Cranial Suture Regeneration Mitigates Skull and Neurocognitive Defects in Craniosynostosis.

Craniosynostosis results from premature fusion of the cranial suture(s), which contain mesenchymal stem cells (MSCs) that are crucial for calvarial expansion in coordination with brain growth. Infants with craniosynostosis have skull dysmorphology, increased intracranial pressure, and complications such as neurocognitive impairment that compromise quality of life. Animal models recapitulating these phenotypes are lacking, hampering development of urgently needed innovative therapies. Here, we show that Twist1+/- mice with craniosynostosis have increased intracranial pressure and neurocognitive behavioral abnormalities, recapitulating features of human Saethre-Chotzen syndrome. Using a biodegradable material combined with MSCs, we successfully regenerated a functional cranial suture that corrects skull deformity, normalizes intracranial pressure, and rescues neurocognitive behavior deficits. The regenerated suture creates a niche into which endogenous MSCs migrated, sustaining calvarial bone homeostasis and repair. MSC-based cranial suture regeneration offers a paradigm shift in treatment to reverse skull and neurocognitive abnormalities in this devastating disease.

Resilient anatomy and local plasticity of naive and stress haematopoiesis.

The bone marrow adjusts blood cell production to meet physiological demands in response to insults. The spatial organization of normal and stress responses are unknown owing to the lack of methods to visualize most steps of blood production. Here we develop strategies to image multipotent haematopoiesis, erythropoiesis and lymphopoiesis in mice. We combine these with imaging of myelopoiesis1 to define the anatomy of normal and stress haematopoiesis. In the steady state, across the skeleton, single stem cells and multipotent progenitors distribute through the marrow enriched near megakaryocytes. Lineage-committed progenitors are recruited to blood vessels, where they contribute to lineage-specific microanatomical structures composed of progenitors and immature cells, which function as the production sites for each major blood lineage. This overall anatomy is resilient to insults, as it was maintained after haemorrhage, systemic bacterial infection and granulocyte colony-stimulating factor (G-CSF) treatment, and during ageing. Production sites enable haematopoietic plasticity as they differentially and selectively modulate their numbers and output in response to insults. We found that stress responses are variable across the skeleton: the tibia and the sternum respond in opposite ways to G-CSF, and the skull does not increase erythropoiesis after haemorrhage. Our studies enable in situ analyses of haematopoiesis, define the anatomy of normal and stress responses, identify discrete microanatomical production sites that confer plasticity to haematopoiesis, and uncover unprecedented heterogeneity of stress responses across the skeleton.

A landmark-free morphometrics pipeline for high-resolution phenotyping: application to a mouse model of Down syndrome.

Characterising phenotypes often requires quantification of anatomical shape. Quantitative shape comparison (morphometrics) traditionally uses manually located landmarks and is limited by landmark number and operator accuracy. Here, we apply a landmark-free method to characterise the craniofacial skeletal phenotype of the Dp1Tyb mouse model of Down syndrome and a population of the Diversity Outbred (DO) mouse model, comparing it with a landmark-based approach. We identified cranial dysmorphologies in Dp1Tyb mice, especially smaller size and brachycephaly (front-back shortening), homologous to the human phenotype. Shape variation in the DO mice was partly attributable to allometry (size-dependent shape variation) and sexual dimorphism. The landmark-free method performed as well as, or better than, the landmark-based method but was less labour-intensive, required less user training and, uniquely, enabled fine mapping of local differences as planar expansion or shrinkage. Its higher resolution pinpointed reductions in interior mid-snout structures and occipital bones in both the models that were not otherwise apparent. We propose that this landmark-free pipeline could make morphometrics widely accessible beyond its traditional niches in zoology and palaeontology, especially in characterising developmental mutant phenotypes.

Assessment of novel surgical procedures using decellularised muscle and bioactive ceramic: a histological analysis.

