Facial development disorders due to inhibition to endochondral ossification of mandibular condyle process caused by malnutrition

Bone formation during growth is related to several factors, including heredity, function, and environment.1 Environmental factors include nutritional deficiencies, which may appear during growth and affect bone development, thereby causing marked variations in bone shape and size.2,3 

Dietary proteins are essential to the biosynthesis of the organic matrix of bone tissue. During fetal and postnatal growth, there is a close relation between overall nutritional status and longitudinal bone growth. A number of studies have looked at how undernourishment affects the development of long bones. It is well documented that protein deprivation affects total epiphyseal growth plate height, the number of chondrocytes per column in the proliferative and hypertrophic zones of the growth cartilage, and the size of hypertrophic chondrocytes.4–6 In addition to causing morphologic alterations in the epiphyseal growth plate in rats, isocaloric protein deprivation after weaning disturbs bone volume and endochondral ossification, in which chondrogenesis is coupled with subsequent bone formation.4 

Although there is evidence of an association between protein deprivation and osteoporosis, systemic bone mineral density (BMD), and alveolar bone BMD, this association seems to be inferior in the mandible compared with that observed in other skeletal sites.7,8 However, studies of the effects of systemic bone loss stimuli on mandibular bone show conflicting results. It is well documented that the mandible is functionally and morphologically different from other bones of the skeleton. The mechanical loading of the mandible during mastication has an influence on the mass, density, and microarchitecture of the mandibular alveolar bone.9 But to our knowledge no reports are available on the effects of isocaloric protein deprivation in growth and bone activity in the mandibular condylar process, in which, like the alveolar, bone is subjected to heavy and intermittent forces during mastication.

Because protein malnutrition severely affects endochondral ossification in long bones,4,5 we hypothesize that, despite the different mechanical function of the mandible, this nutritional disorder may alter bone formation in areas of active ossification in the mandibular condylar process, also altering their growth. Based on the above, the aim of the present experimental work was to assess the influence of chronic protein deprivation on bone remodeling in the subchondral region of the mandibular condyle process, its impact on their length, and the implications that these undernutrition-related alterations would have in the direction of mandibular displacement during facial development.

Twenty newly born Wistar rats were used. The animals were weaned at the age of 21 days and assigned to one of the following groups: a control group (n  =  10), in which rats were fed a biosynthetic regular hard diet ad libitum (Table 1), and protein-restricted (PR) group (n  =  10), in which rats were fed a biosynthetic hard diet lacking protein ad libitum (Table 1). Body weight and food consumed by the animals of both groups was recorded throughout. Five weeks after the onset of the experiment (when the rats were 8 weeks old) the animals were euthanized with diethyl ether inhalation. The mandibles were then carefully dissected and fixed in 10% formalin, after which the soft tissue was removed.

The experimental protocol was approved by the Ethical Committee for Animal Care, and the experimental procedures were carried out in accordance with institutional guidelines. The National Institutes of Health Guidelines for the Care and Use of Laboratory Animals (NHI publication 85-23 Rev. 1985) were observed.

In order to perform radiographic study, the mandibles were hemisected at the symphysis. A metallic landmark consisting of an L-shaped 0.2-mm steel ligature wire10 was placed in the mandibular foramen of one hemimandible of each rat (Figure 1). The marked hemimandibles were positioned laterally on the film alongside a 10-mm-long wire and radiographed using standard X-ray equipment at 70 Kv and 8 mA with a 0.3-secind exposure time; the focus to film distance was 40 cm. Reference points were marked on paper tracings of the projected image of the radiographs (×7 magnification) in order to perform the cephalometric measurements (Figure 1). The projected image of the wire served as a scale, and the measurements were calibrated according to the image of the standard length of wire.

After this, the hemimandibles were decalcified in 10% ethylenediaminetetraacetic acid (pH  =  7.2) and processed following routine procedures for embedding in paraffin. Longitudinal sections were obtained at the level of the mandibular condyle and stained with hematoxylin and eosin. The stained sections were observed under a light microscope and studied histomorphometrically. The area used to perform the histomorphometric determinations is shown in Figure 2.

The following cephalometric parameters were measured on paper tracings of the projected image of the radiographs: (1) the length of the condylar process and (2) the height of the lower alveolar process.

The following histomorphometric parameters were measured in mandibular condyle subchondral bone following standard stereologic methods11: (1) percentage of osteoblast surfaces, which is indicative of active bone formation (OblS/BS %). (2) eroded surfaces (with or without osteoclasts), which are indicative of bone resorption (RS/BS %); (3) quiescent surfaces, which are indicative of bone at rest (FlatOblS/BS %); and (4) bone volume fraction (OblS/BS%) Table 2.

Experimental data were expressed as the mean ± standard deviation. Single comparisons between groups were assessed with Student's t-test. Differences were considered significant at P < .05.

A slight increase in body weight was observed in both groups throughout the first 10 days of the study, after which no further increase was detected in the PR group. Body weight was significantly lower in the PR group than the control group at the end of the experimental period (Figure 3). There was no significant difference in the amount of food consumed between the control and PR groups (Figure 4)

The length of the of the condylar process (A–B) was significantly lower in the PR group (control group 21 ± 2 mm vs PR group 17±3 mm; P < .05). No differences were observed in height of the lower alveolar process (C–D) (control group 10 ± 1 mm vs PR group 9 ± 1 mm; P > .05).

The condylar process histologic sections of the control animals exhibited normal bone trabeculae as regards number and thickness. The primary trabeculae had a cartilage core and mostly cuboidal osteoblasts on the surface (Figure 5).

