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Muenke syndrome is characterized by various craniofacial deformities and is caused by an autosomal-dominant activating mutation in fibroblast growth factor receptor 3 (FGFR3(P250R) ). Here, using mice carrying a corresponding mutation (FgfR3(P244R) ), we determined whether the mutation affects temporomandibular joint (TMJ) development and growth. In situ hybridization showed that FgfR3 was expressed in condylar chondroprogenitors and maturing chondrocytes that also expressed the Indian hedgehog (Ihh) receptor and transcriptional target Patched 1(Ptch1). In FgfR3(P244R) mutants, the condyles displayed reduced levels of Ihh expression, H4C-positive proliferating chondroprogenitors, and collagen type II- and type X-expressing chondrocytes. Primary bone spongiosa formation was also disturbed and was accompanied by increased osteoclastic activity and reduced trabecular bone formation. Treatment of wild-type condylar explants with recombinant FGF2/FGF9 decreased Ptch1 and PTHrP expression in superficial/polymorphic layers and proliferation in chondroprogenitors. We also observed early degenerative changes of condylar articular cartilage, abnormal development of the articular eminence/glenoid fossa in the TMJ, and fusion of the articular disc. Analysis of our data indicates that the activating FgfR3(P244R) mutation disturbs TMJ developmental processes, likely by reducing hedgehog signaling and endochondral ossification. We suggest that a balance between FGF and hedgehog signaling pathways is critical for the integrity of TMJ development and for the maintenance of cellular organization. [ABSTRACT FROM AUTHOR]

Objective: To describe long-term outcomes and complications following mandibular distraction osteogenesis (MDO) in a diverse patient cohort., Design: Cross-sectional study., Setting: Single tertiary-care pediatric center., Patients: Forty-eight patients previously undergoing MDO with minimum 4-year follow-up., Main Outcome Measures: Respiratory outcomes, feeding patterns, dental development, motor/sensory nerve function, temporo-mandibular joint function, and postsurgical scarring., Results: Forty-six patients with a median age of 7 years were evaluated. Of 20 nonsyndromic patients, none required additional airway procedures, none required continuous positive airway pressure (CPAP) during sleep, and 19 (95%) fed exclusively by mouth. Among 26 syndromic patients, 7 (27%) required CPAP and 8 (31%) were tube fed. Permanent first molar differences were seen in the majority of subjects; patterns of damage interfering with function were more common in syndromic (13/28, 46%) compared to nonsyndromic (5/24, 21%; P  = .014) subjects. MDO prior to age two was associated with more frequent and worse dental damage ( P  = .001). Inferior alveolar nerve and marginal mandibular nerve function were fully intact in 37 (80%) and 39 (85%) of patients, respectively. Three patients (6%), all with associated genetic syndromes, demonstrated severe nerve impairment. By the Vancouver scar scale, ≥ 80% of surgical scars were rated in the most favorable category for each quality assessed. Temporomandibular joint dysfunction was rare., Conclusions: MDO shows highly favorable long-term respiratory, feeding, nerve, and scar outcomes in nonsyndromic patients, although permanent molar changes not precluding tooth viability are commonly seen. Patients with associated syndromes demonstrate respiratory and feeding benefits, but higher rates of dental and nerve abnormalities., Competing Interests: Declaration of Conflicting InterestsJWS has received an educational grant and developed educational content for KLS Martin and Depuy Synthes, both of which produce mandibular distraction devices. JAT is co-founder of Ostiio, LLC.

Background: Patients with micrognathia undergoing mandibular distraction osteogenesis (MDO) for functional and aesthetic improvement are at significant risk for dental complications. The authors investigated the association of 2 osteotomy patterns-oblique and inverted-L-with risk to developing dentition., Methods: A senior orthodontist (H.D.N.) performed a retrospective review of dental radiographs of patients undergoing MDO with confirmed oblique or inverted-L osteotomies between 2012 and 2022. Images were assessed for evidence of missing, damaged, or displaced teeth, and proportion of affected hemimandibles by injury type and median number of affected teeth per hemimandible were compared between groups using appropriate statistical methodology., Results: Analysis included 44 patients (23 oblique, 21 inverted-L) and 85 hemimandibles (45 oblique, 40 inverted-L). Mean age at surgery was 3.1 ± 4.6 years, and mean time to imaging was 4.9 ± 4.1 years; there was no difference between groups ( P = 0.23, P = 0.34, respectively). Oblique osteotomy was associated with greater odds of missing teeth (odds ratio [OR], 13.3, P < 0.001), damaged teeth (OR, 3.2; P = 0.02), and any dental injury (OR, 39.9; P < 0.001) compared with inverted-L, as well as greater number of missing teeth (β = 0.6; P < 0.01), damaged teeth (β = 0.3; P = 0.02), and total number of affected teeth (β = 0.9; P < 0.001). There was no difference in incidence ( P = 0.5) or number ( P = 0.4) of displaced teeth between groups., Conclusions: Inverted-L osteotomies were associated with fewer dental complications compared with oblique osteotomy at all ages studied. Although longer-term follow-up and prospective data are needed before definitive recommendations can be made, these data are helpful to surgeons as they plan MDO., Clinical Question/level of Evidence: Therapeutic, III., (Copyright © 2023 by the American Society of Plastic Surgeons.)

Background: Costochondral grafts (CCGs) can be used in mandibular reconstruction of Kaban-Pruzansky IIB/III hemifacial microsomia (HFM). Their growth is variable, occasionally necessitating secondary surgery. This study examined one surgeon's 24-year experience to better quantify long-term outcomes and surgical care required in CCG reconstruction of HFM mandibles., Methods: Serial 3-dimensional computed tomography scans, from preoperative to most recent, were analyzed in patients with a minimum of 4 years of clinical follow-up after CCG reconstruction. Graft/ramus height, length, volume, bilateral mandibular body length, and chin deviation were measured. Changes in measurements were analyzed preoperatively, immediately postoperatively, at the most recent imaging before secondary surgery, and at the most recent imaging overall. Growth rates per measure were calculated using scans after CCG but before secondary surgery., Results: Thirteen patients were analyzed. Median (SD) clinical follow-up was 10.0 (5.1) years. One patient developed temporomandibular joint ankylosis secondary to stacked-graft malposition, which was repaired without further complications. CCG reconstruction led to immediate improvement in graft/ramus height ( P = 0.03), length ( P = 0.002), and volumetric symmetry ( P = 0.02). No difference was found between graft and native ramus height ( P = 0.4) or length measures ( P = 0.5), whereas graft volume and the affected mandibular body grew significantly more slowly. According to the latest imaging, 63% of patients required secondary surgery, including distraction osteogenesis or orthognathic surgery, due to differential graft or hemimandible growth behavior. Based on the most recent clinical follow-up, this proportion increased to 93%., Conclusions: CCGs provide significant short-term mandibular and facial symmetry improvement in HFM IIB/III. Long-term analysis reveals frequent undergrowth requiring secondary intervention to promote and maintain symmetry., Clinical Question/level of Evidence: Therapeutic, IV., (Copyright © 2023 by the American Society of Plastic Surgeons.)

Background: Improving occlusion and aesthetics is the primary objective of orthognathic surgery for patients with cleft lip and palate (CLP). However, these patients often suffer from horizontal, vertical, and rotational asymmetry in addition to maxillary retrusion. This study aims to describe maxillary and mandibular asymmetry in patients with CLP undergoing orthognathic surgery and analyze its anatomic basis., Methods: Patients with isolated CLP undergoing CT imaging prior to orthognathic surgery were retrospectively reviewed. Maxillary and mandibular positioning and dimensional symmetry were evaluated. Incidence of clinically significant asymmetry, correlations between areas of asymmetry, and associations with clinical history were analyzed., Results: Fifty-eight patients, with mean age 17 years were analyzed, including 32 patients with unilateral CLP and 26 with bilateral CLP. Twenty (34%) patients demonstrated chin deviation ≥4mm and 21 (36%) had a ≥5% discrepancy in mandibular ramus lengths. Horizontal occlusal plane cant of ≥2° was seen in 20 (34%) maxillae and 28 (48%) mandibles, with dental arch yaw ≥2° noted in 32 (55%) of both maxillae and mandibles. Chin deviation correlated with maxillary cant, discrepancy in ramus length, discrepancy in mandibular body length, and discrepancy in condylar volume (p<0.05). Bilateral and unilateral CLP did not show significantly different asymmetry on any measure (p>0.05)., Conclusions: Both maxillary and mandibular asymmetry is common in skeletally mature patients with CLP and frequently results in notable chin deviation. Preoperative three-dimensional imaging and virtual surgical planning of orthognathic surgery aid in recognition of facial asymmetries and reveal opportunities to optimize results in this population., Competing Interests: Conflict of Interest: J.A.T. is co-founder of Ostiio, LLC and J.W.S. is a consultant for KLS Martin and Synthes. The other authors have no financial disclosures or conflicts of interest to report., (Copyright © 2024 by the American Society of Plastic Surgeons.)