Tissue regeneration and neovascularisation in cases of major bone loss is a challenge in maxillofacial surgery. The hypothesis of the present study is that the addition of resorbable bioactive ceramic Silica Calcium Phosphate Cement (SCPC) to Declluraized Muscle Scaffold (DSM) can expedite bone formation and maturation. Two surgical defect models were created in 18 nude transgenic mice. Group 1(n = 6), with a 2-mm decortication calvarial defect, was treated with a DSM/SCPC sheet over the corticated bone as an onlay then seeded with human Mesenchymal Stromal Cells hMSC in situ. In Group 2 (n = 6), a critical size (4 mm) calvarial defect was made and grafted with DSM/SCPC/in situ human bone marrow stromal cells (hMSCs). The control groups included Group 3 (n = 3) animals, with a 2-mm decortication defect treated with an onlay DSM sheet, and Group 4 (n = 3) animals, treated with critical size defect grafted with plain DSM. After 8 weeks, bone regeneration in various groups was evaluated using histology, immunohistochemistry and histomorphometry. New bone formation and maturation was superior in groups treated with DSM/SCPC/hMSC. The DMS/SCPC scaffold has the ability to augment and induce bone regeneration and neovascularisation in cases of major bone resorption and critical size defects.

Human amniotic fluid stem cells attract osteoprogenitor cells in bone healing.

Current treatments of large bone defects are based on autologous or allogenic bone transplantation. Human amniotic fluid stem cells (hAFSCs) were evaluated for their potential in bone regenerative medicine. In this study, hAFSCs were transduced with lentiviral vector harboring red fluorescent protein to investigate their role in the regeneration of critical-size bone defects in calvarial mouse model. To distinguish donor versus recipient cells, a transgenic mouse model carrying GFP fluorescent reporter was used as recipient to follow the fate of hAFSCs transplanted in vivo into Healos® scaffold. Our results showed that transduced hAFSCs can be tracked in vivo directly at the site of transplantation. The presence of GFP positive cells in the scaffold at 3 and 6 weeks after transplantation indicates that donor hAFSCs can recruit host cells during the repair process. These observations help clarify the role of hAFSCs in bone tissue repair.

Focal adhesion kinase promotes BMP2-induced osteogenic differentiation of human urinary stem cells via AMPK and Wnt signaling pathways.

Human urine-derived stem cells (hUSCs) serve as favorable candidates for bone transplants due to their efficient proliferative and multipotent differentiation abilities, as well as the capacity to secrete a variety of vasoactive agents to facilitate tissue engineering. The present study aimed to explore the role of focal adhesion kinase (FAK) in bone morphogenetic protein 2 (BMP2)-induced osteogenic differentiation of hUSCs and to investigate the underlying mechanism. The degree of osteogenic differentiation and the correlated signals, following BMP2 overexpression and siRNA-mediated silencing of FAK, were determined in vitro. Moreover, hUSCs induced bone formation in a rat model with cranial defects, in vivo. Our findings revealed that alkaline phosphatase production, calcium deposits, osteocalcin and osteopontin expression, and bone formation were upregulated in vitro and in vivo following BMP2-induced osteogenic differentiation, and AMPK and Wnt signaling pathway activation by FAK could effectively regulate BMP2-enhanced osteogenic differentiation of hUSCs. Taken together, these findings indicated that FAK could mediate BMP2-enhanced osteogenic differentiation of hUSCs through activating adenosine 5'-monophosphate-activated protein kinase and Wnt signaling pathways.

Cranial burr holes in the emergency department: to drill or not to drill?

The practice of trepanning (referred to today as a craniotomy) dates back to the Neolithic period. Reasons for drilling a hole through the skull evolved from releasing evil spirits and curing insanity to practical management of head injuries in ancient Greece and Rome. Today, craniotomy or drilling a burr hole through the skull is very much the purview of the neurosurgeon. Yet one could argue that the procedure itself is more 'bone surgery' than 'brain surgery'. Nevertheless, despite the fact that head injury is a common presentation at district general hospitals and traumatic extra-axial haemorrhages are encountered often, the straightforward skillset required to drill a burr hole as a pretransfer, temporising, life-saving measure is seldom taught and has never gained traction. What we advocate in this article is the adaptation and novel application of an old, tried and tested technique in new hands. The critical pathophysiological turning point of any expanding extra-axial haemorrhage is the inflection point on the volume/Intracranial pressure (ICP) curve beyond which compensation is impossible. The subsequent rising ICP initiates a predictable continuum of clinical signs signalling progressive herniation. There are few emergencies as time-critical as a patient with an isolated, expanding extradural haemorrhage embarking on a trajectory of rostrocaudal deterioration and inevitable death. In many cases, the tragedy is compounded by the knowledge that such a patient probably has a healthy underlying brain, often evidenced by a lucid period after trauma. Our emergency department is attached to a small 300-bed District General Hospital (DGH) on the rural North West coast of Ireland. We are 262 km distant by road from a national neurosciences department that can, at best, be reached in 2 hours and 30 min. Quality improvement review of years of dismal outcomes in patients such as those described earlier with potentially remediable pathology prompted research and development of the skillset we are now able to offer, an old technique in new hands.

