Faculty Bibliography

The influence of dura mater on adjacent cranial sutures is significant. By better understanding the mechanisms of normal suture fusion and the role of the dura mater, it may be possible to delineate the events responsible for the premature suture fusion seen in craniosynostosis. In the Sprague-Dawley rat, the posterior frontal suture normally fuses between 12 and 20 days of postnatal life and has proved to be an excellent model to describe normal suture fusion. The purpose of this study was to document the critical role that the dura mater-suture complex may play on cranial suture biology. Forty Sprague-Dawley rats at 8 days of age were divided into two groups of 20 animals each. The control group (group A) had surgical disruption of the dura mater-calvarial interface. This was accomplished by elevating a strip of cranium inclusive of the posterior frontal and sagittal sutures and replacement of the cranial strip back to its anatomic position, all with the dura mater left intact. The experimental group (group B) had the same calvarial elevation (strip craniectomy), but the sutural anatomy/alignment was rotated 180 degrees. This rotation placed the posterior frontal suture into the sagittal suture's anatomic position and the sagittal suture into the posterior frontal suture's anatomic position. All of these procedures were accomplished by leaving the underlying dura mater intact. Animals were killed at 20, 30, 40, and 50 days (12, 22, 32, and 42 days postoperatively), and tissue sections were examined with hematoxylin and eosin staining. Group A (control) showed normal but delayed suture activity. The posterior frontal suture fused, and the sagittal suture remained patent. Fusion was delayed, not beginning before 20 days (12 days postoperative) and showing complete fusion between 30 and 40 days. Group B (180-degree calvarial rotation) demonstrated that the suture in the posterior frontal anatomic position (actual sagittal suture) fused between 20 and 40 days, whereas the suture in the sagittal anatomic position (actual posterior-frontal suture) remained patent throughout the study. This study demonstrates that the location of the dura mater-suture complex is important in determining either suture patency or closure in this model. Normal closure of the suture overlying the posterior frontal dura mater demonstrates that the dura mater itself, or forces derived in specific cranial locations, determines the overlying suture biology

The mechanisms involved in normal cranial suture development and fusion as well as in the pathophysiology of craniosyostosis are not well understood. The purpose of this study was to investigate the expression of several cytokines--transforming growth factor-beta-1 (TGF-beta1), basic fibroblast growth factor (bFGF), and interleukin-6 (IL-6)--during cranial suture fusion. TGF-beta exists in three mammalian isoforms that are abundant in bone and stimulate calvarial bone formation when delivered locally. Other bone growth factors including basic fibroblast growth factor and the interleukins regulate bone growth and are mitogenic for bone marrow cells and osteoblasts. The involvement of growth factors in the pathophysiology of craniosynostosis is supported by recent genetics data linking fibroblast growth factor receptor mutations to syndromal craniosynostoses. In this experimental study, in situ hybridization was used to localize and quantify the gene expression of TGF-beta1, bFGF, and IL-6 during cranial suture fusion. In the Sprague-Dawley rat, the posterior frontal cranial suture normally undergoes fusion between 12 and 22 days of age, whereas all other cranial sutures remain patent. All in situ analyses of fusing posterior frontal sutures were compared with the patent, control, sagittal sutures. Posterior frontal and sagittal sutures, together with underlying dura, were harvested from rats at 8, 12, 16, and 35 days of postnatal life to analyze posterior frontal suture activity before, during, and after fusion. In situ hybridization was performed on frozen sections of these specimens using DNA probes specific for TGF-beta1, bFGF, and IL-6 mRNA. A negative control probe to IL-6 in the sense orientation was also used to validate the procedure. Cells expressing cytokine-specific mRNA were quantified (in cells positive per 10(-1) mm2) and analyzed using the unpaired Student's t test. Areas encompassing the fibrous suture and the surrounding bone plates were analyzed for cellular mRNA activity. IL-6 mRNA expression showed a minimal rise in the posterior frontal suture at days 12 and 16, with an average count of 10 and 6 cells per 10(-1) mm2, respectively. The sagittal suture remained negative for IL-6 mRNA at all time points. TGF-beta1 and bFGF analyses were most interesting, showing marked increases specifically in the posterior frontal suture during the time of active suture fusion. On postnatal day 8, a 1.5-fold increase in posterior frontal suture TGF-beta1 mRNA was found compared with sagittal sutures (p = 0.1890, unpaired Student's t test). This difference was increased 26-fold on day 12 in posterior frontal suture TGF-beta1 expression (p = 0.0005). By day 35, posterior frontal suture TGF-beta1 mRNA had nearly returned to prefusion levels, whereas TGF-beta1 mRNA levels in the sagittal suture remained low. A similar upregulation of bFGF mRNA, peaking at day 12, was observed in posterior frontal but not sagittal sutures (p = 0.0003). Furthermore, both TGF-beta1 and bFGF mRNA samples with intact dura showed an intense dural mRNA expression in the time preceding and during active posterior frontal suture fusion but not in sagittal tissues. Our data demonstrate that TGF-beta1 and bFGF mRNA are up-regulated in cranial suture fusion, possibly signaling in a paracrine fashion from dura to suture. TGF-beta1 and bFGF gene expression were dramatically increased both in and surrounding the actively fusing suture and followed the direction of fusion from endocranial to epicranial. These experimental data on bone growth factors support the recent human genetics data linking growth factor/fibroblast growth factor receptor deletions to syndromal craniosynostoses. The ultimate aim of these studies is to understand the underlying mechanisms regulating suture growth, development, and fusion so surgeons may one day manipulate the biology of premature cranial suture fusion

