Effects of 4-Hexylresorcinol on Craniofacial Growth in Rats.

4-Hexylresorcinol (4HR) has been used as a food additive, however, it has been recently demonstrated as a Class I histone deacetylase inhibitor (HDACi). Unlike other HDACi, 4HR can be taken through foods. Unfortunately, some HDACi have an influence on craniofacial growth, therefore, the purpose of this study was to evaluate the effects of 4HR on craniofacial growth. Saos-2 cells (osteoblast-like cells) were used for the evaluation of HDACi and its associated activities after 4HR administration. For the evaluation of craniofacial growth, 12.8 mg/kg of 4HR was administered weekly to 4 week old rats (male: 10, female: 10) for 12 weeks. Ten rats were used for untreated control (males: 5, females: 5). Body weight was recorded every week. Serum and head samples were collected at 12 weeks after initial administration. Craniofacial growth was evaluated by micro-computerized tomography. Serum was used for ELISA (testosterone and estrogen) and immunoprecipitation high-performance liquid chromatography (IP-HPLC). The administration of 4HR (1-100 µM) showed significant HDACi activity (p < 0.05). Body weight was significantly different in male rats (p < 0.05), and mandibular size was significantly smaller in 4HR-treated male rats with reduced testosterone levels. However, the mandibular size was significantly higher in 4HR treated female rats with increased growth hormone levels. In conclusion, 4HR had HDACi activity in Saos-2 cells. The administration of 4HR on growing rats showed different responses in body weight and mandibular size between sexes.

Dipyridamole-loaded 3D-printed bioceramic scaffolds stimulate pediatric bone regeneration in vivo without disruption of craniofacial growth through facial maturity.

This study investigates a comprehensive model of bone regeneration capacity of dypiridamole-loaded 3D-printed bioceramic (DIPY-3DPBC) scaffolds composed of 100% beta-tricalcium phosphate (ß -TCP) in an immature rabbit model through the time of facial maturity. The efficacy of this construct was compared to autologous bone graft, the clinical standard of care in pediatric craniofacial reconstruction, with attention paid to volume of regenerated bone by 3D reconstruction, histologic and mechanical properties of regenerated bone, and long-term safety regarding potential craniofacial growth restriction. Additionally, long-term degradation of scaffold constructs was evaluated. At 24 weeks in vivo, DIPY-3DPBC scaffolds demonstrated volumetrically significant osteogenic regeneration of calvarial and alveolar defects comparable to autogenous bone graft with favorable biodegradation of the bioactive ceramic component in vivo. Characterization of regenerated bone reveals osteogenesis of organized, vascularized bone with histologic and mechanical characteristics comparable to native bone. Radiographic and histologic analyses were consistent with patent craniofacial sutures. Lastly, through application of 3D morphometric facial surface analysis, our results support that DIPY-3DPBC scaffolds do not cause premature closure of sutures and preserve normal craniofacial growth. Based on this novel evaluation model, this DIPY-3DPBC scaffold strategy is a promising candidate as a safe, efficacious pediatric bone tissue engineering strategy.

Adaptive correction of craniofacial defects in pre-metamorphic Xenopus laevis tadpoles involves thyroid hormone-independent tissue remodeling.

Although it is well established that some organisms can regenerate lost structures, the ability to remodel existing malformed structures has been less well studied. Therefore, in this study we examined the ability of pre-metamorphic Xenopus laevis tadpoles to self-correct malformed craniofacial tissues. We found that tadpoles can adaptively improve and normalize abnormal craniofacial morphology caused by numerous developmental perturbations. We then investigated the tissue-level and molecular mechanisms that mediate the self-correction of craniofacial defects in pre-metamorphic X. laevis tadpoles. Our studies revealed that this adaptive response involves morphological changes and the remodeling of cartilage tissue, prior to metamorphosis. RT-qPCR and RNA-seq analysis of gene expression suggests a thyroid hormone-independent endocrine signaling pathway as the potential mechanism responsible for triggering the adaptive and corrective remodeling response in these larvae that involves mmp1 and mmp13 upregulation. Thus, investigating how malformed craniofacial tissues are naturally corrected in X. laevis tadpoles has provided valuable insights into the maintenance and manipulation of craniofacial morphology in a vertebrate system. These insights may help in the development of novel therapies for developmental craniofacial anomalies in humans.

