered: young (YC) and old (OC) control groups fed with a normal diet and old caloric restriction group (OCR) composed of 18-month-old animals fed with a 31% less caloric diet from 6-months of age. Articular cartilage was studied through morphometry and immunohistochemistry. Body mass was 12% less in the OCR group than in the OC group. The articular cartilage from OC rats show thinner medial condyles, fewer chondrocytes, smaller chondrocytes nuclear volume and, in both condyles, a predominance of collagen type II and less collagen density compared to both YC and OCR groups (p < .001). In contrast, OCR articular cartilage show thicker medial condyles, larger chondrocytes nuclear volume and increased collagen density compared to OC group (p < 0.001). We concluded that caloric restriction minimizes the effects of aging on medial condyles of the femoral articular cartilage.

Keywords:

• Morphometry
• hipocaloric nutrition
• articular cartilage
• Wistar rats

Introduction

Articular cartilage is a highly specialized tissue that supports forces and reduces friction during movement in the synovial joints [1,2]. Structurally, it consists of chondrocytes and extracellular matrix [3–5].

During aging, occurs chondrocytes loss [1,6] with associated changes in the matrix density, composition, and organization of type II collagen and proteoglycans [2,5,7–9] thus causing significant reduction of the cartilage thickness [10–12].

Studies have shown that the level of caloric intake (CR) could influences the aging of many tissues [13,14]. Caloric restriction, characterized by reduced caloric intake below usual ad libitum without malnutrition [15], was shown to slow aging effects and increase longevity by preventing the onset of chronic diseases [16–19]. Given the degeneration of the articular cartilage during aging and the beneficial effects of CR reported in the literature, the present study aimed to analyze the influence of CR on structural modifications of femoral articular cartilage of aging Wistar rats.

Materials and methods

Twenty-four male Wistar rats were divided into groups: YC (6 months-old fed with a normal diet, from weaning until six months of age, n = 8), OC (18 months-old fed with normal diet from weaning until 18 months of age, n = 8) and OCR (18 months-old fed with a diet containing fewer calories, beginning at six months of age, n = 8). Animals were individually housed in polypropylene boxes, provided with food and water ad libitum and environment-controlled temperature and lighting.

To determine the daily amount of chow provided during experimental period, all animals passed through a 7-day adaptation process. During this process, the animals were given a normal diet and water ad libitum. The same amount of chow consumed per day during the adaptation phase was administered throughout the duration of the first month of the experiment. Thereafter, the amount of chow supplied to the animals in the OCR group was adjusted monthly based on the average consumption of the animals in the OC group. This procedure was necessary to prevent the animals of the OCR group from compensating for the reduced-calorie feed by increasing their consumption to maintain the same caloric intake. After the adjustment period, the animals in the OC group were fed with a normal diet with a chemical composition based on the dietary recommendations for laboratory animals. Data about diet ingredients are published elsewhere [20].

Animals in the OCR group were fed with a diet in which the raw energy was reduced by 31% (Table 1). The animals in the OC and ORC groups began the experiment at 6 months of age and completed the experiment at 18 months of age. The animals were weighted monthly until euthanasia.

Table 1. Data of articular cartilage thickness and chondrocytes numerical density and nuclear volume of the young control (YC), old control (OC), and old restricted groups (OCR).

	Groups
Condyles	YC (n = 8)	OC (n = 8)	OCR (n = 8)
Thickness (μm)
Medial	68.5 ± 22.5	46.5 ± 12.4^a^,^b	69.6 ± 33.4
Lateral	59.4 ± 8.1	55.5 ± 8.9	50.7 ± 9.0
Numerical density (n°/area)
Medial	25.3 ± 3.5	18.9 ± 2.2^a^,^b	26.6 ± 3.5
Lateral	23.8 ± 1.7	23.8 ± 3.9	25.2 ± 3.3
Nuclear volume (n°)
Medial	133.3 ± 16.9	92.3 ± 30.1^a^,^c	108.3 ± 45.6
Lateral	117.2 ± 30.9	109.9 ± 37.6	106.2 ± 19.6

Data are presented as mean and standard error.

^a Significantly different from YC medial condyle (p < .001).

^b Significantly different from OCR medial condyle (p < .001).

^c Significantly different from OCR medial condyle (p < .05).

Euthanasia and tissue preparation

At the end of the experimental period, animals were fasted for 12 h and euthanized using a lethal, intravenous dose (tail vein) of anesthetic (Thiopental^® 40 mg/kg of body weight). The euthanasia protocol of the animals was submitted to the Committee of Bioethics of the College of Veterinary Medicine and Zootechny of the University of São Paulo (Protocol n° 2477/2011).