Tongue reduction surgery is often pursued to manage the adverse effects of macroglossia in patients with Beckwith-Wiedemann syndrome (BWS). This study characterized dental outcomes in patients with BWS based on surgical timing and molecular diagnosis. A retrospective study was designed to include patients with BWS over the age of two who had clinical or radiographic documentation of dental development. Patients were grouped by history of tongue reduction surgery and surgical timing (early: <12 months). One hundred three patients were included (55 no tongue reduction, 18 early, 30 late). Patients who underwent late surgery had lower odds of class I occlusion (OR 0.11, 95% CI 0.02-0.58, p = 0.009) and higher odds of anterior open bite (OR 7.5, 95% CI 1.14-49.4, p = 0.036). Patients with clinical diagnoses and negative molecular testing had anterior open bite less frequently than patients with imprinting center 2 loss of methylation and paternal uniparental isodisomy of 11p15.5 (p = 0.009). Compared to reference values, patients who had tongue reductions had an increased mandibular plane angle (32.0 ± 4.5° versus 36.9 ± 5.0°, p = 0.001), indicative of hyperdivergent growth. The results of this study help to understand the complex nature of dentoskeletal growth in BWS and shed insight on how surgical timing and molecular diagnosis influence prognosis., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2023. Published by Elsevier Ltd.)

Computer-aided surgical planning has become popular for planning orthognathic surgery (OS) as it saves surgeons' time and effort. A recent advancement has been the utilization of patient-specific cutting guides and osteosynthesis. The purpose of this study is to report the postoperative bimaxillary position utilizing custom plates for both jaws versus custom plates used in the maxilla only in 23 consecutive patients., Methods: All patients who underwent bimaxillary OS in 2017-2018 with preoperative computed tomography (CT) scan, postoperative day 1 CT scan, and at least 6 months follow-up were included in the study. Group 1 utilized maxillary preprinted plates (maxilla only). Group 2 utilized bimaxillary preprinted plates (maxillomandibular). Eight cephalometric landmarks to evaluate the movements were chosen. The ranges of the angle between the sella/nasion plane and the nasion/A plane (SNA), the angle between the sella/nasion plane and the nasion/B plane (SNB), and the angle created by the A point' nasion' and B point' which measures the relative position of maxilla to mandible, were analyzed to assess the angular change. Mean-squared displacement and the SD of the distances were used to assess movement in space., Results: Twenty-three patients (nine in group 1 and 14 in group 2) met the inclusion criteria. Results showed interarch relationships using custom plates for both jaws with ANB 0.4 compared to ANB 1.4 for maxillary custom plates only. Mandibular landmarks showed greater variation, and the t test study revealed the right mandibular first molar landmark showing the greatest variation ( P = 0.03)., Conclusions: Custom osteosynthesis plates for OS show good accuracy for the maxilla and higher variation in the mandible. Further studies will determine the margin of error that cannot be corrected with postoperative orthodontics., (Copyright © 2022 The Authors. Published by Wolters Kluwer Health, Inc. on behalf of The American Society of Plastic Surgeons.)

Background: Sagittal craniosynostosis may present with complete or partial fusion of the sagittal suture, but relationships between degree of sagittal suture fusion and head shape are currently poorly described. The aim of this study was to characterize sagittal suture fusion patterns and determine associations with head shape in a cohort of patients with nonsyndromic sagittal craniosynostosis., Methods: Patients with nonsyndromic sagittal craniosynostosis at a tertiary care center with available computed tomography imaging were included in this study. The anterior and posterior distances of sagittal suture patency were measured along 3-dimensional parietal bones. Degree of sagittal suture fusion was compared to head shape characteristics, including cephalic index (CI), frontal bossing, and occipital bulleting., Results: Ninety patients (69 male) were included in this retrospective study. The sagittal suture was on average 85.6±20.1% fused, and 45 (50.0%) patients demonstrated complete fusion of the sagittal suture. CI was associated with increased degree of fusion for the anterior one-half (ρ=0.26, P =0.033) and anterior one-third (ρ=0.30, P =0.012) of the sagittal suture. Complete fusion of the anterior one-third of the sagittal suture predicted higher CI (β=13.86, SE=6.99, z =-0.25, P =0.047). Total degree of sagittal suture fusion was not predictive of CI or head shape in any analysis., Conclusions: Decreased fusion of the anterior one-third of the sagittal suture, but not total suture, may paradoxically predict increased severity of scaphocephaly as quantified by CI in nonsyndromic sagittal craniosynostosis., Competing Interests: The authors report no conflicts of interest., (Copyright © 2022 by Mutaz B. Habal, MD.)

Background: Patients with hemifacial microsomia (HFM) may undergo unilateral mandibular distraction osteogenesis (MDO) before skeletal maturity in an effort to improve facial symmetry. Mandibular distraction osteogenesis's effect on airway volumes have been studied in the past, though to our knowledge, none have accounted for the effect of head and neck posture on airway morphology. This study aimed to tackle this shortcoming, using imaging to analyze the upper airway of patients with HFM before and after surgical intervention with MDO., Methods/description: The authors retrospectively reviewed patients with a diagnosis of unilateral HFM whom underwent unilateral MDO with an oblique vector at age 4 to 14 years at a single institution from 2004 to 2019. Patients with pre- and post-MDO three-dimensional computed tomography scans of the upper airway within 12 months of distractor placement and removal, respectively, were included. Head and neck postures were determined by craniocervical, pitch, roll, and yaw angles. Pre- and post-operative pharyngeal airway volumes, pharyngeal surface area, minimum retropalatal cross-sectional areas (RP CSA) and retroglossal (RG) CSA and associated anteroposterior distances were measured using Mimics 22.0 (Materialise; Leuven, Belgium). Comparison was done using Kruskal-Wallis tests and linear mixed-effects models controlling for head and neck postures., Results: Ten patients met inclusion criteria. Mean age at pre-distractor placement computed tomography scan was 99 ± 35 months, and mean duration between pre- and post-surgery scans was 220 ± 90 days. Head and neck posture were found to be significant predictors of all airway dimensions. After controlling for significant factors with fixed effects linear modeling, surface area was found to be significantly smaller in patients after MDO by 189.48 mm2 (F[10.8] = -3.47, P = 0.0053), compared to their preoperative measurements. Surgery was not a significant predictor of changes in airway volume (F[11.6] = 0.52, P = 0.61), minimum RP CSA (F[12.2] =  -0.64, P = 0.53), minimum RG CSA (F[12.6] =  -1.64, P = 0.13), RP anteroposterior distance (F[14.0] = 0.30, P = 0.77), or RG anteroposterior distance (F[20.0] =  -0.04, P = 0.97)., Conclusions: Oblique vector MDO in patients with HFM is associated only with statistically significant changes in the surface area of the upper airway, and is not associated with statistically significant changes in dimensions like volume, CSA, or anteroposterior dimension. This is an important finding, as it may guide discussions surrounding risk/benefit ratio for MDO in childhood., Competing Interests: The authors report no conflicts of interest., (Copyright © 2021 by Mutaz B. Habal, MD.)

Background: Timing of frontofacial surgery for the syndromic craniosynostosis as it relates to various surgical risks has not been adequately studied. The purpose of this study was to investigate posterior dental complications of midface advancement in patients with syndromic craniosynostosis undergoing surgery at different ages and the effects on subsequent orthognathic surgery., Methods: A retrospective chart review of patients with syndromic craniosynostosis treated with midface advancement (monobloc or Le Fort III) from 1999 to 2018 was carried out. Patient demographics, records, and imaging studies were reviewed. A subanalysis of those patients who were also treated with orthognathic surgery from 2014 to 2018 with imaging studies available for analysis was also performed., Results: Thirty-seven patients met the inclusion criteria. Sixty-four percent of the patients had radiographic evidence of maxillary molar dental abnormality. Older age at the time of surgery was significantly associated with a lower odds of sustaining dental injury (OR, 0.55; p = 0.034). The odds of damaging second or third maxillary molars was significantly higher with a younger age at the time of surgery (p = 0.021 and p = 0.034). The odds of sustaining dental injury increased moving posteriorly, showing the risk of abnormal pattern of M3 greater than M2 greater than M1. Advanced age at the time of surgery was significantly associated with decreased odds of dental injury (OR, 0.55; p = 0.034)., Conclusions: Damage to the developing permanent maxillary molars may affect orthodontic management, mastication, and potentially maxillary development. Delaying frontofacial surgery until development of the permanent maxillary dentition should be considered if other indications do not mandate earlier intervention., Competing Interests: Disclosure:The authors report no conflicts of interest., (Copyright © 2020 by the American Society of Plastic Surgeons.)

