Decreased Tiam1-mediated Rac1 activation is responsible for impaired directional persistence of chondrocyte migration in microtia.

The human auricle has a complex structure, and microtia is a congenital malformation characterized by decreased size and loss of elaborate structure in the affected ear with a high incidence. Our previous studies suggest that inadequate cell migration is the primary cytological basis for the pathogenesis of microtia, however, the underlying mechanism is unclear. Here, we further demonstrate that microtia chondrocytes show a decreased directional persistence during cell migration. Directional persistence can define a leading edge associated with oriented movement, and any mistakes would affect cell function and tissue morphology. By the screening of motility-related genes and subsequent confirmations, active Rac1 (Rac1-GTP) is identified to be critical for the impaired directional persistence of microtia chondrocytes migration. Moreover, Rho guanine nucleotide exchange factors (GEFs) and Rho GTPase-activating proteins (GAPs) are detected, and overexpression of Tiam1 significantly upregulates the level of Rac1-GTP and improves directional migration in microtia chondrocytes. Consistently, decreased expression patterns of Tiam1 and active Rac1 are found in microtia mouse models, Bmp5se/J and Prkralear-3J/GrsrJ. Collectively, our results provide new insights into microtia development and therapeutic strategies of tissue engineering for microtia patients.

Microtia patients: Auricular chondrocyte ECM is promoted by CGF through IGF-1 activation of the IGF-1R/PI3K/AKT pathway.

OBJECTIVE: To explore the effectiveness of the insulin-like growth factor 1 receptor (IGF-1R)/PI3K/AKT pathway in promoting the synthesis of the auricular chondrocyte extracellular matrix (ECM) using concentrated growth factor (CGF). METHODS: Chondrocytes isolated from the remnant auricular cartilage of microtia patients were randomly divided into different experimental and control groups, then stimulated with a reagent. IGF-1 released by CGF was quantified by enzyme-linked immunosorbent assay. Glycosaminoglycan (GAG), proteoglycan, and type II collagen (COLAII) were examined by histological and immunohistological analysis. Expression levels of IGF-1R, pIGF-1R, PI3K, pPI3K, AKT, pAKT, COLAII, and Aggrecan were detected by western blot analysis technique and gene expression was tested by real-time polymerase chain reaction. RESULTS: CGF significantly stimulated the synthesis of COLAII and Aggrecan and increased the phosphorylation levels of IGF-1R, PI3K, and AKT. Small interfering RNA IGF-1 blocked ECM synthesis, COLAII and Aggrecan gene expression, and IGF-1R/PI3K/AKT activation. Inhibitor AG1024 and LY294002 significantly inhibited ECM synthesis and the phosphorylation of IGF-1R, PI3K, and AKT. CONCLUSION: CGF-released IGF-1 stimulates the synthesis of the auricular chondrocyte ECM via the IGF-1R/PI3K/AKT signaling pathway.

Combining Virtual Surgical Planning, Intraoperative Navigation, and 3-Dimensional Printing in Prosthetic-Based Bilateral Microtia Reconstruction.

Reconstructing auricular deformities for bilateral microtia is a demanding challenge especially after failed autologous reconstruction. This case report presents a novel application of virtual surgical planning, computer-assisted design, and intraoperative surgical navigation to preplan and execute placement of custom-tailored silicone auricular prostheses and titanium osseointegrated implants for a bone-anchored hearing aid system in a patient with Treacher Collins syndrome in whom autologous reconstruction had previously failed. Through a collaborative approach between the reconstructive surgeon and anaplastologist, the implementation of advanced digital technologies may offer a superior esthetic and functional outcome to patients with previously failed reconstruction.

Characterization of pediatric microtia cartilage: a reservoir of chondrocytes for auricular reconstruction using tissue engineering strategies.

