MiR-26b-5p/TET3 regulates the osteogenic differentiation of human bone mesenchymal stem cells and bone reconstruction in female rats with calvarial defects.

BACKGROUND: MicroRNAs (miRNAs) play critical roles in the osteogenic differentiation of human bone mesenchymal stem cells (hBMSCs), but the mechanism by which miRNAs indirectly modulate osteogenesis remains unclear. Here, we explored the mechanism by which miRNAs indirectly modulate gene expression through histone demethylases to promote bone regeneration. METHODS AND RESULTS: Bioinformatics analysis was performed on hBMSCs after 7 days of osteogenic induction. The differentially expressed miRNAs were screened, and potential target mRNAs were identified. To determine the bioactivity and stemness of hBMSCs and their potential for bone repair, we performed wound healing, Cell Counting Kit-8 (CCK-8), real-time reverse transcription quantitative polymerase chain reaction (RT‒qPCR), alkaline phosphatase activity, alizarin red S (ARS) staining and radiological and histological analyses on SD rats with calvarial bone defects. Additionally, a dual-luciferase reporter assay was utilized to investigate the interaction between miR-26b-5p and ten-eleven translocation 3 (TET3) in human embryonic kidney 293T cells. The in vitro and in vivo results suggested that miR-26b-5p effectively promoted the migration, proliferation and osteogenic differentiation of hBMSCs, as well as the bone reconstruction of calvarial defects in SD rats. Mechanistically, miR-26b-5p bound to the 3' untranslated region of TET3 mRNA to mediate gene silencing. CONCLUSIONS: MiR-26b-5p downregulated the expression of TET3 to increase the osteogenic differentiation of hBMSCs and bone repair in rat calvarial defects. MiR-26b-5p/TET3 crosstalk might be useful in large-scale critical bone defects.

Fluorinated hydroxyapatite conditions a favorable osteo-immune microenvironment via triggering metabolic shift from glycolysis to oxidative phosphorylation.

BACKGROUND: Biological-derived hydroxyapatite is widely used as a bone substitute for addressing bone defects, but its limited osteoconductive properties necessitate further improvement. The osteo-immunomodulatory properties hold crucial promise in maintaining bone homeostasis, and precise modulation of macrophage polarization is essential in this process. Metabolism serves as a guiding force for immunity, and fluoride modification represents a promising strategy for modulating the osteoimmunological environment by regulating immunometabolism. In this context, we synthesized fluorinated porcine hydroxyapatite (FPHA), and has demonstrated its enhanced biological properties and osteogenic capacity. However, it remains unknown whether and how FPHA affects the immune microenvironment of the bone defects. METHODS: FPHA was synthesized and its composition and structural properties were confirmed. Macrophages were cultured with FPHA extract to investigate the effects of FPHA on their polarization and the related osteo-immune microenvironment. Furthermore, total RNA of these macrophages was extracted, and RNA-seq analysis was performed to explore the underlying mechanisms associated with the observed changes in macrophages. The metabolic states were evaluated with a Seahorse analyzer. Additionally, immunohistochemical staining was performed to evaluate the macrophages response after implantation of the novel bone substitutes in critical size calvarial defects in SD rats. RESULTS: The incorporation of fluoride ions in FPHA was validated. FPHA promoted macrophage proliferation and enhanced the expression of M2 markers while suppressing the expression of M1 markers. Additionally, FPHA inhibited the expression of inflammatory factors and upregulated the expression of osteogenic factors, thereby enhancing the osteogenic differentiation capacity of the rBMSCs. RNA-seq analysis suggested that the polarization-regulating function of FPHA may be related to changes in cellular metabolism. Further experiments confirmed that FPHA enhanced mitochondrial function and promoted the metabolic shift of macrophages from glycolysis to oxidative phosphorylation. Moreover, in vivo experiments validated the above results in the calvarial defect model in SD rats. CONCLUSION: In summary, our study reveals that FPHA induces a metabolic shift in macrophages from glycolysis to oxidative phosphorylation. This shift leads to an increased tendency toward M2 polarization in macrophages, consequently creating a favorable osteo-immune microenvironment. These findings provide valuable insights into the impact of incorporating an appropriate concentration of fluoride on immunometabolism and macrophage mitochondrial function, which have important implications for the development of fluoride-modified immunometabolism-based bone regenerative biomaterials and the clinical application of FPHA or other fluoride-containing materials.