Cleft lip is one of the most common human birth defects. However, there remain a limited number of mouse models of cleft lip that can be leveraged to characterize the genes and mechanisms that cause this disorder. Crosstalk between epithelial and mesenchymal cells underlies formation of the face and palate, but the basic molecular events mediating this crosstalk remain poorly understood. We previously demonstrated that mice lacking the epithelial-specific splicing factor Esrp1 have fully penetrant bilateral cleft lip and palate. In this study, we further investigated the mechanisms leading to cleft lip as well as cleft palate in both existing and new Esrp1 mutant mouse models. These studies included a detailed transcriptomic analysis of changes in ectoderm and mesenchyme in Esrp1 -/- embryos during face formation. We identified altered expression of genes previously implicated in cleft lip and/or palate, including components of multiple signaling pathways. These findings provide the foundation for detailed investigations using Esrp1 mutant disease models to examine gene regulatory networks and pathways that are essential for normal face and palate development - the disruption of which leads to orofacial clefting in human patients., Competing Interests: Competing interestsY.X. is a scientific cofounder of Panorama Medicine. All other authors declare no competing interests., (© 2020. Published by The Company of Biologists Ltd.)

The temporomandibular joint (TMJ) is an intricate structure composed of the mandibular condyle, articular disc, and glenoid fossa in the temporal bone. Apical condylar cartilage is classified as a secondary cartilage, is fibrocartilaginous in nature, and is structurally distinct from growth plate and articular cartilage in long bones. Condylar cartilage is organized in distinct cellular layers that include a superficial layer that produces lubricants, a polymorphic/progenitor layer that contains stem/progenitor cells, and underlying layers of flattened and hypertrophic chondrocytes. Uniquely, progenitor cells reside near the articular surface, proliferate, undergo chondrogenesis, and mature into hypertrophic chondrocytes. During the past decades, there has been a growing interest in the molecular mechanisms by which the TMJ develops and acquires its unique structural and functional features. Indian hedgehog (Ihh), which regulates skeletal development including synovial joint formation, also plays pivotal roles in TMJ development and postnatal maintenance. This review provides a description of the many important recent advances in Hedgehog (Hh) signaling in TMJ biology. These include studies that used conventional approaches and those that analyzed the phenotype of tissue-specific mouse mutants lacking Ihh or associated molecules. The recent advances in understanding the molecular mechanism regulating TMJ development are impressive and these findings will have major implications for future translational medicine tools to repair and regenerate TMJ congenital anomalies and acquired diseases, such as degenerative damage in TMJ osteoarthritic conditions.

Condylar articular cartilage in mouse temporomandibular joint develops from progenitor cells near the articulating surface that proliferate, undergo chondrogenesis and mature into hypertrophic chondrocytes. However, it remains unclear how these processes are regulated, particularly postnatally. Here we focused on the apical polymorphic layer rich in progenitors and asked whether the phenotype and fate of the cells require signaling by Indian hedgehog (Ihh) previously studied in developing long bones. In condyles in newborn mice, the apical polymorphic/progenitor cell layer was ~10 cell layer-thick and expressed the articular matrix marker Tenascin-C (Tn-C), and the underlying thick cell layer expressed Tn-C as well as the chondrogenic master regulator Sox9. By 1 month, condylar cartilage had gained its full width, but became thinner along its main longitudinal axis and displayed hypertrophic chondrocytes. By 3 months, articular cartilage consisted of a 2-3 cell layer-thick zone of superficial cells and chondroprogenitors expressing both Tn-C and Sox9 and a bottom zone of chondrocytes displaying vertical matrix septa. EdU cell tracing in juvenile mice revealed that conversion of chondroprogenitors into chondrocytes and hypertrophic chondrocytes required about 48 and 72 h, respectively. Notably, EdU injection in 3 month-old mice labeled both progenitors and maturing chondrocytes by 96 h. Conditional ablation of Ihh in juvenile/early adult mice compromised chondroprogenitor organization and function and led to reduced chondroprogenitor and chondrocyte proliferation. The phenotype of mutant condyles worsened over time as indicated by apoptotic chondrocyte incidence, ectopic chondrocyte hypertrophy, chondrocyte column derangement and subchondral bone deterioration. In micromass cultures of condylar apical cells, hedgehog (Hh) treatment stimulated chondrogenesis and alkaline phosphatase (APase) activity, while treatment with HhAntag inhibited both. Our findings indicate that the chondroprogenitor layer is continuously engaged in condylar growth postnatally and its organization and functioning depend on hedgehog signaling., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The temporomandibular joint (TMJ) is a diarthrodial joint that relies on lubricants for frictionless movement and long-term function. It remains unclear what temporal and causal relationships may exist between compromised lubrication and onset and progression of TMJ disease. Here we report that Proteoglycan 4 (Prg4)-null TMJs exhibit irreversible osteoarthritis-like changes over time and are linked to formation of ectopic mineralized tissues and osteophytes in articular disc, mandibular condyle and glenoid fossa. In the presumptive layer of mutant glenoid fossa's articulating surface, numerous chondrogenic cells and/or chondrocytes emerged ectopically within the type I collagen-expressing cell population, underwent endochondral bone formation accompanied by enhanced Ihh expression, became entrapped into temporal bone mineralized matrix, and thereby elicited excessive chondroid bone formation. As the osteophytes grew, the roof of the glenoid fossa/eminence became significantly thicker and flatter, resulting in loss of its characteristic concave shape for accommodation of condyle and disc. Concurrently, the condyles became flatter and larger and exhibited ectopic bone along their neck, likely supporting the enlarged condylar heads. Articular discs lost their concave configuration, and ectopic cartilage developed and articulated with osteophytes. In glenoid fossa cells in culture, hedgehog signaling stimulated chondrocyte maturation and mineralization including alkaline phosphatase, while treatment with hedgehog inhibitor HhAntag prevented such maturation process. In sum, our data indicate that Prg4 is needed for TMJ integrity and long-term postnatal function. In its absence, progenitor cells near presumptive articular layer and disc undergo ectopic chondrogenesis and generate ectopic cartilage, possibly driven by aberrant activation of Hh signaling. The data suggest also that the Prg4-null mice represent a useful model to study TMJ osteoarthritis-like degeneration and clarify its pathogenesis., (Copyright © 2016 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.)

Heterotopic cartilage develops in certain pathologic conditions, including those affecting the human temporomandibular joint (TMJ), but the underlying molecular mechanisms remain obscure. This is in part due to the fact that a reliable animal model of such TMJ diseases is not available. Here, we show that aberrant chondrocyte differentiation and ectopic cartilage formation occur spontaneously in proteoglycan 4 (Prg4) mutant TMJ discs without further invasive procedure. By 2 mo of age, mutant disc cells displayed chondrocyte transdifferentiation, accompanied by strong expression of cartilage master gene Sox9 and matrix genes aggrecan and type II collagen. By 6 mo, heterotopic cartilage had formed in the discs and expressed cartilage hypertrophic markers Runx2 and ColX. The ectopic tissue grew in size over time and exhibited regional mineralization by 12 mo. Bone morphogenetic protein (BMP) signaling was activated with the ectopic chondrogenic cells and chondrocytes, as indicated by phosphorylated Smad 1/5/8 nuclear staining and by elevated expression of Bmp2, Bmpr1b, Bmpr2, and BMP signaling target genes. Likewise, we found that upon treatment with recombinant human BMP 2 in high-density micromass culture, mutant disc cells differentiated into chondrocytes and synthesized cartilage matrix more robustly than control cells. Importantly, a specific kinase inhibitor of BMP receptors drastically attenuated chondrogenesis in recombinant human BMP 2-treated mutant disc cultures. Unexpectedly, we found that Prg4 was expressed at joint-associated sites, including disc/muscle insertion and muscle/bone interface, and all these structures were abnormal in Prg4 mutants. Our data indicate that Prg4 is needed for TMJ disc integrity and function and that its absence leads to ectopic chondrogenesis and cartilage formation in conjunction with abnormal BMP signaling. Our findings imply that the BMP signaling pathway could be a potential therapeutic target for prevention or inhibition of ectopic cartilage formation in TMJ disease., (© International & American Associations for Dental Research 2015.)

