Stem cells of the suture mesenchyme in craniofacial bone development, repair and regeneration.

Development of the skeleton is mediated through two distinct ossification mechanisms. Craniofacial bones are formed mainly through intramembranous ossification, a mechanism different from endochondral ossification required for development of the body skeleton. The skeletal structures are quite distinct between the two, thus they are likely to have their unique stem cell populations. The sutures serve as the growth center critical for healthy development of the craniofacial skeleton. Defects in suture morphogenesis cause its premature closure, resulting in development of craniosynostosis, a devastating disease affecting 1 in ~2,500 individuals. The suture mesenchyme has been postulated to act as the niche of skeletal stem cells essential for calvarial morphogenesis. However, very limited knowledge is available for suture biology and suture stem cells (SuSCs) have yet to be isolated. Here we report the first evidence for identification and isolation of a stem cell population residing in the suture midline. Genetic labeling of SuSCs shows their ability to self-renew and continually give rise to mature cell types over a 1-year monitoring period. They maintain their localization in the niches constantly produce skeletogenic descendants during calvarial development and homeostastic maintenance. Upon injury, SuSCs expand drastically surrounding the skeletogenic mesenchyme, migrate to the damaged site and contribute directly to skeletal repair in a cell autonomous fashion. The regeneration, pluripotency and frequency of SuSCs are also determined using limiting dilution transplantation. In vivo clonal expansion analysis demonstrates a single SuSC capable of generating bones. Furthermore, SuSC transplantation into injured calvaria facilitates the healing processes through direct engraftments. Our findings demonstrate SuSCs are bona fide skeletal stem cells ideally suited for cell-based craniofacial bone therapy as they possess abilities to engraft, differentiate.(Presented at the 1980th Meeting, April 16, 2019).

Phenomenological tissue fracture modeling for an Endoscopic Sinus and Skull Base Surgery training system based on experimental data.

The ideal simulator for Endoscopic Sinus and Skull Base Surgery (ESSS) training must be supported by a physical model and provide repetitive behavior in a controlled environment. Development of realistic tissue models is a key part of ESSS virtual reality (VR)-based surgical simulation. Considerable research has been conducted to address haptic or force feedback and propose a phenomenological tissue fracture model for sino-nasal tissue during surgical tool indentation. Mechanical properties of specific sino-nasal regions of the sheep head have been studied in various indentation and relaxation experiments. Tool insertion at different indentation rates into coronal orbital floor (COF) tissue is modeled as a sequence of three events: deformation, fracture, and cutting. The behavior in the deformation phase can be characterized using a non-linear, rate-dependent modified Kelvin-Voigt model. A non-linear model for tissue behavior prior to the fracture point is presented. The overall model shows a non-positive dependency of maximum force on tool indentation rate, which indicates faster tool insertion velocity decreases the maximum final fracture force. The tissue cutting phase has been modeled to characterize the force necessary to slice through the COF. The proposed model in this study can help develop VR-based ESSS base simulators in otolaryngology and ophthalmology surgeries. Such simulators are useful in preoperative planning, accurate surgical simulation, intelligent robotic assistance, and treatment applications.

Deformed Skull Morphology Is Caused by the Combined Effects of the Maldevelopment of Calvarias, Cranial Base and Brain in FGFR2-P253R Mice Mimicking Human Apert Syndrome.

Apert syndrome (AS) is a common genetic syndrome in humans characterized with craniosynostosis. Apert patients and mouse models showed abnormalities in sutures, cranial base and brain, that may all be involved in the pathogenesis of skull malformation of Apert syndrome. To distinguish the differential roles of these components of head in the pathogenesis of the abnormal skull morphology of AS, we generated mouse strains specifically expressing mutant FGFR2 in chondrocytes, osteoblasts, and progenitor cells of central nervous system (CNS) by crossing Fgfr2+/P253R-Neo mice with Col2a1-Cre, Osteocalcin-Cre (OC-Cre), and Nestin-Cre mice, respectively. We then quantitatively analyzed the skull and brain morphology of these mutant mice by micro-CT and micro-MRI using Euclidean distance matrix analysis (EDMA). Skulls of Col2a1-Fgfr2+/P253R mice showed Apert syndrome-like dysmorphology, such as shortened skull dimensions along the rostrocaudal axis, shortened nasal bone, and evidently advanced ossification of cranial base synchondroses. The OC-Fgfr2+/P253R mice showed malformation in face at 8-week stage. Nestin-Fgfr2+/P253R mice exhibited increased dorsoventral height and rostrocaudal length on the caudal skull and brain at 8 weeks. Our study indicates that the abnormal skull morphology of AS is caused by the combined effects of the maldevelopment in calvarias, cranial base, and brain tissue. These findings further deepen our knowledge about the pathogenesis of the abnormal skull morphology of AS, and provide new clues for the further analyses of skull phenotypes and clinical management of AS.