Adiponectin promotes human jaw bone marrow mesenchymal stem cell chemotaxis via CXCL1 and CXCL8.

Adiponectin (APN) is known to promote the osteogenic differentiation of human jaw bone marrow mesenchymal stem cells (h-JBMMSCs). However, the underlying mechanism has not been fully elucidated. Previously, we showed that APN could promote h-JBMMSC osteogenesis via APPL1-p38 by up-regulating osteogenesis-related genes. Here, we aimed to determine whether APN could promote h-JBMMSC chemotaxis through CXCL1/CXCL8. The CCK-8, wound healing and transwell assays were used to evaluate the proliferation, migration and chemotaxis of h-JBMMSCs with or without APN treatment. Chemotaxis-related genes were screened using RNA-seq, and the results were validated using real-time PCR and ELISA. We also performed Western blot using the AMPK inhibitor, WZ4003, and the p38 MAPK inhibitor, SB203580, to identify the signalling pathway involved. We found that APN could promote h-JBMMSC chemotaxis in the co-culture transwell system. CXCL1 and CXCL8 were screened and confirmed as the up-regulated target genes. The APN-induced CXCL1/8 up-regulation to promote chemotaxis could be blocked by CXCR2 inhibitor SB225002. Western blot revealed that the phosphorylation of AMPK and p38 MAPK increased in a time-dependent manner with APN treatment. Additionally, WZ4003 and SB203580 could suppress the APN-induced overexpression of CXCL1 and CXCL8. The results of the transwell chemotaxis assay also supported the above results. Our data suggest that APN can promote h-JBMMSC chemotaxis by up-regulating CXCL1 and CXCL8.

Growth hormone positive effects on craniofacial complex in Turner syndrome.

OBJECTIVE: Turner syndrome occurs in phenotypic females with complete or partial absence of X chromosome. The leading symptom is short stature, while numerous but mild stigmata manifest in the craniofacial region. These patients are commonly treated with growth hormone to improve their final height. The aim of this study was to assess the influence of long-term growth hormone therapy on craniofacial morphology in Turner syndrome patients. DESIGN: In this cross-sectional study cephalometric analysis was performed on 13 lateral cephalograms of patients with 45,X karyotype and the average age of 17.3 years, who have received growth hormone for at least two years. The control group consisted of 13 Turner syndrome patients naive to growth hormone treatment, matched to study group by age and karyotype. Sixteen linear and angular measurements were obtained from standard lateral cephalograms. Standard deviation scores were calculated in order to evaluate influence of growth hormone therapy on craniofacial components. RESULTS: In Turner syndrome patients treated with growth hormone most of linear measurements were significantly larger compared to untreated patients. Growth hormone therapy mainly influenced posterior face height, mandibular ramus height, total mandibular length, anterior face height and maxillary length. While the increase in linear measurements was evident, angular measurements and facial height ratio did not show statistically significant difference. Acromegalic features were not found. CONCLUSIONS: Long-term growth hormone therapy has positive influence on craniofacial development in Turner syndrome patients, with the greatest impact on posterior facial height and mandibular ramus. However, it could not compensate X chromosome deficiency and normalize craniofacial features.

Adiponectin Promotes Human Jaw Bone Marrow Stem Cell Osteogenesis.