Introduction: The purpose of this study was to investigate odontogenic and osteogenic cell adhesion, proliferation, and survival on the surface of a newly developed bioceramic material (EndoSequence Root Repair Material [RRM]; Brasseler USA, Savannah, GA) and compare it with mineral trioxide aggregate (gray MTA) (ProRoot MTA; Dentsply Tulsa Dental, Tulsa, OK). A potential role of extracellular signal-regulated kinase (ERK) signaling in the RRM/MTA-induced cellular activities was also investigated., Methods: Human bone marrow mesenchymal stem cells, periodontal ligament stem cells, and dental pulp stem cells were cultured on RRM- or MTA-coated slides. Cell proliferation was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays after 1, 3, and 5 days of growth. Cell survival was assessed under serum starvation (0.5% and 0.2% serum) using MTT assays. RRM and MTA surface characteristics and cell morphology were studied using a scanning electron microscope. The role of ERK signaling in RRM/MTA-induced cell proliferation/survival was studied using an ERK-specific inhibitor., Results: All cell types firmly attached to RRM- and MTA-coated plates. The coated surfaces had a granular appearance under the scanning electron microscope. Compared with those grown on uncoated plates, the cells on MTA/RRM-coated plates appeared healthy and smaller. Cell proliferation was significantly higher on RRM/MTA-coated surfaces (2- to 3-fold in cell number). The mitogenic effect on periodontal ligament stem cells and dental pulp stem cells was more pronounced with RRM than MTA (49% and 26% higher, respectively), but human bone marrow mesenchymal stem cells responded to both materials similarly. In serum-deprived conditions, significantly more cells (2- to 3-fold) survived on RRM/MTA surfaces. The cells grown on RRM/MTA surfaces showed sustained up-regulation of ERK phosphorylation, and blocking ERK signaling with U0126 significantly reduced RRM- and MTA-dependent cell survival., Conclusions: MTA and RRM are biocompatible and promote cell proliferation and survival in an ERK-dependent manner., (Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Introduction: The purpose of this study was to compare healing after root-end surgery by using grey mineral trioxide aggregate (MTA) and EndoSequence Root Repair Material (RRM) as root-end filling material in an animal model., Methods: Apical periodontitis was induced in 55 mandibular premolars of 4 healthy beagle dogs. After 6 weeks, root-end surgeries were performed by using modern microsurgical techniques. Two different root-end filling materials were used, grey MTA and RRM. Six months after surgery, healing of the periapical area was assessed by periapical radiographs, cone-beam computed tomography (CBCT), micro computed tomography (CT), and histology., Results: Minimal or no inflammatory response was observed in the majority of periapical areas regardless of the material. The degree of inflammatory infiltration and cortical plate healing were not significantly different between the 2 materials. However, a significantly greater root-end surface area was covered by cementum-like, periodontal ligament-like tissue, and bone in RRM group than in MTA group. When evaluating with periapical radiographs, complete healing rate in RRM and MTA groups was 92.6% and 75%, respectively, and the difference was not statistically significant (P = .073). However, on CBCT and micro CT images, RRM group demonstrated significantly superior healing on the resected root-end surface and in the periapical area (P = .000 to .027)., Conclusions: Like MTA, RRM is a biocompatible material with good sealing ability. However, in this animal model RRM achieved a better tissue healing response adjacent to the resected root-end surface histologically. The superior healing tendency associated with RRM could be detected by CBCT and micro CT but not periapical radiography., (Copyright © 2015 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Background: Bone transport osteogenesis (BTO), distraction of a free portion of bone across a defect, offers an autologous solution to large cranial defects that may allow treatment without permanent hardware implantation. This study establishes a sheep model to evaluate the feasibility and distraction kinetics of BTO., Methods: Subtotal cranial defects (3.5 × 3.5 cm) were created in 10 young adult sheep and a transport segment (3.5 × 2 cm) traversed the defect at varying distraction rates (0, 0.5, 1.0, and 1.5 mm/day) using semi-buried cranial distractors. After a 6-week consolidation period, sheep were euthanized and the resultant bone was analyzed by CT, histology, and mechanical testing., Results: Gross examination, histology, and 3D CT revealed that control animals had fibrous nonunion whereas distraction animals had ossified defects with fibrous nonunion at the distal docking site. There was one premature consolidation in the 0.5 mm/day group. The volume of bony regenerate in the 0.5, 1.0, and 1.5 mm/day distraction rate groups was statistically indistinct (P = 0.16). The mean flexural moduli (MPa) of non-decalcified samples from the control cranium, transport segment, and bone regenerate were found to be 4.50 ± 4.9, 6.17 ± 2.1, and 4.14 ± 4.8, respectively (P = 0.24)., Conclusions: This experiment provides proof of concept for BTO for large calvarial defects in a sheep model. Distraction at a rate of 0.5 mm per day may place individuals at higher risk for premature consolidation, but distraction rates did not have significant effects on regenerate quantity or quality. Future work will include the use of curvilinear distraction devices for 3-dimensional contour.

Background: The spheno-occipital synchondrosis is an important driver of facial and cranial base growth. The current study characterizes its fusion in patients with Apert, Crouzon, and Pfeiffer syndromes and correlates early fusion with the presence, and degree, of midface hypoplasia., Methods: A retrospective case-control study was performed of all syndromic patients treated between 1996 and 2012. Case computed tomographic scans and age- and sex-matched control scans were analyzed as demonstrating either open, partially fused, or completely fused synchondroses, and patient age at each scan was recorded. Midface hypoplasia as determined by sella-nasion-A point angle measurement at the time of midface surgery was correlated to fusion status., Results: Fifty-four patients with 206 computed tomographic scans met inclusion criteria. Two hundred six age- and sex-matched control scans were also identified. Average age at computed tomographic scanning was 6.1 years. The earliest ages of partial and complete fusion were 1.1 and 7.0 years, respectively, among cases; and 6.2 and 12.7 years, respectively, among controls. The odds of synchondrosis fusion in case computed tomographic scans was 66.0 times that of controls (95 percent CI, 9.2 to 475.5 times that of controls; p < 0.000001). Average age of synchondrosis fusion was 3.5 years (range, 0.5 to 6.0 years). Average sella-nasion-A point angle at the time of midface surgery was 67.5 degrees (range, 58 to 76 degrees), with a positive correlation between earlier age of fusion and more severe midface hypoplasia (p = 0.028)., Conclusions: The spheno-occipital synchondrosis fuses earlier in syndromic patients compared with age-matched controls. Moreover, there is a positive correlation between earlier fusion and degree of midface hypoplasia, although definitive causality cannot be concluded. This is the first study to demonstrate such a correlation in human subjects., Clinical Question/level of Evidence: Risk, II.

Background: Commercially available recombinant human bone morphogenetic protein 2 (rhBMP2) has demonstrated efficacy in bone regeneration, but not without significant side effects. The authors used rhBMP2 encapsulated in poly(lactic-co-glycolic acid) (PLGA) microspheres placed in a rabbit cranial defect model to test whether low-dose, sustained delivery can effectively induce bone regeneration., Methods: The rhBMP2 was encapsulated in 15% PLGA using a double-emulsion, solvent extraction/evaporation technique, and its release kinetics and bioactivity were tested. Two critical-size defects (10 mm) were created in the calvaria of New Zealand white rabbits (5 to 7 months of age, male and female) and filled with a collagen scaffold containing either (1) no implant, (2) collagen scaffold only, (3) PLGA-rhBMP2 (0.1 μg per implant), or (4) free rhBMP2 (0.1 μg per implant). After 6 weeks, the rabbits were killed and defects were analyzed by micro-computed tomography, histology, and finite element analysis., Results: The rhBMP2 delivered by means of bioactive PLGA microspheres resulted in higher volumes and surface area coverage of new bone than an equal dose of free rhBMP2 by micro-computed tomography (p=0.025 and p=0.025). Finite element analysis indicated that the mechanical competence using the regional elastic modulus did not differ with rhBMP2 exposure (p=0.70). PLGA-rhBMP2 did not demonstrate heterotopic ossification, craniosynostosis, or seroma formation., Conclusions: Sustained delivery by means of PLGA microspheres can significantly reduce the rhBMP2 dose required for de novo bone formation. Optimization of the delivery system may be a key to reducing the risk for recently reported rhBMP2-related adverse effects.