Roles of the primary cilium component Polaris in synchondrosis development.

Primary cilia regulate several developmental processes and mediate hedgehog signaling. To study their roles in cranial base development, we created conditional mouse mutants deficient in Polaris, a critical primary cilium component, in cartilage. Mutant post-natal cranial bases were deformed, and their synchondrosis growth plates were disorganized. Expression of Indian hedgehog, Patched-1, collagen X, and MMP-13 was reduced and accompanied by decreases in endochondral bone. Interestingly, there was excessive intramembranous ossification along the perichondrium, accompanied by excessive Patched-1 expression, suggesting that Ihh distribution was wider and responsible for such excessive response. Indeed, expression of heparan sulfate proteoglycans (HS-PGs), normally involved in restricting hedgehog distribution, was barely detectable in mutant synchondroses. Analyses of the data provides further evidence for the essential roles of primary cilia and hedgehog signaling in cranial base development and chondrocyte maturation, and point to a close interdependence between cilia and HS-PGs to delimit targets of hedgehog action in synchondroses.

In vitro regulation of proliferation and differentiation within a postnatal growth plate of the cranial base by parathyroid hormone-related peptide (PTHrP).

Parathyroid hormone-related peptide (PTHrP) is known to be an important regulator of chondrocyte differentiation in embryonic growth plates, but little is known of its role in postnatal growth plates. The present study explores the role of PTHrP in regulating postnatal chondrocyte differentiation using a novel in vitro organ culture model based on the ethmoidal growth plate of the cranial base taken from the postnatal day 10 mouse. In vitro the ethmoidal growth plate continued to mineralize and the chondrocytes progressed to hypertrophy, as observed in vivo, but the proliferative zone was not maintained. Treatment with PTHrP inhibited mineralization and reduced alkaline phosphatase (ALP) activity in the hypertrophic zone in the ethmoidal growth plates grown ex vivo, and also increased the proliferation of non-hypertrophic chondrocytes. In addition, exogenous PTHrP reduced the expression of genes associated with terminal differentiation: type X collagen, Runx2, and ALP, as well as the PTH/PTHrP receptor (PPR). Activation of the protein kinase A pathway using 8-Br-cAMP mimicked some of these pro-proliferative/anti-differentiative effects of PTHrP. PTHrP and PPR were found to be expressed within the ethmoidal growth plate using semi-quantitative PCR, and in other cranial growth plates such as the spheno-occipital and pre-sphenoidal synchondroses. These results provide the first functional evidence that PTHrP regulates proliferation and differentiation within the postnatal, cranial growth plate. J. Cell. Physiol. 219: 688-697, 2009. (c) 2009 Wiley-Liss, Inc.

Development and tissue origins of the mammalian cranial base.