The external ear is composed of elastic cartilage. Microtia is a congenital malformation of the external ear that involves a small reduction in size or a complete absence. The aim of tissue engineering is to regenerate tissues and organs clinically implantable based on the utilization of cells and biomaterials. Remnants from microtia represent a source of cells for auricular reconstruction using tissue engineering. To examine the macromolecular architecture of microtia cartilage and behavior of chondrocytes, in order to enrich the knowledge of this type of cartilage as a cell reservoir. Auricular cartilage remnants were obtained from pediatric patients with microtia undergoing reconstructive procedures. Extracellular matrix composition was characterized using immunofluorescence and histological staining methods. Chondrocytes were isolated and expanded in vitro using a mechanical-enzymatic protocol. Chondrocyte phenotype was analyzed using qualitative PCR. Microtia cartilage preserves structural organization similar to healthy elastic cartilage. Extracellular matrix is composed of typical cartilage proteins such as type II collagen, elastin and proteoglycans. Chondrocytes displayed morphological features similar to chondrocytes derived from healthy cartilage, expressing SOX9, COL2 and ELN, thus preserving chondral phenotype. Cell viability was 94.6 % during in vitro expansion. Elastic cartilage from microtia has similar characteristics, both architectural and biochemical to healthy cartilage. We confirmed the suitability of microtia remnant as a reservoir of chondrocytes with potential to be expanded in vitro, maintaining phenotypical features and viability. Microtia remnants are an accessible source of autologous cells for auricular reconstruction using tissue engineering strategies.

Sociodemographic and hispanic acculturation factors and isolated anotia/microtia.

BACKGROUND: It has been observed in several studies that infants with anotia/microtia are more common among Hispanics compared with other racial/ethnic groups. We examined the association between selected Hispanic ethnicity and acculturation factors and anotia/microtia in the National Birth Defects Prevention Study. METHODS: We examined data from mothers of 351 infants with isolated anotia/microtia and 8435 unaffected infants from the National Birth Defects Prevention Study with an expected delivery date from 1997 to 2007. Sociodemographic, maternal, and acculturation factors (e.g., age, maternal education, household income, body mass index, gestational diabetes, folic acid, smoking, alcohol intake, study center, parental birthplace, and years lived in the United States, maternal language) were assessed as overall risk factors and also as risk factors among subgroups of Hispanics (United States- and foreign-born) versus non-Hispanic whites. RESULTS: Compared with non-Hispanic whites, both United States- and foreign-born Hispanic mothers demonstrated substantially higher odds of delivering infants with anotia/microtia across nearly all strata of sociodemographic and other maternal factors (adjusted odds ratios range: 2.1-11.9). The odds of anotia/microtia was particularly elevated among Hispanic mothers who emigrated from Mexico after age five (adjusted odds ratios = 4.88; 95% confidence interval = 2.93-8.11) or who conducted the interview in Spanish (adjusted odds ratios = 4.97; 95% confidence interval = 3.00-8.24). CONCLUSION: We observed that certain sociodemographic and acculturation factors are associated with higher risks of anotia/microtia among offspring of Hispanic mothers.

Interrater reliability of a phenotypic assessment tool for the ear morphology in microtia.

The Elements of Morphology Standard Terminology working group published standardized definitions for external ear morphology. The primary objective of our study was to use these descriptions to evaluate the interrater reliability for specific features associated with microtia. We invited six raters from three different subspecialities to rate 100 ear photographs on 32 features. We calculated overall and within specialty and professional experience intraclass correlation coefficients (ICC) and 95% confidence intervals. A total of 600 possible observations were recorded for each feature. The overall interrater reliability ranged from 0.04 (95% CI: 0.00-0.14) for the width of the antihelix inferior crus to 0.93 (95% CI: 0.91-0.95) for the presence of the inferior crux of the antihelix. The reliability for quantitative characteristics such as length or width of an ear structure was generally lower than the reliability for qualitative characteristics (e.g., presence or absence of an ear structure). Categories with very poor interrater reliability included anti-helix inferior crux width (0.04, 95% CI: 0.00-0.14), crux helix extension (0.17, 95% CI 0.00-0.37), and shape of the incisura (0.14, 95% CI: 0.01-0.27). There were no significant differences in reliability estimates by specialty or professional experience for most variables. Our study showed that it is feasible to systematically characterize many of structures of the ear that are affected in microtia. We incorporated these descriptions into a standardized phenotypic assessment tool (PAT-Microtia) that might be used in multicenter research studies to identify sub-phenotypes for future studies of microtia.

