Cranial sutures separate neighboring skull bones and are sites of bone growth. A key question is how osteogenic activity is controlled to promote bone growth while preventing aberrant bone fusions during skull expansion. Using single-cell transcriptomics, lineage tracing, and mutant analysis in zebrafish, we uncover key developmental transitions regulating bone formation at sutures during skull expansion. In particular, we identify a subpopulation of mesenchyme cells in the mid-suture region that upregulate a suite of genes including BMP antagonists (e.g. grem1a) and pro-angiogenic factors. Lineage tracing with grem1a:nlsEOS reveals that this mid-suture subpopulation is largely non-osteogenic. Moreover, combinatorial mutation of BMP antagonists enriched in this mid-suture subpopulation results in increased BMP signaling in the suture, misregulated bone formation, and abnormal suture morphology. These data reveal establishment of a non-osteogenic mesenchyme population in the mid-suture region that restricts bone formation through local BMP antagonism, thus ensuring proper suture morphology.

BACKGROUND: Palatal expansion is a common way of treating maxillary transverse deficiency. Under mechanical force, the midpalatal suture is expanded, causing local immune responses. This study aimed to determine whether macrophages participate in bone remodeling of the midpalatal suture during palatal expansion and the effects on bone remodeling.
METHODS: Palatal expansion model and macrophage depletion model were established. Micro-CT, histological staining, and immunohistochemical staining were used to investigate the changes in the number and phenotype of macrophages during palatal expansion as well as the effects on bone remodeling of the midpalatal suture. Additionally, the effect of mechanically induced M2 macrophages on palatal osteoblasts was also elucidated in vitro.
RESULTS: The number of macrophages increased significantly and polarized toward M2 phenotype with the increase of the expansion time, which was consistent with the trend of bone remodeling. After macrophage depletion, the function of osteoblasts and bone formation at the midpalatal suture were impaired during palatal expansion. In vitro, conditioned medium derived from M2 macrophages facilitated osteogenic differentiation of osteoblasts and decreased the RANKL/OPG ratio.
CONCLUSIONS: Macrophages through polarizing toward M2 phenotype participated in midpalatal suture bone remodeling during palatal expansion, which may provide a new idea for promoting bone remodeling from the perspective of regulating macrophage polarization.

BACKGROUND: The purpose of this study was to assess the effects of systemically given krill oil (KO) on the development of new bone formation in the sutura palatina media following rapid maxillary expansion (RME).
METHODS: 28 4-5 week-old male Wistar albino rats were randomly divided into 4 groups: Control (C), Only Expansion (OE) (no supplement but undergoing expansion and retention), KE (supplemented during both the expansion and retention phases), Krill Oil Nursery Group (KN) (supplemented during the 40-day nursery phase as well as during the expansion and retention phases). A 5-day RME was followed by a 12-day retention period. All rats were euthanized simultaneously. Micro-computerized tomography (Micro-CT), hemotoxylen-eosin (H&E) staining, and immunohistochemical analysis were conducted. Kruskal-Wallis and Dunn tests with Bonferonni corrrection were applied (p < 0.05).
RESULTS: Expansion and KO supplementation did not cause a statistically significant change in bone mineral density (BMD), bone volume fraction (BV/TV), spesific bone surface (BS/BV) and trabecular thickness (Tb.Th). While the expansion prosedure increased the trabecular seperation (Tb.Sp), KO supplemantation mitigated this effect. The KE group exhibited a statistically significantly increase in trabecular number (Tb.N) compared to the OE group. Although receptor activator of nuclear factor-kappa-Β ligand (RANKL)/osteoprotegerin (OPG) ratios did not show significant differences between groups, the KE and OE groups demonstrated the lowest and highest value, respectively. KE showed a reduced amount of tartrate-resistant acid phosphatase (TRAP) compared to the OE.
CONCLUSIONS: KO positively affected the architecture of the new bone formed in the mid-palatal suture. In this rat model of RME, results support the idea that administering of KO during the expansion period or beginning before the RME procedure may reduce relapse and enhance bone formation within the mid-palatal suture.

Aplasia cutis congenita (ACC) manifests at birth as a defect of the scalp skin. New findings answer 2 longstanding questions: why ACC forms and why it affects mainly the midline scalp skin. Dominant-negative mutations in the genes KCTD1 or KCTD15 cause ACC owing to loss of function of KCTD1/KCTD15 complexes in cranial neural crest cells (NCCs), which normally form midline cranial suture mesenchymal cells that express keratinocyte growth factors. Loss of KCTD1/KCTD15 function in NCCs impairs the formation of normal midline cranial sutures and, consequently, the overlying skin, resulting in ACC. Moreover, KCTD1/KCTD15 complexes in keratinocytes regulate skin appendage morphogenesis.