The Proteoglycan 4 (Prg4) product lubricin plays essential roles in boundary lubrication and movement in limb synovial joints, but its roles in temporomandibular joint (TMJ) are unclear. Thus, we characterized the TMJ phenotype in wild-type and Prg4(-/-) mouse littermates over age. As early as 2 weeks of age, mutant mice exhibited hyperplasia in the glenoid fossa articular cartilage, articular disc, and synovial membrane. By 1 month of age, there were fewer condylar superficial tenascin-C/Col1-positive cells and more numerous apoptotic condylar apical cells, while chondroprogenitors displayed higher mitotic activity, and Sox9-, Col2-, and ColX-expressing chondrocyte zones were significantly expanded. Mutant subchondral bone contained numerous Catepsin K-expressing osteoclasts at the chondro-osseous junction, increased invasive marrow cavities, and suboptimal subchondral bone. Mutant glenoid fossa, disc, synovial cells, and condyles displayed higher Hyaluronan synthase 2 expression. Mutant discs also lost their characteristic concave shape, exhibited ectopic chondrocyte differentiation, and occasionally adhered to condylar surfaces. A fibrinoid substance of unclear origin often covered the condylar surface. By 6 months of age, mutant condyles displayed osteoarthritic degradation with apical/mid-zone separation. In sum, lubricin exerts multiple essential direct and indirect roles to preserve TMJ structural and cellular integrity over post-natal life., (© International & American Associations for Dental Research.)

The efficiency of hematopoietic stem cell (HSC) engraftment after bone marrow (BM) transplantation depends largely on the capacity of the marrow microenvironment to accept the transplanted cells. While radioablation of BM damages osteoblastic stem cell niches, little is known about their restoration and mechanisms governing their receptivity to engraft transplanted HSCs. We previously reported rapid restoration and profound expansion of the marrow endosteal microenvironment in response to marrow radioablation. Here, we show that this reorganization represents proliferation of mature endosteal osteoblasts which seem to arise from a small subset of high-proliferative, relatively radio-resistant endosteal cells. Multiple layers of osteoblasts form along the endosteal surface within 48 hours after total body irradiation, concomitant with a peak in marrow cytokine expression. This niche reorganization fosters homing of the transplanted hematopoietic cells to the host marrow space and engraftment of long-term-HSC. Inhibition of insulin-like growth factor (IGF)-1-receptor tyrosine kinase signaling abrogates endosteal osteoblast proliferation and donor HSC engraftment, suggesting that the cytokine IGF-1 is a crucial mediator of endosteal niche reorganization and consequently donor HSC engraftment. Further understanding of this novel mechanism of IGF-1-dependent osteoblastic niche expansion and HSC engraftment may yield clinical applications for improving engraftment efficiency after clinical HSC transplantation., (© AlphaMed Press.)

Long bone is an anatomically complicated tissue with trabecular-rich metaphyses at two ends and cortical-rich diaphysis at the center. The traditional flushing method isolates only mesenchymal progenitor cells from the central region of long bones and these cells are distant from the bone surface. We propose that mesenchymal progenitors residing in endosteal bone marrow that is close to the sites of bone formation, such as trabecular bone and endosteum, behave differently from those in the central bone marrow. In this report, we separately isolated endosteal bone marrow using a unique enzymatic digestion approach and demonstrated that it contained a much higher frequency of mesenchymal progenitors than the central bone marrow. Endosteal mesenchymal progenitors express common mesenchymal stem cell markers and are capable of multi-lineage differentiation. However, we found that mesenchymal progenitors isolated from different anatomical regions of the marrow did exhibit important functional differences. Compared with their central marrow counterparts, endosteal mesenchymal progenitors have superior proliferative ability with reduced expression of cell cycle inhibitors. They showed greater immunosuppressive activity in culture and in a mouse model of inflammatory bowel disease. Aging is a major contributing factor for trabecular bone loss. We found that old mice have a dramatically decreased number of endosteal mesenchymal progenitors compared with young mice. Parathyroid hormone (PTH) treatment potently stimulates bone formation. A single PTH injection greatly increased the number of endosteal mesenchymal progenitors, particularly those located at the metaphyseal bone, but had no effect on their central counterparts. In summary, endosteal mesenchymal progenitors are more metabolically active and relevant to physiological bone formation than central mesenchymal progenitors. Hence, they represent a biologically important target for future mesenchymal stem cell studies., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Cleft palate is a developmental defect resulting from the failure of embryonic palatal shelves to fuse with each other at a critical time. Immediately before and during palatal fusion (E13-E15 in mice), transforming growth factor β3 (TGFβ3) is expressed in the palatal shelf medial edge epithelium (MEE) and plays a pivotal role in palatal fusion. Using Tgfβ3(-/-) mice, which display complete penetrance of the cleft palate phenotype, we tested the hypothesis that intra-amniotic gene transfer could be used to prevent cleft palate formation by restoring palatal midline epithelial function. An adenoviral vector encoding Tgfβ3 was microinjected into the amniotic sacs of mouse embryos at successive developmental stages. Transduced Tgfβ3(-/-) fetuses showed efficient recovery of palatal fusion with mesenchymal confluence following injection at E12.5 (100%), E13.5 (100%), E14.5 (82%), and E15.5 (75%). Viral vectors injected into the amniotic sac transduced the most superficial and transient peridermal cell layer but not underlying basal epithelial cells. TGFβ3 transduction of the peridermdal cell layer was sufficient to induce adhesion, fusion, and disappearance of the palatal shelf MEE in a cell nonautonomous manner. We propose that intra-amniotic gene transfer approaches have therapeutic potential to prevent cleft palate in utero, especially those resulting from palatal midline epithelial dysfunction.

Background: The spheno-occipital synchondrosis is a driver of cranial base and facial growth. Its premature fusion has been associated with midface hypoplasia in animal models. The authors reviewed computed tomographic scans of patients with Apert and Muenke syndrome, craniosynostosis syndromes with midface hypoplasia, to assess premature fusion of the spheno-occipital synchondrosis when compared with normal controls., Methods: Ninety head computed tomographic scans of Apert syndrome patients and 31 head scans of Muenke syndrome patients were assessed, in addition to an equal number of control scans. Spheno-occipital synchondrosis fusion on axial images was graded as open, partially closed, or closed. Analysis focused on ages 7 to 14 years, as no control patient fused before age 7 or had failed to fuse after age 14., Results: All 38 Apert syndrome patients aged 7 to 14 had some degree of spheno-occipital synchondrosis closure, compared with 29 of 38 matched controls (p = 0.0023). Seventeen of 20 Muenke syndrome patients showed closure, compared with 14 of 20 matched controls (p = 0.4506). Partial fusion was seen as early as age 2 in Apert syndrome and age 6 in Muenke syndrome patients; the earliest fusion was seen at age 7 in the control group., Conclusions: Compared with matched controls, the spheno-occipital synchondrosis closes significantly earlier in patients with Apert syndrome but not Muenke syndrome. This correlates well to reported incidences of midface hypoplasia in these syndromes. Although causality cannot be concluded from this study, an association exists between midface phenotype and degree of spheno-occipital synchondrosis closure., Clinical Question/level of Evidence: Risk, II.

Purpose: The Muenke syndrome mutation (FGFR3 (P250R)), which was discovered 15 years ago, represents the single most common craniosynostosis mutation. Muenke syndrome is characterized by coronal suture synostosis, midface hypoplasia, subtle limb anomalies, and hearing loss. However, the spectrum of clinical presentation continues to expand. To better understand the pathophysiology of the Muenke syndrome, we present collective findings from several recent studies that have characterized a genetically equivalent mouse model for Muenke syndrome (FgfR3 (P244R)) and compare them with human phenotypes., Conclusions: FgfR3 (P244R) mutant mice show premature fusion of facial sutures, premaxillary and/or zygomatic sutures, but rarely the coronal suture. The mice also lack the typical limb phenotype. On the other hand, the mutant mice display maxillary retrusion in association with a shortening of the anterior cranial base and a premature closure of intersphenoidal and spheno-occipital synchondroses, resembling human midface hypoplasia. In addition, sensorineural hearing loss is detected in all FgfR3 (P244R) mutant mice as in the majority of Muenke syndrome patients. It is caused by a defect in the mechanism of cell fate determination in the organ of Corti. The mice also express phenotypes that have not been previously described in humans, such as reduced cortical bone thickness, hypoplastic trabecular bone, and defective temporomandibular joint structure. Therefore, the FgfR3 (P244R) mouse provides an excellent opportunity to study disease mechanisms of some classical phenotypes of Muenke syndrome and to test novel therapeutic strategies. The mouse model can also be further explored to discover previously unreported yet potentially significant phenotypes of Muenke syndrome.

Aim: We report an in vitro technique to establish alignment of collagen fibres and cells within a three-dimensional tissue equivalent that mimics the natural periodontal ligament (PDL) using a novel custom-designed bioreactor., Material and Methods: Shear stress was applied to the tissue equivalent prepared with collagen solution and seeded with human PDL cells. Stress-strain dynamics and the alignment of collagen fibres and PDL cells in tissue equivalents were analysed., Results: Shear stress aligned collagen fibres and PDL cells in a direction parallel to the principle strain vector. PDL cells and Collagen fibres aligned in strained tissue equivalents with higher uniformity than in unstrained tissue equivalents., Conclusions: The cell and fibre alignment of the engineered PDL was precisely guided by mechanical shear stress along the direction of principal strain vector using a custom-designed bioreactor, suggesting that the enhanced functional property of engineered PDL constructs could be achieved with this technique., (© 2011 John Wiley & Sons A/S.)

