Norman M. Rowe

Rat Mandibular Distraction Osteogenesis: II. Molecular Analysis of Transforming Growth Factor Beta-1 and Osteocalcin Gene Expression

Distraction osteogenesis is a powerful technique capable of generating viable osseous tissue by the gradual separation of osteotomized bone edges. Although the histologic and ultrastructural changes associated with this process have been extensively delineated, the molecular events governing these changes remain essentially unknown. We have devised a rat model of mandibular distraction osteogenesis that facilitates molecular analysis of this process. Such information has significant clinical implications because it may enable targeted therapeutic manipulations designed to accelerate osseous regeneration. In this study, we have evaluated the expression of transforming growth factor beta-1, a major regulator of osteogenesis during endochondral bone formation and development, and osteocalcin, an abundant noncollagenous extracellular matrix protein implicated in the regulation of mineralization and bone turnover. The right hemimandible of 36 adult male rats was osteotomized, and a customized distraction device was applied. Animals were allowed to recover and, after a 3-day latency period, were distracted at a rate of 0.25 mm twice daily for 6 days followed by a 2- or 4-week consolidation period. Distraction regenerate was harvested after the latency period, days 2, 4, or 6 of distraction, and after 2 or 4 weeks of consolidation and processed for Northern analysis (n = 4 at each time point) and immunohistochemical localization of TGF-beta1 (n = 2 at each time point). Six sham-operated animals (i.e., skin incision without osteotomy) were also killed (immediately postoperatively), and the mandibles were harvested and prepared in a similar fashion. Equal loading and transfer of RNA for Northern analysis was ensured by stripping and probing membranes with a probe against GAPDH (a housekeeping gene). Our results demonstrate that the spatial and temporal patterns of TGF-beta1 mRNA expression and protein production coincide with osteoblast migration, differentiation, and extracellular matrix synthesis. In addition, we demonstrate that TGF-beta1 production may be an important regulator of vasculogenesis during mandibular distraction osteogenesis. Finally, we have shown that osteocalcin gene expression coincides temporally with mineralization during rat mandibular distraction osteogenesis.

Rat mandibular distraction osteogenesis : Part I. Histologic and radiographic analysis

&NA; The application of distraction osteogenesis to craniofacial surgery has altered the approach and treatment of congenital and acquired craniofacial defects. Although the histologic and ultrastructural changes associated with distraction osteogenesis have been described extensively, relatively little is known about the molecular regulation of this process. The elucidation of the molecular mechanisms of distraction osteogenesis has important clinical implications because it may facilitate the use of recombinant proteins or gene therapy to accelerate bone regeneration. Molecular analysis of distraction osteogenesis has been hindered by the use of large animal models in which only limited genetic information is available. In this study, a rat model of mandibular distraction osteogenesis is described. This report includes a pilot study (n = 50) to develop an appropriate distraction device and to determine the optimal placement of the osteotomy. The study subsequently included 80 animals, 35 of which were distracted at a rate of 0.25 mm per day for 6 days (1.5 mm total) and 35 that were distracted at a rate of 0.25 mm twice per day (3.0 mm total). These animals were killed at various time points (after latency and during the distraction and consolidation periods) and displayed histologic and radiographic findings of membranous bone distraction osteogenesis that were consistent with those in large animal and clinical models. In addition, five animals each were acutely lengthened 1.5 mm and 3.0 mm and demonstrated a fibrous nonunion. Furthermore, the utility of this model is demonstrated in the analysis of the molecular mechanisms of distraction osteogenesis by applying the polymerase chain reaction to total cellular RNA isolated from normal and distracted rat mandibles. In conclusion, it is believed that the rat model of distraction osteogenesis has significant advantages over traditional models, including decreased costs and facilitation of molecular analysis. (Plast. Reconstr. Surg. 102: 2022, 1998.)

Angiogenesis during mandibular distraction osteogenesis.