Long-Term Clinical Results of Two-Stage Total Ear Reconstruction of Microtia Using Autologous Cell-Engineered Chondrocytes.

BACKGROUND: Microtia repair requires a large volume of reconstruction material.In pediatric patients, the collectable volume of autologous cartilage is limited, and the impact of surgical invasion and donor-site morbidity can be particularly severe. The authors developed a new treatment method using cultured autologous human auricular chondrocytes that provides a sufficiently large volume of reconstruction material. METHODS: Approximately 1 cm2 of auricular cartilage was collected from the affected site. Chondrocytes were isolated and cultured with autologous serum to accelerate cell proliferation. The cells were subcultured and formed a gel-form mass without a scaffold. In our two-stage implantation, the cultured chondrocytes were first injected into the patient's lower abdomen, where the cells grew into a large, newly generated cartilage in 6 months. Thereafter, this cartilage was sculpted into an ear framework and subcutaneously reimplanted into the new ear location. Clinical outcomes were assessed over a long-term follow-up. RESULTS: Eight patients underwent surgery using cultured autologous auricular chondrocytes from 2002 to 2008. The patients' ages ranged from 6 to 10 years. The follow-up period ranged from 11 to 18 years. None of the patients experienced absorption of cultured chondrocytes after the second stage. Complications included one case of absorption and one case of allergic reaction in the first stage. CONCLUSIONS: The authors' patients represent the first successful cases of regenerative surgery for microtia using cultured chondrocytes. No malignant transformation, change in size, deformation, or other abnormalities were observed during the long-term follow-up, demonstrating the safety of cultured cartilage. No major complications occurred. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.

Fabrication of 3D Models for Microtia Reconstruction Using Smartphone-Based Technology.

OBJECTIVE: Microtia reconstruction is technically challenging due to the intricate contours of the ear. It is common practice to use a two-dimensional tracing of the patient's normal ear as a template for the reconstruction of the affected side. Recent advances in three-dimensional (3D) surface scanning and printing have expanded the ability to create surgical models preoperatively. This study aims to describe a simple and affordable process to fabricate patient-specific 3D ear models for use in the operating room. STUDY DESIGN: Applied basic research on a novel 3D optical scanning and fabrication pathway for microtia reconstruction. SETTING: Tertiary care university hospital. METHODS: Optical surface scanning of the patient's normal ear was completed using a smartphone with facial recognition capability. The Heges application used the phone's camera to capture the 3D image. The 3D model was digitally isolated and mirrored using the Meshmixer software and printed with a 3D printer (MonopriceTM Select Mini V2) using polylactic acid filaments. RESULTS: The 3D model of the ear served as a helpful intraoperative reference and an adjunct to the traditional 2D template. Collectively, time for imaging acquisition, editing, and fabrication was approximately 3.5 hours. The upfront cost was around $210, and the recurring cost was approximately $0.35 per ear model. CONCLUSION: A novel, low-cost approach to fabricate customized 3D models of the ear is introduced. It is feasible to create individualized 3D models using currently available consumer technology. The low barrier to entry raises the possibility for clinicians to incorporate 3D printing into various clinical applications.

Whole-exome sequencing analysis in 10 families of sporadic microtia with thoracic deformities.

BACKGROUND: Microtia is a congenital malformation of the external ear and may occur as an isolated deformity or as part of a syndrome. Our previous study found a high correlation between microtia and thoracic deformities, thus, we propose that external ear and thorax development may be regulated by certain genes in common. METHODS: We performed exome sequencing on 10 families of sporadic microtia with thoracic abnormalities. We identified mutated genes under different models of inheritance, and checked them through Mouse Genome Informatics and association analysis. RESULTS: We identified 45 rare mutations, including 9 de novo mutations, 20 heterozygous mutations, 3 homozygous mutations, and 13 hemizygous mutations, of which 2 are likely to be causative. They are de novo missense variant in PHF5A and compound heterozygous mutations in CYP26B1, of which CYP26B1 mutation is highly likely pathogenic. CONCLUSION: The results indicate that certain genes may affect both external ear and thorax development, and demonstrate the benefits of whole-exome sequencing in identifying candidate genes of microtia. This study provides a new way for genetic exploration in microtia.