Human jaw bone marrow mesenchymal stem cells (h-JBMMSCs) are multipotent progenitor cells with osteogenic differentiation potential. The relationship between adiponectin (APN) and the metabolism of h-JBMMSCs has not been fully elucidated, and the underlying mechanism remains unclear. The aim of the study was to investigate the effect and mechanism of APN on h-JBMMSC metabolism. h-JBMMSCs were obtained from the primary culture of human jaw bones and treated with or without APN (1 µg/mL). Osteogenesis-related gene expression was evaluated by real-time polymerase chain reaction (PCR), alkaline phosphatase (ALP) activity assay, and enzyme-linked immunosorbent assay (ELISA). To further investigate the signaling pathway, mechanistic studies were performed using Western blotting, immunofluorescence, lentiviral transduction, and SB202190 (a specific p38 inhibitor). Alizarin Red staining showed that APN promoted h-JBMMSC osteogenesis. Real-time PCR, ALP assay, and ELISA showed that ALP, osteocalcin (OCN), osteopontin, and integrin-binding sialoprotein were up-regulated in APN-treated cells compared to untreated controls. Immunofluorescence revealed that adaptor protein containing a pleckstrin homology domain, phosphotyrosine domain, and leucine zipper motif (APPL1) translocated from the nucleus to the cytoplasm with APN treatment. Additionally, the phosphorylation of p38 mitogen-activated protein kinase (MAPK) increased over time with APN treatment. Moreover, knockdown of APPL1 or p38 MAPK inhibition blocked the expression of APN-induced calcification-related genes including ALP, Runt-related transcription factor 2 (RUNX2), and OCN. Furthermore, Alizarin Red staining of calcium nodes was not increased by the knockdown of APPL1 or p38 inhibition. Our data suggest that this regulation is mediated through the APPL1-p38 MAPK signaling pathway. These findings collectively provide evidence that APN induces the osteogenesis of h-JBMMSCs through APPL1-mediated p38 MAPK activation.

The chick embryo as a model for the effects of prenatal exposure to alcohol on craniofacial development.

Prenatal exposure to ethanol results in fetal alcohol spectrum disorder (FASD), a syndrome characterised by a broad range of clinical manifestations including craniofacial dysmorphologies and neurological defects. The characterisation of the mechanisms by which ethanol exerts its teratogenic effects is difficult due to the pleiotropic nature of its actions. Different experimental model systems have been employed to investigate the aetiology of FASD. Here, I will review studies using these different model organisms that have helped to elucidate how ethanol causes the craniofacial abnormalities characteristic of FASD. In these studies, ethanol was found to impair the prechordal plate-an important embryonic signalling centre-during gastrulation and to negatively affect the induction, migration and survival of the neural crest, a cell population that generates the cartilage and most of the bones of the skull. At the cellular level, ethanol appears to inhibit Sonic hedgehog signalling, alter levels of retionoic acid activity, trigger a Ca(2+)-CamKII-dependent pathway that antagonises WNT signalling, affect cytoskeletal dynamics and increase oxidative stress. Embryos of the domestic chick Gallus gallus domesticus have played a central role in developing a working model for the effects of ethanol on craniofacial development because they are easily accessible and because key steps in craniofacial development are particularly well established in the avian embryo. I will finish this review by highlighting some potential future avenues of fetal alcohol research.

The effects of denervation and formoterol administration on facial growth.

OBJECTIVE: To identify and demonstrate possible alterations of skeletal structures which might follow either unilateral surgical denervation of the masseter muscle, unilateral intramuscular injection of formoterol directly into the masseter muscle, or intramuscular formoterol injection after surgical denervation. MATERIALS AND METHODS: Male Sprague Dawley rats (N = 16; four weeks of age) were prepared as four groups: 1. surgical sham + saline injection into the masseter muscle (sham); 2. surgical denervation of the masseter muscle only (den.); 3. surgical denervation of the masseter muscle plus intramuscular formoterol injection into the affected muscle (den.+form.); 4. intramuscular formoterol injection into the masseter muscle only (form.). The specimens were submitted for CT examination, the skulls and hemimandibles were photographed and measurements of craniofacial bones were made. RESULTS: In this relatively small sample, comparisons between non-experimental and experimental sides revealed differences, both within the groups and for the same measurements between groups, with the den. and den.+form. groups showing the most change. Relative increases in the gonial angle shown in these groups occurred bilaterally, with the change on the experimental side always greater in magnitude than the change on the contralateral side. CONCLUSIONS: Surgical denervation of the masseter muscle leads to an alteration in the size and shape of the skeletal structures close to the zygoma and the mandible. The intramuscular injection of formoterol into denervated masseter muscle seems to limit this skeletal alteration after surgical denervation.

Late effects of orbital enucleation and radiation on maxillofacial prosthetic rehabilitation: A clinical report.

The retinoblastoma is one of the most common tumors of the eye diagnosed in childhood. The treatment for patients with retinoblastoma includes surgical removal of the lesion along with adjunctive chemotherapy and radiotherapy. If the tumor is controlled, these treatments can have secondary adverse effects pertaining to the growth and development of orofacial structures in young patients. This clinical report describes the prosthetic rehabilitation of a patient who underwent enucleation followed by radiation therapy and adjunctive chemotherapy to treat the primary diagnosis of retinoblastoma. This therapy resulted in a combination of dental and facial growth and developmental abnormalities.