The Not-So-Soft Spot: Pathophysiology of the Bulging Fontanelle in Association With Roseola.

Roseola infantum is a clinical syndrome characterized by high fever followed by the emergence of a rash. Case reports have documented an association between bulging fontanelles and roseola. We propose a novel mechanism for the development of intracranial hypertension caused by human herpesvirus 6-induced cytokine elevation leading to increased cerebrospinal fluid production.

Orthotopic Bone Formation by Streamlined Engineering and Devitalization of Human Hypertrophic Cartilage.

Most bones of the human body form and heal through endochondral ossification, whereby hypertrophic cartilage (HyC) is formed and subsequently remodeled into bone. We previously demonstrated that HyC can be engineered from human mesenchymal stromal cells (hMSC), and subsequently devitalized by apoptosis induction. The resulting extracellular matrix (ECM) tissue retained osteoinductive properties, leading to ectopic bone formation. In this study, we aimed at engineering and devitalizing upscaled quantities of HyC ECM within a perfusion bioreactor, followed by in vivo assessment in an orthotopic bone repair model. We hypothesized that the devitalized HyC ECM would outperform a clinical product currently used for bone reconstructive surgery. Human MSC were genetically engineered with a gene cassette enabling apoptosis induction upon addition of an adjuvant. Engineered hMSC were seeded, differentiated, and devitalized within a perfusion bioreactor. The resulting HyC ECM was subsequently implanted in a 10-mm rabbit calvarial defect model, with processed human bone (Maxgraft®) as control. Human MSC cultured in the perfusion bioreactor generated a homogenous HyC ECM and were efficiently induced towards apoptosis. Following six weeks of in vivo implantation, microcomputed tomography and histological analyses of the defects revealed an increased bone formation in the defects filled with HyC ECM as compared to Maxgraft®. This work demonstrates the suitability of engineered devitalized HyC ECM as a bone substitute material, with a performance superior to a state-of-the-art commercial graft. Streamlined generation of the devitalized tissue transplant within a perfusion bioreactor is relevant towards standardized and automated manufacturing of a clinical product.

Difficult Intubation due to Penetrating Trauma from a Crossbow Bolt.

We present the case of a patient with penetrating neck and craniofacial trauma from a self-inflicted crossbow bolt injury. This case highlights the challenges involved in prehospital airway management related to an in situ foreign object penetrating the oral cavity. We review the complications associated with such injuries and considerations for effective prehospital airway management.

Ocular congenital cranial dysinnervation disorders (CCDDs): insights into axon growth and guidance.

Unraveling the genetics of the paralytic strabismus syndromes known as congenital cranial dysinnervation disorders (CCDDs) is both informing physicians and their patients and broadening our understanding of development of the ocular motor system. Genetic mutations underlying ocular CCDDs alter either motor neuron specification or motor nerve development, and highlight the importance of modulations of cell signaling, cytoskeletal transport, and microtubule dynamics for axon growth and guidance. Here we review recent advances in our understanding of two CCDDs, congenital fibrosis of the extraocular muscles (CFEOM) and Duane retraction syndrome (DRS), and discuss what they have taught us about mechanisms of axon guidance and selective vulnerability. CFEOM presents with congenital ptosis and restricted eye movements, and can be caused by heterozygous missense mutations in the kinesin motor protein KIF21A or in the ß-tubulin isotypes TUBB3 or TUBB2B. CFEOM-causing mutations in these genes alter protein function and result in axon growth and guidance defects. DRS presents with inability to abduct one or both eyes. It can be caused by decreased function of several transcription factors critical for abducens motor neuron identity, including MAFB, or by heterozygous missense mutations in CHN1, which encodes α2-chimaerin, a Rac-GAP GTPase that affects cytoskeletal dynamics. Examination of the orbital innervation in mice lacking Mafb has established that the stereotypical misinnervation of the lateral rectus by fibers of the oculomotor nerve in DRS is secondary to absence of the abducens nerve. Studies of a CHN1 mouse model have begun to elucidate mechanisms of selective vulnerability in the nervous system.