BACKGROUND: Craniofacial skeletal deformities can be addressed by applying tensile force to sutures to prompt sutural bone formation. The intricate process of mechanical modulation in craniofacial sutures involves complex biomechanical signal transduction. The small GTPase Ras homolog gene family member A (RhoA) functions as a key mechanotransduction protein, orchestrating the dynamic assembly of the cytoskeleton by activating the Rho-associated coiled-coil containing protein kinase (ROCK). Transcriptional coactivator with PDZ-binding motif (TAZ) serves as a crucial mediator in the regulation of genes and the orchestration of biological functions within the mechanotransduction signaling pathway. However, the role of RhoA/ROCK-TAZ in trans-sutural distraction osteogenesis has not been reported.
METHODS: We utilized pre-osteoblast-specific RhoA deletion mice to establish an in vivo calvarial trans-sutural distraction model and an in vitro mechanical stretch model for pre-osteoblasts isolated from neonatal mice. Micro-CT and histological staining were utilized to detect the formation of new bone in the sagittal suture of the skull as well as the activation of RhoA, Osterix and TAZ. The activation of ROCK-limk-cofilin and the nuclear translocation of TAZ in pre-osteoblasts under mechanical tension were detected through Western blot, qRT-PCR, and immunofluorescence.
RESULTS: The osteogenic differentiation of pre-osteoblasts was facilitated by mechanical tension through the activation of RhoA and Rho-associated kinase (ROCK), while ablation of RhoA impaired osteogenesis by inhibiting pre-osteoblast differentiation after suture expansion. Furthermore, inhibiting RhoA expression could block tensile-stimulated nuclear translocation of TAZ by preventing F-actin assembly through ROCK-LIM-domain kinase (LIMK)-cofilin pathway. In addition, the TAZ agonist TM-25659 could attenuate impaired osteogenesis caused by ablation of RhoA in pre-osteoblasts by increasing TAZ nuclear accumulation.
CONCLUSIONS: This study demonstrates that mechanical stretching promotes the osteogenic differentiation of pre-osteoblasts in trans-sutural distraction osteogenesis, and this process is mediated by the RhoA/ROCK-TAZ signaling axis. Overall, our results may provide an insight for potential treatment strategies for craniosynostosis patients through trans-sutural distraction osteogenesis.

BACKGROUND: Trans-sutural distraction osteogenesis (TSDO) involves the application of distraction force to facial sutures to stimulate osteogenesis. Gli1+ cells in the cranial sutures play an important role in bone growth. However, whether Gli1+ cells in facial sutures differentiate into bone under distraction force is unknown.
METHODS: 4-week-old Gli1ER/Td and C57BL/6 mice were used to establish a TSDO model to explore osteogenesis of zygomaticomaxillary sutures. A Gli1+ cell lineage tracing model was used to observe the distribution of Gli1+ cells and explore the role of Gli1+ cells in facial bone remodeling.
RESULTS: Distraction force promoted bone remodeling during TSDO. Fluorescence and two-photon scanning images revealed the distribution of Gli1+ cells. Under distraction force, Gli1-lineage cells proliferated significantly and co-localized with Runx2+ cells. Hedgehog signaling was upregulated in Gli1+ cells. Inhibition of Hedgehog signaling suppresses the proliferation and osteogenesis of Gli1+ cells induced by distraction force. Subsequently, the stem cell characteristics of Gli1+ cells were identified. Cell-stretching experiments verified that mechanical force promoted the osteogenic differentiation of Gli1+ cells through Hh signaling. Furthermore, immunofluorescence staining and RT-qPCR experiments demonstrated that the primary cilia in Gli1+ cells exhibit Hedgehog-independent mechanosensitivity, which was required for the osteogenic differentiation induced by mechanical force.
CONCLUSIONS: Our study indicates that the primary cilia of Gli1+ cells sense mechanical stimuli, mediate Hedgehog signaling activation, and promote the osteogenic differentiation of Gli1+ cells in zygomaticomaxillary sutures.

Fragments of two skulls of young cetotheriid baleen whales were described from the Fortep\'yanka 2 locality (Russia, Republic of Adygea, Maikop district, Fortep\'yanka River valley, Upper Miocene, Upper Sarmatian, Blinovskaya Formation). The finds were attributed to Kurdalagonus maicopicus (Spasskii, 1951) based on the morphology of the posterior (mastoid) process of the petrosal bone, the structure of the posterior edge of the temporal fossa, and the S-like shape of the supraoccipital ridges. The skull proportions and the degree of suture closure made it possible to determine the individual age of the whales within a year. New finds significantly complement the data on the structure of the sutures of the lateral wall of the skull and age-related variability of cranial morphology in representatives of the genus Kurdalagonus.