Growth hormone therapy and craniofacial bones: a comprehensive review.

Growth hormone (GH) has significant effects on linear bone growth, bone mass and bone metabolism. The primary role of GH supplementation in children with GH deficiency, those born small for gestational age or with other types of disorders in somatic development is to increase linear growth. However, GH therapy seems to elicit varying responses in the craniofacial region. Whereas the effects of GH administration on somatic development are well documented, comparatively little is known of its effects on the craniofacial region. The purpose of this review was to search the literature and compile results from both animal and human studies related to the impact of GH on craniofacial growth.

Ontogenetic changes of craniofacial complex in Turner syndrome patients treated with growth hormone.

OBJECTIVE: The present study assessed changes of craniofacial complex in Turner syndrome (TS) patients treated with growth hormone (GH) during development. The objective was to examine the growth rate and pattern of craniofacial structures and to establish effects of GH on craniofacial development. MATERIALS AND METHODS: The study population consisted of 15 TS patients treated with GH aged 5-18.5 years (13.3 ± 4.4) and corresponding control group of 45 females aged 6.8-18.7 (11.4 ± 2.6). According to the stage of cervical vertebral maturation, subjects were categorized into pre-growth (5 TS and 15 controls) and growth (10 TS and 30 controls) subgroups. The cephalometric analysis comprised angular and linear variables, measured on lateral cephalometric radiographs. RESULTS: The mandibular corpus/anterior cranial base ratio increased significantly only in controls during development. In growth period, ramus/corpus ratio was significantly larger in TS group. SNA and SNB angles were significantly smaller in TS growth subgroup compared to corresponding controls. Among other variables, no statistically significant differences were revealed. CONCLUSIONS: In TS patients treated with GH, growth capacities of cranial base and maxilla are adequate which can be attributed to GH treatment. Shape of mandible is altered due to decreased growth of corpus and overdeveloped ramus. Both maxillary and mandibular retrognathism are becoming more expressed during development. CLINICAL RELEVANCE: Favorable influence of GH on craniofacial complex growth rate and altered growth pattern revealed in this study should be considered while planning both orthodontic treatment and retention.

Disturbances in dental development and craniofacial growth in children treated with hematopoietic stem cell transplantation.

OBJECTIVES: To investigate the correlation between age, degree of disturbances in dental development, and vertical growth of the face in children treated with hematopoietic stem cell transplantation (HSCT). PATIENTS: 39 long-term survivors of HSCT performed in childhood and transplanted before the age of 12, at a mean age of 6.8±3.3 years. METHODS: Panoramic and cephalometric radiographs were taken at a mean age of 16.2 years. For each patient two age- and sex-matched healthy controls were included. The area of three mandibular teeth was measured and a cephalometric analysis was performed. RESULTS: The mean area of the mandibular central incisor, first and second molar was significantly smaller in the HSCT group, and the vertical growth of the face was significantly reduced, especially in the lower third, compared to healthy controls. A statistically significant correlation between age at HSCT, degree of disturbances in dental development, and vertical growth of the face was found. Children subjected to pre-HSCT chemotherapy protocols had significantly more growth reduction in vertical craniofacial variables compared to children without pre-HSCT chemotherapy. Conditioning regimens including busulfan or total body irradiation had similar deleterious effects on tooth area reduction and craniofacial parameters. CONCLUSIONS: The younger the child is at HSCT, the greater the impairment in dental and vertical facial development. This supports the suggestion that the reduction in lower facial height found in SCT children mainly is a result of impaired dental development and that young age is a risk factor for more severe disturbances.

Effects of phenytoin on Satb2 and Hoxa2 gene expressions in mouse embryonic craniofacial tissue.

