Removal of mandibular tooth follicles before distraction osteogenesis

Distraction osteogenesis is an innovative technique that has transformed the treatment of craniofacial malformations in young children. Bone generation obviates the need for graft material, which is in short supply in young patients, thus making possible surgical procedures on the craniofacial skeleton in young children. Sufficient mandibular volume is required for the osteotomy and placement of the device screws and/or pins. To have adequate bone stock and to facilitate distraction, the authors preoperatively examined all patients radiographically and selected those with tooth follicles that precluded successful osteotomy and pin placement for planned mandibular distraction. This report is of the first 13 children, aged 9 months to 6 years, who underwent predistraction enucleation. The osteotomy and device placement were performed successfully at least 4 months after enucleation. The described procedure has minimal morbidity and has resulted in successful subsequent distraction. The advantages, disadvantages, and cost-benefit issues are discussed.     

Transport distraction osteogenesis: a new method to heal adult calvarial defects

Popularized by Gavril Ilizarov in the 1960s, monofocal distraction osteogenesis has become a well-established method of endogenous bone engineering. This revolutionary surgical technique has significantly augmented the available reconstructive orthopedic and craniomaxillofacial procedures. Bifocal distraction osteogenesis, or bone transportation, is a modification of monofocal distraction that involves moving a free segment of living bone to fill an intercalary bone defect. Bifocal distraction has been applied successfully to reconstruct complex mandibular and long bone defects. Because traumatic or postsurgical calvarial defects do not spontaneously heal in humans older than 18 to 24 months of age, we hypothesized that bifocal distraction osteogenesis could be applied to the skull to close critical size calvarial defects. Critical size (15 x 15 mm) calvarial defects were created in eight New Zealand White rabbits. Next, a 15-mm x 10-mm calvarial box osteotomy was created just anterior to the skull defect. This osteotomy created a free bone segment that could be transported. A custom-made transport distraction device was fixed into place and the skin incision was closed. After a 4-day latency period, the distraction device was activated (0.5 mm once daily for 30 days) in seven animals; the distraction device in one animal was not activated and served as a control. All animals underwent 30 days of consolidation and were then killed. Radiographs and computed tomographic scans were performed at the following time points: end of latency period (postoperative day 4), mid-distraction (postoperative day 19), and end of consolidation period (postoperative day 64). Gross and histologic analysis was performed to evaluate the quality of the bony regenerate. The control animal healed with a fibrous union. Complete closure of the skull defects was observed in five of seven rabbits at the end of the consolidation period. One animal was removed from the study because of an early loosening of the distraction device, and one was removed because of device failure. Of the remaining five animals that completed the distraction protocol, radiographs and computerized tomographic scans showed successful ossification in all five rabbits at the end of the consolidation period. This study suggests that transport distraction osteogenesis is a promising technique that may be applied to a variety of commonly encountered craniofacial problems such as nonhealing calvarial defects.     

Adjustable Stiffness,External Fixator for the Rat Femur Osteotomy and Segmental Bone Defect Models

The mechanical environment around the healing of broken bone is very important as it determines the way the fracture will heal. Over the past decade there has been great clinical interest in improving bone healing by altering the mechanical environment through the fixation stability around the lesion. One constraint of preclinical animal research in this area is the lack of experimental control over the local mechanical environment within a large segmental defect as well as osteotomies as they heal. In this paper we report on the design and use of an external fixator to study the healing of large segmental bone defects or osteotomies. This device not only allows for controlled axial stiffness on the bone lesion as it heals, but it also enables the change of stiffness during the healing process in vivo. The conducted experiments have shown that the fixators were able to maintain a 5 mm femoral defect gap in rats in vivo during unrestricted cage activity for at least 8 weeks. Likewise, we observed no distortion or infections, including pin infections during the entire healing period. These results demonstrate that our newly developed external fixator was able to achieve reproducible and standardized stabilization, and the alteration of the mechanical environment of in vivo rat large bone defects and various size osteotomies. This confirms that the external fixation device is well suited for preclinical research investigations using a rat model in the field of bone regeneration and repair.     

