Pthlha and mechanical force control early patterning of growth zones in the zebrafish craniofacial skeleton

Secreted signals in patterning systems often induce repressive signals that shape their distributions in space and time. In developing growth plates (GPs) of endochondral long bones, Parathyroid hormone-like hormone (Pthlh) inhibits Indian hedgehog (Ihh) to form a negative feedback loop that controls GP progression and bone size. Whether similar systems operate in other bones and how they arise during embryogenesis remain unclear. We show that Pthlha expression in the zebrafish craniofacial skeleton precedes chondrocyte differentiation and restricts where cells undergo hypertrophy, thereby initiating a future GP. Loss of Pthlha leads to an expansion of cells expressing a novel early marker of the hypertrophic zone (HZ), entpd5a, and later HZ markers such as ihha, while local Pthlha misexpression induces ectopic entpd5a expression. Formation of this early pre-HZ correlates with onset of muscle contraction and requires mechanical force; paralysis leads to loss of entpd5a and ihha expression in the pre-HZ, mislocalized pthlha expression, and no subsequent ossification. These results suggest that local Pthlh sources combined with force determine HZ locations, establishing the negative feedback loop that later maintains GPs.

Self-Reported Ancestry and Craniofacial SNPs

Genotype-phenotype studies increasingly link single nucleotide polymorphism (SNPs) to the dimensions of the face for presumed homogeneous populations. To appreciate the significance of these findings, it is essential to investigate how these results differ between the genetic and phenotypic profiles of individuals. In prior work, we investigated the connection between SNPs previously identified as informative of soft tissue expression and measurements of the craniofacial skeleton. Using matched genetic and skeletal information on 17 individuals who self-identified as White with presumed common continental ancestry (European), we obtained significant Spearman correlations for 11 SNPs. In the present study, we looked at self-identified ancestry to understand the intersectional background of the individual’s phenotype and genotype. We integrated our samples within a diverse dataset of 2,242 modern Americans and applied an unsupervised model-based clustering routine to 13 craniometrics. We generated a mean estimate of 69.65% (±SD = 18%) European ancestry for the White sample under an unsupervised cluster model. We estimated higher quantities of European ancestry, 88.5%–93%, for our subset of 17 individuals. These elevated estimates were of interest with respect to the distribution of population-informative SNPs; we found, for example, that one of our sampled self-identified White individuals displayed SNPs commonly associated with Latin American populations. These results underscore the complex interrelationship between environment and genetics, and the need for continued research into connections between population affinity, social identity, and morphogenetic expression.

Basic Biomechanics for Craniofacial Surgeons: The Responses of Alloplastic Materials and Living Tissues to Mechanical Forces

Many terms such as twist, compress, bend, and stretch, describe how materials behave when subjected to mechanical stresses. Subjective adjectives to describe the property of materials such as hard or brittle are imprecise and impedes proper understanding of important principles needed in planning and performing surgical treatments. The viability of tissue and time dependent variables effect healing and compound the issue. Some parameters are time dependent (strain rate), while others are nearly independent of time (Young’s modulus). The craniofacial skeleton and enveloping soft tissues are viscoelastic composite materials which undergo time-dependent changes upon loading. The ability to remodel and respond to environmental changes makes them “smart,” reenforcing where needed and removing where not required based on a set of predetermined upper and lower thresholds. This mini review has 7 sections on engineering principles that underpin craniofacial surgery: (1) The general concept of mechanics: load, force, stress, strain, compression, tension, shear, stress-strain curves and values derived from them such as Young’s modulus, fatigue damage, and load- shearing. (2) Material properties of bone and suture and structural engineering of the craniofacial skeleton in normal and pathological conditions. (3) Fixation using wires, screws, and plates: anatomy and function of screws and plates, locking plates, lag screws, internal and external fixators. (4) Biomechanics of distraction osteogenesis and the effects of radiation. (5) Finite element analysis and other computational biomechanical tools. (6) Virtual surgical planning, cutting guides, and intra-operative navigation. (7) Tissue engineering: design goals, criteria, and constraints. An appreciation and understanding of these biomechanical principles will help craniofacial surgeons to facilitate intrinsic optimization and better treat complex morphological problems, helping one achieve the most favorable and durable results. The biological responses to mechanical stress are extremely important as well, but due to space constraints, they will be the subject of a separate dedicated review.