[Topographic study of the parietal region for the elaboration of revascularized cranium vault autograft]. / Izuchenie topografo-anatomicheskikh osobennostei temennoi oblasti pri razrabotke revaskulyariziruemogo kostno-fastsial'nogo temennogo autotransplantata.

OBJECTIVE: Topographic and blood vessel architecture study of the parietal area and distal regional pool of the superficial temporal artery (STA) to assess the possibility of revascularized cranium vault bone autograft formation. MATERIAL AND METHODS: For the topographic and anatomical study, 30 non-fixed corpses (17 male and 13 female) were selected, the average age of which was 59±5 years. In the anamnesis and catamnesis, there were no indications of trauma or other pathology of the head and neck, including vascular. STA was contrasted with a non-radiocontrast dye (brilliant green) with the introduction of the dye into the STA with preliminary ligation of the frontal branch of the STA. The area of blood supply to soft tissue and bone structures was studied. The angioarchitectonics of the parietal region was studied, the feeding vessel of the studied flap was identified. RESULTS: The obtained anatomical landmarks for the collection of CPFP flap make it possible to form a flap with high accuracy and minimize the morbidity of the donor area.

Cranial Repair in Children: Techniques, Materials, and Peculiar Issues.

Cranial repair in children deserves particular attention since many issues are still controversial. Furthermore, literature data offer a confused picture of outcome of cranioplasty, in terms of results and complication rates, with studies showing inadequate follow-up and including populations that are not homogeneous by age of the patients, etiology, and size of the bone defect.Indeed, age has merged in the last years as a risk factor for resorption of autologous bone flap that is still the most frequent complication in cranial repair after decompressive craniectomy.Age-related factors play a role also when alloplastic materials are used. In fact, the implantation of alloplastic materials is limited by skull growth under 7 years of age and is contraindicated in the first years if life. Thus, the absence of an ideal material for cranioplasty is even more evident in children with a steady risk of complications through the entire life of the patient that is usually much longer than surgical follow-up.As a result, specific techniques should be adopted according to the age of the patient and etiology of the defect, aiming to repair the skull and respect its residual growth.Thus, autologous bone still represents the best option for cranial repair, though limitations exist. As an alternative, biomimetic materials should ideally warrant the possibility to overcome the limits of other inert alloplastic materials by favoring osteointegration or osteoinduction or both.On these grounds, this paper aims to offer a thorough overview of techniques, materials, and peculiar issues of cranial repair in children.

Evolutionary trends in the elasmobranch neurocranium.

The neurocranium (braincase) is one of the defining vertebrate characters. Housing the brain and other key sensory organs, articulating with the jaws and contributing to the shape of the anteriormost portion of the body, the braincase is undoubtedly of great functional importance. Through studying relationships between braincase shape and ecology we can gain an improved understanding of form-function relationships in extant and fossil taxa. Elasmobranchii (sharks and rays) represent an important case study of vertebrate braincase diversity as their neurocranium is simplified and somewhat decoupled from other components of the cranium relative to other vertebrates. Little is known about the associations between ecology and braincase shape in this clade. In this study we report patterns of mosaic cranial evolution in Elasmobranchii that differ significantly from those present in other clades. The degree of evolutionary modularity also differs between Selachii and Batoidea. In both cases innovation in the jaw suspension appears to have driven shifts in patterns of integration and modularity, subsequently facilitating ecological diversification. Our results confirm the importance of water depth and biogeography as drivers of elasmobranch cranial diversity and indicate that skeletal articulation between the neurocranium and jaws represents a major constraint upon the evolution of braincase shape in vertebrates.

Evaluation and 3D imaging of mineral structure changes of the rabbit (New Zealand) skull during developmental periods.