A new transplant bone for maxillary alveolar cleft

An innovative technique with distraction osteogenesis has been developed in our research group to explore autogenous bone transplantation into craniofacial bone defects. This technique was designed to investigate bone-marrow transplantion using a chondroid or fibula bone graft into simulated alveolar bone defects in mice in terms of the osteogenic process and activity. As an experimental model of maxillary alveolar bone cleft available for testing bone-inductive materials, a critical-size defect was formed in the pre-maxillary bone of male mice using a surgical trephine bur with a low-speed dental engine. Distraction osteogenesis was performed using an external fixation device. The osteotomy site was occupied by an external callus consisting of hyaline cartilage with a large quantity of chondroid bone. Moreover, bone remodelling with new bone formation was demonstrated 30 days after the transplantation. Bone adhesion was better in chondroid bone grafting than in fibula bone grafting. The present findings are the first to demonstrate the potential of chondroid bone transplantation as a new therapeutic system of bone grafting, suitable for bone substitutes in craniofacial bone defects.     

Feminization of the forehead: contour changing to improve female aesthetics

Anthropologists have identified those characteristics which enable them to differentiate the male from the female skull. Some women have masculine skeletal characteristics which, if changed, would improve their facial appearance. Changing the skeletal configuration through bony contouring of the craniofacial skeleton for aesthetic purposes is a natural spinoff of craniomaxillofacial surgery. Techniques for sculpturing the masculine characteristics present in the foreheads of some females are discussed. The deformity has been divided into three subdivisions. Group 1 patients can be treated through bony contouring alone; group 2 patients require bony contouring in conjunction with a methyl methacrylate cranioplasty; and group 3 are those patients with a more severe deformity requiring osteotomies. The technique, results, complications, and patient acceptance are discussed.     

Distraction osteogenesis of costochondral bone grafts in the mandible

Costochondral grafting for reconstruction of the Pruzansky type III mandible has given variable results. Lengthening of the rib graft by means of distraction had been advocated when subsequent growth of the grafted mandible is inadequate. This retrospective study reviews a series of patients with mandibular costochondral grafts who underwent subsequent distraction osteogenesis of the graft. A retrospective review identified two patient groups: group 1 consisted of individuals (n = 9) who underwent costochondral rib grafting of the mandible followed by distraction osteogenesis several months later at a rate of 1 mm/day. Group 2 consisted of patients with Pruzansky type II mandibles who had distraction osteogenesis without prior rib grafting (n = 9). The biomechanical parameters, orthodontic treatment regimens, and complications were examined versus patient age and quality of the rib graft. Distraction osteogenesis was successfully performed in six of the rib graft patients (group 1) and in all of the group 2 individuals. On the basis of the Haminishi scale, the computed tomographic scan appearance of the regenerate was classified as "standard or external" in six of the group 1 patients and as either "agenetic" or "pillar" (fibrous union) in the remaining three patients. In group 1, the average device was expanded 23 mm (range, 20 to 30 mm). Group 2 mandibular distraction results were all classified as either standard or external, and there was an average device expansion of 22.4 mm (range, 16 to 30 mm). The length of consolidation averaged 12.6 weeks in group 1, compared with 8.5 weeks in the traditional mandibular distraction patients (group 2). The mean shift of the dental midline to the contralateral side was 2.5 mm in group 1 versus 4.0 mm in group 2. Complex multiplanar and transport distractions were successfully performed on grafts of adequate bony volume. All four patients in group 1 with tracheostomies were successfully decannulated after consolidation. Rib graft distraction complications included pin tract infections in two patients, hardware failure with premature pin pullout in one patient, and evidence of fibrous nonunions in three young patients with single, diminutive rib grafts. In group 2, there were no distraction failures. Distraction osteogenesis can be successfully performed on costochondral rib grafts of the mandible; however, the complication rate is higher than in non-rib-graft patients. Performing the technique on older, more cooperative individuals seems to reduce this risk. In addition, placement of a double rib graft or an iliac bone graft of sufficient volume to create a neomandible with greater bone stock is an absolute requirement to decrease the risk of fibrous nonunion and provide a bone base of sufficient size for retention of the distraction device and manipulation of the regenerate.     

