Topical matrix-based siRNA silences local gene expression in a murine wound model.

The ability to affect gene expression via topical therapy has profound therapeutic implications for conditions characterized by open wounds including cutaneous neoplasms, thermal injury, skin disorders and dysfunctional wound healing. Specifically targeting local gene expression avoids systemic toxicity and simplifies treatment. We have developed a new method of topical matrix-based short interfering RNA application to precisely and effectively silence local gene expression in nondelimited wounds.

Hyponatremia in the postoperative craniofacial pediatric patient population: a connection to cerebral salt wasting syndrome and management of the disorder.

Hyponatremia after cranial vault remodeling has been noted in a pediatric patient population. If left untreated, the patients may develop a clinical hypoosmotic condition that can lead to cerebral edema, increased intracranial pressure, and eventually, to central nervous system and circulatory compromise. The hyponatremia has traditionally been attributed to the syndrome of inappropriate secretion of antidiuretic hormone (SIADH); however, in our patients the treatment has been resuscitation with normal saline as opposed to fluid restriction (the accepted treatment of SIADH), thus placing the diagnosis of SIADH in question. Patients who developed hyponatremia after intracranial injury or surgery were, until recently, grouped together as having SIADH. However, there are diagnosis and treatment differences between SIADH and another distinct but poorly understood disorder that is designated cerebral salt wasting syndrome (CSW). CSW is associated with increased urine output and increased urine sodium concentration and volume contraction, and it is frequently seen after a central nervous system trauma. We therefore developed a prospective study to evaluate the cause of the sodium imbalance.Ten consecutive pediatric patients who underwent intracranial surgery for various craniosynostotic disorders were postoperatively monitored in the pediatric intensive care unit for hemodynamic, respiratory, and fluid management. The first four patients were evaluated for electrolyte changes and overall fluid balance to determine the consistency with which these changes occurred. The remaining six patients had daily (including preoperative) measurement of serum electrolytes, urine electrolytes, urine osmolarity, serum antidiuretic hormone (ADH), aldosterone, and atrial natriuretic hormone (ANH). All patients received normal saline intravenous replacement fluid in the postoperative period. All of the patients developed a transient hyponatremia postoperatively, despite normal saline resuscitation. Serum sodium levels as low as 128 to 133 mEq per liter (normal, 137 to 145 mEq per liter) were documented in the patients. All patients had increased urine outputs through the fourth postoperative day (>1 cc/kg/h). The six patients who were measured had an increased ANH level, with a peak value as high as 277 pg/ml (normal, 25 to 77 pg/ml). ADH levels were low or normal in all but one patient, who had a marked increase in ADH and ANH. Aldosterone levels were variable. On the basis of these results, all but one patient showed evidence of CSW characterized by increased urine output, normal or increased urine sodium, low serum sodium, and increased ANH levels. The other patient had similar clinical findings consistent with CSW but also had an increase in ADH, thus giving a mixed laboratory picture of SIADH and CSW. The association of CSW to cranial vault remodeling has previously been ignored. This study should prompt reevaluation of the broad grouping of SIADH as the cause of all hyponatremic episodes in our postoperative patient population. An etiologic role has been given to ANH and to other, as yet undiscovered, central nervous system natriuretic factors. All of the patients studied required normal saline resuscitation, a treatment approach that is contrary to the usual management of SIADH. These findings should dictate a change in the postoperative care for these patients. After cranial vault remodeling, patients should prophylactically receive normal saline, rather than a more hypotonic solution, to avoid sodium balance problems.

Auricular reconstruction: indications for autogenous and prosthetic techniques.

