Faculty Bibliography

Premature cranial suture fusion, or craniosynostosis, can result in gross aberrations of craniofacial growth. The biology underlying cranial suture fusion remains poorly understood. Previous studies of the Sprague-Dawley rat posterior frontal suture, which fuses at between 12 and 20 days, have suggested that the regional dura mater beneath the cranial suture directs the overlying suture's fusion. To address the dura-suture paracrine signaling that results in osteogenic differentiation and suture fusion, the authors investigated the possible role of insulin-like growth factors (IGF) I and II. The authors studied the temporal and spatial patterns of the expression of IGF-I and IGF-II mRNA and IGF-I peptide and osteocalcin (bone morphogenetic protein-4) protein in fusing posterior frontal rat sutures, and they compared them with patent coronal (control) sutures. Ten Sprague-Dawley rats were studied at the following time points: 16, 18, and 20 days of gestation and 2, 5, 10, 15, 20, 30, 50, and 80 days after birth (n = 110). Posterior frontal and coronal (patent, control) sutures were analyzed for IGF-I and IGF-II mRNA expression by in situ hybridization by using 35S-labeled IGF-I and IGF-II antisense riboprobes. Levels of IGF-I and IGF-II mRNA were quantified by counting the number of autoradiograph signals per cell. IGF-I and osteocalcin immunoreactivity were identified by avidin-biotin peroxidase immunohistochemistry. IGF-I and IGF-II mRNA were expressed in dural cells beneath fusing sutures, and the relative mRNA abundance increased between 2 and 10 days before initiation of fusion. Subsequently, IGF-I and IGF-II mRNA were detected in the suture connective tissue cells at 15 and 20 days during the time of active fusion. In contrast, within large osteoblasts of the osteogenic front, the expression of IGF-I and IGF-II mRNA was minimal. However, IGF-I peptide and osteocalcin protein were intensely immunoreactive within these osteoblasts at 15 days (during the period of suture fusion). These data suggest that the dura-suture interaction may be signaled in a paracrine fashion by dura-derived growth factors, such as IGF-I and IGF-II. These peptides, in turn, stimulate nearby osteoblasts to produce bone-promoting growth factors, such as osteocalcin

The cleft nasal deformity, a combination of malpositioned cartilage and tissue and postrepair scarring, is a difficult problem to correct. To harness the potential of scarless fetal wound healing, in utero repair of cleft lip and palate deformities has been studied but the fetal cleft nose deformity has not been addressed. The purpose of this study was to manipulate the fetal nasal shape in utero as a first step toward restoration of normal nasal form in cleft nasal deformities. To do this, preformed hypertonic sponges were placed into the right nostril of eight fetal lambs during the second trimester (when scarless cutaneous wound repair is known to occur). Then, the size and shape of fetal nasal structures were analyzed after selected time periods (1, 2, and 6 weeks) with measurements, routine histologic examination, and three-dimensional computed tomographic scans of the experimentally expanded noses compared with the control nonexpanded noses of the birth twins or age-matched specimens. Results showed that experimentally expanded nasal structures had markedly increased in septal length measurement, in nostril area (doubled), and in intranasal volume (more than doubled). Histology showed normal cellular elements without scarring in the tissue sections from the expanded nasal areas. In conclusion, the shape of nasal tissue can be manipulated without scarring in second-trimester fetal lambs after placement of a nasal expansion device. This study is an experimental first step toward restoring normal nasal form by repositioning alar cartilages and soft tissue during fetal cleft repair

The application of distraction osteogenesis in craniofacial surgery has significantly altered the treatment of congenital mandibular deficiencies. However, evaluation of results in both animal studies and clinical cases has revealed deficiencies, particularly in two areas. First, distraction using a uniplanar device in an anteroposterior direction can result in a persistent anterior open bite. Second, the lateralization of the distracted hemimandible was often limited, with insufficient incremental gain in the bigonial distance. To overcome these shortcomings, a multiplanar distraction device was developed and tested in the canine model. This report details canine studies addressing the first problem: combined anteroposterior or sagittal (z-axis) and superoinferior or vertical (y-axis) movements. Six dogs underwent bilateral mandibular distraction with an external (extraoral), multiplanar device and completed sagittal plus vertical distraction. Evaluation included clinical examination (facial form, jaw position, and occlusion), photography, cephalograms (posteroanterior, basilar, and lateral), three-dimensional computed tomography reconstructions, and examination of dry skulls. The dogs averaged 18.5 mm (range, 15-20 mm) of sagittal distraction and 41.0 degrees (range, 21-50 degrees) of vertical distraction. Marked anterior open bites were produced after vertical distraction secondary to premature contact of the maxillary and mandibular molars. Distraction in the vertical direction also had the additive effect of increasing the sagittal gains by approximately 5% to 10%. In conclusion, a multiplanar distraction device (with the potential for distraction in three planes) was effective in increasing mandibular anteroposterior thrust (sagittal distraction) and also in creating an anterior open bite (vertical or superoinferior distraction). Vertical distraction probably requires bilateral osteotomies to obtain optimal results. The preliminary gains in sagittal length are modified (reduced or increased) after distraction in a second plane (vertical and horizontal). Specifically, vertical distraction in the inferior direction (creating an open bite) also leads to isolated increases in the anteroposterior plane. Conversely, vertical distraction in the superior direction (closing an open bite), as seen in a human malocclusion, may lead to isolated decreases in the anteroposterior plane, but this question remains to be investigated in the laboratory

The requirements for reconstruction in patients with midface hypoplasia can be formidable: a bicoronal scalp incision, Le Fort III or monobloc skeletal advancement, harvesting and insertion of bone grafts, application of rigid (and occasionally intermaxillary) fixation, blood transfusions, and prolonged operative time and hospitalization. The introduction of the endoscope offers the possibility of minimally invasive surgery with improved visualization of the osteotomy sites. The development of distraction osteogenesis as a surgical technique allows controlled and gradual advancement of the osteotomized skeletal segment and associated soft tissue. The purpose of this study was to develop a canine model of an endoscopically assisted Le Fort III osteotomy with attendant midface distraction. Four mongrels (20 kg in weight) were study subjects. Three 2-cm skin incisions were made (two perpendicular to the zygomaticomaxillary suture and one perpendicular to the nasofrontal suture). The soft tissue and periosteum were evaluated bluntly. Retractors specially designed for the project created a space for endoscopic visualization. Bilateral zygomatic, nasofrontal, and medial orbital wall osteotomies, corticotomies, or both were performed under endoscopic visualization using a reciprocating saw; the medial orbital wall sectioning was specifically not completed (i.e., corticotomy) to avoid laceration of the mucosa and attendant bleeding. The pterygomaxillary osteotomy was completed with an osteotome and mallet. Finally, the nasal septum was only partially divided with an osteotome to avoid excessive blood loss. Four distraction devices were placed across the above-noted osteotomies (two across the nasofrontal osteotomy and one across each lateral osteotomy). The animals were distracted 1 mm per day for 16 to 40 days after surgery (16-40 mm of linear distraction). Cephalograms and computed tomography scans were obtained before and after distraction. The animals were killed after remaining in fixation for 4 to 6 weeks after distraction. All soft tissue was removed and the skull was examined. Photos were obtained throughout the experiment for documentation. The study demonstrated that Le Fort III osteotomies can be performed successfully via small incisions with endoscopic assistance in canine subjects with excellent visualization and minimal bleeding. The advancement of the midface segment can be achieved by activation of an external distraction device

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