Immediate versus delayed midface distraction in a primate model using a new intraoral internal device

The theoretic advantage of distraction osteogenesis of the craniofacial skeleton, especially in cases of severe midface retrusion and in the presence of maxillary scarring, is prevention of relapse following significant advancements. The purpose of this study is to demonstrate the utility of a new low-profile, intraoral, internal device for midface distraction at the conventional or high Le Fort I level. In addition, the present study compares the efficacy of immediate versus delayed distraction on subsequent maxillary relapse. Four adult rhesus Macaca mulatta monkeys were divided into two groups. Group 1 underwent immediate midface distraction; group 2 underwent delayed distraction. All four monkeys underwent a modified Le Fort I osteotomy through an upper buccal sulcus incision and bilateral application of the intraoral midface distraction devices. No other osteotomies or incisions were necessary. Immediate distraction, performed in group 1, entailed intraoperative activation of the devices and distraction of 10 mm followed by a 5-day lag period before postoperative activation and distraction of an additional 10 mm at the rate of 1 mm/day. Delayed distraction, performed in group 2, entailed a 5-day postoperative lag period before device activation and distraction of 20 mm at the rate of 1 mm/day. Both groups thus underwent 20 mm of midface distraction. All devices were removed 6 weeks after completion of distraction. All monkeys tolerated the devices and daily distraction uneventfully. On the basis of serial cephalograms and dental models obtained throughout the experimental period, there was no evidence of relapse in either the immediate or delayed groups 6 months after distraction. In addition, on the basis of histologic, ultrastructural, and dry skull analysis, no significant differences were observed in the quality of regenerate bone obtained when comparing the immediate and delayed distraction groups. Significant midface advancement is thus feasible using this new internal, intraoral distraction device, which presents several advantages over other internal devices that require coronal incisions and additional osteotomies to achieve midface advancement. In addition, immediate distraction may abbreviate the distraction period without adverse sequelae.     

Distraction osteogenesis of the craniofacial skeleton

In summary, distraction osteogenesis is a safe and effective means of achieving bone lengthening. These techniques were originally applied to the long bones of the extremities; over the past 10 years they have been effectively applied to the bones of the craniofacial skeleton. The new bone regenerate that is observed after distraction osteogenesis is stable, and relapse rates after skeletal advancement are believed to be lower than with conventional osteotomy and bone graft techniques. There is considerable variability in distraction protocols employed in clinical practice, including differences in the types of devices used and in the rate, rhythm, latency, and period of consolidation for distraction osteogenesis. The greatest application for distraction osteogenesis in the craniofacial skeleton has been with mandible lengthening, for which there is presently a 10-year clinical experience. Midfacial advancement is a newer application of distraction osteogenesis, for which clinical experience has been accrued over the past 5 years. This latter experience indicates that distraction osteogenesis is a viable treatment option for lengthening of the hypoplastic mandible and midface. These techniques have advantages over conventional means of bone graft and rigid fixation because of the quality of the bone regenerate, the decrease in the long-term relapse rate of the advanced bone segments in both the mandible and the midface, and the simultaneous soft-tissue elongation that accompanies the distraction process. Distraction osteogenesis is particularly applicable to the correction of severe deformities of the mandible and midface in children with developmental hypoplasia and syndromic craniosynostosis. However, growth is an added variable in this patient population. The amount of overcorrection in lengthening of the hypoplastic bone required to compensate for continued growth discrepancy of the adjacent facial bones is difficult to predict. Therefore, the families of these patients should be informed that many children will require repeated operations at a later age as they reach skeletal maturity.     

Development and clinical application of an external fixator monitoring system

A monitoring system for measuring movement occurring in a dynamic external fixator used to treat fractures is described. The system measures shortening during fracture healing, micromovement at the fracture site on weight bearing and detects pin loosening. The method of calibration including cadaver experiments is presented. The clinical application is described and the reasons for measuring movement are discussed.     