Muenke syndrome caused by the FGFR3(P250R) mutation is an autosomal dominant disorder mostly identified with coronal suture synostosis, but it also presents with other craniofacial phenotypes that include mild to moderate midface hypoplasia. The Muenke syndrome mutation is thought to dysregulate intramembranous ossification at the cranial suture without disturbing endochondral bone formation in the skull. We show in this study that knock-in mice harboring the mutation responsible for the Muenke syndrome (FgfR3(P244R)) display postnatal shortening of the cranial base along with synchondrosis growth plate dysfunction characterized by loss of resting, proliferating and hypertrophic chondrocyte zones and decreased Ihh expression. Furthermore, premature conversion of resting chondrocytes along the perichondrium into prehypertrophic chondrocytes leads to perichondrial bony bridge formation, effectively terminating the postnatal growth of the cranial base. Thus, we conclude that the Muenke syndrome mutation disturbs endochondral and perichondrial ossification in the cranial base, explaining the midface hypoplasia in patients., (Copyright © 2011 Wiley Periodicals, Inc.)

We report on the novel association of trigonocephaly and polysyndactyly in two unrelated patients due to mutations within the last third (exon 14) and first third (exon 6) of the GLI3 gene, respectively. GLI3 acts as a downstream mediator of the Sonic hedgehog signal-transduction pathway which is essential for early development; and plays a role in cell growth, specialization, and patterning of structures such as the brain and limbs. GLI3 mutations have been identified in patients with Pallister-Hall, Grieg cephalopolysyndactyly syndrome (GCPS), postaxial polydactyly type A1, preaxial polydactyly type IV, and in one patient with acrocallosal syndrome (ACLS). Furthermore, deletions including the GLI3 gene have been reported in patients with features of GCPS and ACLS. To date, trigonocephaly has not been associated with abnormalities of GLI3 and craniosynostosis is not a feature of GCPS. However, Hootnick and Holmes reported on a father with polysyndactyly and son with trigonocephaly, polysyndactyly, and agenesis of the corpus callosum, considered GCPS thereafter. Guzzetta et al. subsequently described a patient with trigonocephaly, polysyndactyly, and agenesis of the corpus callosum postulating a diagnosis of GCPS, later considered ACLS. In retrospect, these two patients, evaluated prior to mutational analysis, and our patients, with confirmed mutations, likely fall within the GLI3 morphopathy spectrum and may provide a bridge to better understanding those patients with overlapping features of GCPS and ACLS. Based on this observation, we suggest GLI3 studies in patients presenting with this constellation of findings, specifically metopic craniosynostosis with polysyndactyly, in order to provide appropriate medical management and genetic counseling., ((c) 2010 Wiley-Liss, Inc.)

Craniometaphyseal dysplasia is caused by mutations in ANKH (ankylosis, progressive homolog [mouse]) in the majority of cases, and all of the reported mutations are single amino acid changes. Genomic DNA from an affected patient, his biological parents, and a sibling was amplified and ANKH was sequenced. The affected patient had a complex heterozygous mutation in exon 7 (c.936T > C, c.938C > G, c.942&#95;953delTGGTTGACGGAA), predicting p.Try290Gln and p.Trp292&#95;Glu295del. We studied the effect of the predicted mutation on the subcellular distribution of ANKH protein. Immunofluorescent labeling of COS-7 cells transduced with normal or mutant Ank (murine progressive ankylosis), showed that normal Ank localized to both the plasma membrane and cytoplasm, whereas mutant Ank was detected only in the cytoplasmic compartment. We propose that this craniometaphyseal dysplasia mutation causes a loss of ANKH protein expression and activity in the plasma membrane as a result of aberrant intracellular protein trafficking., ((c) 2010 Wiley-Liss, Inc.)

Mutations in fibroblast growth factor receptor 2 (FGFR2), a transmembrane receptor expressed in suture mesenchyme, osteogenic fronts, and dura, have been implicated in the etiopathogenesis of craniosynostosis syndromes. The C278F- and P253R-FGFR2 mutations result in Crouzon and Apert syndromes, respectively. The dura mater plays a critical role in the formation and maintenance of cranial sutures. However, its role in syndromic craniosynostosis remains unclear. This study examines the influence of FGFR2 mutations in dural cells on osteoblast proliferation and differentiation. Primary cultures of dural cells and osteoblasts were established, and adenoviral-FGFR2 constructs were prepared by subcloning mutant (C278F and P253R) FGFR2 constructs into adenovirus. Dural cells were infected with adenovirus, and dural protein expression was confirmed by immunostaining. Infected dural cells were cocultured with osteoblasts using a transwell system for 7 days. Dural cells infected with null adenovirus served as the negative control. In separate cultures, osteoblasts were directly infected with the adenoviral-FGFR2 constructs. Osteoblast proliferation was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and differentiation was analyzed by alkaline phosphatase assay, histochemical staining, and gene expression studies. Osteoblasts directly infected with the Crouzon (C278F-FGFR2) mutation demonstrated an increase in cell proliferation (P < 0.05). Osteoblasts directly infected with the Apert (P253R-FGFR2) mutation demonstrated an increase in alkaline phosphatase activity. In coculture experiments, osteoblasts cocultured with Crouzon-transformed dural cells demonstrated increased cell proliferation (P < 0.05), whereas osteoblasts cocultured with Apert-transformed dural cells showed an increase in alkaline phosphatase activity (P < 0.05). In addition, osteogenic gene expression (alkaline phosphatase, osteopontin, and runx2) were up-regulated in osteoblasts cocultured with Apert-expressing dural cells. These experiments suggest that FGFR2 mutations in dural cells alter normal dural signaling. Apert mutations promote osteodifferentiation, whereas Crouzon mutations result in enhanced cell proliferation. These mutations may induce craniosynostosis in part through the influence of mutation-induced constitutive signaling in the dura, with subsequent enhancement of dural-mediated osteogenesis.

Objective: To identify, in a non-hypothesis manner, novel genetic factors associated with nonsyndromic cleft lip with or without cleft palate (NSCL/P)., Study Design: We performed a genome-wide association study in a pediatric cohort of European decent consisting of 111 NSCL/P cases and 5951 control subjects. All subjects were consecutively recruited from the Greater Philadelphia area from 2006 to 2009. High throughput genome-wide single nucleotide polymorphism genotyping was carried out with the Illumina Infinium II HumanHap550 BeadChip technology., Results: We observed association at the genome-wide significance level with SNP rs987525 at a locus on 8q24, which harbors no characterized genes to date (P = 9.18 x 10(-8); odds ratio = 2.09, 95% confidence interval = 1.59 to 2.76). While searching for a replication cohort, the same genetic determinant was established through a genome-wide association study of NSCL/P in Germany, so this previous report acts as a de novo replication for our independent observation outlined here., Conclusions: These results strongly suggest that a locus on 8q24 is involved in the pathogenesis of NSCL/P.

Background: Infant dura mater plays a critical role in calvarial development. This investigation examines the expression of nitric oxide synthase isoforms in the craniofacial skeleton and the influence of nitric oxide signaling on the growth and differentiation of fetal dural and calvarial bone cells., Methods: Sections of fetal and adult calvaria were evaluated for endothelial and inducible nitric oxide synthase expression by immunohistochemistry. Primary fetal (E18) murine dural cell and calvarial osteoblast cultures were treated with 1 microM or 10 microM DETA-NONOate, a nitric oxide donor compound, or 1 mM N-monomethyl-l-arginine (l-NMMA), a nitric oxide synthase inhibitor. Controls were left untreated. Cell proliferation was measured at 48 hours, and mRNA transcripts for Runx2, alkaline phosphatase, and osteopontin were measured by reverse transcription and quantitative real-time polymerase chain reaction at 2 to 18 days. Experiments were performed in triplicate., Results: Fetal, but not adult, dural cells express endothelial nitric oxide synthase. DETA-NONOate stimulated osteoblast mitogenesis by 16 percent (p < 0.05) but did not affect proliferation of dural cells. l-NMMA inhibited proliferation of dural cells and calvarial osteoblasts by 35 percent (p < 0.01) and 17 percent (p = 0.05), respectively. Exogenous nitric oxide increased dural cell transcription of Runx2, alkaline phosphatase (p = 0.03), and osteopontin (p = 0.09) and calvarial osteoblast transcription of Runx2 (p = 0.02) and osteopontin (p < 0.01). Fetal calvarial osteoblasts and dural cells treated with l-NMMA demonstrated reduced transcription of Runx2 and alkaline phosphatase (p < 0.05)., Conclusions: Fetal dural cells and calvarial osteoblasts express endothelial nitric oxide synthase. Nitric oxide enhances proliferation and differentiation of fetal dural cells and calvarial osteoblasts. These results suggest that endothelial nitric oxide synthase-derived nitric oxide may play an important role in development of the fetal craniofacial skeleton.