The vertebrate cranial base is a complex structure composed of bone, cartilage and other connective tissues underlying the brain; it is intimately connected with development of the face and cranial vault. Despite its central importance in craniofacial development, morphogenesis and tissue origins of the cranial base have not been studied in detail in the mouse, an important model organism. We describe here the location and time of appearance of the cartilages of the chondrocranium. We also examine the tissue origins of the mouse cranial base using a neural crest cell lineage cell marker, Wnt1-Cre/R26R, and a mesoderm lineage cell marker, Mesp1-Cre/R26R. The chondrocranium develops between E11 and E16 in the mouse, beginning with development of the caudal (occipital) chondrocranium, followed by chondrogenesis rostrally to form the nasal capsule, and finally fusion of these two parts via the midline central stem and the lateral struts of the vault cartilages. X-Gal staining of transgenic mice from E8.0 to 10 days post-natal showed that neural crest cells contribute to all of the cartilages that form the ethmoid, presphenoid, and basisphenoid bones with the exception of the hypochiasmatic cartilages. The basioccipital bone and non-squamous parts of the temporal bones are mesoderm derived. Therefore the prechordal head is mostly composed of neural crest-derived tissues, as predicted by the New Head Hypothesis. However, the anterior location of the mesoderm-derived hypochiasmatic cartilages, which are closely linked with the extra-ocular muscles, suggests that some tissues associated with the visual apparatus may have evolved independently of the rest of the "New Head".

A new model of skull base reconstruction following expanded endonasal or transoral approaches--long-term results in primates.

OBJECTIVE: The direct endonasal or transoral transclival approaches to the skull base permit effective minimally invasive surgery along the clivus region. Developing consistently effective techniques to prevent cerebrospinal fluid (CSF) leaks and their consequences (infections and healing processes with long and complicated recoveries) remains a major challenge. In this study, we tested over a long period a method of bone reconstruction newly developed by us, which makes use of a specially designed elastic silicone plug that can be employed for bone replacement after minimally invasive skull base surgery without risk of postoperative CSF leaks. After acute testing of plug efficiency in a pig model, which showed a 100% closure of the bone defect without CSF leak, we now tested the long-term accuracy of the plugs. METHODS: In 3 primates, we used an endoscope-controlled transoral transclival approach and after opening the dura we simulated a CSF leakage. We inserted the plug into the bone defect and closed the mucosa of the oral cavity with stitches. The follow-up included blood, weight, and wound control 1, 4 and 8 weeks postoperatively. Social behavior, such as reintegration and postoperative eating abnormalities, was also studied. The aims of this study were: (1) testing the biocompatibility of the material; (2) development of infection against the foreign body; (3) effects of the plug on the surrounding bone, and (4) development of CSF leakages during the postoperative phase. RESULTS: Clinically no infection was seen. Wound healing, immediate and long-term postoperative social behavior of the animals, feeding and body weight were normal. No CSF leakages developed. The histological examination of the clivus bone showed no abnormalities. The implant was covered by fibrous layer; there was no bone atrophy but osteoid formation. CONCLUSION: This novel medical device allows easy, fast and uncomplicated, leak-proof closure of bone defects after minimally invasive craniotomies as seen in transsphenoidal or transoral skull base approaches.

Neural-dural transition at the medial anterior cranial base: an anatomical and histological study with clinical applications.

OBJECT: Few anatomical studies have been focused on the morphological features and microscopic anatomy of the transition from the intracranial space to the medial anterior cranial base. The authors of the current study performed histological analyses to define the structure of the transition from neural foramina to the cranial base (neural-dural transition) at the cribriform plate, particularly as related to cerebrospinal fluid (CSF) fistula formation and surgical intervention in the region. METHODS: The medial anterior cranial base was resected in six cadaveric specimens. Histological methods were used to study the anatomy of the region on the microscopic level. Results of these examinations revealed a multilayered neural-dural transition at the cribriform plate, which consisted of an arachnoid membrane and a potential subarachnoid space as well as dura mater, periosteum, ethmoid bone, and associated layers of submucosa and mucosa of the paranasal air spaces. A subarachnoid space was identified around the olfactory nerves as they exited the neural foramina of the cribriform plates. The dura mater eventually thinned out and became continuous with the periosteum in the ethmoid bone. The dura, arachnoid membrane, and associated potential subarachnoid space were obliterated at a place 1 to 2 mm into the olfactory foramen. The authors present a case of recurrent CSF rhinorrhea successfully treated using a technique of multilayered reconstruction with pericranium, fat, and bone. CONCLUSIONS: The findings provide an anatomical basis for CSF fistula formation in the region of the cribriform plate and help to explain the unusual presentations in patients who have CSF rhinorrhea and meningitis. These results may facilitate the treatment of CSF fistulas, repair of defects in the medial anterior cranial base, and approaches to tumors and other pathological entities in the region.