Cleft lip and cleft palate are common congenital craniofacial birth defects in humans. Phenytoin (PHT) is a risk factor of cleft palate formation; however, the molecular mechanisms by which phenytoin exerts its teratogenic effects resulting in cleft palate remain unknown. The Satb2 gene mutation is associated with cleft palate. Satb2-deficient mice exhibit cleft palate deformity and an up-regulation of Hoxa2 in the fronto-nasal region. In this study, phenytoin was administered intraperitoneally to pregnant C57BL/6 mice on the 10th day of gestation. Real-time PCR results showed that the expressions of Satb2 and Hoxa2 in craniofacial tissues of mouse embryos were obviously different at different time points. The Satb2 gene was down-regulated and the Hoxa2 gene was up-regulated in phenytoin-treated mouse embryonic craniofacial tissue. We conclude that phenytoin may regulate the expression of these two genes in C57BL/6 mice and it may also be involved in the formation of cleft palate.

Delayed osteoprogenitor differentiation in cleft-palate models.

Failure of palatal shelf fusion results in cleft palate (CP) and may lead to malformation of palatal bones and undergrowth of the maxilla. It is not known whether defects in bone formation may contribute to this phenotype. We tested the hypothesis that impaired fusion of developing palatal shelves affects palatal bone development using palate organotypic cultures. Using two different approaches, we show that induction of cleft results in increased expression of pre-osteoblast and early osteoblast markers, Twist1, Snai1 and Runx2, and decreased expression of more mature markers of bone differentiation, collagen-1 and osteopontin, indicating delayed osteoblast differentiation in CPs. This, together with the increase in immature osteoblasts and proliferation observed in non-fused palatal shelves, suggests that palatal osteoblast differentiation is at least partly modulated by shelf fusion. Delayed osteoblast differentiation may therefore contribute to defects in gross morphology and function of the maxilla in CP patients.

The effect of the Botulinum toxin-A on craniofacial development: an experimental study.

In this study, we developed a novel experimental model to evaluate muscular action on bone formation and remodeling by the help of Botulinum toxin-A (BTX). Forty-nine 15-day-old male Wistar rats were put into 4 groups randomly. Group 1 was the control group. BTX 0.4 IU (0.05 mL) was injected into the right masseter muscle in group 2 and into right temporalis muscle in group 3. The same volume of sterile saline was given into the both above-mentioned muscles in group 4. At the end of the fourth month, all animals were killed. Histology and weight of the masseter and temporalis muscles were studied. Thirty different osteometric measurements were also taken from skulls. Significant atrophy in BTX injected muscles was observed in groups 2 and 3. In group 4 (saline injection), only few osteometric measurements were significantly reduced, indicating the effect of the injection itself. Both groups 2 and 3 have apparent decrease in nasal bone, premaxilla, maxilla, and zygomatic dimensions on the injected side. When masseter group was compared with control and saline groups, no significant difference was found in skull base dimensions and mandibular length. In contrast, temporal group has also shown significant decrease in skull base dimensions. Our conclusions are as follows: (1) With this model, it is possible to study muscular action on bone formation and modeling without any surgical intervention, that is, by avoiding surgical artifacts, such as scar and contracture; (2) denervation of the skeletal muscles with BTX during the growing phase does effect bone development in a negative way; (3) pediatric use of the BTX deserves reevaluation under the light of these findings.

Effects on craniofacial growth and development of unilateral botulinum neurotoxin injection into the masseter muscle.

INTRODUCTION: The effects of botulinum neurotoxin type A (BoNT/A) on masseter muscles, when injected for cosmetic purposes (volumetric reduction) or treatment of excessive muscle activity (bruxism), have been investigated. However, the full anatomic effects of treatment are not known, particularly with respect to the mandible and relevant anthropometric measurements. The intent of this study was to use unilaterial BoNT/A injections to induce localized masseter atrophy and paresis and then to measure the effects of muscle influence on craniofacial growth and development. METHODS: Growing male Wistar rats, 30 days old, were studied. The experimental group consisted of 8 rats. One side of the masseter muscle was injected with BoNT/A and the other side of the masseter muscle was injected with saline. The side with BoNT/A belonged to 1 group and the side with saline was the sham group. Three rats without injections was the control. After 45 days, the masseter muscles were dissected and weighed. Dry skulls were prepared, and anthropometric measurements determined. RESULTS: One-way ANOVA showed that the animals maintained their weight in both groups; however, the muscles injected with BoNT/A were smaller than the sham or control muscles. Anthropometric measurements of the bony structures attached to the masseter muscle showed a significant treatment effect. CONCLUSIONS: After localized masseter muscle atrophy induced by BoNT/A injection, alterations of craniofacial bone growth and development were seen. The results agree with the functional matrix theory that soft tissues regulate bone growth.