The complete healing of wounds is the final step in a highly regulated response to injury. Although many of the molecular mediators and cellular events of healing are known, their manipulation for the enhancement and acceleration of wound closure has not proven practical as yet. We and others have established that adenosine is a potent regulator of the inflammatory response, which is a component of wound healing. We now report that ligation of the G alpha s-linked adenosine receptors on the cells of an artificial wound dramatically alters the kinetics of wound closure. Excisional wound closure in normal, healthy mice was significantly accelerated by topical application of the specific A2A receptor agonist CGS-21680 (50% closure by day 2 in A2 receptor antagonists. In rats rendered diabetic (streptozotocin-induced diabetes mellitus) wound healing was impaired as compared to nondiabetic rats; CGS-21680 significantly increased the rate of wound healing in both nondiabetic and diabetic rats. Indeed, the rate of wound healing in the CGS-21680-treated diabetic rats was greater than or equal to that observed in untreated normal rats. These results appear to constitute the first evidence that a small molecule, such as an adenosine receptor agonist, accelerates wound healing in both normal animals and in animals with impaired wound healing

Craniosynostosis results in alterations in craniofacial growth that create cosmetic abnormalities and functional deficits, yet the biology underlying cranial suture fusion remains unknown. The purpose of the present study was to show that regional dura mater can induce suture fusion while in an organ culture system in cranial sutures programmed to remain patient. To accomplish this, we studied mouse cranial sutures, since in this model the posterior frontal suture (analogous to the human metopic suture) fuses in both in vivo and in vitro environments while all other sutures remain patent. We demonstrated that when mouse sagittal sutures (programmed to remain patent) were rotated or translocated to overlie the posterior frontal dura then grown in organ culture systems, suture fusion occurred. Twenty-four-day-old CD-1 mice (time when the posterior frontal suture was patent) were divided into three groups of 50 (n = 165: three groups of 50 cultured and three groups of 5 uncultured controls). Group A (unrotated control group) was characterized by a strip of posterior frontal and sagittal suture with underlying dural tissue grown in organ culture systems for up to 30 days and resulted in persistent patency of the sagittal suture and fusion of the posterior frontal suture in an anterior-to-posterior direction. Group B (rotated experimental group) was characterized by 180-degree suture rotation while in vitro and resulted in patency of the posterior frontal suture over the sagittal dura and fusion of the sagittal suture over the posterior frontal dura in a posterior-to-anterior suture direction. Group C (translocated experimental group) was characterized by translocation or shifting of sutures while in vitro and resulted in patency of the posterior frontal suture over the sagittal dura and fusion of the sagittal suture over the posterior frontal dura in an anterior-to-posterior suture direction. These data from the in vitro rotation and translocation experiments indicate that the 'regional' posterior frontal dura determined in vitro cranial suture fusion. Molecular mechanisms behind this process are thought to involve inductive tissue interactions of the dural cells with the suture cells by means of growth factor-mediated signal pathways