Recruitment of a blood supply is critical for successful bone induction and fracture healing. Despite the clinical success of distraction osteogenesis (DO), an analysis of angiogenesis during membranous bone DO has not been performed. The purpose of this study was to evaluate the temporal and spatial pattern of angiogenesis during mandibular DO. The right hemimandible of adult male rats was osteotomized, and a customized distraction device was applied. Following a 3-day latency period, distraction was begun at a rate of 0.25 mm twice daily for 6 days (3.0 mm total; 12% increase in mandibular length). Three animals each were sacrificed on days 2, 4, and 6 of distraction (D1, D2, and D3 respectively), or after 1, 2, or 4 weeks of consolidation (C1, C2, and C3 respectively). Two experienced pathologists reviewed the regenerate histology, and angiogenesis was assessed by counting the number of blood vessels per intermediate-power field (IPF). Statistical analysis was performed using analysis of variance, with p < or = 0.05 considered significant. Results demonstrate that mandibular DO was associated with an intense vascular response during the early stages of distraction (D1). On average, 31.5+/-7.9 vessels were noted in each IPF examined during this time point. The number of blood vessels in the distraction regenerate decreased significantly during the later distraction time points, with approximately 14.0+/-2.0 and 14.7+/-3.5 blood vessels per IPF in sections obtained after days 4 and 6 of distraction (D2, D3) respectively. However, blood vessels at these time points took on a more mature histological pattern. During the consolidation period, the number of blood vessels noted in the regenerate decreased with 8.0+/-2.6, 9.3+/-2.1, and 4.0+/-2.0 vessels per IPF in sections obtained after 1, 2, or 4 weeks of consolidation (C1, C2, C3) respectively (p < 0.05 compared with vessel counts during the earliest distraction time point). This study demonstrates for the first time that an intense vascular response associated with mandibular DO occurs primarily during the early stages of distraction. The authors hypothesize that as distraction continues, newly formed vessels likely undergo consolidation, thus forming more mature vessels capable of withstanding distraction forces. Future studies will assess the effects of therapeutic interventions designed to increase angiogenesis during DO on bony regenerate formation.

Hypoxia upregulates VEGF production in keloid fibroblasts.

The etiology of keloid formation is diverse. They are characterized grossly as thick scar tissue that extends beyond the boundaries of the original wound. Histologically, keloids are composed of excessive collagen with an abnormally large number of partially or totally occluded microvessels. This occlusion of keloid microvessels has been hypothesized to contribute to a hypoxic microenvironment within these pathological scars. Vascular endothelial growth factor (VEGF), a potent endothelial cell mitogen, and proangiogenic cytokine have been implicated in normal and pathological wound healing. The purpose of this study was to evaluate the amount of VEGF protein production by fibroblast cell lines derived from keloids and normal human dermal skin in hypoxic compared with normoxic culture conditions. By enzyme-linked immunosorbent protein assay, VEGF was increased in both keloid and normal human dermal fibroblasts in hypoxia over normoxic controls. There was not, however, a significant difference between upregulation of VEGF protein when comparing the keloid and normal fibroblast groups. As the result of the data, alternative hypotheses for hypoxia-induced keloid formation were explored: (1) downstream modulation or signal transduction of VEGF, (2) VEGF production from cells other than fibroblasts, (3) the importance of matrix accumulation stimulated by hypoxia, or (4) increased migration of cells (other than fibroblasts) specific to keloid biology. These hypotheses may help explain the possible role of hypoxia in the pathogenesis of keloid formation. Future studies involving in situ hybridization or immunohistochemical analysis may offer greater insight into the mechanisms underlying keloid formation. Ultimately, our therapeutic goal is the utilization of biomolecular approaches for the suppression of keloid formation.

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 &bgr;1 (TGF-&bgr;1), its receptor (T&bgr;-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-&bgr;1 and T&bgr;-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-&bgr;1 and T&bgr;-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-&bgr;1 expression 7 days postoperatively; TGF-&bgr;1 expression returned thereafter to near baseline levels. T&bgr;-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 I&agr;I 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-&bgr;1 protein localized to inflammatory cells and extracellular matrix within the fracture gap, periosteum, and peripheral soft tissues. On postoperative day 7, TGF-&bgr;1 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-&bgr;1 staining was localized to the newly formed bone matrix and areas of remodeling. On postoperative day 3, T&bgr;-RII immunostaining localized to inflammatory cells within the fracture gap, periosteal cells, and surrounding soft tissues. By day 7, T&bgr;-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, T&bgr;-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-&bgr;1 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. (Plast. Reconstr. Surg. 105: 2028, 2000.)