Background: The higher prevalence of metopic and sagittal suture synostosis in male infants suggests a role for androgens in early craniofacial development. These experiments characterize the influence of androgen stimulation on growth and differentiation of fetal dural and calvarial bone cells and on cranial suture fusion., Methods: Primary murine fetal (E18) dural cells and calvarial osteoblasts were isolated and cultured. Cells were treated for 48 hours with 5alpha-dihydrotestosterone (0 to 1000 nM). Cell proliferation was examined by nonradioactive proliferation assay; mRNA expression of alkaline phosphatase, transforming growth factor (TGF)-beta1, and the bone matrix proteins osteopontin, osteocalcin, and type 1 collagen was determined by reverse-transcriptase polymerase chain reaction. In separate experiments, intact fetal calvariae were grown in tissue culture with 10 nM 5alpha-dihydrotestosterone for 7 and 14 days and then examined histologically., Results: Androgen stimulation at 5 nM increased proliferation of fetal dural cells by 46.0 percent and of fetal calvarial osteoblasts by 20.5 percent. Dural expression of osteopontin, osteocalcin, and type 1 collagen was enhanced by 5alpha-dihydrotestosterone, as was that of TGF-beta1 and alkaline phosphatase. Androgen stimulation increased calvarial osteoblast expression of alkaline phosphatase and TGF-beta1 but induced little change in expression of osteocalcin, osteopontin, and type 1 collagen. In tissue culture, 5alpha-dihydrotestosterone stimulated osteoid formation and fusion of sagittal sutures., Conclusions: Androgen stimulation of dural cells and osteoblasts isolated from fetal calvaria promotes cell proliferation and osteoblastic differentiation and can induce cranial suture fusion. These results suggest that sex steroid hormone signaling may stimulate sutural osteogenesis by means of osteodifferentiation of dural cells, thus explaining the male prevalence of nonsyndromic craniosynostosis.

Mutations in the Notch1 receptor and delta-like 3 (Dll3) ligand cause global disruptions in axial segmental patterning. Genetic interactions between members of the notch pathway have previously been shown to cause patterning defects not observed in single gene disruptions. We examined Dll3-Notch1 compound mouse mutants to screen for potential gene interactions. While mice heterozygous at either locus appeared normal, 30% of Dll3-Notch1 double heterozygous animals exhibited localized, segmental anomalies similar to human congenital vertebral defects. Unexpectedly, double heterozygous mice also displayed statistically significant reduction of mandibular height and decreased length of the [corrected] maxillary hard palate. Examination of somite-stage embryos and perinatal anatomy and histology did not reveal any organ defects, so we used microarray-based analysis of Dll3 and Notch1 mutant embryos to identify gene targets that may be involved in notch-regulated segmental or craniofacial development. Thus, Dll3-Notch1 double heterozygous mice model human congenital scoliosis and craniofacial disorders., (2007 Wiley-Liss, Inc.)

Background: Proteins within the transforming growth factor (TGF)-beta family play a central role in both normal and pathologic calvarial morphogenesis. Previous work has suggested differential functions of the TGF-beta isoforms in these processes. Little is known, however, about effects of TGF-betas on the underlying dura. Furthermore, studies on the effects of TGF-beta isoforms on osteoblasts have been conflicting. The purpose of this study was to determine the effect of TGF-beta isoforms, specifically TGF-beta1 and TGF-beta3, on fetal calvarial osteoblast and dural cell differentiation, proliferation, and apoptosis., Methods: Primary cultures of fetal calvarial osteoblasts and dural cells were established from embryonic day-18 CD-1 mice. Cells were treated for 48 hours with TGF-beta1 or TGF-beta3. Northern blot analysis, cell counts, and apoptosis assays were performed., Results: In dural cells, TGF-beta1 stimulated the expression of early osteodifferentiation genes and resulted in a slight decrease in cell number and no effect on apoptosis. Similar results were observed in osteoblasts. TGF-beta3 had little or no effect on the genes studied in both cell types but resulted in increased apoptosis and concomitant decreases in cell number in both cell types., Conclusions: This study demonstrates that dural cells respond to TGF-beta and that this response is isoform-specific. TGF-beta1 stimulates osteodifferentiation of previously uncommitted cells in the dura. It also stimulates early events in bone matrix deposition and has little effect on late markers of bone differentiation in osteoblasts and dural cells. Both isoforms result in decreases in cell number. TGF-beta3 results in greater decreases in cell number and isoform-specific stimulation of apoptosis in both dural cells and calvarial osteoblasts.

Calvarial bone is formed by the intramembranous bone-forming process, which involves many signaling molecules. The constitutive activation of the fibroblast growth factor (FGF) signaling pathway accelerates osteoblast differentiation and results in premature cranial suture closure. Bone morphogenetic protein (BMP) signaling pathways, which involve the downstream transcription factors Dlx5 and Msx2, are also involved in the bone-forming processes. However, the relationships between these two main signaling cascades are still unclear. We found that FGF2 treatment of developing bone fronts stimulated Bmp2 gene expression but that BMP2 treatment could not induce Fgf2 expression. Moreover, the disruption of the Runx2 gene completely eliminated the expression of Bmp2 and its downstream genes Dlx5 and Msx2 in the developing primordium of bone, while the expression of Fgf2 was maintained. In addition, cultured Runx2-/- cells expressed very low baseline levels of Bmp2 that were up-regulated by transfection with a Runx2-expressing plasmid. These levels in turn were markedly elevated by FGF2 treatment. FGF2 treatment also strongly enhanced the Bmp2 expression in MC3T3-E1 cells, whose endogenous Runx2 gene is intact and which express Bmp2 at low baseline levels as well. These results indicate that Runx2 is an important mediator of the expression of Bmp2 in response to FGF stimulation in cranial bone development., (Copyright 2005 Wiley-Liss, Inc.)

Systemically administered fibroblast growth factors (FGFs) show anabolic effects on bone formation in animals, whereas in vitro cell culture studies have demonstrated that FGFs block mineralized bone nodule formation. These apparently contradictory outcomes indicate that the nature of FGF action is complex and that the biological effect of FGFs may depend on the differentiation stage of osteoblasts, interaction with other cytokines, or the length and mode of exposure to factors. Thus, we have utilized primary calvarial bone cell populations at different maturation phases to determine their responses to 2, FGF-9, and BMP-2, the factors expressed in bone. FGF-2 and FGF-9 stimulated proliferation of the cell populations consisting of more mature osteoblasts, but not those with undifferentiated precursor cells. Continuous treatment with FGF-2/-9 inhibited expression of several osteoblast marker genes and mineralization. However, brief pretreatment with FGF-2/-9 or sequential treatment with FGF-2/-9 followed by BMP-2 led to marked stimulation of mineralization, suggesting that FGFs enhance the intrinsic osteogenic potential. Furthermore, FGF-2 and FGF-9 increased expression of other osteogenic factors BMP-2 and TGFbeta-1. Meanwhile, blocking endogenous FGF signaling, using a virally transduced dominant-negative FGF receptor (FgfR), resulted in drastically reduced expression of the BMP-2 gene, demonstrating for the first time that endogenous FGF/FgfR signaling is a positive upstream regulator of the BMP-2 gene in calvarial osteoblasts. In contrast, expression of a BMP antagonist noggin was inhibited by FGF-2 and FGF-9. Thus, collective data from this study suggest that FGF/FgfR signaling enhances the intrinsic osteogenic potential by selectively expanding committed osteogenic cell populations as well as inversely regulating BMP-2 and noggin gene expression.

Growth and development is the net result of environmental modulation of genetic inheritance. Mesenchymal cells differentiate into chondrogenic, osteogenic, and fibrogenic cells: the first 2 are chiefly responsible for endochondral ossification, and the last 2 for sutural growth. Cells are influenced by genes and environmental cues to migrate, proliferate, differentiate, and synthesize extracellular matrix in specific directions and magnitudes, ultimately resulting in macroscopic shapes such as the nose and the chin. Mechanical forces, the most studied environmental cues, readily modulate bone and cartilage growth. Recent experimental evidence demonstrates that cyclic forces evoke greater anabolic responses of not only craniofacial sutures, but also cranial base cartilage. Mechanical forces are transmitted as tissue-borne and cell-borne mechanical strain that in turn regulates gene expression, cell proliferation, differentiation, maturation, and matrix synthesis, the totality of which is growth and development. Thus, hereditary and mechanical modulations of growth and development share a common pathway via genes. Combined approaches using genetics, bioengineering, and quantitative biology are expected to bring new insight into growth and development, and might lead to innovative therapies for craniofacial skeletal dysplasia including malocclusion, dentofacial deformities, and craniofacial anomalies such as cleft palate and craniosynostosis, as well as disorders associated with the temporomandibular joint.