Early reconstruction of large osseous defects in children is often delayed due to limited availability of autogenous bone graft donor sites. With the advent of growth factors, osteoinductive proteins, and delivery matrices, it is possible to fabricate new bone at extraskeletal sites. Due to their own blood supply, adequate bony volume, and decreased resorption, vascularized bone flaps have demonstrated greater success in restoring large bony defects compared with nonvascularized bone grafts. The purpose of this study is to prefabricate a vascularized bone flap in the immature-age rabbit using the auricularis anterior muscle as a muscle pedicle. Sixteen female New Zealand White rabbits, 2.0 to 2.5 kg, were divided into two groups. Group 1 contained 8 animals that had T-shaped, 10 x 6 x 4-mm hydroxyapatite (HA) implants combined with 100-microgram bovine-derived bone morphogenetic protein (BMP) placed supraperiosteally and fixed deep to the auricularis anterior muscle. Implants with HA alone were placed in the same animal and secured to the contralateral auricularis anterior muscle. Group 2 contained 8 animals that had HA/BMP placed subperiosteally and fixed deep to the auricularis anterior muscle, while implants with HA alone were secured in the same animal to the contralateral auricularis anterior muscle. In each group, 4 animals were sacrificed at 4 and 8 weeks. The animals underwent randomized bilateral carotid artery injection with micropaque barium suspension just prior to sacrifice to help maintain vascularity. At harvest the implants and surrounding muscle and cranium were removed en bloc. New bone formation in the HA implants was examined by using routine histology and scanning electron microscopic backscattering image (quantitative) analysis. Microradiographs were performed on representative specimens. At 4 weeks postimplantation, backscattering analysis in the subperiosteal HA/BMP showed a mean 17.1% bone ingrowth vs. 11.3% of HA alone (p < 0.05). Supraperiosteal HA/BMP showed a mean 12.9% bone ingrowth vs. 0% of HA alone (p < 0.05). At 8 weeks, backscatter analysis of supraperiosteal HA/BMP showed a mean 19.33% bone ingrowth vs. 0% of HA alone (p < 0.05). Subperiosteal HA/BMP showed a mean 22% bone ingrowth vs. 20.85% of HA alone. This was the only group that did not have statistically significant results. Implant histology demonstrated woven bone within the interstices of HA/BMP placed either supra- or subperiosteally. In the HA-alone implants placed supraperiosteally, fibrovascular ingrowth was seen without any evidence of bone formation. In the HA-alone implant placed subperiosteally, woven bone was seen at the calvarium-implant junction. Microradiographs also demonstrated vascularization and bone formation similar to that seen on scanning electron microscopy. BMP-treated specimens appeared to have slightly greater vascularity than the nontreated specimens. The greatest bone formation occurred with the HA/BMP implant placed subperiosteally in the immature rabbit. Furthermore, these results demonstrate the potential prefabrication of vascularized bone flaps as early as 4 to 8 weeks. The clinical advantage of HA permits the surgeon to design osseous flaps that are customized in shape, fill all contour defects, and have little resorptive properties. Such prefabricated bone with an axial blood supply may allow for ultimate transfer as a pedicle or free flap to reconstruct osseous defects in children

The mechanisms involved in normal cranial suture development and fusion as well as the pathophysiology of craniosynostosis, a premature fusion of the cranial sutures, are not well understood. Transforming growth factor-beta isoforms (TGF-beta 1, beta 2, and beta 3) are abundant in bone and stimulate calvarial bone formation when injected locally in vivo. To gain insight into the role of these factors in normal growth and development of cranial sutures and the possible etiology of premature cranial suture fusion, we examined the temporal and spatial expression of TGF-beta isoforms during normal cranial suture development in the rat. In the Sprague-Dawley rat, only the posterior frontal cranial suture undergoes fusion between 12 and 22 days of age, while all other cranial sutures remain patent. Therefore, immunohistochemical analysis of the fusing posterior frontal suture was compared with the patent sagittal suture at multiple time points from the fetus through adult. Whereas the intensity of immunostaining was the same in the posterior frontal and sagittal sutures in the fetal rat, there was increased immunoreactivity for TGF-beta isoforms in the actively fusing posterior frontal suture compared with the patent sagittal suture starting 2 days after birth and continuing until approximately 20 days. There were intensely immunoreactive osteoblasts present during fusion of the posterior frontal suture. In contrast, the patent sagittal suture was only slightly immunoreactive. A differential immunostaining pattern was observed among the TGF-beta isoforms; TGF-beta 2 was the most immunoreactive isoform and was also most strongly associated with osteoblasts adjacent to the dura and the margin of the fusing suture. Since the increased expression of TGF-beta 2 during suture fusion suggested a possible regulatory role, recombinant TGF-beta 2 was added directly to the posterior frontal and sagittal sutures in vivo to determine if suture fusion could be initiated. Exogenously added TGF-beta 2 stimulated fusion of the ectocranial surface of the posterior frontal suture. These data provide evidence for a regulatory role for these growth factors in cranial suture development and fusion. Additionally, the intense immunostaining for TGF-beta 2 in the dura mater underlying the fusing suture supports a role for the dura mater in suture fusion. It is possible that premature or excessive expression of these factors may be involved in the etiopathogenesis of craniosynostosis and that modulation of the growth factor profile at the suture site may have potential therapeutic value