The use of miniplates in craniomaxillofacial surgery

Miniplates were used in craniomaxillofacial surgery for fixation in the skull, maxilla, and/or mandible in 74 patients with minimal or no intermaxillary wiring. Procedures included forehead and orbital repositioning, frontofacial advancement, Le Fort III and particularly Le Fort I osteotomies, as well as mandibular osteotomies and fracture repair. The miniplates provided stable fixation and, compared with other techniques, improved airway safety. The complication rate was low: there were no infections, but two plates (1 percent) became exposed in the buccal sulcus. Although application of miniplates lengthened surgery and increased the cost of the procedure, the savings in intensive care monitoring more than offset these costs. The stability of fixation minimizes the opportunity to reposition the fragments postoperatively with training elastics. Therefore, meticulous technique is mandatory, with particular emphasis on passive fitting of the plates and precise drilling of screw holes.     

The structural elements responsible for contraction in the ciliateSpirostomum

Summary The surface of the contractile ciliateSpirostomum contains continuous spiral ciliated grooves traversing its length. Bundles of microtubules run parallel to the base of the grooves and appear to be part of a cohesive, semi-rigid cortex. Beneath this, a network of microfilament bundles occurs which is attached to the peristomal membranelle apparatus, also part of the cortex. The possible roles played by these structures during contraction of the organism were examined.During contraction, the entire cortex twists and the spiral arrangement of the grooves and microtubules decreases in pitch and increases in diameter. Simultaneously, the bundles of microfilaments change in distribution and appearance so as to suggest that they are undergoing an active shortening process. Based on these observations, two models for contraction are presented. In one, shortening of the animal arises from a smooth muscle-like contraction of the microfilament network whose attachment to the basal bodies of the membranellar cilia guarantees shortening and widening of the cortex and consequently the whole animal. In the second, a shortening (or sliding) of external microtubules relative to internal ones in the cortical microtubule bundles would result in an increase in diameter, a decrease in pitch, and a decrease in axial length of the bundles, resulting in contraction of the animal. The observations do not allow a choice between these alternatives to be made, and they may not, in fact, be mutually exclusive.     

Effects of ACh and adenosine mediated by Kir3.1 and Kir3.4 on ferret ventricular cells

The inotropic effects of ACh and adenosine on ferret ventricular cells were investigated with the action potential-clamp technique. Under current clamp, both agonists resulted in action potential shortening and a decrease in contraction. Under action potential clamp, both agonists failed to decrease contraction substantially. In the absence of agonist, application of the short action potential waveform (recorded previously in the presence of agonist) also resulted in a decrease in contraction. Under action potential clamp, application of ACh resulted in a Ba(2+)-sensitive outward current with the characteristics of muscarinic K+ current (I(K,ACh)); the presence of the muscarinic K+ channel was confirmed by PCR and immunocytochemistry. In the absence of agonist, on application of the short ACh action potential waveform, the decrease in contraction was accompanied by loss of the inward Na(+)/Ca(2+) exchange current (I(NaCa)). ACh also inhibited the background inward K+ current (I(K,1)). It is concluded that ACh activates I(K,ACh), inhibits I(K,1), and indirectly inhibits I(NaCa); this results in action potential shortening, decrease in contraction, and, as a result of the inhibition of I(K,1), minimum decrease in excitability.     

Craniofacial growth in subjects with unilateral complete cleft lip and palate, and unilateral incomplete cleft lip, from 2 to 22 months of age