Analysis of craniofacial and extremity growth in patients with growth hormone deficiency during growth hormone therapy.

BACKGROUND/AIMS: There are many controversies regarding side effects on craniofacial and extremity growth due to growth hormone (GH) treatment. Our aim was to estimate GH action on craniofacial development and extremity growth in GH-deficient patients. METHODS: Twenty patients with GH deficiency with a chronological age ranging from 4.6 to 24.3 years (bone age from 1.5 to 13 years) were divided in 2 groups: group 1 (n = 6), naive to GH treatment, and group 2 (n = 14), ongoing GH treatment for 2-11 years. GH doses (0.1-0.15 U/kg/day) were adjusted to maintain insulin-like growth factor 1 and insulin-like growth factor binding protein 3 levels within the normal range. Anthropometric measurements, cephalometric analyses and facial photographs to verify profile and harmony were performed annually for at least 3 years. RESULTS: Two patients with a disharmonious profile due to mandibular growth attained harmony, and none of them developed facial disharmony. Increased hand or foot size (>P97) was observed in 2 female patients and in 4 patients (1 female), respectively, both not correlated with GH treatment duration and increased levels of insulin-like growth factor 1. CONCLUSIONS: GH treatment with standard doses in GH-deficient patients can improve the facial profile in retrognathic patients and does not lead to facial disharmony although extremity growth, mainly involving the feet, can occur.

Timing effects of growth hormone supplementation on rat craniofacial growth.

Growth hormone (GH) supplementation has become a popular treatment approach for GH normal children with short stature. To investigate how the timing of GH supplementation affects the growth of the craniofacial region, three groups of GH-normal, 28-day-old female Wistar rats were examined over 4 weeks: the early group (n = 10) received two daily injections of rhGH (2 mg/kg/day) from days 1 to 28, the late group (n = 10) received two daily saline injections from days 1 to 14 (Phase I) followed by two daily injections of rhGH from days 14 to 28 (Phase II), and the control group (n = 12) received two daily saline injections from days 1 to 28. Lateral cephalometric, forelimb and hindlimb radiographs obtained weekly were scanned, standardized points digitized, and distances were measured using the Viewbox software. Growth curves between groups were compared using multilevel iterative generalized least squares curve fitting procedures. Supplementation during Phase I in the early group produced significant treatment effects in cranial and cranial base, midface, posterior corpus, and limb lengths which varied inversely with relative maturity (percentage growth completed at the start of the study). During Phase II, GH supplementation in the early and late groups showed treatment effects as above and additional viscerocranial and mandibular measurements, but these effects were unrelated to the relative maturity of the variables. These latter results are at variance with earlier findings in GH-deficient rats, raising the possibility that that GH-normal rats may not respond to GH supplementation in a similar fashion to GH-deficient rats.

Growth effects of botulinum toxin type A injected into masseter muscle on a developing rat mandible.

OBJECTIVES: Botulinum toxin type A (BTX-A) reduces the muscular contractions by temporarily inhibiting the release of acetylcholine at the neuromuscular junction. The purpose of this study was to investigate the effects of the BTX-A injected into the masseter muscle of a developing rat mandible. MATERIALS AND METHODS: Four-week-old male (no. 80) Sprague-Dawley rats were divided into four groups: control group, saline group, BTX-A group and baseline control group. Rats of baseline group were sacrificed at 0 day to provide baseline values of the mandibular measurements. The masseter muscle of rats in the saline and the BTX-A group were administered with saline and BTX-A solutions respectively. Experimental animals were sacrificed after 4 weeks. RESULTS: The BTX-A group demonstrated smaller mandibular dimension compared with the other groups (P < 0.05). Their condylar cartilages showed increased apoptosis at the proliferation stage of the reserve zone and masseter muscle fibers demonstrated atrophic changes. CONCLUSIONS: The result demonstrated BTX-A influence on inhibitory action of the developing mandible because of apoptosis at the proliferation stage of the reserve zone of the condylar cartilage in developing rat mandible.