This study aimed to determine the morphometric measurements anatomically and CT images of skulls of healthy male and female rabbits during postnatal development, to analyse the data statistically and to demonstrate the structural changes in bone. A total of 40 rabbits (20 females and 20 males) were divided into four groups including prepubertal period (group I (0-1 month)), period between adolescence and adulthood (group II (3-5 month)) and later (young adult period as group III (1-3 years) and old adult period as group IV (3-5 years)), with five animals in each group. After the morphometric measurements, the surface area and volume values of the skull were calculated. The skulls were reconstructed using a 3D Slicer (5.0.2), which is used for 3D modelling. The cranial bones in each group were then crushed using a grinder so that the powdered samples were obtained for XRF (X-ray fluorescence technique). The p-value was statistically highly significant between group and gender (p < 0.001). In morphometric measurements, males were generally higher than females. Only PL, GBOC and GNB measurements were higher in females. The p-value between groups (in all measurements), between genders (in TL, GLN, FL, VL, OZB and GBN parameters) and between groups and genders (in TL, DL and VL parameters) was statistically highly significant (p < 0.001). The p-value between the groups, p-value between sexes and p-value between group and sex in Si, P, K, Ca, Ni, Zn, Sr, Sr and Ca/P elements were statistically significant (p < 0.001). Consequently, metric, volume and surface area measurements were taken through 3D modelling of skull bone in prepubertal period (group I), period between adolescence and adulthood (group II) and later (young adult period as group III and old adult period as group IV) of rabbits and the change in the mineral structure during postnatal development and effect of sex on this change were investigated. This might be the first study to assess both metric and mineral changes at four age intervals taken during the life span of rabbits.

Does cranial bone ossification differ in children with developmental dysplasia of the hip?

OBJECTIVES: This study aims to compare cranial bone ossification between patients with developmental dysplasia of the hip (DDH) and healthy individuals. PATIENTS AND METHODS: Between September 2021 and April 2022, a total of 60 healthy female individuals (median age: 24.5 months; range, 18 to 36 months) and 56 female DDH patients (median age: 23 months; range, 18 to 35 months) were included. Age, head circumference, weight, height, and patency of the anterior fontanel were measured in groups. Percentiles were classified as very low, low, normal, high and very high. All patients were female and those with abnormal thyroid function test, vitamin D, calcium, phosphate and alkaline phosphatase values were not included in the study. For those diagnosed with DDH, they were included in the group regardless of the type of treatment. RESULTS: No statistically significant difference was found between the groups in terms of age and weight (p>0.05). The very low and very high head circumferences were more frequent, and the normal head circumferences were less frequent in the DDH group (p<0.05). There was no significant difference between groups in terms of fontanel closure (p>0.05). In open fontanels, no significant difference was found in both groups in terms of age (p>0.05). CONCLUSION: Our study results showed no significant difference between the fontanel ossifications of children with and without DDH; however, we found that the ossification of the skull bones of children with DDH was different compared to healthy children.

Craniofacial identification standards: A review of reliability, reproducibility, and implementation.

There are numerous anatomical and anthropometrical standards that can be utilised for craniofacial analysis and identification. These standards originate from a wide variety of sources, such as orthodontic, maxillofacial, surgical, anatomical, anthropological and forensic literature, and numerous media have been employed to collect data from living and deceased subjects. With the development of clinical imaging and the enhanced technology associated with this field, multiple methods of data collection have become accessible, including Computed Tomography, Cone-Beam Computed Tomography, Magnetic Resonance Imaging, Radiographs, Three-dimensional Scanning, Photogrammetry and Ultrasound, alongside the more traditional in vivo methods, such as palpation and direct measurement, and cadaveric human dissection. Practitioners often struggle to identify the most appropriate standards and research results are frequently inconsistent adding to the confusion. This paper aims to clarify how practitioners can choose optimal standards, which standards are the most reliable and when to apply these standards for craniofacial identification. This paper describes the advantages and disadvantages of each mode of data collection and collates published research to review standards across different populations for each facial feature. This paper does not aim to be a practical instruction paper; since this field encompasses a wide range of 2D and 3D approaches (e.g., clay sculpture, sketch, automated, computer-modelling), the implementation of these standards is left to the individual practitioner.

A xenogeneic extracellular matrix-based 3D printing scaffold modified by ceria nanoparticles for craniomaxillofacial hard tissue regeneration via osteo-immunomodulation.