A synonymous variant in a non-canonical exon of CDC45 disrupts splicing in two affected sibs with Meier-Gorlin syndrome with craniosynostosis.

Disruption of the initiation of DNA replication is significantly associated with Meier-Gorlin syndrome (MGORS), an autosomal recessive condition of reduced growth, microtia and patellar a/hypoplasia. Biallelic mutations in CDC45, a member of the pre-initiation complex in DNA replication, cause a spectrum of phenotypes ranging from MGORS with craniosynostosis, through to isolated short stature and craniosynostosis. Here we report two affected sibs with MGORS and craniosynostosis, with biallelic variants in CDC45 identified by 10X Chromium whole genome sequencing. One variant is a frameshift mutation, predicted to be pathogenic, and is inherited in trans with a synonymous variant in a non-canonical exon (exon 7) of CDC45. An in vitro splicing assay showed that while the canonical CDC45 exon 6-exon 8 transcript (with skipping of exon 7; numbering as per NM001178010.2) remained as the predominant transcript, the variant allele induced the use of novel splice acceptor sites in intron 6, all of which produced transcripts harbouring premature stop codons. This perturbation of canonical splicing provides evidence that this synonymous variant is indeed a deleterious alteration in this family. This report adds to the initial patient cohort in which several synonymous variants were also described, further highlighting the contribution of this variant type in CDC45. It also reiterates the true potential pathogenicity of synonymous variants, which is a mutation type that is commonly ignored in variant prioritization strategies.

MYT1 role in the microtia-craniofacial microsomia spectrum.

BACKGROUND: Craniofacial microsomia (CFM), also known as the oculo-auriculo-vertebral spectrum, comprises a variable phenotype with the most common features including microtia and mandibular hypoplasia on one or both sides, in addition to lateral oral clefts, epibulbar dermoids, cardiac, vertebral, and renal abnormalities. The etiology of CFM is largely unknown. The MYT1 gene has been reported as a candidate based in mutations found in three unrelated individuals. Additional patients with mutations in this gene are required to establish its causality. We present two individuals with CFM that have rare variants in MYT1 contributing to better understand the genotype and phenotype associated with mutations in this gene. METHODS/RESULTS: We conducted genetic analysis using whole-exome and -genome sequencing in 128 trios with CFM. Two novel MYT1 mutations were identified in two participants. Sanger sequencing was used to confirm these mutations. CONCLUSION: We identified two additional individuals with CFM who carry rare variants in MYT1, further supporting the presumptive role of this gene in the CFM spectrum.

[Anthropometric measurements of moderate concha-type microtia after auricular cartilage unfolding].

OBJECTIVE: To explore the anthropometric changes of the auricle after auricular cartilage unfolding in moderate concha-type microtia patients, so as to provide the basis to help evaluate surgical timing and prognostic. METHODS: A total of 33 children with moderate concha-type microtia, who were treated with auricular cartilage unfolding between October 2016 and September 2018 and met the inclusive criteria, were included in the study. There were 24 boys and 9 girls with an average age of 1.4 years (range, 1-3 years). Sixteen cases were left ears and 17 cases were right ears. The follow-up time was 12-23 months (mean, 17.5 months). The affected auricular detailed structures were observed and quantitatively analyzed before operation and at immediate after operation. The width, length, and perimeter of auricle before operation and at immediate after operation and at last follow-up were noted with three dimensional-scanning technology. The normal auricle was noted as control. RESULTS: There were (7.5±1.0) and (11.3±0.8) structures of the affected auricle at pre- and post-operation, respectively, showing significant difference between pre- and post-operation ( t=23.279, P=0.000). The length, width, and perimeter of the affected auricle constantly increased after operation, and there were significant differences between pre-operation and immediately after operation and between immediately after operation and last follow-up ( P<0.05). The differences of length, width, and perimeter of the affected auricle between immediately after operation and last follow-up were (3.13±1.44), (2.44±0.92), and (8.50±3.76) mm, respectively. And the differences of length, width, and perimeter of the normal auricle between pre-operation and last follow-up were (3.16±1.54), (2.35±0.86), and (9.79±4.60) mm, respectively. There was no significant difference in the differences of length, width, and perimeter between the affected auricle and the normal auricle ( P>0.05). CONCLUSION: The auricular cartilage unfolding in treatment of the moderate concha-type microtia can receive more ear structures and increase auricle sizes, which make it possible for free composite tissue transplantation. In addition, the affected and the contralateral normal auricles have a very similar growth rate and it offers the theoretical foundation for the early treatment for moderate concha-type microtia.