Mosmo Is Required for Zebrafish Craniofacial Formation

Hedgehog (Hh) signaling is a highly regulated molecular pathway implicated in many developmental and homeostatic events. Mutations in genes encoding primary components or regulators of the pathway cause an array of congenital malformations or postnatal pathologies, the extent of which is not yet fully defined. Mosmo (Modulator of Smoothened) is a modulator of the Hh pathway, which encodes a membrane tetraspan protein. Studies in cell lines have shown that Mosmo promotes the internalization and degradation of the Hh signaling transducer Smoothened (Smo), thereby down-modulating pathway activation. Whether this modulation is essential for vertebrate embryonic development remains poorly explored. Here, we have addressed this question and show that in zebrafish embryos, the two mosmo paralogs, mosmoa and mosmob, are expressed in the head mesenchyme and along the entire ventral neural tube. At the cellular level, Mosmoa localizes at the plasma membrane, cytoplasmic vesicles and primary cilium in both zebrafish and chick embryos. CRISPR/Cas9 mediated inactivation of both mosmoa and mosmob in zebrafish causes frontonasal hypoplasia and craniofacial skeleton defects, which become evident in the adult fish. We thus suggest that MOSMO is a candidate to explain uncharacterized forms of human congenital craniofacial malformations, such as those present in the 16p12.1 chromosomal deletion syndrome encompassing the MOSMO locus.

Management of Panfacial Trauma: Sequencing and Pitfalls

Panfacial trauma refers to high-energy mechanism injuries involving two or more areas of the craniofacial skeleton, the frontal bone, the midface, and the occlusal unit. These can be distracting injuries in an unstable patient and, as in any trauma, Advanced Trauma Life Support (ATLS) protocols should be followed. The airway should be secured, bleeding controlled, and sequential examinations should take place to avoid overlooking injuries. When indicated, neurosurgery and ophthalmology should be consulted as preservation of brain, vision, and hearing function should be prioritized. Once the patient is stabilized, reconstruction aims to reduce panfacial fractures, restore the horizontal and vertical facial buttresses, and resuspend the soft tissue to avoid the appearance of premature aging. Lost or comminuted bone can be replaced with bone grafts, although adequate reduction should be ensured prior to any grafting. Operative sequencing can be performed from top-down and outside-in or from bottom-up and inside-out depending on patient presentation. All protocols can successfully manage panfacial injuries, and the emphasis should be placed on a systematic approach that works from known areas to unknown areas.

Mandibular osteoma as a cause of ankylosis and progressive trismus

Osteomas are benign tumours of bone tissue restricted to the craniofacial skeleton. The aim of this article is to present and discuss the demographic and clinical aspects and the management of craniomaxillofacial osteomas. When the patient was submitted from primary care to our hospital, he was 68 years old, and he had ankylosis of the temporomandibular joint for the previos 4 years. A CT scan was performed, finding a giant mandibular osteoma. Conservative treatment and radiological follow-up were carried out with clinical stability. Osteomas more often are seen in the paranasal sinuses and in young adults, with no differences in gender. Most are asymptomatic, but they can cause local problems. For its diagnosis, CT is usually performed. Treatment options are conservative management and follow-up or surgery. Although rarely, they can recur. Mandibular peripheral osteoma is a rare entity. Depending on the symptoms, a conservative or surgical treatment can be chosen. A clinical and radiological follow-up is necessary to detect possible recurrences or enlargement.

An efficient miRNA knockout approach using CRISPR-Cas9 in Xenopus

In recent years CRISPR-Cas9 knockouts (KO) have become increasingly ultilised to study gene function. MicroRNAs (miRNAs) are short non-coding RNAs, 20-22 nucleotides long, which affect gene expression through post-transcriptional repression. We previously identified miRNAs-196a and -219 as implicated in the development of Xenopus neural crest (NC). The NC is a multipotent stem-cell population, specified during early neurulation. Following EMT NC cells migrate to various points in the developing embryo where they give rise to a number of tissues including parts of the peripheral nervous system and craniofacial skeleton. Dysregulation of NC development results in many diseases grouped under the term neurocristopathies. As miRNAs are so small it is difficult to design CRISPR sgRNAs that reproducibly lead to a KO. We have therefore designed a novel approach using two guide RNAs to effectively drop out a miRNA. We have knocked out miR-196a and miR-219 and compared the results to morpholino knockdowns (KD) of the same miRNAs. Validation of efficient CRISPR miRNA KO and phenotype analysis included use of whole-mount in situ hybridization of key NC and neural plate border markers such as Pax3, Xhe2, Sox10 and Snail2, q-RT-PCR and Sanger sequencing. miRNA-219 and miR-196a KOs both show loss of NC, altered neural plate and hatching gland phenotypes. Tadpoles show gross craniofacial and pigment phenotypes.