Hard tissue engineering scaffolds especially 3D printed scaffolds were considered an excellent strategy for craniomaxillofacial hard tissue regeneration, involving crania and facial bones and teeth. Porcine treated dentin matrix (pTDM) as xenogeneic extracellular matrix has the potential to promote the stem cell differentiation and mineralization as it contains plenty of bioactive factors similar with human-derived dentin tissue. However, its application might be impeded by the foreign body response induced by the damage-associated molecular patterns of pTDM, which would cause strong inflammation and hinder the regeneration. Ceria nanoparticles (CNPs) show a great promise at protecting tissue from oxidative stress and influence the macrophages polarization. Using 3D-bioprinting technology, we fabricated a xenogeneic hard tissue scaffold based on pTDM xenogeneic TDM-polycaprolactone (xTDM/PCL) and we modified the scaffolds by CNPs (xTDM/PCL/CNPs). Through series ofin vitroverification, we found xTDM/PCL/CNPs scaffolds held promise at up-regulating the expression of osteogenesis and odontogenesis related genes including collagen type 1, Runt-related transcription factor 2 (RUNX2), bone morphogenetic protein-2, osteoprotegerin, alkaline phosphatase (ALP) and DMP1 and inducing macrophages to polarize to M2 phenotype. Regeneration of bone tissues was further evaluated in rats by conducting the models of mandibular and skull bone defects. Thein vivoevaluation showed that xTDM/PCL/CNPs scaffolds could promote the bone tissue regeneration by up-regulating the expression of osteogenic genes involving ALP, RUNX2 and bone sialoprotein 2 and macrophage polarization into M2. Regeneration of teeth evaluated on beagles demonstrated that xTDM/PCL/CNPs scaffolds expedited the calcification inside the scaffolds and helped form periodontal ligament-like tissues surrounding the scaffolds.

Ontogenetic changes in jaw leverage and skull shape in tufted and untufted capuchins.

The ontogeny of feeding is characterized by shifting functional demands concurrent with changes in craniofacial anatomy; relationships between these factors will look different in primates with disparate feeding behaviors during development. This study examines the ontogeny of skull morphology and jaw leverage in tufted (Sapajus) and untufted (Cebus) capuchin monkeys. Unlike Cebus, Sapajus have a mechanically challenging diet and behavioral observations of juvenile Sapajus suggest these foods are exploited early in development. Landmarks were placed on three-dimensional surface models of an ontogenetic series of Sapajus and Cebus skulls (n = 53) and used to generate shape data and jaw-leverage estimates across the tooth row for three jaw-closing muscles (temporalis, masseter, medial pterygoid) as well as a weighted combined estimate. Using geometric morphometric methods, we found that skull shape diverges early and shape is significantly different between Sapajus and Cebus throughout ontogeny. Additionally, jaw leverage varies with age and position on the tooth row and is greater in Sapajus compared to Cebus when calculated at the permanent dentition. We used two-block partial least squares analyses to identify covariance between skull shape and each of our jaw muscle leverage estimates. Sapajus, but not Cebus, has significant covariance between all leverage estimates at the anterior dentition. Our findings show that Sapajus and Cebus exhibit distinct craniofacial morphologies early in ontogeny and strong covariance between leverage estimates and craniofacial shape in Sapajus. These results are consistent with prior behavioral and comparative work suggesting these differences are a function of selection for exploiting mechanically challenging foods in Sapajus, and further emphasize that these differences appear quite early in ontogeny. This research builds on prior work that has highlighted the importance of understanding ontogeny for interpreting adult morphology.

Two surgical instruments.

The only instruments for opening the cranium considered in this chapter are drills, and in some cases facilitated with a special chisel called a lenticular. There were two kinds of trepan. The modiolus was the Latin name for a crown trepan which had a circular base with teeth which sawed a hole. Then there were the non-penetrating trepans which had a bit shaped to prevent unwanted penetration. They made small openings which could be joined by chisels to remove altogether larger areas of bone than were accessible to modioli. They were the favored instrument from the ancient world up to the Renaissance. At the beginning of the Renaissance, there was a move toward greater use of crown trepans and various methods were applied to stop them sinking too far inward. These included wings in the outer wall and changing the shape of the bit from cylindrical to conic. In time preferences returned to the cylindrical shape and larger diameters. There was also two instruments called lenticulars, the illustrations of which have been confused in the literature. It is now clear that the Roman instrument was shaped to cut the cranium and minimize the need for trepanation. The Renaissance instrument had a different shape and was used to smooth rough bone edges and excise spicules penetrating the meninges. They were simply two different instruments to which the same name was applied.

Modified Mouse Model of Repetitive Mild Traumatic Brain Injury Incorporating Thinned-Skull Window and Fluid Percussion.