Identification of loss-of-function HOXA2 mutations in Chinese families with dominant bilateral microtia.

HOX genes are important regulatory genes patterning head formation, including development of the ear. Microtia is a congenital ear anomaly characterized by lacking all or part of the structures of the outer ear. To date, only four HOXA2 mutations were reported in families with autosomal-recessive or dominant microtia, with or without hearing impairment. More identified mutations are needed to confirm the correlation between genotype and phenotype. Here, we collect two Chinese families with non-syndromic bilateral microtia. Next generation sequencing identified two heterozygous nonsense HOXA2 mutations, one in each family. One mutation (c.637A > T, p.Lys213*) is newly reported, while the other one (c.703C > T,p.Gln235*) is consistent with a previous report. In mouse, Hoxa2 can bind to a long-range enhancer and regulate expression of the Hmx1 gene, which is a crucial transcription factor in eye and ear development. Using dual luciferase reporter assays, we showed that both HOXA2 mutations have impaired activation of the long-range enhancer of HMX1. In the present study, we report the first two bilateral non-syndromic microtia cases with HOXA2 mutations of Chinese origin and identify a novel mutation. Our results also provide molecular insights about how these nonsense HOXA2 mutations could affect the activation of its downstream target HMX1 by interacting with the long-range enhancer.

Duplication in ECR Near HMX1 and a SNP in GATA6 Genes Regulate Microtia in Awassi Sheep.

Microtia and anotia are hereditary traits characterized by an underdevelopment or complete absence of the outer ear. These congenital malformations observed in many species can exist as part of various syndromes or as an isolated trait as seen in the fat-tailed Awassi sheep breed. Our study aims to identify the genetic mutations causing microtia in Awassi sheep by DNA sequencing. DNA was extracted from blood samples randomly collected from 84 Awassi sheep (16 earless, 41 short ear and 27 normal ear) across different farms. GATA6 exons 1, 2, 4, 6 and 7, CLRN1 intron 3, DCC intron 2, ECR near HMX1 and the intergenic region between GATA6 and MIB1 genes were screened, amplified and sequenced. Allele and genotype frequencies were calculated by direct counting. Association was performed using chi-squared test for goodness-of-fit. Results showed mutations in only two genes significantly associated with microtia in Awassi: duplication in part of ECR near HMX1 (6:114293121-6:114293196) and a SNP at GATA6 exon 7 (23:34498242). Association results revealed that the ECR locus accounts for the microtia phenotype, while GATA6 exon 7 acts as a modifier gene. Genetic screening for these loci can be used to improve selection against microtia in Awassi sheep.

Three New Mutations and Mild, Asymmetrical Phenotype in the Highly Distinctive LAMM Syndrome: A Report of Eight Further Cases.

Labyrinthine aplasia, microtia, and microdontia (LAMM) is an autosomal recessive condition causing profound congenital deafness, complete absence of inner ear structures (usually Michel's aplasia), microtia (usually type 1) and microdontia. To date, several families have been described with this condition and a number of mutations has been reported. We report on eight further cases of LAMM syndrome including three novel mutations, c. 173T>C p.L58P; c. 284G>A p.(Arg95Gln) and c.325_327delinsA p.(Glu109Thrfs*18). Congenital deafness was the primary presenting feature in all affected individuals and consanguinity in all but two families. We compare the features in our patients to those previously reported in LAMM, and describe a milder, asymmetrical phenotype associated with FGF3 mutations.