LEARNING OBJECTIVES: After studying this article, the participant should be able to: 1. Describe the alternatives for auricular reconstruction. 2. Discuss the pros and cons of autogenous reconstruction of total or subtotal auricular defects. 3. Enumerate the indications for prosthetic reconstruction of total or subtotal auricular defects. 4. Understand the complexity of and the expertise required for prosthetic reconstruction of auricular defects. The indications for autogenous auricular reconstruction versus prosthetic reconstruction with osseointegrated implant-retained prostheses were outlined in Plastic and Reconstructive Surgery in 1994 by Wilkes et al. of Canada, but because of the relatively recent Food and Drug Administration approval (1995) of extraoral osseointegrated implants, these indications had not been examined by a surgical unit in the United States. The purpose of this article is to present an evolving algorithm based on an experience with 98 patients who underwent auricular reconstruction over a 10-year period. From this experience, the authors conclude that autogenous reconstruction is the procedure of choice in the majority of pediatric patients with microtia. Prosthetic reconstruction of the auricle is considered in such pediatric patients with congenital deformities for the following three relative indications: (1) failed autogenous reconstruction, (2) severe soft-tissue/skeletal hypoplasia, and/or (3) a low or unfavorable hairline. A fourth, and in our opinion the ideal, indication for prosthetic ear reconstruction is the acquired total or subtotal auricular defect, most often traumatic or ablative in origin, which is usually encountered in adults. Although prosthetic reconstruction requires surgical techniques that are less demanding than autogenous reconstruction, construction of the prosthesis is a time-consuming task requiring experience and expertise. Although autogenous reconstruction presents a technical challenge to the surgeon, it is the prosthetic reconstruction that requires lifelong attention and may be associated with late complications. This article reports the first American series of auricular reconstruction containing both autogenous and prosthetic methods by a single surgical team.

Restoration of abdominal wall integrity as a salvage procedure in difficult recurrent abdominal wall hernias using a method of wide myofascial release.

The management of primary and recurrent giant incisional hernias remains a complex and frustrating challenge even with multiple alloplastic and autogenous closure options. The purpose of this study was to develop a reconstructive technique of restoring abdominal wall integrity to a subcategory of patients, who have failed initial hernia therapy, by performing superior and lateral myofascial release. Over a 1.5-year period, 10 patients with previously unsuccessful treatment of abdominal wall hernias, using either primary repair or placement of synthetic material, were studied. The patients had either recurrence of the hernia or complications such as infections requiring removal of synthetic material. The hernias were not able to be treated with standard primary closure techniques or synthetic material. The average defect size was 19 x 9 cm. Each patient underwent wide lysis of bowel adhesions releasing the posterior abdominal wall fascia to the posterior axillary line, subcutaneous release of the anterior abdominal wall fascia to a similar level, and complete removal of any synthetic material (if present). The abdominal domain was reestablished by releasing the laterally retracted abdominal wall. The amount of available abdominal wall tissue was increased by wide release of the cephalic abdominal wall fascia overlying the costal margin and the external oblique fascia and muscle laterally. If needed, partial thickness of the internal oblique muscle and its anterior fascia were also released laterally to perform a tension-free primary closure of the defect. All repairs were closed with satisfactory functional and aesthetic results. All alloplastic material was removed. Fascial release was limited so as to close only the hernia defect without tension. No significant release of the rectus sheath and muscle was needed. Good, dynamic muscle function was noted postoperatively. All repairs have remained intact, and no further abdominal wall hernias have been noted on follow-up.

Osteoporosis. Recommended guidelines and New Jersey legislation.

More than 28 million Americans aged 50 and over have osteoporosis or low bone density. While 80% of those affected are women, one in eight men also suffer from the disease. This number is expected to increase as men live longer. Commonly called the "silent disease," osteoporosis exhibits no symptoms or pain until a fracture occurs. The most common fractures involve the wrist, vertebra, and hip. Half of all women and 20% of men will have at least one osteoporosis fracture in their lifetime. This article describes statewide efforts to prevent and manage osteoporosis and the recommended practice guidelines for the diagnosis and treatment of the disease.

Growth restriction of cranial sutures in the fetal lamb causes deformational changes, not craniosynostosis.