This paper reports a longitudinal quantitative cephalometric analysis of the craniofacial growth in subjects with unilateral complete cleft lip and palate (UCCLP), and unilateral incomplete cleft lip (UICL), from 2 to 22 months of age. The purpose of the study was to determine the amount and direction of growth in UCCLP compared to UICL (control group) from 2 months of age (just prior to lip repair) to 22 months of age, 20 months later. The sample comprised of 49 subjects with UCCLP (37 males and 11 females) and 45 with UICL (29 males and 16 females). The cephalometric analysis of the craniofacial morphology included lateral, frontal, and axial projections. The data were presented as mean plots of the craniofacial region including the calvaria, cranial base, orbits, nasal bone, maxilla, mandible, cervical column, pharynx, and soft-tissue profile. A valid common coordinate system (registration according to the n-s line in the lateral projection, latero-orbitale line in the frontal projection, and meatus acusticus externus line in the axial projection for the landmark positions at examination 1 and 2) was ascertained. The growth at a specific anatomical location in a patient was defined as the displacement vector from the coordinate of the corresponding landmark in the X-ray at examination 1 to its coordinate at examination 2, corrected for X-ray magnification. The growth of an anatomical region in a patient was assessed by investigating the growth pattern formed by a collection of individual growth vectors in that region. The amount of growth in the UCCLP and UICL group was very similar. The general craniofacial growth pattern, in terms of the direction of growth, was also fairly similar in the UCCLP group and the control group. However, the maxilla and mandible showed a more vertical growth pattern than that observed in the control group. This study confirms that UCCLP is a localized deviation, and not a craniofacial anomaly, due to the fact that a normal growth potential has been observed in all craniofacial regions, except where the growth had been directly influenced by surgical intervention. Furthermore, the vertical growth pattern of the maxilla and mandible supports the hypothesis of a special facial type in cleft lip and palate individuals, and the facial type as a liability factor increasing the probability of cleft lip and palate.     

Linkage between parasternals and external intercostals during resting breathing

To assess the mechanical coupling between the parasternal and external intercostals in the cranial portion of the rib cage, we measured the respiratory changes in length and the electromyograms of the two muscles in the same third or fourth intercostal space in 24 spontaneously breathing dogs. We found that 1) the amount of inspiratory shortening of the external intercostal was considerably smaller than the amount of shortening of the parasternal; 2) after selective denervation of the parasternal, the inspiratory shortening of both the parasternal and the external intercostal was almost abolished; 3) on the other hand, after selective denervation of the external intercostal, the inspiratory shortening of the parasternal was unchanged, and the inspiratory shortening of the external intercostal was reduced but not suppressed; and 4) this persistent shortening of the external intercostal was reversed into a clear-cut inspiratory lengthening when the parasternal was subsequently denervated. We conclude that in the dog 1) the inspiratory contraction of the external intercostals in the cranial portion of the rib cage is agonistic in nature as is the contraction of the parasternals; 2) during resting breathing, however, the changes in length of these external intercostals are largely determined by the action of the parasternals. These observations are consistent with the idea that in the dog, the parasternals play a larger role than the external intercostals in elevating the ribs during resting inspiration.     

Rotation advancement of the midface by distraction osteogenesis

A wide variety of disease processes produce alteration of midfacial skeletal growth, resulting in moderate-to-severe midface deficiency presenting as retrusion associated with Angle's class III malocclusion. Le Fort III osteotomies with advancement can provide an excellent tool for correction of this deformity. Recently, the corrective procedure of choice for advancement of midfacial segments has been distraction osteogenesis after osteotomy. Straight linear advancement is the most common choice for corrective movement of the midfacial segment, whether accomplished through acute surgical advancement or through the progressive distraction technique. Unfortunately, linear advancement can produce abnormal configurations, both at the nasal root and lateral orbits, regardless of the technique used. Enophthalmos, caused by orbital enlargement, may limit the advancement necessary to achieve class I occlusion.The authors have extended the utility of the Le Fort III procedure and have improved the final outcome by creating a controlled rotation advancement of the midfacial segment using distraction. The application of an existing internal distraction device is modified to control the movement of the midfacial segment in a rotation advancement path. Included in the series were 10 patients with severe midface retrusion secondary to multiple congenital syndromes, along with cleft lip and palate. The ages of the patients ranged from 6 to 14 years. An internal distraction system was used in all cases. Application of the distractor was substantially modified to simplify both fixation and removal and to produce controlled rotation advancement. The team orthodontist determined the final occlusal relationship. Percutaneous distractor drive rods were removed 4 to 6 weeks after active distraction to increase patient comfort. The distractors and all associated hardware were removed after 12 to 16 weeks of consolidation; follow-up periods ranged from 1 to 3 years.By using the modified distractor application to produce rotation advancement, the contour abnormalities at the nasal root and lateral orbit and the enophthalmos produced by linear advancement were eliminated. Significant improvement in facial contour and class I occlusion was obtained in all cases. Complications consisted of near exposure of the device in one patient. Stability has been excellent, with no relapse reported by the orthodontist.Rotational advancement of facial segments by distraction allows successful early intervention in patients with significant midface retrusion. The abnormal nasal root and lateral orbital configurations produced by direct linear advancement are avoided, and a stable and normalized facial configuration is produced.     