Acellular dermal composite allografts for reconstruction of the radial forearm donor site.

Purpose:Since its description in the 1970s, the radial forearm free flap has earned a clearly defined role in the armamentarium of reconstructive head and neck surgery. Three decades later, the donor site remains an intrinsic drawback primarily due to its esthetic impact, although functional morbidity is significant in a minority. These points do not outweigh significant advantages but are occasionally reasons for the choice of alternative flaps. Modifications evolved in an effort to improve these undesirable features include primary closure, rotation-advancement, proximal paddle placement, full-thickness skin graft (FTSG) and suprafascial dissection. We describe a novel technique of engineering a composite graft of cadaveric acellular dermal matrix and autologous split-thickness skin graft (STSG) for a better donor-site closure. Methods:From December 1995 to August 2003, 23 patients underwent radial forearm reconstruction of head and neck defects. Control patients (Group I; n = 5) had donor sites closed by conventional STSG technique (0.014–0.016 inch). In 18 patients (Group II), the donor site was closed with a composite technique (dermal allograft, 0.020–0.030 inch, and an ultrathin STSG, 0.0080 inch). Both groups of patients were retrospectively studied for comparative defects. Contralateral upper extremities also served as controls. All patients underwent a standardized functional examination of the donor and contralateral extremities, as well as an outcome questionnaire. All extremities were photographed for visual comparison by the author. Results:Three of the 5 group I patients were available for follow-up, which averaged 64 months (60–72 months). Thirty-three percent had a decrease in functional parameters and 67% complained of paresthesia. Patient satisfaction was 3.5/5. Six of the 18 patients were excluded from Group II due to insufficient follow-up or inability to follow. Follow-up averaged 8 months (1–24 months). Functional parameters in all patients were comparable to the contralateral extremity, except in 1 patient. In this case, a 0.030-inch allograft was used which never revascularized, inhibiting wrist motion. Other patients exhibited excellent range of motion of the wrist and fingers. This was the only patient in this group that exhibited paresthesia of the donor site. Patient satisfaction was 4.6/5. Esthetic results were extremely gratifying as judged by the author. Esthetic results were better than those observed in Group I. Conclusions/Significance:Composite grafting with acellular dermal matrix and STSG provides a comparable (trending to superior) result with traditional STSG for the treatment of radial forearm graft donor sites. Even if functionally equivalent, it is esthetically superior and therefore a technique warranting further investigation.

Remodeling of the temporomandibular joint following mandibular distraction osteogenesis in the transverse dimension

Transverse mandibular distraction osteogenesis involves moving the osteotomized segments of the mandible in either a varus or valgus direction. This maneuver allows for widening of the bigonial distance or for a lateral shift of an asymmetric mandibular midline. During this process, a significant amount of torque is placed on the mandibular condyles, because they act as the pivot point for the mandibular translation. Although standard linear distraction osteogenesis induces transient, reversible changes in the temporomandibular joint, it is not known what effect the varus and valgus stresses of transverse distraction have on the temporomandibular joint. We therefore designed a study to document the temporomandibular joint changes following various degrees of transverse distraction. Bilateral transverse mandibular distraction was performed on 10 adult, female mongrel dogs using an external, multiplanar mandibular distraction device. The distraction protocol was as follows: (1) complete osteotomy at the angle of the mandible, (2) 5‐day latency period, (3) distraction rate of 1 mm/day, (4) rhythm of one turn per day, (5) linear activation 16 to 30 mm bilaterally, and (6) 8‐week consolidation period. A variety of varus and valgus distraction vectors were applied to the mandible only after 10 mm of initial linear distraction had been achieved. Posteroanterior and lateral cephalograms were performed throughout the entire process. Pre‐distraction and post‐consolidation computed tomographic scans were also performed. Changes in mandibular conformation, axis of rotation, temporomandibular joint structure, and glenoid fossa changes were directly assessed by evaluating the postmortem craniofacial skeleton. The findings were compared with those of normal, age‐matched mongrel dog skulls. Significant remodeling changes were observed in the temporomandibular joints of all animals involved in the study. The mandibular condyles demonstrated varying degrees of flattening and erosion at all contact points with the craniofacial skeleton. In some cases, the condyle became part of the distraction regenerate process and was hypertrophied in all dimensions. The condyles were frequently displaced out of the glenoid fossa, particularly on the side in the direction of varus distraction. When the latter occurred, a new fossa was created on the undersurface of the zygomatic arch. Varying degrees of mandibular rotation in the sagittal plane were also observed, which led to abnormal torquing of the condyles in the coronal plane, depending on whether the axis of rotation occurred primarily around the condyle or around the distraction regenerate zone. In conclusion, transverse mandibular distraction is an effective means of producing a varus or valgus shift in the gonion relative to the midsagittal plane. However, unlike linear or angular mandibular distraction, transverse distraction has a multitude of nontransient effects on the temporomandibular joint. Therefore it must be emphasized that in clinical practice, transverse distraction should be used cautiously. One must also be aware that such a maneuver in distraction can have negative effects on the temporomandibular joint. (Plast. Reconstr. Surg. 107: 647, 2001.)