Newborns with in utero cranial vault molding can present with severe forms of plagiocephaly. Intrauterine constraint has been proposed as one cause for craniosynostosis. The purpose of this experiment was to investigate whether rigid plate fixation across a fetal cranial suture, representing a severe form of growth restriction in utero, would lead to cranial suture fusion in a fetal lamb model. Six fetal lambs at 85 to 95 days gestation (term = 145 days) underwent laparotomy, hysterotomy, fetal coronal scalp incision, and miniplate screw fixation across the right coronal suture in utero. Two unoperated twins and four unoperated age-matched lambs were used as controls (n = 12). Animals were killed at both 4 and 8 weeks postoperatively. Fetal head analysis consisted of gross examination, photography, basilar and lateral radiographs, and three-dimensional computed tomographic scans. Cranial suture analysis consisted of imaging by computed tomographic scan (axial and sagittal cuts) and histology of experimentally plated coronal sutures, contralateral nonplated coronal sutures and twin control coronal sutures. Gross examination, radiographs, and three-dimensional computed tomographic analysis of heads with cranial suture plating showed ipsilateral forehead flattening, contralateral forehead bossing, superiorly displaced ipsilateral orbital rim, anterolateral projection of ipsilateral malar eminence, and anterior position of the ipsilateral ear point compared with the contralateral side of the same animal and normal controls. There was no change in nasal root, chin point, or predentition occlusal plane. Although analysis of the plated coronal sutures by computed tomographic scans showed diminished width or even stenosis, the histology revealed narrowed but patent experimental coronal sutures at 4 and 8 weeks. Contralateral, nonplated coronal sutures were not only patent, but widened compared with normal control sutures. This finding may have represented compensatory changes in the contralateral coronal suture caused by growth restriction at the plated suture. These data demonstrate that intrauterine growth restriction across a cranial suture caused by compression plate fixation resulted in deformational skull changes, not craniosynostosis. In addition, these data strongly support a role for in utero positional molding secondary to growth restriction in the maternal pelvis as a cause for nonsynostotic plagiocephaly seen in newborns.

Increased IGF-I and IGF-II mRNA and IGF-I peptide in fusing rat cranial sutures suggest evidence for a paracrine role of insulin-like growth factors in suture fusion.

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.

Nasal expansion in the fetal lamb: a first step toward management of cleft nasal deformity in utero.

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 combination of endoscopy and distraction osteogenesis in the development of a canine midface advancement model.

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.

Studies in cranial suture biology: up-regulation of transforming growth factor-beta1 and basic fibroblast growth factor mRNA correlates with posterior frontal cranial suture fusion in the rat.

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.

Studies in cranial suture biology: regional dura mater determines overlying suture biology.

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.

Controlled multiplanar distraction of the mandible, Part II: Laboratory studies of sagittal (anteroposterior) and vertical (superoinferior) movements.

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.

Peripheral progesterone (P) levels and endometrial response to various dosages of vaginally administered P in estrogen-primed women.

OBJECTIVE: To compare the pharmacokinetics and pharmacodynamics of 100 mg/d, 200 mg/d, and 400 mg/d (200 mg two times per day) of P administered vaginally for 14 days to estrogen-primed postmenopausal women. DESIGN: Randomized, open-label, three-way crossover study. SETTING: Two university-based investigative sites. PATIENT(S): Twenty healthy postmenopausal women with histologically normal endometria. INTERVENTION(S): Oral 17 beta-E2 was given each day of a 28-day cycle; a P vaginal suppository was inserted daily according to the randomization schedule during days 15-28 of each cycle; blood samples were collected; an endometrial biopsy was obtained on day 25; and patients were crossed over to the next treatment cycle after a washout period of at least 30 days. MAIN OUTCOME MEASURE(S): Mean P blood levels, endometrial dating/conversion. RESULT(S): There was good vaginal absorption of P for all dosages. Endometrial conversion occurred in all 200- and 400-mg/d P-dosed cycles, whereas the 100-mg/d dosage failed to convert primed endometria consistently. There also was a significantly increased tendency for earlier bleeding and spotting with the 100-mg/d dosage. CONCLUSION(S): Both the 200- and 400-mg/d dosage regimens consistently convert an estrogen-primed endometrium, and yield appropriate endometrial dating and bleeding patterns. However, the 400-mg/d dosage attains the highest sustained blood levels and may be the best dosage regimen for further study.

Studies in cranial suture biology: regional dura mater determines in vitro cranial suture fusion.

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.

Bone morphogenetic protein promotes vascularization and osteoinduction in preformed hydroxyapatite in the rabbit.

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.

Studies in cranial suture biology: Part I. Increased immunoreactivity for TGF-beta isoforms (beta 1, beta 2, and beta 3) during rat cranial suture fusion.

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.

Studies in cranial suture biology: IV. Temporal sequence of posterior frontal cranial suture fusion in the mouse.

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).

Studies in cranial suture biology: part II. Role of the dura in cranial suture fusion.