Rat mandibular distraction osteogenesis: part III. Gradual distraction versus acute lengthening

Distraction osteogenesis is a well-established method of endogenous tissue engineering. This technique has significantly augmented our armamentarium of reconstructive craniofacial procedures. Although the histologic and ultrastructural changes associated with distraction osteogenesis have been extensively described, the molecular mechanisms governing successful membranous distraction remain unknown. Using an established rat model, the molecular differences between successful (i.e., osseous union with gradual distraction) and ineffective (i.e., fibrous union with acute lengthening) membranous bone lengthening was analyzed. Herein, the first insight into the molecular mechanisms of successful membranous bone distraction is provided. In addition, these data provide the foundation for future targeted therapeutic manipulations designed to improve osseous regeneration. Vertical mandibular osteotomies were created in 52 adult male Sprague-Dawley rats, and the animals were fitted with customized distraction devices. Twenty-six animals underwent immediate acute lengthening (3 mm; a length previously shown to result in fibrous union) and 26 animals were gradually distracted (after a 3-day latency period, animals were distracted 0.25 mm twice daily for 6 days; total = 3 mm). Four mandibular regenerates were harvested from each group for RNA analysis on 5, 7, 9, 23, and 37 days postoperatively (n = 40). Two mandibular regenerates were also harvested from each group and prepared for immunohistochemistry on postoperative days 5, 7, and 37 (n = 12). In addition to the 52 experimental animals, 4 control rats underwent sham operations (skin incision only) and mandibular RNA was immediately collected. Control and experimental specimens were analyzed for collagen I, osteocalcin, tissue inhibitor of metalloproteinase-1, and vascular endothelial growth factor mRNA and protein expression. In this study, marked elevation of critical extracellular matrix molecules (osteocalcin and collagen I) during the consolidation phase of gradual distraction compared with acute lengthening is demonstrated. In addition, the expression of an inhibitor of extracellular matrix turnover, tissue inhibitor of metalloproteinase-1, remained strikingly elevated in gradually distracted animals. Finally, this study demonstrated that neither gradual distraction nor acute lengthening appreciably alters vascular endothelial growth factor expression. These results suggest that gradual distraction osteogenesis promotes successful osseous bone repair by regulating the expression of bone-specific extracellular matrix molecules. In contrast, decreased production or increased turnover of bone scaffolding proteins (i.e., collagen) or regulators of mineralization (i.e., osteocalcin) may lead to fibrous union during acute lengthening.     

Distraction osteogenesis of the porcine mandible: histomorphometric evaluation of bone