Subsequently, types of collagen were assessed by immunohistochemistry and picrosirius staining. Morphometric study was carried out, using histological sections of femoral articular cartilage stained with picrosirius and hematoxylin–eosin.

For immunohistochemical analysis of cartilage matrix, collagen type II was used as six sections of each animal’s tissue. This type of collagen was chosen by being predominantly in articular cartilage [21,22]. The cuts in silanized slides were unmasked in a water bath in borate buffer at 80° C for 30 min. Then, the sections were submitted to the blocking of peroxidase in 3% H₂O₂ solution, diluted in methanol for 5 min. After washing, the antigenic blocking of the slides was performed in acid5-thio-2-nitrobenzoic acid (TNB) for 30 min. Subsequently, the sections were washed and incubated in a humidified chamber overnight at 4 °C with mouse anti-human collagen II [2150–0040 (Serotec) Oxford, England]. Then, the slides were incubated with secondary antibodies [(Dako) Glostrup, Denmark] for 30 min in humid chamber. After washing, the revelation was performed using diamino benzidine (DAB). Subsequently, background staining was performed with Harris hematoxylin for 5 s. The slides were mounted and immediately photographed with a magnification of 400×. Negative controls were carried out to prevent the nonspecific markings by secondary antibody.

Picrosirius-stained histological sections were examined with polarizing filters in conventional light microscope. When analyzed by this method, type I collagen presents an orange or red color, while type III collagen presents a green color. The type II collagen in general, presents a yellow color, but can be found in varying colors; however, these colors are always clearly distinct from type I and III collagens [23].

Quantitative analysis

Articular cartilage thickness, chondrocytes numerical density (number per area) and volume of nuclei, [24] and volume density of collagen fibers were obtained by light microscopy, using the AxioVision 4.8 digital analyzing computer program (Zeiss, Germany).

Statistical analysis

Descriptive statistics were given as mean ± standard error of mean (SEM). Data were not normally distributed. The parameters of three groups of rats were compared using the ANOVA and post hoc Tukey’s test. When not specified, p ≤ .05 was considered as significant.

Results

Body mass

At six months of age, there was no difference between the OC and OCR groups (p > .05) (Figures 1 and 2). From 8 months of age, we observed an increase in body weight of the animals from the OC group and at 9 months of age in the OCR. In OCR group, there was weight loss at 8 months with gain by the end of the experiment. Nevertheless, this gain was significantly lower than that found in OC animals up to 18 months of age (p < .05).

Figure 1. Mean values of body mass (g) obtained monthly during the experimental period (12 months) for animals of OC group.

Figure 2. Mean values of body mass (g) obtained monthly during the experimental period (12 months) for animals of OCR group.

For the OC group, the exponential growth coefficient (e) and the coefficient of determination (R²) of body weight were and .97, respectively (p < .0001). For OCR, the exponential growth coefficient and the coefficient of determination were e .81, respectively (p < .0001).

Articular cartilage thickness

Medial condyle of OC group was thinner than YC and OCR groups (p < .001).

Density of chondrocytes

The number of chondrocytes in the medial condyle was fewer in the OC compared to YC and OCR groups (p < .001).

Nuclear volume of chondrocytes

The chondrocyte nuclear volume was fewer in the medial condyle from OC group relative to YC (p < .001) and OCR groups (p < .05). Data are presented in Table 1.

Collagen

There was fewer volume of collagen in the OC group in relation to the YC in both femoral condyles (p < .01), while the OCR animals had larger collagen volume in relation to the OC only in the medial condyle of the femur (p < .05) (Figure 3).

Figure 3. Volumetric proportion of collagen in the extracellular matrix (% by area) from the lateral and medial femoral condyle in the studied groups (YC, OC, OCR). *Significant in relation to the lateral condyle of YC group (p < .001); **Significant in relation to the lateral condyle of YC group (p < .05); #Significant in relation to the medial condyle of YC group (p < .001); ##Significant in relation to the medial condyle of OC group (p < .05).

Types of collagen

In the YC group, at both condyles, we note the presence of collagen type I fibers (orange) and type II (yellow and brown), with a predominance of type II collagen (Figure 4)

Figure 4. Histological sections of the articular cartilage of the femur showing the distribution of collagen fibers (arrows) in the layers of cartilage in the three studied groups (YC, OC, OCR); SB: subchondral bone. Picrosirius staining.

In the OC group, at both condyles, we note the presence of collagen type I fibers (red), type II (yellow and brown) and type III (green) with a predominance of type II collagen. In OCR group, we note the presence of collagen type I fibers (orange), type II (yellow and brown), and type III (green) with predominance of collagen type II. The type II collagen fibers predominated in both condyles of the YC group compared to OC. However, we found equilibrium in the presence of collagen type II fibers between OC and OCR groups.