Sections corresponding to the PR animals showed alterations in the subchondral bone exhibiting fewer, shorter, thinner, and wider bone trabeculae compared with those observed in the control sections, reflecting an imbalance of bone formation and resorption. In addition, none of the sections of the undernourished group presented bone trabeculae with osteoid on the surface. The few osteoblasts that were encountered failed to exhibit the typical cuboidal shape and were irregular. In addition, angiogenesis was clearly evident, and a large number of trabeculae had no lining cells on the surface (Figure 6).

The results of the morphometric study are shown in Table 2. The histomorphometric analysis of bone activity shows a statistically significant decrease in bone formation surfaces covered with active osteoblasts associated with an increase in bone resorption surfaces in the PR animals. Bone volume was significantly lower in this group.

In the present work the cephalometric study shows that dietary protein deficiency after weaning alters the length of the condylar process (A–B). However, the diet used did not affect the height of the lower alveolar process (C–D). The results presented herein also show that protein restriction alters subchondral bone remodeling in the mandibular condylar process; these alterations are characterized by a decrease in bone surfaces covered by active osteoblasts, indicating a severe decrease in bone formation.

The histomorphometric study of subchondral bone activity shows that the decrease in bone formation surfaces in subchondral bone of the condyle in the PR group was associated with an increase in erosive surfaces, whereas rest surfaces were not significantly different compared with those of the control group. The increase in erosive areas in bone tissue under conditions of protein undernutrition may be explained by the fact that osteoblasts play a major role in their onset.12 Thus, the increase observed in bone resorption surfaces may be due to damage to the osteoblasts, which in turn may release soluble mediators of bone resorption13 or lose contact with the bone surface, as was observed in the present study, resulting in the presence of erosive surfaces.14 Although in the present study a quantitative analysis of the osteoclast population was not performed, the presence of functional, well-differentiated osteoclasts in the mandibular condyle under conditions of severe protein undernutrition was evident. It could therefore be inferred that osteoclastogenesis regulation in PR animals may be affected by other factors besides osteoblast-osteoclast interaction mediated by RANK-RANKL (receptor activator of nuclear factor-Kappa B-RANK ligand)15 and TGFβ₁ (transforming growth factor beta 1) associated with an increase in RANK RNA messenger and protein.16 

On the other hand, it is well documented that severe protein undernutrition affects visceral proteins, particularly albumin and carrier proteins. Protein malnutrition also produces severe hypocalcemia.17,18 This hypocalcemia is produced, first, because the intestinal absorptive capacity is impaired, and second, because calcium is not transported due to the lack of albumin, which carries 40% of the calcium absorbed.17,18 In this context osteoclast number and bone resorption surfaces may be increased, probably via parathormone.19 

The histomorphometric study shows that isocaloric protein restriction negatively affects the osteogenic activity in the mandibular condylar process in rats, resulting in a longitudinal growth arrest, which is demonstrated in this work by the cephalometric study. However, the cellular and molecular mechanisms by which PR affects bone remodeling in the condylar process are still unclear.

These results are also in agreement with other findings that demonstrate that protein undernutrition affects bone formation and microstructure,20 as shown in studies of BMD and bone mineral content. This study shows a highly diet-dependent effect on mandibular and humeral BMD and on humeral bone mineral content. Another study21 shows that undernourished rats present proximal tibia and alveolar process BMD that is lower than that in controls. Moreover, undernutrition negatively affects bone volume, trabecular thickness, and trabecular number in the proximal tibia and the alveolar process. The aforementioned results show that protein restriction affects bone mineral content, such as calcium, phosphorus, magnesium, and potassium,22 which are associated with differentiation and activity of osteoblasts and osteoclasts.23 

Despite the different mechanical function, mandibular bone and axial or peripheral skeleton are negatively affected by nutritional deficiencies.21 Protein undernutrition has been associated with decreased bone growth in humans24 and animals.25,26 According to the literature and the results obtained in this study, it could be stated that a lack of condylar process development in malnourished children may be found. In this context, it is necessary to take into account that these changes could have an immediate impact on facial development.

During normal facial growth the mandibular symphysis moves down and forward with respect to the other facial structures along Rickett's facial axis.1,27 This movement is the result of vertical and sagittal growth vectors. However, the vertical growth vector has a crucial effect on the sagittal (anteroposterior) growth direction of the mandibular symphysis. This growth pattern results from the descent of the glenoid fossae and, essentially, from the increase in condyle length.27,28 The development of both components compensates simultaneously for the vertical growth of the upper jaw and the upper alveolar process as well as of the lower alveolar process.27 In this context the adequate development of the lower alveolar process, together with the lack of development of the condylar process in undernourished subjects, may alter the normal pattern of facial growth. This alteration could be characterized by the loss of vertical control and the loss of the spatial position of the mandible with respect to the base of the skull, a growth pattern characterized by a downward and backward displacement of the mandibular symphysis and a posterior rotation of the mandible. On the other hand, an increase in the angle to the base of the skull and a downward inclination of the occlusal plane that results in a dolichofacial growth pattern could also cause this type of nutritional disorder. Under these conditions in which there is a marked occlusal curve it would be plausible to posit that the lower incisors would drift upward and forward to compensate for the posterior rotation of the mandible. In addition, it is likely that the lack of condylar growth would result in premature contact given our finding that the lower alveolar process suffered no alterations; thus, the anterior open bite could be a frequent finding in this type of nutrition deficiency.29 

• Protein restriction inhibits bone formation and longitudinal growth in the mandibular condylar process.

• However, the height of the lower alveolar process is not affected by the diet used in this experiment.

• These results suggest that protein restriction can alter normal facial development; however, extrapolation of these results to humans should be done with caution.

This research work was supported by grants from the National University of Tucumán Research Council (26/J412).