Distraction osteogenesis is a technique for skeletal lengthening that exploits the body's innate capacity for bone formation in response to tension forces on the repair callus. The authors developed a distraction osteogenesis model with a semiburied device in the Yucatan minipig mandible because of similarities between human and porcine mandibular anatomy, temporomandibular function, chewing patterns, and bone turnover rates. The purpose of this study was to measure histomorphometric bone fill after different latency periods, rates of distraction, and duration of neutral fixation in the minipig mandible. In addition, the relationship between histomorphometric bone fill and clinical stability was investigated. Mandibular osteotomies in 20 female Yucatan minipigs weighing 25 to 30 kg were distracted with modified semiburied distraction devices. Variables included 0-day or 4-day latency; 1-mm, 2-mm, or 4-mm daily distraction rates; gap size of 7 or 12 mm; and evaluation after neutral fixation for various lengths of time. Specimens were fixed in 2% paraformaldehyde, pH 7.4, before being embedded in methylmethacrylate. Sections were prepared from the region just below the inferior alveolar canal. The area of new bone formation within the gap was measured and expressed as a percentage of the total area of the distraction gap. Bone fill ranged from 0 to 100 percent. A pilot study with 7-mm advancements showed similar bone fill with 0-day or 4-day latency, but with poor reproducibility. Mandibles that were distracted to 12 mm at 1 mm per day exhibited nearly complete bone fill, either with 0-day latency (average, 93 percent) or 4-day latency (average, 100 percent). Mandibles that had been distracted for 3 days at 4 mm per day showed moderate osteogenesis and clinical stability with increasing time of neutral fixation. Bone fill was significantly correlated with clinical stability (Spearman r = 0.801, p = 0.001). Histological examination showed exuberant periosteal osteogenesis in distracted mandibles, even in those that showed poor bone fill and clinical stability. Thus, the periosteum appears to be a major source of new bone formation. These results show that osteogenesis was nearly complete with 1 mm per day and 0-day or 4-day latency. These results are consistent with the authors' previously reported clinical and radiographic observations that a latency period is not necessary for successful healing of the mandibular distraction osteogenesis wound.     

Palatal distraction in a canine cleft palate model

The purpose of this study was to determine whether the canine hard palate can be lengthened by distraction osteogenesis in a cleft palate model using a mostly submucosal distractor. Five mongrel dogs were used. After raising mucoperiosteal flaps, a midline strip of bone was removed from the hard palate of each dog to simulate the bony defect seen in a cleft palate. A transverse osteotomy was then made to separate the posterior segment of the hard palate from the anterior segment. Posterior osteotomies were also made laterally parallel to the teeth so that the 2 posterior segments (one on either side of the bony cleft) were mobile. An intraoral distractor that was mostly submucosal was attached to the anterior hard palate and both segments of the mobilized posterior hard palate. Radiopaque bone markers were placed, and x-rays were obtained. After a 10-day latency period, the distractor was expanded 0.675 mm per day until it had been lengthened 10.125 mm. Distractors were left in place for an additional 8 weeks. After distractor removal, animals were observed for an additional 8 weeks before euthanization. Follow-up x-rays and histologic examinations were performed. New bone formation was found at the site of distraction in all dogs at the time of death. This new bone was seen on the follow-up x-rays and on histologic examination of the hard palates using both hematoxylin and eosin staining and Masson's trichrome stain. Distraction osteogenesis using a mostly submucosal device is an effective technique for lengthening the hard palate in a canine cleft palate model. The technique may eventually provide an alternative treatment for velopharyngeal incompetence in humans that is more precise and involves less morbidity than existing treatments.     

Calcium and contractions of isolated smooth muscle cells from rat myometrium

Smooth muscle cells were isolated from estrogenized rat myometrium by collagenase digestion. Electron microscopic examination and measurement of cell lengths by image-splitting micrometry were carried out after fixation with acrolein. Mean lengths of cells before and after isolation were 81.7 and 66.9 micron, respectively. Responses of cells were compared with contractions of isolated strips recorded isometrically. Effects of carbachol and KCl were examined in 2 mM Ca, 2 mM Ca + 4 mM EGTA, and 2 mM Ca + 10(-8) M nitrendipine solution. Carbachol and KCl produced concentration-dependent shortening of isolated cells maximal at 30 s after addition. The concentrations of carbachol required to produce shortenings were about 100-fold less than those required to produce isometric contractions; but no major difference was observed in the concentration dependence of cell shortening and isometric contraction produced by potassium-induced depolarization. In 2 mM Ca solution, there was a phasic response, followed by a tonic response such that more than 50% of maximum cell shortening was maintained for 10 min. However, in 2 mM Ca + 4 mM EGTA or 10(-8) M nitrendipine, the tonic contraction was abolished and cells rapidly relaxed after 30 s. If carbachol was added to cells after varying times in the EGTA-containing solution, the ability to initiate a contraction declined exponentially with a half-time of 160 s. Effects of depolarization by KCl were examined in 2 mM Ca plus nitrendipine and 2 mM Ca + 4 mM EGTA solution. Shortening occurred in 2 mM Ca solution by depolarization but not if nitrendipine was added. Though shortening was not observed in 2 mM Ca + 4 mM EGTA solution by KCl, subsequent addition of carbachol induced shortening. These results suggested that there was an intracellular Ca store site from which Ca was released by carbachol and which was not affected by depolarization in the absence of external Ca. No evidence was obtained that the contraction persists in Ca2+-free medium in isolated cells, which is in agreement with previous findings in small muscle strips in which only a similar transient response was obtained.     