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 contalateral 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. (Plast. Reconstr. Surg. 105: 2416, 2000.)

Sonographically guided percutaneous carpal tunnel release: An anatomic and cadaveric study

Minimally invasive techniques have become the standard of care for multiple procedures. This paper demonstrates both the surgeons’ capacity to perform an accurate anatomic evaluation of the hand and forearm (n = 10) and the use of this anatomic information to accurately perform sonographically guided, percutaneous carpal tunnel release using a single-portal endoscope without direct or indirect visualization in a cadaver model (n = 6). Open dissection was then performed to confirm complete ligament transection and to evaluate the surrounding structures for injury. In all 6 cadavers, the transverse carpal ligament was transected completely without injury to any surrounding structures. With further investigation, this novel technique may offer a less invasive, office-based method for the surgical treatment of carpal tunnel syndrome that may offer patients an expedited recovery.

Molding of the Regenerate in Mandibular Distraction : Part 1 : Laboratory Study

Distraction osteogenesis has evolved as a mainstream surgical technique for lengthening and augmentation of the hypoplastic mandible. As clinical experience accumulated, there developed the need to “mold” the bony regenerate to avoid the development of postdistraction malocclusion and to achieve the desired craniofacial form. Although the potential to mold the regenerate has important clinical implications, the safety and efficacy of such an acute manipulation of the bony regenerate form have not yet been investigated in the laboratory. The purpose of this study was to determine if the distraction regenerate could be molded and result in a bony union. Four adult female dogs underwent bilateral mandibular distraction with an external multiplanar device (Stryker, Osteonics). After a latency period of 5 days, the mandibles underwent linear (anteroposterior) and angular (superoinferior) distraction to produce an anterior open bite of approximately 30°. At the conclusion of the distraction procedure, the distraction sites were molded to close the open bite. In two dogs, the maneuver was performed over 3 days by changing the angulation of the devices (gradual molding), and in the other two dogs, molding was achieved with a single movement (acute molding). In the latter, the distraction devices were adjusted and reapplied to allow for anatomical fixation during the consolidation period of 49 days. According to the research protocol, the mandibles were assessed serially by cephalograms and computed tomography (CT) scans. All dogs survived the study without complications. The bony regenerate was easily molded in both groups to close the surgically created open bite. After molding, all the regenerates showed CT scan evidence of solid bone (consolidation), which was classified as “extended” on the Hamanishi scale. After the dogs were killed and soft tissue was removed, the regenerate seemed to be robust on gross examination without any evidence of fibrous nonunion. In addition, histological study of the regenerate confirmed the bony union. The study demonstrates that the mandible can be successfully molded into a desired anatomical position immediately after distraction without producing a fibrous union. Furthermore, it has been demonstrated that the bony regenerate is sufficiently malleable before consolidation to undergo either acute or gradual angular molding without disturbing osteogenic potential. The ability to mold the regenerate without the fear of creating a fibrous union or destroying bony potential provides the surgeon the capability to optimize the dental occlusion and mandibular form as part of the distraction treatment process.