Long-Term Characterization of Cranial Defects After Surgical Correction for Single-Suture Craniosynostosis.

INTRODUCTION: Craniosynostosis is typically corrected surgically within the first year of life through cranial vault reconstruction. These procedures often leave open calvarial defects at the time of surgery, which are anticipated to close over time in a large proportion of cases. However, residual calvarial defects may result as long-term sequelae from cranial vault remodeling. When larger defects are present, they may necessitate further reconstruction for closure.Better understanding of the calvarial osseous healing process may help to identify which defects will resolve or shrink to acceptable size and which will require further surgery. Our study aims to assess the long-term changes in defect size after cranial vault reconstruction for craniosynostosis. METHODS: One-year postoperative and long-term computed tomography scans were retrieved from the craniofacial anomalies archive. Analysis used custom software. All defects above the size of 1 cm were analyzed and tracked for calvarial location, surface area, and circularity. Monte Carlo simulation was performed to model the effect of initial defect size on the rate of defect closure. RESULTS: We analyzed a total of 74 defects. The mean ± SD initial defect surface area was 3.27 ± 3.40 cm. The mean ± SD final defect surface area was 1.71 ± 2.54 cm. The mean ± SD percent decrease was 55.06% ± 28.99%. There was a significant difference in the percentage decrease of defects in the parietal and frontoparietal locations: 68.4% and 43.7%, respectively (P = 0.001). Monte Carlo simulation results suggest that less than 10% of defects above the size of 9 cm will close to the size of 2.5 cm or less. CONCLUSIONS: We describe and make available a novel validated method of measuring cranial defects. We find that the large majority of initial defects greater than 9 cm remain at least 1 in in size (2.5 cm) 1 year postoperatively. In addition, there appear to be regional differences in closure rates across the cranium, with frontoparietal defects closing more slowly than those in the parietal region. This information will aid surgeons in the decision-making process regarding cranioplasty after craniosynostosis correction.

A targeted sequencing panel identifies rare damaging variants in multiple genes in the cranial neural tube defect, anencephaly.

Neural tube defects (NTDs) affecting the brain (anencephaly) are lethal before or at birth, whereas lower spinal defects (spina bifida) may lead to lifelong neurological handicap. Collectively, NTDs rank among the most common birth defects worldwide. This study focuses on anencephaly, which despite having a similar frequency to spina bifida and being the most common type of NTD observed in mouse models, has had more limited inclusion in genetic studies. A genetic influence is strongly implicated in determining risk of NTDs and a molecular diagnosis is of fundamental importance to families both in terms of understanding the origin of the condition and for managing future pregnancies. Here we used a custom panel of 191 NTD candidate genes to screen 90 patients with cranial NTDs (n = 85 anencephaly and n = 5 craniorachischisis) with a targeted exome sequencing platform. After filtering and comparing to our in-house control exome database (N = 509), we identified 397 rare variants (minor allele frequency, MAF < 1%), 21 of which were previously unreported and predicted damaging. This included 1 frameshift (PDGFRA), 2 stop-gained (MAT1A; NOS2) and 18 missense variations. Together with evidence for oligogenic inheritance, this study provides new information on the possible genetic causation of anencephaly.

Fluid-structure interaction analysis of cerebrospinal fluid with a comprehensive head model subject to a rapid acceleration and deceleration.

