INTRODUCTION

The heavy metal hexavalent chromium (CrVI), which can be found in the soil, air and water in industrial areas in large cities around the world, is very dangerous to human health^1-3. Hexavalent chromium has been found in inadequately treated wastes from petrochemical and leather industries that are discharged into rivers⁴. Exposure to CrVI can affect the population at large⁵,⁶ and children in particular⁷. In lactating infants, exposure can occur through breast milk from contaminated mothers⁸ or by ingesting infant formula prepared with contaminated water⁹,¹⁰. A child’s behavior and life-style also influence exposure. Children crawl on the floor, put things in their mouths, and eat inappropriate things such as dirt or paint chips. It has been reported that materials containing CrVI (plastics, dyes and paints) used to manufacture toys can be another source of exposure¹¹.

Dental eruption involves the movement of a tooth from its site of development in the jaw bone to its functional position in the oral cavity. Andreasen et al.¹² divided this continuous process into five stages: preeruptive movements, intraosseous eruption, mucosal penetration, preocclusal eruption, and postocclusal eruption. Each stage involves interactions between the developing tooth and the surrounding periodontal tissues, and it is temporally and spatially controlled to coordinate the growth of the jaws and the position of other adjacent and antagonist teeth¹³. The most widely accepted theory at present explaining the tooth eruption process under normal physiological conditions involves the action of bone modeling and remodeling¹⁴. The mechanism of bone modeling involves formation and resorption processes of different and often opposite bone surfaces. Bone formation occurs at the base of the developing alveolus, facilitating tooth eruption, whereas bone resorption occurs at the lateral walls and especially the bone overlying the crown of the tooth¹⁵. Simultaneously, the developing alveolar bone begins to undergo a remodeling process¹⁶. Bone remodeling involves the coordinated and coupled action of osteoclasts and osteoblasts at specific sites on the bone surface with the final aim of replacing and renewing the bone matrix¹⁷. Pathological conditions, as is CrVI exposure, impair the tooth eruption process¹⁸,¹⁹. The effects of CrVI exposure on bone tissue in rats include alterations in bone formation²⁰ and resorption²¹, and in bone growth²²,²³.

Moreover, defects in skeletal development in children born in contaminated areas close to leather tanning processing plants have been reported²⁴. Previous studies conducted by our research group have shown that tooth eruption is delayed in suckling rats exposed to hexavalent chromium¹⁸,¹⁹. Nevertheless, the effects of CrVI exposure on modeling and remodeling of the walls of the developing alveolus at the different stages of tooth eruption remain unknown. Therefore, the aim of the present work was to study the effects of CrVI on bone formation and resorption of the alveolus of the developing first lower molar of 9- and 15-day-old rats, which correspond to rats in the intraosseous and mucosa penetration stages of tooth eruption, respectively.

MATERIALS AND METHODS

Thirty-two suckling Wistar rats aged 4 days at the onset of the experiment were used. Experimental animals received daily 12.5 mg/kg of body weight of potassium dichromate (Biopack, Argentina) dissolved in saline solution by oral gavage until the day prior to euthanasia. Both the experimental and control groups were divided into two sub-sets of eight animals each according to age at euthanasia: 9 and 15 days, which correspond to the intraosseous and mucosa penetration stages of tooth eruption, respectively.

All the animals were housed throughout the experiment with their corresponding dam (8 pups/ dam) in individual metal cages 30 cm high, 40 cm wide, 40 cm deep, with wood chip bedding. The animals were kept under controlled housing conditions: 7-hour light/dark cycles, 20 to 26ºC temperature, and 40 to 70% humidity. The dams were allowed free access to solid chow pellets and water ad libitum. The pups were returned to their cage with their dam after each procedure.

This study was approved by the Institutional Ethics Committee for the Care and Use of Laboratory Animals of the School of Dentistry of the University of Buenos Aires (Res. No. 27/03/2013-51), and all the experiments were conducted in keeping with The National Institutes of Health Guidelines for the Care and Use of Laboratory Animals (NIH publication 85-123 Rev. 2010).

