Cellular and extracellular matrix aspects of the nasal cartilage in the fetus and early infant
Demographics Supplementary Figure is a flow chart of samples included. We had access to the corpses of 34 children under 2 years of age during the study period. Twenty-two samples could not be collected either because the family member responsible for the body was unwilling or unable to sign the informed consent in 6 cases or the pathologist on duty considered the foetus macerated. There were no samples with septum perforation. Thus, we collected the NSC from twelve individuals comprising 5, 4, and 3 from fetuses, newborns, and early infants, respectively. There were 7 (57.8%) and 5 (42.2%) male and female samples, respectively, with 0 day and 12 h median age (0–600 days range). Mean harvest time was 19.9 (± 11.4) hours, being 3 and 41 h the earliest and longest times, respectively. The ages of the infants, fetal gestational ages, and the causes of death of each individual are shown in supplementary Table . We believe that the causes of death did not impact the histopathology of the NSC given its avascular nature and the very short post-mortem time for removal. We should also remark that 16 fetal samples, judged to be macerated, were excluded, as shown in supplementary Fig. . Figure a illustrates an NSC specimen, with its digitalized version in Fig. b, showing the division in 9 sections, as described above. Careful attention was paid to correctly identify the anterior and inferior regions of the NSC, as well as the anterior/middle/posterior and superior/middle/inferior regions along the antero-posterior and supero-inferior axes, respectively (Fig. ). Cellular component Figure a illustrates the cell density of the NSC of the 5 fetus samples according to the anterior-posterior axis. Although there was a trend for a higher cell density in the anterior and middle regions isolated, as compared to the posterior region, it did not reach statistical significance. However, cell density in the combined anterior and middle regions was significantly higher ( p = 0.044), as compared to the posterior region, meaning that cell density was increased in the anterior two-thirds of the NSC of fetuses. Similarly, cell density in the superior and medium regions along the supero-inferior axis exhibited a trend for being higher as compared to the lower region, though not reaching statistical significance. However, contrary to the antero-posterior axis, there was no difference in the combined cell density of the supero-middle as compared to the inferior regions ( p = 0.2315) (Fig. b). In the newborn group, cell density was significantly higher in the anterior and middle regions along the antero-posterior axis as compared to the posterior region of the NSC. Cell density of the combined antero-middle regions of the newborn group was also significantly higher, as compared to the posterior region ( p = 0.0022) (Fig. c). Similar to the fetal group, there were no differences in the cell density of regions along the supero-inferior axis (Fig. d). In the infant group, although there was a trend for a reduced cell density in the posterior region as compared to the antero-middle region, it did not reach statistical significance ( p = 0.1972) (Fig. e). There was also no difference when comparing the various regions across the supero-inferior axis (Fig. f). 

Supplementary Figure is a flow chart of samples included. We had access to the corpses of 34 children under 2 years of age during the study period. Twenty-two samples could not be collected either because the family member responsible for the body was unwilling or unable to sign the informed consent in 6 cases or the pathologist on duty considered the foetus macerated. There were no samples with septum perforation. Thus, we collected the NSC from twelve individuals comprising 5, 4, and 3 from fetuses, newborns, and early infants, respectively. There were 7 (57.8%) and 5 (42.2%) male and female samples, respectively, with 0 day and 12 h median age (0–600 days range). Mean harvest time was 19.9 (± 11.4) hours, being 3 and 41 h the earliest and longest times, respectively. The ages of the infants, fetal gestational ages, and the causes of death of each individual are shown in supplementary Table . We believe that the causes of death did not impact the histopathology of the NSC given its avascular nature and the very short post-mortem time for removal. We should also remark that 16 fetal samples, judged to be macerated, were excluded, as shown in supplementary Fig. . Figure a illustrates an NSC specimen, with its digitalized version in Fig. b, showing the division in 9 sections, as described above. Careful attention was paid to correctly identify the anterior and inferior regions of the NSC, as well as the anterior/middle/posterior and superior/middle/inferior regions along the antero-posterior and supero-inferior axes, respectively (Fig. ). 