PRIMARY OBJECTIVE: Closed brain injuries are a common danger in contact sports and motorized vehicular collisions. Mild closed brain injuries, such as concussions, are not easily visualized by computed imaging or scans. Having a comprehensive head/brain model and using fluid-structure interaction (FSI) simulations enable us to see the exact movement of the cerebrospinal fluid (CSF) under such conditions and to identify the areas of brain most affected. RESEARCH DESIGN: The presented work is based on the first FSI model capable of simulating the interaction between the CSF flow and brain. METHODS AND PROCEDURES: FSI analysis combining smoothed-particle hydrodynamics and high-order finite-element method is used. MAIN OUTCOMES AND RESULTS: The interaction between the CSF and brain under rapid acceleration and deceleration is demonstrated. The cushioning effect of the fluid and its effect on brain are shown. CONCLUSIONS: The capability to locate areas (down to the exact gyri and sulci) of the brain the most affected under given loading conditions, and therefore assess the possible damage to the brain and consequently predict the symptoms, is shown.

Porous Poly(ε-caprolactone)-Poly(l-lactic acid) Semi-Interpenetrating Networks as Superior, Defect-Specific Scaffolds with Potential for Cranial Bone Defect Repair.

The treatment of irregular cranial bone defects is currently limited due to the graft resorption that can occur when an ill-fitting interface exists between an autograft and the surrounding tissue. A tissue engineering scaffold able to achieve defect-specific geometries could improve healing. This work reports a macroporous, shape memory polymer (SMP) scaffold composed of a semi-interpenetrating network (semi-IPN) of thermoplastic poly(l-lactic acid) (PLLA) within cross-linked poly(ε-caprolactone) diacrylate (PCL-DA) that is capable of conformal fit within a defect. The macroporous scaffolds were fabricated using a fused salt template and were also found to have superior, highly controlled properties needed for regeneration. Specifically, the scaffolds displayed interconnected pores, improved rigidity, and controlled, accelerated degradation. Although slow degradation rates of scaffolds can limit healing, the unique degradation behavior observed could prove promising. Thus, the described SMP semi-IPN scaffolds overcome two of the largest limitations in bone tissue engineering: defect "fit" and tailored degradation.

Effects of induced placental and fetal growth restriction, size at birth and early neonatal growth on behavioural and brain structural lateralization in sheep.

Poor perinatal growth in humans results in asymmetrical grey matter loss in fetuses and infants and increased functional and behavioural asymmetry, but specific contributions of pre- and postnatal growth are unclear. We therefore compared strength and direction of lateralization in obstacle avoidance and maze exit preference tasks in offspring of placentally restricted (PR: 10M, 13F) and control (CON: 23M, 17F) sheep pregnancies at 18 and 40 weeks of age, and examined gross brain structure of the prefrontal cortex at 52 weeks of age (PR: 14M, 18F; CON: 23M, 25F). PR did not affect lateralization direction, but 40-week-old PR females had greater lateralization strength than CON (P = .021). Behavioural lateralization measures were not correlated with perinatal growth. PR did not alter brain morphology. In males, cross-sectional areas of the prefrontal cortex and left hemisphere correlated positively with skull width at birth, and white matter area correlated positively with neonatal growth rate of the skull (all P < .05). These studies reinforce the need to include progeny of both sexes in future studies of neurodevelopmental programming, and suggest that restricting in utero growth has relatively mild effects on gross brain structural or behavioural lateralization in sheep.

Assessing the osteoblast transcriptome in a model of enhanced bone formation due to constitutive Gs-G protein signaling in osteoblasts.