Mild traumatic brain injury is a clinically highly heterogeneous neurological disorder. Highly reproducible traumatic brain injury (TBI) animal models with well-defined pathologies are urgently needed for studying the mechanisms of neuropathology after mild TBI and testing therapeutics. Replicating the entire sequelae of TBI in animal models has proven to be a challenge. Therefore, the availability of multiple animal models of TBI is necessary to account for the diverse aspects and severities seen in TBI patients. CHI is one of the most common methods for fabricating rodent models of rmTBI. However, this method is susceptible to many factors, including the impact method used, the thickness and shape of the skull bone, animal apnea, and the type of head support and immobilization utilized. The aim of this protocol is to demonstrate a combination of the thinned-skull window and fluid percussion injury (FPI) methods to produce a precise mouse model of CHI-associated rmTBI. The primary objective of this protocol is to minimize factors that could impact the accuracy and consistency of CHI and FPI modeling, including skull bone thickness, shape, and head support. By utilizing a thinned-skull window method, potential inflammation due to craniotomy and FPI is minimized, resulting in an improved mouse model that replicates the clinical features observed in patients with mild TBI. Results from behavior and histological analysis using hematoxylin and eosin (HE) staining suggest that rmTBI can lead to a cumulative injury that produces changes in both behavior and gross morphology of the brain. Overall, the modified CHI-associated rmTBI presents a useful tool for researchers to explore the underlying mechanisms that contribute to focal and diffuse pathophysiological changes in rmTBI.

Skull asymmetry in various sheep breeds: Directional asymmetry and fluctuating asymmetry.

Sheep (Ovis aries) play an important role in the economy of Turkey and the Balkan Peninsula due to their use in farming. As a domesticated species, sheep's morphometric and morphological diversity is likely determined by selective breeding practices rather than geographic distribution. This study aimed to analyse four different sheep breed skulls and reveal skull asymmetry using geometric morphometric methods. For this purpose, 2D images of 52 sheep skulls from different breeds were analysed from the dorsal view of the skull, using 28 landmarks. In the comparison of sheep skulls from the dorsal view, the first principal components for directional asymmetry (DA) and fluctuating asymmetry (FA) were 32.98% and 39.62% of the total variation, respectively. Sharri and Ivesi (Awassi) sheep breeds had the broadest distribution of skull shapes among the breeds, while Lara e Polisit was the most conservative breed. DA was used as a measure of biomechanical constraints, and FA was used as an indicator of environmental stress. Consistent with DA, both differences in centroid size and shape between breeds were statistically significant. No differences between males and females related to asymmetry were revealed. Ivesi sheep revealed the highest fluctuating asymmetry. Geometric morphometric methods proved to be a useful tool for distinguishing differences in the shape of the skull of different sheep breeds and also can be useful for taxonomic purposes.

Comparative skull osteology of Amphisbaena arda and Amphisbaena vermicularis (Squamata: Amphisbaenidae).

The skull anatomy of amphisbaenians directly influences their capacity to burrow and is crucial for the study of their systematics, which ultimately contributes to our comprehension of their evolution and ecology. In this study, we employed three-dimensional X-ray computed tomography to provide a detailed description and comprehensive comparison of the skull anatomy of two amphisbaenian species with similar external morphology, Amphisbaena arda and Amphisbaena vermicularis. Our findings revealed some differences between the species, especially in the sagittal crest of the parietal bone, the ascendant process, and the transverse occipital crest of the occipital complex. We also found intraspecific variation within A. vermicularis, with some specimens displaying morphology that differed from their conspecifics but not from A. arda. The observed intraspecific variation within A. vermicularis cannot be attributed to soil features because all specimens came from the same locality. Specimen size and soil type may play a role in the observed differences between A. arda and A. vermicularis, as the single A. arda specimen is the largest of our sample and soil type and texture differ between the collection sites of the two species.

Using your head - cranial steering in pterosaurs.

The vast majority of pterosaurs are characterized by relatively large, elongate heads that are often adorned with large, elaborate crests. Projecting out in front of the body, these large heads and any crests must have had an aerodynamic effect. The working hypothesis of the present study is that these oversized heads were used to control the left-right motions of the body during flight. Using digital models of eight non-pterodactyloids ("rhamphorhyncoids") and ten pterodactyloids, the turning moments associated with the head + neck show a close and consistent correspondence with the rotational inertia of the whole body about a vertical axis in both groups, supporting the idea of a functional relationship. Turning moments come from calculating the lateral area of the head (plus any crests) and determining the associated lift (aerodynamic force) as a function of flight speed, with flight speeds being based on body mass. Rotational inertias were calculated from the three-dimensional mass distribution of the axial body, the limbs, and the flight membranes. The close correlation between turning moment and rotational inertia was used to revise the life restorations of two pterosaurs and to infer relatively lower flight speeds in another two.