Figure a illustrates the cell density of the NSC of the 5 fetus samples according to the anterior-posterior axis. Although there was a trend for a higher cell density in the anterior and middle regions isolated, as compared to the posterior region, it did not reach statistical significance. However, cell density in the combined anterior and middle regions was significantly higher ( p = 0.044), as compared to the posterior region, meaning that cell density was increased in the anterior two-thirds of the NSC of fetuses. Similarly, cell density in the superior and medium regions along the supero-inferior axis exhibited a trend for being higher as compared to the lower region, though not reaching statistical significance. However, contrary to the antero-posterior axis, there was no difference in the combined cell density of the supero-middle as compared to the inferior regions ( p = 0.2315) (Fig. b). In the newborn group, cell density was significantly higher in the anterior and middle regions along the antero-posterior axis as compared to the posterior region of the NSC. Cell density of the combined antero-middle regions of the newborn group was also significantly higher, as compared to the posterior region ( p = 0.0022) (Fig. c). Similar to the fetal group, there were no differences in the cell density of regions along the supero-inferior axis (Fig. d). In the infant group, although there was a trend for a reduced cell density in the posterior region as compared to the antero-middle region, it did not reach statistical significance ( p = 0.1972) (Fig. e). There was also no difference when comparing the various regions across the supero-inferior axis (Fig. f). 

Isogenous groups were semiquantitatively counted across the different regions, as mentioned above. Figure a shows that the number of isogenous groups was significantly higher in the antero-middle regions as compared to the posterior region of the NSC, regardless of age among the evaluated groups. We did also find a higher number of isogenous groups in the antero-middle portion of the NSC in infants as compared to samples from fetuses or newborns. Isogenous groups were also more frequent in fetal samples as compared to those from newborn groups (Fig. b). Representative illustrations of the number of isogenous groups in the antero-middle and posterior regions, respectively, in a sample from the infant group, are shown in Fig. c,d. Sulfated glycosaminoglycan production A semiquantitative evaluation of the safranin O staining of the extracellular matrix is shown in Fig. . The mean percentage difference of safranin O staining in each individual in the anterior-middle (AM) as compared to the posterior (P) region along the antero-posterior axis is shown in Fig. a. Safranin O staining exhibited a trend to progressively increase in the AM region of the NSC thus suggesting that this region is comparably more metabolically active as the individual thrives. However, this increase did not reach statistical significance ( p > 0.05). The comparison of the intensity of safranin O staining in the supero-middle (SM) against the inferior region of the NSC regions was also similar across age groups (Fig. b). Representative images of safranin O-stained sections are shown in Fig. c,d. In the panoramic view (c) it can be seen that staining of the extracellular matrix appears homogenous, with minimal variation across different regions. However, at higher magnification, the presence of isogenous groups can be easily seen, together with areas of more intense staining, which as appear as darker shadows around the periphery of the cells, indicating active ( de novo ) synthesis of sGAG. 

A semiquantitative evaluation of the safranin O staining of the extracellular matrix is shown in Fig. . The mean percentage difference of safranin O staining in each individual in the anterior-middle (AM) as compared to the posterior (P) region along the antero-posterior axis is shown in Fig. a. Safranin O staining exhibited a trend to progressively increase in the AM region of the NSC thus suggesting that this region is comparably more metabolically active as the individual thrives. However, this increase did not reach statistical significance ( p > 0.05). The comparison of the intensity of safranin O staining in the supero-middle (SM) against the inferior region of the NSC regions was also similar across age groups (Fig. b). Representative images of safranin O-stained sections are shown in Fig. c,d. In the panoramic view (c) it can be seen that staining of the extracellular matrix appears homogenous, with minimal variation across different regions. However, at higher magnification, the presence of isogenous groups can be easily seen, together with areas of more intense staining, which as appear as darker shadows around the periphery of the cells, indicating active ( de novo ) synthesis of sGAG. 