G protein-coupled receptor (GPCR) signaling in osteoblasts (OBs) is an important regulator of bone formation. We previously described a mouse model expressing Rs1, an engineered constitutively active Gs-coupled GPCR, under the control of the 2.3 kb Col I promoter. These mice showed a dramatic age-dependent increase in trabecular bone of femurs. Here, we further evaluated the effects of enhanced Gs signaling in OBs on intramembranous bone formation by examining calvariae of 1- and 9-week-old Col1(2.3)/Rs1 mice and characterized the in vivo gene expression specifically occurring in osteoblasts with activated Gs G protein-coupled receptor signaling, at the cellular level rather than in a whole bone. Rs1 calvariae displayed a dramatic increase in bone volume with partial loss of cortical structure. By immunohistochemistry, Osterix was detected in cells throughout the inter-trabecular space while Osteocalcin was expressed predominantly in cells along bone surfaces, suggesting the role of paracrine mediators secreted from OBs driven by 2.3 kb Col I promoter could influence early OB commitment, differentiation, and/or proliferation. Gene expression analysis of calvarial OBs revealed that genes affected by Rs1 signaling include those encoding proteins important for cell differentiation, cytokines and growth factors, angiogenesis, coagulation, and energy metabolism. The set of Gs-GPCRs and other GPCRs that may contribute to the observed skeletal phenotype and candidate paracrine mediators of the effect of Gs signaling in OBs were also determined. Our results identify novel detailed in vivo cellular changes of the anabolic response of the skeleton to Gs signaling in mature OBs.

Validation of a New Minimally Invasive Intracranial Pressure Monitoring Method by Direct Comparison with an Invasive Technique.

In this chapter we present in vivo experiments with a new minimally invasive method of monitoring intracranial pressure (ICP). Strain gauge deformation sensors are externally glued onto the exposed skull. The signal from these sensors is amplified, filtered, and sent to a computer with appropriate software for analysis and data storage. Saline infusions into the spinal channel of rats were performed to produce ICP changes, and minimally invasive ICP and direct Codman intraparenchymal ICP were simultaneously acquired in six animals. The similarity between the invasive and minimally invasive methods in response to ICP increase was assessed using Pearson's correlation coefficient. It demonstrated good agreement between the two measures < r > = 0.8 ± 0.2, with a range of 0.31-0.99.

Validation of a New Noninvasive Intracranial Pressure Monitoring Method by Direct Comparison with an Invasive Technique.

The search for a completely noninvasive intracranial pressure (ICPni) monitoring technique capable of real-time digitalized monitoring is the Holy Grail of brain research. If available, it may facilitate many fundamental questions within the range of ample applications in neurosurgery, neurosciences and translational medicine, from pharmaceutical clinical trials, exercise physiology, and space applications. In this work we compare invasive measurements with noninvasive measurements obtained using the proposed new noninvasive method. Saline was infused into the spinal channel of seven rats to produce ICP changes and the simultaneous acquisition of both methods was performed. The similarity in the invasive and noninvasive methods of ICP monitoring was calculated using Pearson's correlation coefficients (r). Good agreement between measures < r > = 0.8 ± 0.2 with a range 0.28-0.96 was shown.

The Effects of Molding Helmet Therapy on Spring-Mediated Cranial Vault Remodeling for Sagittal Craniosynostosis.

There is no clear consensus for the optimal treatment of sagittal craniosynostosis; however, recent studies suggest that improved neurocognitive outcomes may be obtained when surgical intervention imparts active cranial expansion or remodeling and is performed before 6 months of age. The authors consider spring-mediated cranioplasty (SMC) to optimally address these imperatives, and this is an investigation of how helmet orthoses before or after SMC affect aesthetic outcomes.The authors retrospectively evaluated patients treated with SMC and adjunct helmeting for sagittal synostosis. Patients were stratified into 4 cohorts based on helmet usage: preop, postop, both, and neither. The cephalic index (CI) was used to assess head shape changes and outcomes. Twenty-six patients met inclusion criteria: 6 (23%) had preop, 11 (42%) had postop, 4 (15%) had preop and postop, and 5 (19%) had no helmeting. Average age at surgery was 3.6 months. Overall, CI improved from a mean 69.8 to 77.9 during an average 7-month course of care. Mean preoperative change in CI showed greater improvement with preop helmet (1.3) versus not (0.0), (P = 0.029), despite similar initial CI in these cohorts (70.4 and 69.6 respectively, P = 0.69). Nonetheless, all patient cohorts regardless of helmeting status achieved similar final CIs (range 76.4-80.4; P = 0.72).In summary, preoperative molding helmet therapy leads to improved CI at the time of spring-mediated cranioplasty. However, this benefit does not necessarily translate into overall improved CI after surgery and in follow-up, calling into question the benefits of molding helmet therapy in this setting.