Gene expression of TGF-beta, TGF-beta receptor, and extracellular matrix proteins during membranous bone healing in rats

Poorly healing mandibular fractures and osteotomies can be troublesome complications of craniomaxillofacial trauma and reconstructive surgery. Gene therapy may offer ways of enhancing bone formation by altering the expression of desired growth factors and extracellular matrix molecules. The elucidation of suitable candidate genes for therapeutic intervention necessitates investigation of the endogenously expressed patterns of growth factors during normal (i.e., successful) fracture repair. Transforming growth factor beta1 (TGF-beta1), its receptor (Tbeta-RII), and the extracellular matrix proteins osteocalcin and type I collagen are thought to be important in long-bone (endochondral) formation, fracture healing, and osteoblast proliferation. However, the spatial and temporal expression patterns of these molecules during membranous bone repair remain unknown. In this study, 24 adult rats underwent mandibular osteotomy with rigid external fixation. In addition, four identically treated rats that underwent sham operation (i.e., no osteotomy) were used as controls. Four experimental animals were then killed at each time point (3, 5, 7, 9, 23, and 37 days after the procedure) to examine gene expression of TGF-beta1 and Tbeta-RII, osteocalcin, and type I collagen. Northern blot analysis was used to compare gene expression of these molecules in experimental animals with that in control animals (i.e., nonosteotomized; n = 4). In addition, TGF-beta1 and T-RII proteins were immunolocalized in an additional group of nine animals killed on postoperative days 3, 7, and 37. The results of Northern blot analysis demonstrated a moderate increase (1.7 times) in TGF-beta1 expression 7 days postoperatively; TGF-beta1 expression returned thereafter to near baseline levels. Tbeta-RII mRNA expression was downregulated shortly after osteotomy but then increased, reaching a peak of 1.8 times the baseline level on postoperative day 9. Osteocalcin mRNA expression was dramatically downregulated shortly after osteotomy and remained low during the early phases of fracture repair. Osteocalcin expression trended slowly upward as healing continued, reaching peak expression by day 37 (1.7 times the control level). In contrast, collagen type IalphaI mRNA expression was acutely downregulated shortly after osteotomy, peaked on postoperative days 5, and then decreased at later time points. Histologic samples from animals killed 3 days after osteotomy demonstrated TGF-beta1 protein localized to inflammatory cells and extracellular matrix within the fracture gap, periosteum, and peripheral soft tissues. On postoperative day 7, TGF-beta1 staining was predominantly localized to the osteotomized bone edges, periosteum, surrounding soft tissues, and residual inflammatory cells. By postoperative day 37, complete bony healing was observed, and TGF-beta1 staining was localized to the newly formed bone matrix and areas of remodeling. On postoperative day 3, Tbeta-RII immunostaining localized to inflammatory cells within the fracture gap, periosteal cells, and surrounding soft tissues. By day 7, Tbeta-RII staining localized to osteoblasts of the fracture gap but was most intense within osteoblasts and mesenchymal cells of the osteotomized bone edges. On postoperative day 37, Tbeta-RII protein was seen in osteocytes, osteoblasts, and the newly formed periosteum in the remodeling bone. These observations agree with those of previous in vivo studies of endochondral bone formation, growth, and healing. In addition, these results implicate TGF-beta1 biological activity in the regulation of osteoblast migration, differentiation, and proliferation during mandibular fracture repair. Furthermore, comparison of these data with gene expression during mandibular distraction osteogenesis may provide useful insights into the treatment of poorly healing fractures because distraction osteogenesis has been shown to be effective in the management of these difficult clinical cases.