Immediately after euthanasia, the mandibles were resected and fixed in 4% formaldehyde in PBS at 4°C for 48 hours, decalcified in 10% EDTA, and embedded in paraffin. Buccolingually oriented sections were obtained at the level of the mesial root of the first lower molars under a stereoscopic microscope (Nikon, Japan). Serial sections were stained with routine H&E and alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) histochemical techniques, used as markers of bone formation and bone resorption, respectively. Photomicrographs of the histologic sections were taken using a light field microscope (Axioskop 2; Carl Zeiss, Jena, Germany) and a digital camera (Nikon CoolPix 12 Mp). The images were used to perform the histomorphometric analysis of the developing alveolus using Image Pro® Plus software, version 5.1 (Media Cybernetics), following the stereological principles described by Weibel and Elias²⁵, and using the nomenclature employed by Parfitt et al.²⁶ and revised by Depster et al.²⁷.

Histomorphometric Analysis

Digital images of the TRAP-stained sections were used to assess the resorption activity of the inner wall of the forming socket. During tooth eruption, bone resorption occurs only in the most superficial third. Therefore, TRAP+ edges were measured at a length of 500 μm from the point closest to the tip of the crown in both the buccal and lingual directions (See Fig. 1 for orientation), and expressed as percentage. Digital images of ALP-stained sections were used to measure the percentage of ALP+ bone formation in the total bone edge of the developing alveolus. Data were analyzed statistically using Student’s t test, setting statistical significance at p<0.05.

Fig.1 Buccolingual sections at the level of the mesial root of the first lower molar of 9- and 15-day-old animals. A. Control animals. B. CrVI-exposed animals. Nine-day-old exposed animals showed a less erupted tooth germ covered with thicker bone and less developed crown tissues than controls. Molars of 15-day-old exposed animals were less erupted with less developed crown tissues and less developed roots than control molars. 

RESULTS

The histologic features of the first molars in the intraosseous and mucosa penetration stages of both experimental and control animals are shown in Fig. 1. At age 9 days, the tooth germs of both groups were covered with bone and oral mucosa. The distance between the tooth germs and the surface of the oral mucosa was greater in CrVI-exposed animals, which thus showed a decreased eruption rate. Thickness of developing dental tissues was less and the entire germ was smaller and less developed. The walls of the developing alveolus were thinner and showed lower bone density than in controls.

At 15 days of age, the first molars were in the mucosa penetration stage, in which the crown is only covered by the oral mucosa and the bone overlying the crown has resorbed completely so that the tooth can open its eruption pathway. The upper portion of the cortical plates of the alveolus was separated from the enamel organ remnant by primitive connective tissue. Periodontal ligament fibers were inserted in the cortical bone plate and in the developing tooth root. The first molars of animals exposed to CrVI, however, had erupted less and were less developed, and the periodontal space around both the developing crown and root was smaller than in controls. The crown and roots were smaller, the Hertwig’s sheath was longer, and the bone volume of the developing bony crypt was lower.

Nine-day-old experimental animals showed fewer erosion surfaces on the buccal and lingual sides of the developing alveolus than their age-matched controls. Conversely, 15-day-old experimental animals showed more erosion surfaces at the aforementioned sites than their matched controls (Fig. 2). In addition, the trabeculae of the walls of the developing alveolus of experimental animals were thicker and larger than those of controls at both stages of tooth eruption studied.

Fig 2 Buccolingual sections at the level of the mesial root of the first lower molar of 9- and 15-day-old rats. TRAP detection, hematoxylin counterstained. The upper and lower images correspond to 9- and 15-day-old animals, respectively. A. Control rat. B. CrVI-exposed rat. Nine-day-old CrVI animals showed fewer TRAP+ resorption surfaces (red arrows) than their age-matched controls. Conversely, 15-day-old CrVI animals showed more TRAP+ resorption surfaces (red arrows) than their matched controls. In addition, bone trabeculae of both 9- and 15-day-old CrVI animals were thicker and larger. 

The histomorphometric study showed that the percentage of TRAP+ surfaces at the study sites was significantly lower in 9-day-old experimental animals and significantly higher in 15-day-old animals than in the corresponding sites in their agematched controls (Table 1).