This is the first study showing the distribution of chondrocytes as well as sGAG content across various regions of the NSC of fetuses, newborns, and early infants. The NSC of these age groups exhibited a rather uniform arrangement with a higher predominance of cells over the extracellular matrix. After quantification of cells in divided regions of the NSC, as described above, we were able to see that chondrocyte density actually varies across regions of the cartilage. Specifically, there was a significant decrease along the antero-posterior direction in the fetus and newborn samples. In the early infant group, a similar, though non-significant, trend toward lower cell density in the posterior region was also observed. On the other hand, chondrocyte density did not significantly vary along the supero-inferior axis. Previous studies on cartilage cellular distribution have focused mainly in joints, as it was shown in an excellent review from Buckwalter and Mankin . A prior study analysing five adult NSC specimens obtained postmortem showed that chondrocyte distribution varied in number and shape in this age group, being more numerous in the peripheral areas, assuming a flat shape, whereas in deeper zones the number of chondrocytes, which now appeared oval, was lower. However, that study did not provide a detailed cell count, which makes it impossible to compare with our data . Our finding that cell density was higher in the anterior two-thirds of the cartilage in these age groups suggests that this is an area of higher metabolic activity, particularly concerning cell division. This was reinforced when we counted the number of isogenous groups, which were clearly higher in the antero-middle as compared to the posterior region of the NSC. In addition, we also found that the number of isogenous groups increase on average 30.6% within the first year of age in the antero-middle portions of samples from early infants as compared to those from fetal or newborn individuals. Regarding sGAG production, we did not find significant differences across the various regions of the NSC. Actually, there was a rather uniform safranin O staining across all cartilage regions in the three age groups studied. However, there was a trend for increased safranin O staining as the individuals thrive, meaning an increase in the newborn and early infant groups, compared to fetuses. A previous study measured the proteoglycan content of the NSC in adults and elderly individuals using the dimethylmethylene blue assay. The proteoglycan content was relatively stable among age groups in that study with a significant reduction in the amount per mg of dried cartilage in individuals older than 60 years. Those authors also reported that cell content was similar across age groups, based on the total DNA content . We have to stress that we evaluated sGAG content in the entire NSC, whereas their analysis was restricted to the inferior region along the maxillary crest, harvested during surgery. Also, cell content and distribution in our study was based on cell counting, as opposed to DNA content in that study . Our data show that safranin O staining had a trend towards being less intense in the posterior regions of the NSC and staining did also appear to be increased as the individuals thrive in the first year of age. We also have to consider that we found a large predominance of cells as compared to the amount of extracellular matrix in the NSC across the age groups studied. We believe these characteristics have impacted the quantitation of sGAG production. Assuming safranin O staining as an indicator of metabolic activity, our results are in line with the increase in cellular activity suggested by the higher number of isogenous groups in the same areas that had a trend towards higher sGAG production. Whether these changes are sustained or will increase in older children needs to be addressed in future studies. Our data thus add to the bulk of the literature by suggesting that metabolic activity, at least in this very early age group, varies depending on the region of the cartilage. Specifically, we found an increase in the metabolic activity in the NSC in the first year of age, as compared to the prenatal period. Also, the anterior two-thirds of the NSC in this age group present a higher metabolic cellular activity represented by a higher presence of isogenous chondrocyte groups and sGAG production. We may speculate that growth factors and/or hormones acting shortly in the postnatal period impact the biological activity of nasal chondrocytes thereby influencing development of the NSC, an issue that needs further studies. There are claims that surgical interventions on the NSC should be performed after puberty, when full development is achieved. However, children and adolescents in need of repair to that structure suffer from respiratory, aesthetic as well as social issues , . Current knowledge suggests that the anterior portion of the nasal septum drives the development of the face. In keeping with this assumption, a previous work using stored embryos and fetuses from Asian individuals below 14 weeks’ gestation indicated that the development of the nasal septum is driven by its anterior portion . Our results add support to this, indicating that the anterior two-thirds of the NSC display higher cell density and metabolic activity. Hence, we may propose that surgical interventions in this structure should preserve the anterior two-thirds, aiming to decrease a potential impact in the development of the nasal septum. The reduced cell density and lower number of isogenous groups in the posterior region of the NSC, as compared to the anterior two-thirds, may present a lower risk to compromise development of the nasal septum when resected, even if performed in very young individuals. The higher proliferative rate in the anterior two-thirds of the NSC potentially means a higher capacity for healing. Furthermore, interventions in the posterior third could facilitate the mobilization and correct repositioning of the anterior two-thirds of the NSC, thus allowing proper contact with the underlying maxillary crista. Further studies are needed to determine the areas and the extent of tissue that can be safely resected without compromising development of the nasal septum in very young individuals. We did not find differences regarding cell density and sGAG production when comparing data from male and female samples. A previous study using computed tomography of the nasal septum showed that males had a significantly larger area, as compared to females. However, although they compared data from 4 to 99 years old individuals, there were only 4 in the 0–9 age group, which were all males. They also found that nasal septum increased until puberty remaining relatively stable regarding total area during adulthood, decreasing in the elderly, meaning those older than 70 years old. The authors also commented that the cartilage area of the septum rapidly develops in the postnatal period, being relatively stable after two years of age . Contrary to our study, the authors did not include individuals under 2 years old thus not allowing direct comparison. It might well be that gender differences, as expected, would not be apparent in such young individuals, which probably explain our similar results regardless of gender. Another study that focused on components of the NSC obtained from the inferior portion of the septum of adults also found no significant differences regarding water, cell density or sGAG content when comparing male and female samples. However, those authors reported a significant reduction of cell density and sGAG with aging . There is also a previous study reporting higher cell density in the anterior portion of the NSC of adults, which aligns with our findings in very young individuals . Taken together, our data reveal that metabolic activity in the NSC of individuals under two years of age is higher in the anterior two-thirds of this structure. Children in their first year of age exhibit an increased metabolic activity in the NSC as compared to the prenatal period. Further studies on the cellular composition and biological activity in individuals over 2 years old until late adolescence are needed, given that such data may improve safety when performing interventions in the developing nasal septum. There is also a need to clarify whether gender issues impact the metabolism of the nasal cartilage in children over two years old as well as during adolescence. In conclusion, the anterior two-thirds of the postnatal NSC exhibits differences regarding cell density. The proliferation of chondrocytes in the NSC in this age group varies across regions, being higher in the anterior two-thirds. Children in their first year of age exhibit a prominent metabolic activity in the NSC. 