In Vitro Regeneration of Patient-specific Ear-shaped Cartilage and Its First Clinical Application for Auricular Reconstruction.

Microtia is a congenital external ear malformation that can seriously influence the psychological and physiological well-being of affected children. The successful regeneration of human ear-shaped cartilage using a tissue engineering approach in a nude mouse represents a promising approach for auricular reconstruction. However, owing to technical issues in cell source, shape control, mechanical strength, biosafety, and long-term stability of the regenerated cartilage, human tissue engineered ear-shaped cartilage is yet to be applied clinically. Using expanded microtia chondrocytes, compound biodegradable scaffold, and in vitro culture technique, we engineered patient-specific ear-shaped cartilage in vitro. Moreover, the cartilage was used for auricle reconstruction of five microtia patients and achieved satisfactory aesthetical outcome with mature cartilage formation during 2.5years follow-up in the first conducted case. Different surgical procedures were also employed to find the optimal approach for handling tissue engineered grafts. In conclusion, the results represent a significant breakthrough in clinical translation of tissue engineered human ear-shaped cartilage given the established in vitro engineering technique and suitable surgical procedure. This study was registered in Chinese Clinical Trial Registry (ChiCTR-ICN-14005469).

Predictors for Unfavorable Projection of the Constructed Auricle following Ear Elevation Surgery in Microtia Reconstruction.

BACKGROUND: Nagata's two-stage technique for microtia reconstruction using autologous costal cartilage consists of the implantation of a fabricated cartilage framework and the elevation of the constructed auricle. Achieving long-lasting, sufficient projection of the auricle is a goal of second-stage surgery; however, unfavorable outcomes have been seen in some patients, with suboptimal long-term elevation. The present study aimed to investigate prognostic factors that might contribute to adverse outcomes following the ear elevation operation. METHODS: Outcomes of patients with congenital microtia who underwent auricular reconstruction with a modified Nagata technique between January of 2007 and June of 2015 were reviewed. Unfavorable elevation was defined as cases with an auriculocephalic angle less than 20 degrees or with a shallow auricular sulcus requiring revision operations. Univariate and multivariate analyses were conducted to identify independent predictors for the unfavorable elevation. RESULTS: A total of 309 patients representing 323 cases were analyzed with a median follow-up period of 26 months. The mean age of the patients was 15.1 years. The majority of cases had lobule-type microtia. Hemifacial microsomia was present in 114 cases. Canalplasty was performed in 117 cases before the auricular elevation. Unfavorable elevation was observed in 72 cases (22.3 percent). Multivariate analyses showed that the presence of hemifacial microsomia and performance of canalplasty less than or equal to 12 months before the ear elevation operation were independent risk factors for the unfavorable elevation. CONCLUSION: Several preoperative factors, including combined anomalies and operative timing and sequences, might influence the outcomes of the ear elevation operation. CLINICAL QUESTION/LEVEL OF EVIDENCE: Risk, III.

Chondrocytes from congenital microtia possess an inferior capacity for in vivo cartilage regeneration to healthy ear chondrocytes.

The remnant auricular cartilage from microtia has become a valuable cell source for ear regeneration. It is important to clarify the issue of whether the genetically defective microtia chondrocytes could engineer cartilage tissue comparable to healthy ear chondrocytes. In the current study, the histology and cell yield of native microtia and normal ear cartilage were investigated, and the biological characteristics of derived chondrocytes examined, including proliferation, chondrogenic phenotype and cell migration. Furthermore, the in vivo cartilage-forming capacity of passaged microtia and normal auricular chondrocytes were systematically compared by seeding them onto polyglycolic acid/polylactic acid scaffold to generate tissue engineered cartilage in nude mice. Through histological examinations and quantitative analysis of glycosaminoglycan, Young's modulus, and the expression of cartilage-related genes, it was found that microtia chondrocytes had a slower dedifferentiation rate with the decreased expression of stemness-related genes, and weaker migration ability than normal ear chondrocytes, and the microtia chondrocytes-engineered cartilage was biochemically and biomechanically inferior to that constructed using normal ear chondrocytes. This study provides valuable information for the clinical application of the chondrocytes derived from congenital microtia to engineer cartilage. Copyright © 2016 John Wiley & Sons, Ltd.