Table 1 Percentage of bone resorption surfaces in 9- and 15-day-old animals 

The buccal and the lingual sides of the developing alveolus of 9- and 15-day-old CrVI-exposed animals showed fewer osteoblast-covered bone surfaces than controls. The histomorphometric study showed that the percentage of ALP+ surfaces was lower in controls than in the corresponding sites of agematched experimental rats. The differences observed between groups at the intraosseous stage of tooth eruption were statistically significant (Table 2).

Table 2 Percentage of bone formation surfaces in 9- and 15-day-old animals. 

DISCUSSION

To our knowledge, this is the first experimental study to analyze the effects of CrVI on tooth eruption. Our results showed that exposing suckling rats to CrVI delays resorption of the bone overlying the crown and bone formation on the lateral walls of the developing alveolus, delaying tooth eruption.

Bone resorption must take place for the tooth to move through the eruption pathway in all three spatial planes. Marks and Cahill²⁸ reported that the dental follicle plays an essential role in regulating this process. It has been shown that the dental follicle produces chemotactic molecules, such as MCP-1 (Monocyte chemotactic protein-1) and CSF-1 (Colony stimulating factor-1), responsible for recruiting mononuclear cells that fuse to form osteoclasts, which are necessary for the alveolar bone to resorb²⁹. According to reports in the literature, osteoclastogenesis in the developing alveolus of the first molar of rats increases towards day three after birth and then decreases towards 10 – 16 age period ³⁰,³¹.

Our results in the control group agree with these data. In the CrVI group, we found a significantly lower percentage of TRAP+ bone resorption surfaces in 9-day-old rats and a higher percentage of bone resorption surfaces in 15-day-old rats. This indicates alteration of the sequence of bone resorption in the walls of the developing alveolus that is necessary for the tooth to move through the eruption pathway. Exposure to CrVI is likely to affect the regulating function of the dental follicle, altering monocyte recruitment and fusion, consequently altering osteoclastogenesis and resorption of the developing alveolus, and causing a delay in the eruption.

Our observations are in agreement with studies reported in the literature. CrVI affects osteoclastogenesis and osteoclast function in vivo in rats³² and in humans³³, since chromium interferes with monocyte differentiation into osteoclasts and inhibits the Ca²⁺ receptors in the osteoclast cell membrane. The binding site of the receptor is highly sensitive to divalent and trivalent cations, and because CrVI reduces to trivalent chromium, the latter could bind to the receptor, increasing the cytosolic concentration of calcium and decreasing bone resorption. In an in vivo study, Sankaramanivel et al.²⁰ showed a significant decrease in TRAP activity in the skull of exposed rats, which undergoes the same ossification mechanism as the jaws.

In addition, we found a lower percentage of ALP+ formation surfaces on the lateral walls of the developing alveolus of the first molar of CrVIexposed animals as compared to controls at both experimental times. The lower degree of bone formation in the walls of the tooth alveolus observed in our study are in agreement with the effects of CrVI exposure on osteoblasts described by other authors. Reports on in vitro cytotoxic effects of CrVI on mature osteoblasts include alterations in their synthesis function, cell morphology, and capacity to differentiate into osteocytes and bone lining cells^34-36 as well as inhibition of bone mineralization³⁷.

An interesting finding of the present study is that CrVI-exposed animals exhibited thicker and larger bone trabeculae than their matched controls at both experimental times. There are no reports in the literature to date on intoxication with CrVI or other heavy metals showing this very particular alveolar histoarchitecture, which may occur as a consequence of an alteration of the bone remodeling process with the ensuing delay in alveolar bone resorption during tooth eruption.

Although the dose of 12.5 mg/kg of body weight used in our study is lower than the median lethal CrVI dose (LD50) for rats, it is higher than the maximum allowed dose (0.1 mg/kg) for humans³⁸. Environmental levels of CrVI in some large cities around the world are higher than the dose used here^1-3. In fact, the daily dose of CrVI administered to the experimental animals in this study was similar to the dose that growing children are exposed to through drinking formula prepared with contaminated water.

By delaying tooth eruption, exposure to CrVI could cause impaired occlusion and facial growth and development in exposed children. All dentists should therefore be well informed about these CrVI exposure-related disorders for early detection and prompt referral to the corresponding specialist. In addition, having information about the environment in which their patients have grown up helps dentists to diagnose these alterations accurately.