The biology underlying normal and premature cranial suture fusion remains unknown. To develop a model for normal cranial suture fusion, the temporal sequence of the posterior frontal cranial suture fusion in the mouse was determined. To do this, all the cranial sutures of three distinct strains of mice (CD-1, CF-1, and C57bl-6) were studied histologically for fusion at sequential time points. Two studies were set up using group A mice (n = 72, all sutures studied) and group B mice (n = 78, only the posterior frontal suture studied, but more precisely along its anatomic length). In the group A cranial suture study, mice were sacrificed starting at newborn age and then every 5 days until age 50 days. In addition, two mature mice (250 days old) from each strain were sacrificed. In all three mouse strains, histologic examinations showed that the anterior frontal, sagittal, coronal, lambdoid, and occipitointerparietal sutures remained patent at up to 50 days of age and were patent in the 250-day mature mice. However, examination of the midpoint of the posterior frontal suture showed patency at 30 days, partial fusion at 35 days, and complete fusion by 40 days. These data prompted the posterior frontal suture fusion study. In the group B posterior frontal suture fusion study, mice were sacrificed at age 23 days and then every 2 days until 47 days of age. The anterior, midpoint, and posterior aspects of the posterior frontal suture were examined: The anterior aspect fused between 25 and 29 days; the midpoint fused between 31 and 37 days; and the posterior aspect fused between 39 and 45 days. These data indicate that fusion of the posterior frontal cranial suture in the mouse proceeds in a defined temporal sequence from an anterior to posterior direction in three distinct strains of mice, while in the same mice all other cranial sutures remain patent. By describing and understanding the fusion of the normal posterior frontal suture, a biologic basis of normal suture development and fusion can be established and used as a comparison for murine cranial sutures altered surgically, biochemically (with growth factors), or genetically (with craniosynostotic phenotypes)

A neonate with a unilateral cleft lip and palate usually presents with a deviated nasal septum due to the asymmetric bony base associated with cleft palate. Prior to repair, the facial cleft offers a wide open breathing passage despite the septal deviation. Cleft lips are traditionally repaired in neonates at about 3 months of age. These patients usually do not present with significant symptoms of nasal obstruction following repair, except in unusual cases. Severe septal deviation may cause obstructive sleep apnea. Repair of septal deformities in children is controversial due to the potential alteration of facial growth. We present two patients with documented obstructive sleep apnea that began after cleft lip repair. Conservative surgical correction of the septal deviation resulted in relief of the sleep apnea

Plagiocephaly is a term commonly used to describe congenital forehead asymmetry. Previous classification systems based on the various etiologies of dysmorphic crania have been used in an effort to categorize the patients into groups and to assist in treatment planning. The system most commonly used today was described by Bruneteau and Mulliken in 1992. The authors separated frontal plagiocephaly into three types: synostotic, compensational, and deformational. The present study was undertaken in order to define a simple system for classifying plagiocephaly based on Bruneteau and Mulliken's system using the patients' preoperative craniofacial computed tomography scans. The involvement of the entire coronal ring in synostotic plagiocephaly led to the choice of 20 skull base landmarks as the basis of the analysis. Nine lateral landmarks (the superior orbital fissure, the optic foramen, the zygomatic arch, the greater palatine foramen, the foramen ovale, the mastoid tip, the hypoglossal canal, the external auditory canal, and the internal auditory canal) and two midline landmarks (the crista galli and the internal occipital protuberance) were used. The changes that occurred in these landmarks were analyzed in 30 patients. The results demonstrated that Bruneteau and Mulliken's classification system underestimated the number of different subtypes of plagiocephaly. As a result, three major types of frontal plagiocephaly and several different subtypes based on the different etiologies were described. Type I plagiocephaly includes plagiocephaly resulting from cranial suture synostosis. Type II includes those with a nonsynostotic etiology. Type III describes patients with craniofacial microsomia-associated plagiocephaly. Statistical analysis was unavailable because of the small number of patients in each subtype. With a larger number of patients, we hope to refine this system for use by the surgeon in preoperative diagnosis and surgical planning. The analysis is unique in its ability to quantitate changes from normal on the x-, y-, and z-coordinates, and therefore allows for identification of both horizontal (frontal bone deviation) and vertical (ear shear) growth disturbances