Rat mandibular distraction osteogenesis: latency, rate, and rhythm determine the adaptive response.

Distraction osteogenesis is a well-established technique of endogenous tissue engineering. The biomechanical factors thought to affect the quality of the distraction regenerate include the latency, rate, rhythm, and consolidation period. In an effort to understand the impact of these parameters on regenerate bone formation, this study was designed to decipher the most adaptive response in a rat model of mandibular distraction osteogenesis. Ninety-six adult Sprague-Dawley rats were divided into 16 subgroups (n = 6 per subgroup) based on variations in the distraction parameters (i.e., latency, rate, and rhythm). After a 28-day consolidation period, the mandibles were harvested, decalcified, and sectioned. A standardized histologic ranking system was used to evaluate the effect of each protocol on the adaptive response of the regenerate bone. In this study, we have demonstrated that the latency period dramatically affects the success of distraction osteogenesis. Furthermore, distraction rates up to 0.50 mm per day stimulated excellent regenerate bone formation, whereas greater distraction rates produced a fibrous union. Finally, higher frequency distraction (i.e., increased rhythm) appeared to accelerate regenerate bone formation. We believe that defining the critical parameters of this model will improve future analysis of gene expression during rat mandibular distraction osteogenesis and may facilitate the development of biologically based strategies designed to enhance regenerate bone formation.

Expression of bone morphogenetic proteins during membranous bone healing.

For the reconstructive plastic surgeon, knowledge of the molecular biology underlying membranous fracture healing is becoming increasingly vital. Understanding the complex patterns of gene expression manifested during the course of membranous fracture repair will be crucial to designing therapies that augment poor fracture healing or that expedite normal osseous repair by strategic manipulation of the normal course of gene expression. In the current study, we present a rat model of membranous bone repair. This model has great utility because of its technical simplicity, reproducibility, and relatively low cost. Furthermore, it is a powerful tool for analysis of the molecular regulation of membranous bone repair by immunolocalization and/or in situ hybridization techniques. In this study, an osteotomy was made within the caudal half of the hemimandible, thus producing a stable bone defect without the need for external or internal fixation. The healing process was then catalogued histologically in 28 Sprague-Dawley rats that were serially killed at 1, 2, 3, 4, 5, 6, and 8 weeks after operation. Furthermore, using this novel model, we analyzed, within the context of membranous bone healing, the temporal and spatial expression patterns of several members of the bone morphogenetic protein (BMP) family, known to be critical regulators of cells of osteoblast lineage. Our data suggest that BMP-2/-4 and BMP-7, also known as osteogenic protein-1 (OP-1), are expressed by osteoblasts, osteoclasts, and other more primitive mesenchymal cells within the fracture callus during the early stages of membranous fracture healing. These proteins continue to be expressed during the process of bone remodeling, albeit less prominently. The return of BMP-2/-4 and OP-1 immunostaining to baseline intensity coincides with the histological appearance of mature lamellar bone. Taken together, these data underscore the potentially important regulatory role played by the bone morphogenetic proteins in the process of membranous bone repair.

Amyand's hernia: prospective CT diagnosis.

We report two cases of Amyand's hernia, which is the development of acute appendicitis within an inguinal hernia. Both patients were clinically thought to have incarcerated inguinal hernias, but were correctly prospectively diagnosed as having Amyand's hernia on the basis of preoperative computed tomography (CT) examinations. Our cases again show the utility of CT of the acute abdomen and pelvis in revealing a previously unsuspected diagnosis and rapidly triaging patients to the appropriate management.

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.

"Pumping the regenerate": an evaluation of oscillating distraction osteogenesis in the rodent mandible.

Mandibular distraction osteogenesis (DO) has become an important technique to lengthen the hypoplastic mandible and to reconstruct osseous defects after ablative surgery. The hallmark of successful DO is the creation of new bone within the distraction gap. Several anecdotal reports have described alternating compressing and lengthening protocols (i.e., "pumping the regenerate") to augment regenerate bone formation. The purpose of this experiment was to analyze formally the effects of an alternating compression/distraction protocol with a traditional distraction protocol. Ten adult male rats underwent unilateral mandibular osteotomy with placement of a custom distractor. After a latency period of 5 days, distraction was initiated at a rate of 0.25 mm twice daily. Animals in the control group (N = 5) were distracted to a length of 5.0 mm for 10 days at a rate of 0.25 mm twice daily. In contrast, animals in the experimental group (N = 5) were distracted to a length of 2.5 mm (at a rate of 0.25 mm twice daily) for 5 days, then compressed 1.0 mm for a 2-day period, and redistracted to a length of 5.0 mm. Regenerate cross-sectional area was evaluated by computed tomography performed after 5 weeks of consolidation. Gross examination and histological analysis were performed by a panel of experienced reviewers. Radiological as well as histological analysis of regenerate cross-sectional area demonstrated no significant differences between experimental (i.e., "pumped") and control groups. Both groups demonstrated excellent regenerate bone formation with no evidence of fibrous union. This study represents the first attempt to investigate the anecdotal technique of pumping the mandibular regenerate. The authors have demonstrated that pumping the regenerate leads to no substantial differences in radiological or histological appearance of regenerate bone formation.