The NSC from fetuses and infants up to 2 years old was collected post-mortem at the Serviço de Verificação de Óbito (SVO) in Fortaleza-CE, Brazil, between June 2022 and April 2023. SVO is the official public service for death verification that covers the state of Ceará, Brazil, which has 8,791,688 inhabitants, according to the last official Brazilian census ( www.https://cidades.ibge.gov.br/brasil/ce/panorama ). Briefly, after a columellar-septal transfixing incision, the NSC was completely removed with identification of the anterior and inferior margins (Supplementary Fig. a) followed by fixation in 10% buffered formalin solution for further processing. The reconstruction of the nasal pyramids was done using cardboard and internal suture in order not to leave external deformities or signs of incisions or suture. All procedures were conducted only after an adult individual legally responsible for the cadaver signed a written informed consent presented by a board certified otorhynolaryngologist (RBBJ), who performed the surgical procedures. The protocol was approved by 2 review boards that follow the orientation of the Brazilian Committee on Ethics in Human Research (CAAE Protocol numbers: 2.332.307 and 2.820.461 ). Signed informed consent was obtained from a parent and/or legal guardian before any collection procedure. All procedures were performed in accordance with relevant guidelines and regulations. After paraffin embedding, the NSC was serially sectioned at 5 μm sections, with a maximum of 5 sections prepared from each sample, followed by routine hematoxylin-eosin (HE) and safranin O staining for analysis under optical microscopy (Olympus CX31, Biolab Equipamentos Brasil, São Paulo, SP, Brazil) by two blinded observers (GFMS, FT). Slides were digitalized and divided into 9 sections following the antero-posterior and supero-inferior axes, as illustrated in Supplementary Fig. b. Cell density was expressed as the number of cells counted in 5 high-power fields (hpf; original x400) and expressed as the mean number of cells in each of the 9 sections. Cellular proliferation, used as a surrogate for metabolic cellular activity, was assessed by counting the number of isogenous groups in 50 hpf within the divided areas of the NSC. These groups are clusters of cells that undergone mitosis, differentiating into chondrocytes. sGAG production was evaluated with semiquantitative Safranin O staining, as described previously , using ImageJ™ software version 1.45k (Wayne Rasband, NIH, USA). Special care was taken regarding safranin O staining, with samples processed simultaneously by the same technician, following a specific protocol, in order to minimize staining bias. Statistical analysis Data are described as mean ± SD, medians (range) or percentages, as appropriate. Assessment of normality of the safranin O staining data was done using the D’Agostino-Pearson Omnibus test. Differences between means were compared using unpaired Student’s “t” test or one-way ANOVA followed by Tukey’s test, as appropriate; P < 0.05 was considered significant. Analyzes were performed using GraphPad Prism 10.0.0™, San Diego, CA, USA. 

Data are described as mean ± SD, medians (range) or percentages, as appropriate. Assessment of normality of the safranin O staining data was done using the D’Agostino-Pearson Omnibus test. Differences between means were compared using unpaired Student’s “t” test or one-way ANOVA followed by Tukey’s test, as appropriate; P < 0.05 was considered significant. Analyzes were performed using GraphPad Prism 10.0.0™, San Diego, CA, USA. 

Below is the link to the electronic supplementary material. Supplementary Material 1 Supplementary Material 2 