The biology underlying normal and premature cranial suture fusion remains unknown. The purpose of this study was to investigate the role of the dura mater in cranial suture fusion. In the Sprague Dawley rat model, the posterior frontal cranial suture fuses between 10 and 20 days of postnatal life. The effect of separating the posterior frontal cranial suture from its underlying dura mater with an intervening silastic sheet was studied. Sixty rat pups, age 8 days, were divided into four groups of 15. Group A served as unoperated controls. Group B, the experimental group, underwent craniotomy, dural elevation, and insertion of a silicone sheet between the posterior frontal cranial suture and the underlying dura. Two operative sham groups were included. Group C underwent craniotomy and dural deflection only. Group D underwent craniotomy alone without dural deflection. The rats were sacrificed at 15, 22, and 30 days of age. The results showed that the unoperated animals (group A) demonstrated normal initiation of suture fusion at 15 days and complete fusion by 22 days. Group B animals, with silicone sheet barriers placed, showed persistent patency of sutures at 22 days. Initiation of suture fusion was delayed until 30 days. Sham group C, animals with craniotomy and dural deflection, showed that initiation of fusion was delayed until 22 days with complete fusion by 30 days of age. Sham group D, craniotomy alone, had the same normal temporal sequence of suture fusion as the unoperated control group A. These data indicate that normal cranial suture fusion is delayed when the suture-dural interaction is interrupted by a surgically place barrier or by simple dural deflection. Furthermore, interaction between the dura and the overlying suture appears to direct suture fusion.

Studies in cranial suture biology: in vitro cranial suture fusion.

The biology underlying craniosynostosis remains unknown. Previous studies have shown that the underlying dura mater, not the suture itself, signals a suture to fuse. The purpose of this study was to develop an in vitro model for cranial-suture fusion that would still allow for suture-dura interaction, but without the influence of tensional forces transmitted from the cranial base. This was accomplished by demonstrating that the posterior frontal mouse cranial suture, known to be the only cranial suture that fuses in vivo, fuses when plated with its dura in an organ-culture system. In such an organ-culture system, the sutures are free from both the influence of dural forces transmitted from the cranial base and from hormonal influences only available in a perfused system. For the cranial-suture fusion in vitro model study, the sagittal sutures (controls that remain patent in vivo) and posterior frontal sutures (that fuse in vivo) with the underlying dura were excised from 24-day-old euthanized mice, cut into 5 x 4 x 2-mm specimens, and cultured in a chemically defined, serum-free media. One hundred sutures were harvested at the day of sacrifice, then every 2 days thereafter until 30 days in culture, stained with H & E, and analyzed. A subsequent cranial-suture without dura in vitro study was performed in a similar fashion to the first study, but only the calvariae with the posterior frontal or sagittal sutures (without the underlying dura) were cultured. Results from the cranial-suture fusion in vitro model study showed that all sagittal sutures placed in organ culture with the underlying dura remained patent. More importantly, the posterior frontal sutures with the underlying dura, which were plated-down as patent at 24 days of age, demonstrated fusion after various growth periods in organ culture. In vitro posterior frontal mouse-suture fusion occurred in an anterior-to-posterior direction but in a delayed fashion, 4 to 7 days later than in vivo posterior frontal mouse-suture fusion. In contrast, the subsequent cranial-suture without dura in vitro study showed patency of all sutures, including the posterior frontal suture. These data from in vitro experiments indicate that: (1) mouse calvariae, sutures, and the underlying dura survive and grow in organ-culture systems for 30 days; (2) the local dura, free from external influences transmitted from the cranial base and hormones from distant sites, influences the cells of its overlying suture to cause fusion; and (3) without dura influence, all in vitro cranial sutures remained patent. By first identifying the factors involved in dural-suture signaling and then regulating these factors and their receptors, the biologic basis of suture fusion and craniosynostosis may be unraveled and used in the future to manipulate pathologic (premature) suture fusion.

Sexual abuse and premenstrual syndrome: comparison between a lower and higher socioeconomic group.

Of 174 women presenting symptoms of premenstrual syndrome (PMS), 40% had a history as victims of sexual abuse. Thirty-three percent were of a high-SES (socioeconomic status) group and 52% of a low-SES group. The authors found a connection between sexual abuse and psychiatric hospitalization for women seeking treatment for PMS. Beck Depression Inventory scores were high, supporting current research indicating a prevalence of affective disorders in women seeking treatment for PMS. However, the authors found few differences between the high- and low-SES groups. Specific questioning regarding a history of adverse sexual experiences is critical in evaluating all patients.