Expression of adenovirally delivered gene products in healing osseous tissues.

Gene therapy has moved from the promise of laboratory investigation to the reality of clinical practice in just the last decade. Various methods for delivery of genes to host cells have been developed and utilized both in vitro and in vivo. From the perspective of the plastic surgeon, gene therapy holds the promise to augment healing in clinical situations that remain difficult to treat, such as chronic wounds, osteoradionecrosis, or possibly to expedite current clinical practices, such as distraction osteogenesis. The authors chose to investigate the potential for gene therapy in osseous tissues using a replication-deficient adenovirus vector to deliver the marker transgene beta-galactosidase. An adenovirus vector is ideal for use in situations in which transgene expression is desired for only a relatively short period of time, such as wound and fracture healing. Utilizing a rat mandibular osteotomy model, they demonstrated that, using an adenoviral vector, foreign genes can be delivered in a simple fashion and can be expressed in a reliable manner within and around the osteotomy site for at least 10 days. Furthermore, there was no evidence of transfection of distant tissues associated with local application of the adenovirus vector. With this information, clinicians may now attempt to deliver osteogenic and angiogenic genes in a site-specific fashion to improve and expedite osseous healing.

Biomolecular mechanisms of calvarial bone induction: immature versus mature dura mater.

The ability of newborns and immature animals to reossify calvarial defects has been well described. This capacity is generally lost in children greater than 2 years of age and in mature animals. The dura mater has been implicated as a regulator of calvarial reossification. To date, however, few studies have attempted to identify biomolecular differences in the dura mater that enable immature, but not mature, dura to induce osteogenesis. The purpose of these studies was to analyze metabolic characteristics, protein/gene expression, and capacity to form mineralized bone nodules of cells derived from immature and mature dura mater. Transforming growth factor beta-1, basic fibroblast growth factor, collagen type IalphaI, osteocalcin, and alkaline phosphatase are critical growth factors and extracellular matrix proteins essential for successful osteogenesis. In this study, we have characterized the proliferation rates of immature (6-day-old rats, n = 40) and mature (adult rats, n = 10) dura cell cultures. In addition, we analyzed the expression of transforming growth factor beta-1, basic fibroblast growth factor-2, proliferating cell nuclear antigen, and alkaline phosphatase. Our in vitro findings were corroborated with Northern blot analysis of mRNA expression in total cellular RNA isolated from snap-frozen age-matched dural tissues (6-day-old rats, n = 60; adult rats, n = 10). Finally, the capacity of cultured dural cells to form mineralized bone nodules was assessed. We demonstrated that immature dural cells proliferate significantly faster and produce significantly more proliferating cell nuclear antigen than mature dural cells (p < 0.01). Additionally, immature dural cells produce significantly greater amounts of transforming growth factor beta-1, basic fibroblast growth factor-2, and alkaline phosphatase (p < 0.01). Furthermore, Northern blot analysis of RNA isolated from immature and mature dural tissues demonstrated a greater than 9-fold, 8-fold, and 21-fold increase in transforming growth factor beta-1, osteocalcin, and collagen IalphaI gene expression, respectively, in immature as compared with mature dura mater. Finally, in keeping with their in vivo phenotype, immature dural cells formed large calcified bone nodules in vitro, whereas mature dural cells failed to form bone nodules even with extended culture. These studies suggest that differential expression of growth factors and extracellular matrix molecules may be a critical difference between the osteoinductive capacity of immature and mature dura mater. Finally, we believe that the biomolecular bone- and matrix-inducing phenotype of immature dura mater regulates the ability of young children and immature animals to heal calvarial defects.

Transforming growth factor-beta1 modulates the expression of vascular endothelial growth factor by osteoblasts.

Angiogenesis is essential to both normal and pathological bone physiology. Vascular endothelial growth factor (VEGF) has been implicated in angiogenesis, whereas transforming growth factor-beta1 (TGF-beta1) modulates bone differentiation, matrix formation, and cytokine expression. The purpose of this study was to investigate the relationship between TGF-beta1 and VEGF expression in osteoblasts and osteoblast-like cells. Northern blot analysis revealed an early peak of VEGF mRNA (6-fold at 3 h) in fetal rat calvarial cells and MC3T3-E1 osteoblast-like cells after stimulation with TGF-beta1 (2.5 ng/ml). The stability of VEGF mRNA in MC3T3-E1 cells was not increased after TGF-beta1 treatment. Actinomycin D inhibited the TGF-beta1-induced peak in VEGF mRNA, whereas cycloheximide did not. Blockade of TGF-beta1 signal transduction via a dominant-negative receptor II adenovirus significantly decreased TGF-beta1 induction of VEGF mRNA. Additionally, TGF-beta1 induced a dose-dependent increase in VEGF protein expression by MC3T3-E1 cells (P < 0.01). Dexamethasone similarly inhibited VEGF protein expression. Both TGF-beta1 mRNA and VEGF mRNA were concurrently present in rat membranous bone, and both followed similar patterns of expression during rat mandibular fracture healing (mRNA and protein). In summary, TGF-beta1-induced VEGF expression by osteoblasts and osteoblast-like cells is a dose-dependent event that may be intimately related to bone development and fracture healing.

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.