UNASSIGNED: Vigilant monitoring for postoperative complications, including bleeding and dysrhythmia, is crucial in patients with craniosynostosis syndromes like Crouzon syndrome undergoing craniofacial surgery, with a thorough evaluation, including coagulation tests, assisting in diagnosing underlying conditions such as von Willebrand disease subtype 1 to inform appropriate management strategies.
UNASSIGNED: Crouzon syndrome is a rare genetic disorder affecting craniofacial structures. Its etiology is the premature fusion of cranial sutures. The LeFort III advancement surgery is a commonly used approach to correct malformations related to midface hypoplasia. Complications following surgical treatment of craniosynostosis and craniofacial syndromes can include both intracranial and extracranial problems. Reporting of this syndrome and the surgery complications, in addition to consideration of other differential diagnoses, can help improve the treatment plan and surgery outcomes. The aim of the article is to report a 14-year-old female with Crouzon syndrome who underwent the modified LeFort III osteotomy and developed unexpected massive bleeding during the surgery. Post-surgery, she experienced complications including dysrhythmia, hypothermia, and cyanosis. Treatment included fluid therapy, blood transfusions, and antibiotic therapy for suspected septic shock. Differential diagnosis was disseminated intravascular coagulation but was ruled out. Post-discharge, coagulation tests suggested von Willebrand disease subtype 1 as the diagnosis. Excessive bleeding during surgery for craniosynostosis syndromes is a significant and concerning issue in the surgical management of Crouzon syndrome. For patients with von Willebrand disease who are candidates for elective surgeries, von Willebrand factor concentrates or recombinant von Willebrand factor can be used.

OBJECTIVE: This study aimed to determine the presence and incidence of accessory sutures and bipartite zygomatic bone types, and their effects on population affinity and clinical significance.
METHODS: We examined 120 dry skulls and 50 zygomatic bones to evaluate the presence, frequency, and location of accessory zygomatic sutures as well as subtypes of bipartite zygomatic bones. Morphometric analysis included measuring the total width and length of the zygomatic bone with accessory sutures (ASs), the total length of the AS, and the shortest distance between the AS and various anatomical landmarks.
RESULTS: Bipartite zygomatic bone was observed in 14 of 120 dry skulls (11.6%) and 1 of 50 zygomatic bones (2%), indicating an overall incidence of 16 occurrences (8.82%). The ASs were predominantly located posterolaterally in 11 cases (six males, five females), anteromedially in two cases (one male, one female), superiorly in one case (male), and superolaterally in one case (male). Significant differences were noted in the distribution of the ASs (p < 0.001). Notably, a vertical inferior bipartite zygomatic type, which has not been reported in the literature, was identified. Correlations were observed between the various anatomical landmarks. Among females, the length of AS was significantly different (p = 0.038). Significant differences were also noted in the shortest distance between the AS and the zygomaticofacial foramen (ZFF) based on the body side (p = 0.034).
CONCLUSIONS: Our study suggests that the bipartite zygomatic bone is not a common occurrence, indicating its significance as a morphological variation present in certain individuals. The type VII bipartite zygomatic bone exhibited the highest incidence rate, suggesting potential ethnic-specific differences in the prevalence of certain subtypes. The consistent pattern of suture distribution, along with the asymmetry and variability in suture patterns, emphasizes its potential clinical relevance.

The regeneration of the mammalian skeleton\'s craniofacial bones necessitates the action of intrinsic and extrinsic inductive factors from multiple cell types, which function hierarchically and temporally to control the differentiation of osteogenic progenitors. Single-cell transcriptomics of developing mouse calvarial suture recently identified a suture mesenchymal progenitor population with previously unappreciated tendon- or ligament-associated gene expression profile. Here, we developed a Mohawk homeobox (MkxCG; R26RtdT) reporter mouse and demonstrated that this reporter identifies an adult calvarial suture resident cell population that gives rise to calvarial osteoblasts and osteocytes during homeostatic conditions. Single-cell RNA sequencing (scRNA-Seq) data reveal that Mkx+ suture cells display a progenitor-like phenotype with expression of teno-ligamentous genes. Bone injury with Mkx+ cell ablation showed delayed bone healing. Remarkably, Mkx gene played a critical role as an osteo-inhibitory factor in calvarial suture cells, as knockdown or knockout resulted in increased osteogenic differentiation. Localized deletion of Mkx in vivo also resulted in robustly increased calvarial defect repair. We further showed that mechanical stretch dynamically regulates Mkx expression, in turn regulating calvarial cell osteogenesis. Together, we define Mkx+ cells within the suture mesenchyme as a progenitor population for adult craniofacial bone repair, and Mkx acts as a mechanoresponsive gene to prevent osteogenic differentiation within the stem cell niche.