Various activating mutations of FgfR2 have been linked to a number of craniosynostosis syndromes, suggesting that FGFR2-mediated signaling plays significant roles in intramembranous bone formation. To define (i) the roles of FGFR2-mediated signaling in osteogenesis and (ii) bone cell functions affected by abnormal signaling induced by craniosynostosis mutations, chicken calvarial osteoblasts were infected with replication competent avian sarcoma viruses expressing FgfR2 with dominant negative (DN), P253R (Apert), or C278F (Pfeiffer and Crouzon) mutation. Analyses of the infected osteoblasts revealed that attenuated FGF/FGFR signaling by DN-FgfR2 resulted in a decrease in cell proliferation and accelerated mineralization. In contrast, the C278F mutation, which causes ligand-independent activation of the receptor, significantly stimulated cell proliferation and inhibited mineralization. Interestingly, the P253R mutation, which does not cause ligand-independent activation of the receptor, showed a weaker mitogenic effect than the C278F mutation and did not inhibit mineralization. Gene expression analysis also revealed diverse effects of C278F and P253R mutations on expression of several osteogenic genes. Based on these results, we conclude that one of the major functions of FGFR2 is to mediate mitogenic signals in osteoblasts and that distinctively different cellular mechanisms underlie the pathogenesis of craniosynostosis phenotypes resulting from P253R and C278F mutations of the FGFR2 gene.

Type IIA procollagen is an alternatively spliced product of the type II collagen gene and uniquely contains the cysteine (cys)-rich globular domain in its amino (N)-propeptide. To understand the function of type IIA procollagen in cartilage development under normal and pathologic conditions, the detailed expression pattern of type IIA procollagen was determined in progressive stages of development in embryonic chicken limb cartilages (days 5-19) and in human adult articular cartilage. Utilizing the antibodies specific for the cys-rich domain of the type IIA procollagen N-propeptide, we localized type IIA procollagen in the pericellular and interterritorial matrix of condensing pre-chondrogenic mesenchyme (day 5) and early cartilage (days 7-9). The intensity of immunostaining was gradually lost with cartilage development, and staining became restricted to the inner layer of perichondrium and the articular cap (day 12). Later in development, type IIA procollagen was re-expressed at the onset of cartilage hypertrophy (day 19). Different from type X collagen, which is expressed throughout hypertrophic cartilage, type IIA procollagen expression was transient and restricted to the zone of early hypertrophy. Immunoelectron microscopic and immunoblot analyses showed that a significant amount of the type IIA procollagen N-propeptide, but not the carboxyl (C)-propeptide, was retained in matrix collagen fibrils of embryonic limb cartilage. This suggests that the type IIA procollagen N-propeptide plays previously unrecognized roles in fibrillogenesis and chondrogenesis. We did not detect type IIA procollagen in healthy human adult articular cartilage. Expression of type IIA procollagen, together with that of type X collagen, was activated by articular chondrocytes in the upper zone of moderately and severely affected human osteoarthritic cartilage, suggesting that articular chondrocytes, which normally maintain a stable phenotype, undergo hypertrophic changes in osteoarthritic cartilage. Based on our data, we propose that type IIA procollagen plays a significant role in chondrocyte differentiation and hypertrophy during normal cartilage development as well as in the pathogenesis of osteoarthritis., (Copyright 2001 Wiley-Liss, Inc.)

Annexins II, V, and VI are major components of matrix vesicles (MV), i.e. particles that have the critical role of initiating the mineralization process in skeletal tissues. Furthermore, types II and X collagen are associated with MV, and these interactions mediated by annexin V stimulate Ca(2+) uptake and mineralization of MV. However, the exact roles of annexin II, V, and VI and the interaction between annexin V and types II and X collagen in MV function and initiation of mineralization are not well understood. In this study, we demonstrate that annexin II, V, or VI mediate Ca(2+) influx into phosphatidylserine (PS)-enriched liposomes, liposomes containing lipids extracted from authentic MV, and intact authentic MV. The annexin Ca(2+) channel blocker, K-201, not only inhibited Ca(2+) influx into fura-2-loaded PS-enriched liposomes mediated by annexin II, V, or VI, but also inhibited Ca(2+) uptake by authentic MV. Types II and X collagen only bound to liposomes in the presence of annexin V but not in the presence of annexin II or VI. Binding of these collagens to annexin V stimulated its Ca(2+) channel activities, leading to an increased Ca(2+) influx into the liposomes. These findings indicate that the formation of annexin II, V, and VI Ca(2+) channels in MV together with stimulation of annexin V channel activity by collagen (types II and X) binding can explain how MV are able to rapidly take up Ca(2+) and initiate the formation of the first crystal phase.

Calvarial and facial bones form by intramembranous ossification, in which bone cells arise directly from mesenchyme without an intermediate cartilage anlage. However, a number of studies have reported the emergence of chondrocytes from in vitro calvarial cell or organ cultures and the expression of type II collagen, a cartilage-characteristic marker, in developing calvarial bones. Based on these findings we hypothesized that a covert chondrogenic phase may be an integral part of the normal intramembranous pathway. To test this hypothesis, we analyzed the temporal and spatial expression patterns of cartilage characteristic genes in normal membranous bones from chick embryos at various developmental stages (days 12, 15 and 19). Northern and RNAse protection analyses revealed that embryonic frontal bones expressed not only the type I collagen gene but also a subset of cartilage characteristic genes, types IIA and XI collagen and aggrecan, thus resembling a phenotype of prechondrogenic-condensing mesenchyme. The expression of cartilage-characteristic genes decreased with the progression of bone maturation. Immunohistochemical analyses of developing embryonic chick heads indicated that type II collagen and aggrecan were produced by alkaline phosphatase activity positive cells engaged in early stages of osteogenic differentiation, such as cells in preosteogenic-condensing mesenchyme, the cambium layer of periosteum, the advancing osteogenic front, and osteoid bone. Type IIB and X collagen messenger RNAs (mRNA), markers for mature chondrocytes, were also detected at low levels in calvarial bone but not until late embryonic stages (day 19), indicating that some calvarial cells may undergo overt chondrogenesis. On the basis of our findings, we propose that the normal intramembranous pathway in chicks includes a previously unrecognized transient chondrogenic phase similar to prechondrogenic mesenchyme, and that the cells in this phase retain chondrogenic potential that can be expressed in specific in vitro and in vivo microenvironments.

Chicken alpha1(V) collagen cDNAs have been cloned by a variety of methods and positively identified. We present here the entire translated sequence of the chick polypeptide and compare selected regions to other collagen chains in the type V/XI family.

The mechanism(s) by which the cells within the calvaria tissue are restricted into the osteogenic versus the chondrogenic lineage during intramembranous bone formation were examined. Cells were obtained from 12-day chicken embryo calvariae after tissue condensation, but before extensive osteogenic differentiation, and from 17-day embryo calvariae when osteogenesis is well progressed. Only cell populations from the younger embryos showed chondrogenic differentiation as characterized by the expression of collagen type II. The chondrocytes underwent a temporal progression of maturation and endochondral development, demonstrated by the expression of collagen type II B transcript and expression of collagen type X mRNA. Cell populations from both ages of embryos showed progressive osteogenic differentiation, based on the expression of osteopontin, bone sialoprotein, and osteocalcin mRNAs. Analysis using lineage markers for either chondrocytes or osteoblasts demonstrated that when the younger embryonic cultures were grown in conditions that were permissive for chondrogenesis, the number of chondrogenic cells increased from approximately 15 to approximately 50% of the population, while the number of osteogenic cells remained almost constant at approximately 35-40%. Pulse labeling of the cultures with BrdU showed selective labeling of the chondrogenic cells in comparison with the osteogenic cells. These data indicate that the developmental restriction of skeletal cells of the calvaria is not a result of positive selection for osteogenic differentiation but a negative selection against the progressive growth of chondrogenic cells in the absence of a permissive or inductive environment. These results further demonstrate that while extrinsic environmental factors can modulate the lineage progression of skeletal cells within the calvariae, there is a progressive restriction during embryogenesis in the number of cells within the calvaria with a chondrogenic potential. Finally, these data suggest that the loss of cells with chondrogenic potential from the calvaria may be related to the progressive limitation of the reparative capacity of the cranial bones.