Collagen type II immunohistochemistry

In the femoral articular cartilage, the YC group showed homogeneous labeling (++++/++++) of type II collagen while OC (+++/++++) and OCR groups (+++/++++) showed heterogenous labeling, with no difference between the condyles (Figure 5).

Figure 5. Histological sections of the articular cartilage of the femur showing the labeling of type II collagen in beige color (*) by Immunohistochemistry, in the three groups studied (YC, OC, OCR). SB: subchondral bone.

Discussion

Several references demonstrated an increase of weight mass during the process of aging [17,25–27]. Here, we presented data showing that OC group was heavier than OCR. At eight months of experimentation, OCR group showed loss of mass, with subsequent regain by the end of the experiment. The coefficient of exponential growth was higher in the OC group, indicating that they gained weight faster than the OCR. The coefficient of determination indicates how much the data obtained had the same behavior. Thus, OC and OCR groups presented 97% and 81%, respectively, which are similar to the regression model.

Data for body weight obtained for the OCR group agreed with Athanasiou et al. [17], which showed less body weight gain between 6 and 18 months with food restriction. Nevertheless, data diverge from Holloszy [26] that demonstrated there was no significant difference in mass between 6 and 18 months in the animals subjected to food restriction. In addition, Bailey et al. [25] noted an increase in mass up to 15 months in animals with restricted feeding, with subsequent loss up to 18 months. These differences may be due to the dietary restriction model, possibly using food restriction instead caloric restriction.

The reduction in body weight gain of rats between 8 and 18 months of age is a protective factor for the articular cartilage, once overweight and obesity are associated with inflammatory diseases such as osteoarthritis [28–30]. Here, we observed a tuning of the cartilage as age progressed, which was most visible in the medial condyle. Contrary, Wang et al. [27] and Stockwell [31] demonstrated that the thickness of the cartilage of the distal femur did not change with advancing age. Probably, this setback is associated with the decrease in activity of chondrocytes with aging [7,32].

The asymmetry between the condyles may be due to mechanical factors [7,32]. According to Buckwalter and Mankin [33], overloading the joint creates mechanical, electrical, physical, and chemical signals that stimulate the activity of synthesis or degradation of chondrocytes. Caloric restriction maintained cartilage thickness of the medial condyles showing that it is useful to prevent the reduction caused by aging.

In this work, the total number of chondrocytes was lower in the medial condyle than lateral, confirming the findings of Stockwell [31] and Oda et al. [5]. Possibly, this is due to the apoptosis related to inflammatory conditions [10,11]. The loss of chondrocytes leaves the cartilage more vulnerable to degenerative diseases, causing reduction of strength and matrix stiffness [10,34]. In turn, caloric restriction leads to increase in cell density in the medial condyle.

Perhaps, aging influence on chondrocytes nuclei volume of medial condyles indicates a reduction of their metabolic activity. Knowing that the metabolism of the chondrocytes is responsible for maintaining the matrix by a balance between anabolism and catabolism [1,2,10,22,33,35–38], our results agree with the Karvonen et al. [32], Hudelmaier et al. [7] and Martin and Buckwalter [6]. Further, caloric restriction also influenced chondrocytes nuclei volume without any morphoquantitative differences between both condyles. Caloric restriction mitigated the effects of aging, which agrees with literature, showing that caloric intake surely alters cellular metabolic activity [14,39].

Collagen type II was chose being predominant in articular cartilage [21,22]. As known, changes occur in density, composition and/or organization of collagen during the aging process [12,17,28]. Here, we found decreased density of collagen type II in the articular cartilage of the femur with aging.

The caloric restriction prevented the effects of aging on the medial condyle but not on the lateral. This difference may be seen in some pathological conditions such as osteoarthritis. According to Felson et al. [40], patients with knee osteoarthritis tend to support progressively greater weight on the lateral condyle.

The results of polarization method and immunohistochemistry suggested that larger collagen content in the medial condyle is not predominantly type II. Changes of type II collagen observed in the experimental groups OC and OCR may be due to increased weight bearing on the joint, resulting in weight gain in these animals, especially the OC animals. In fact, Roth and Mow [41] and Volpi and Katz [42] quoted that changes in the cartilage collagen depend on the forces acting, which influence remodeling and reorientation of collagen fibers. In conclusion, the present study found that aging of femoral articular cartilage can be slowed down by caloric restriction.

Disclosure statement

None of the authors disclose any conflict of interest.