Overexpression of Fgfr2c causes craniofacial bone hypoplasia and ameliorates craniosynostosis in the Crouzon mouse.

FGFR2c regulates many aspects of craniofacial and skeletal development. Mutations in the FGFR2 gene are causative of multiple forms of syndromic craniosynostosis, including Crouzon syndrome. Paradoxically, mouse studies have shown that the activation (Fgfr2cC342Y; a mouse model for human Crouzon syndrome), as well as the removal (Fgfr2cnull), of the FGFR2c isoform can drive suture abolishment. This study aims to address the downstream effects of pathogenic FGFR2c signalling by studying the effects of Fgfr2c overexpression. Conditional overexpression of Fgfr2c (R26RFgfr2c;ßact) results in craniofacial hypoplasia as well as microtia and cleft palate. Contrary to Fgfr2cnull and Fgfr2cC342Y, Fgfr2c overexpression is insufficient to drive onset of craniosynostosis. Examination of the MAPK/ERK pathway in the embryonic sutures of Fgfr2cC342Y and R26RFgfr2c;ßact mice reveals that both mutants have increased pERK expression. The contrasting phenotypes between Fgfr2cC342Y and R26RFgfr2c;ßact mice prompted us to assess the impact of the Fgfr2c overexpression allele on the Crouzon mouse (Fgfr2cC342Y), in particular its effects on the coronal suture. Our results demonstrate that Fgfr2c overexpression is sufficient to partially rescue craniosynostosis through increased proliferation and reduced osteogenic activity in E18.5 Fgfr2cC342Y embryos. This study demonstrates the intricate balance of FGF signalling required for correct calvarial bone and suture morphogenesis, and that increasing the expression of the wild-type FGFR2c isoform could be a way to prevent or delay craniosynostosis progression.

Long-Term Comparison between Human Normal Conchal and Microtia Chondrocytes Regenerated by Tissue Engineering on Nanofiber Polyglycolic Acid Scaffolds.

BACKGROUND: Previous regeneration studies of auricle-shaped cartilage by tissue engineering leave unresolved whether the chondrocyte phenotype from human auricular chondrocytes seeded onto polymeric scaffolds is retained over the long term and whether microtia remnants may be a viable cell source for auricular reconstruction. METHODS: Chondrocytes were isolated from human ears, either normal conchal ear or microtia cartilage remnants, expanded in vitro, and seeded onto nanoscale-diameter polyglycolic acid sheets. These tissue-engineered constructs were implanted into athymic mice for up to 40 weeks. At harvest times of 5, 10, 20, and 40 weeks, samples were documented by gross morphology, histology, and reverse transcription-quantitative polymerase chain reaction analysis. RESULTS: Neocartilages generated from the two types of surgical tissues were similar in appearance of their extracellular matrices and positive staining for elastin and proteoglycans. In the 5- to 40-week time interval, there was an increasing trend in gene expression for type II collagen, elastin, and sex determining region Y box 5, important to normal cartilage phenotype, and a decreasing trend in gene expression for type III collagen, a fibroblast and dedifferentiation marker. Over 40 weeks of implantation, the original nanoscale-diameter polyglycolic acid scaffold dimensions (1 cm × 1 cm × 80 µm) were generally maintained in tissue-engineered cartilage length and width, and thickness was statistically significantly increased. CONCLUSIONS: Auricular cartilage can be regenerated over the long term (40 weeks) from surgical remnants by tissue-engineering techniques incorporating nanoscale-diameter polyglycolic acid scaffolds. Based on the present assays, microtia neocartilage very closely resembles tissue-engineered cartilage regenerated from chondrocytes isolated from normal conchal cartilage.