Paleoneurology of stem palaeognaths clarifies the plesiomorphic condition of the crown bird central nervous system.

Lithornithidae, an assemblage of volant Palaeogene fossil birds, provide our clearest insights into the early evolutionary history of Palaeognathae, the clade that today includes the flightless ratites and volant tinamous. The neotype specimen of Lithornis vulturinus, from the early Eocene (approximately 53 million years ago) of Europe, includes a partial neurocranium that has never been thoroughly investigated. Here, we describe these cranial remains including the nearly complete digital endocasts of the brain and bony labyrinth. The telencephalon of Lithornis is expanded and its optic lobes are ventrally shifted, as is typical for crown birds. The foramen magnum is positioned caudally, rather than flexed ventrally as in some crown birds, with the optic lobes, cerebellum, and foramen magnum shifted further ventrally. The overall brain shape is similar to that of tinamous, the only extant clade of flying palaeognaths, suggesting that several aspects of tinamou neuroanatomy may have been evolutionarily conserved since at least the early Cenozoic. The estimated ratio of the optic lobe's surface area relative to the total brain suggests a diurnal ecology. Lithornis may provide the clearest insights to date into the neuroanatomy of the ancestral crown bird, combining an ancestrally unflexed brain with a caudally oriented connection with the spinal cord, a moderately enlarged telencephalon, and ventrally shifted, enlarged optic lobes.

Mechanically robust and personalized silk fibroin-magnesium composite scaffolds with water-responsive shape-memory for irregular bone regeneration.

The regeneration of critical-size bone defects, especially those with irregular shapes, remains a clinical challenge. Various biomaterials have been developed to enhance bone regeneration, but the limitations on the shape-adaptive capacity, the complexity of clinical operation, and the unsatisfied osteogenic bioactivity have greatly restricted their clinical application. In this work, we construct a mechanically robust, tailorable and water-responsive shape-memory silk fibroin/magnesium (SF/MgO) composite scaffold, which is able to quickly match irregular defects by simple trimming, thus leading to good interface integration. We demonstrate that the SF/MgO scaffold exhibits excellent mechanical stability and structure retention during the degradative process with the potential for supporting ability in defective areas. This scaffold further promotes the proliferation, adhesion and migration of osteoblasts and the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in vitro. With suitable MgO content, the scaffold exhibits good histocompatibility, low foreign-body reactions (FBRs), significant ectopic mineralisation and angiogenesis. Skull defect experiments on male rats demonstrate that the cell-free SF/MgO scaffold markedly enhances bone regeneration of cranial defects. Taken together, the mechanically robust, personalised and bioactive scaffold with water-responsive shape-memory may be a promising biomaterial for clinical-size and irregular bone defect regeneration.

Correlated evolution of beak and braincase morphology is present only in select bird clades.

Complex morphological structures, such as skulls or limbs, are often composed of multiple morphological components (e.g., bones, sets of bones) that may evolve in a covaried manner with one another. Previous research has reached differing conclusions on the number of semi-independent units, or modules, that exist in the evolution of structures and on the strength of the covariation, or integration, between these hypothesized modules. We focus on the avian skull as an example of a complex morphological structure for which highly variable conclusions have been reached in the numerous studies analyzing support for a range of simple to complex modularity hypotheses. We hypothesized that past discrepancies may stem from both the differing densities of data used to analyze support for modularity hypotheses and the differing taxonomic levels of study. To test these hypotheses, we applied a comparative method to 3D geometric morphometric data collected from the skulls of a diverse order of birds (the Charadriiformes) to test support for 11 distinct hypotheses of modular skull evolution. Across all Charadriiformes, our analyses suggested that charadriiform skull evolution has been characterized by the semi-independent, but still correlated, evolution of the beak from the rest of the skull. When we adjusted the density of our morphometric data, this result held, but the strength of the signal varied substantially. Additionally, when we analyzed subgroups within the order in isolation, we found support for distinct hypotheses between subgroups. Taken together, these results suggest that differences in the methodology of past work (i.e., statistical method and data density) as well as clade-specific dynamics may be the reasons past studies have reached varying conclusions.