Faculty Bibliography

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

Vascular disruption secondary to fracture leads to a hypoxic zone of injury where the oxygen tension at the center of the wound is quite low. In this dynamic microenvironment, a number of growth factors are elaborated to stimulate the synthetic processes of fracture repair. Previously the authors have shown the hypoxia-induced increase of vascular endothelial growth factor expression in osteoblasts. The purpose of these experiments was to examine osteoblast expression of insulinlike growth factors (IGF) I and II--cytokines believed to play a role in increased collagen synthesis, chemotaxis, and proliferation of osteoblasts in response to hypoxia. Primary cell cultures of osteoblasts isolated from neonatal rat calvaria were subjected to hypoxia (PO2 = 35 mmHg) for 0, 3, 6, 24, and 48 hours. Northern blot analysis of ribonucleic acid (RNA) from resulting cultures demonstrated a more than 60% increase in IGF-II messenger RNA (mRNA) expression after 3 hours of hypoxia. IGF-II mRNA expression continued to increase through later time points to 200% and 260% of baseline at 24 and 48 hours respectively. In contrast, IGF-I demonstrated no significant change in mRNA expression compared with baseline control (normoxia) cultures. In these experiments the authors have demonstrated a hypoxia-induced increase in IGF-II but not IGF-I in primary osteoblasts. The differential expression of these two growth factors may underscore important differences in the behavior of osteoblasts in the hypoxic fracture microenvironment. Taken together, these data add additional support to the theory that hypoxia induces gene-specific changes in expression of molecules important to extracellular matrix formation for successful bone healing

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

Angiogenesis is essential for the increased delivery of oxygen and nutrients required for the reparative processes of bone healing. Vascular endothelial growth factor (VEGF), a potent angiogenic growth factor, has been implicated in this process. We have previously shown that hypoxia specifically and potently regulates the expression of VEGF by osteoblasts. However, the molecular mechanisms governing this interaction remain unknown. In this study, we hypothesized that the hypoxic regulation of VEGF expression by osteoblasts occurs via an oxygen-sensing mechanism similar to the regulation of the erythropoietin gene (EPO). To test this hypothesis, we examined the kinetics of oxygen concentration on osteoblast VEGF expression. In addition, we analyzed the effects of nickel and cobalt on the expression of VEGF in osteoblastic cells because these metallic ions mimic hypoxia by binding to the heme portion of oxygen-sensing molecules. Our results indicated that hypoxia potently stimulates VEGF mRNA expression. In addition, we found that nickel and cobalt both stimulate VEGF gene expression in a similar time- and dose-dependent manner, suggesting the presence of a hemelike oxygen-sensing mechanism similar to that of the EPO gene. Moreover, actinomycin D, cycloheximide, dexamethasone, and mRNA stabilization studies collectively established that this regulation is predominantly transcriptional, does not require de novo protein synthesis, and is not likely mediated by the transcriptional activator AP-1. These studies demonstrate that hypoxia, nickel, and cobalt regulate VEGF expression in osteoblasts via a similar mechanism, implicating the involvement of a heme-containing oxygen-sensing molecule. This may represent an important mechanism of VEGF regulation leading to increased angiogenesis in the hypoxic microenvironment of healing bone

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

The ability to avoid a subsequent bone graft makes the use of gingivoperiosteoplasty (GPP) at the time of cleft lip repair an attractive technique. The use of GPP, in combination with presurgical orthodontics, has been shown to result in successful bony union in the majority of patients. However, secondary bone grafting is still necessary in 30% to 40% of patients due to persistent alveolar bony defects. The elucidation of methods to improve the success rates of these procedures has been hampered by the lack of reproducible animal models. The purpose of this study was, therefore, to develop a rodent model of GPP that would facilitate the investigation of methods to improve osteogenesis in alveolar defects. We report a surgically produced rat model (9 x 5 x 3-mm alveolar defect) that is reproducible, inexpensive (relative to large-animal models), and simple technically. In addition, healing in this model occurs in a predictable manner during a 12-week period, thus enabling analysis of methods designed to accelerate or facilitate osseous regeneration

Normal and abnormal extracellular matrix turnover is thought to result, in part, from the balance in the expression of metalloproteinases and tissue inhibitors of metalloproteinases (TIMPs). The clinical manifestations of an imbalance in these relationships are evident in a variety of pathologic states, including osteoarthritis, deficient long-bone growth, rheumatoid arthritis, tumor invasion, and inadequate cartilage repair. Articular cartilage defects commonly heal as fibrocartilage, which is structurally inferior to the normal hyaline architecture of articular cartilage. Transforming growth factor-beta 1 (TGF-beta1), a cytokine central to growth, repair, and inflammation, has been shown to upregulate TIMP-1 expression in human and bovine articular cartilage. Additionally, members of the TGF-beta superfamily are thought to play key roles in chondrocyte growth and differentiation. Bone morphogenetic protein-2 (BMP-2), a member of this superfamily, has been shown to regulate chondrocyte differentiation states and extracellular matrix composition. It was proposed that, by optimizing extracellular matrix composition, BMP-2 would enhance articular cartilage healing. After determining the release kinetics of BMP-2 from a collagen type I implant (Long-Evans male rats; two implants/rat, n = 14), it was found that, in a tissue engineering application, BMP-2 induced a hyaline-like repair of New Zealand White rabbit knee articular cartilage defects (3-mm full-thickness defects in the femoral trochlea; 2 defects/rabbit, n = 36). The quality of cartilage repair with BMP-2 (with or without chondrocytes) was significantly better than defects treated with BMP-2, as assessed by a quantitative scoring scale. Immunohistochemical staining revealed TIMP-1 production in the cartilage defects treated with BMP-2. When studied in vitro, it was found that BMP-2 markedly increased TIMP-1 mRNA by both bovine articular and human rib chondrocytes. Additionally, increased TIMP-1 mRNA was translated into increased TIMP-1 protein production by bovine chondrocytes. Taken together, these data suggest that BMP-2 may be a useful cytokine to improve healing of cartilaginous defects. Furthermore, these data suggest that the beneficial effects of BMP-2 may be, in part, related to alterations in extracellular matrix turnover

The well-described detrimental effects of ionizing radiation on the regeneration of bone within a fracture site include decreased osteocyte number, suppressed osteoblast activity, and diminished vascularity. However, the biologic mechanisms underlying osteoradionecrosis and the impaired fracture healing of irradiated bone remain undefined. Ionizing radiation may decrease successful osseous repair by altering cytokine expression profiles resulting from or leading to a change in the osteoblastic differentiation state. These changes may, in turn, cause alterations in osteoblast proliferation and extracellular matrix formation. The purpose of this study was to investigate the effects of ionizing radiation on the proliferation, maturation, and cytokine production of MC3T3-E1 osteoblast-like cells in vitro. Specifically, the authors examined the effects of varying doses of ionizing radiation (0, 40, 400, and 800 cGy) on the expression of transforming growth factor-beta1 (TGF-beta1), vascular endothelial growth factor (VEGF), and alkaline phosphatase. In addition, the authors studied the effects of ionizing radiation on MC3T3-E1 cellular proliferation and the ability of conditioned media obtained from control and irradiated cells to regulate the proliferation of bovine aortic endothelial cells. Finally, the authors evaluated the effects of adenovirus-mediated TGF-beta1 gene therapy in an effort to 'rescue' irradiated osteoblasts. The exposure of osteoblast-like cells to ionizing radiation resulted in dose-dependent decreases in cellular proliferation and promoted cellular differentiation (i.e., increased alkaline phosphatase production). Additionally, ionizing radiation caused dose-dependent decreases in total TGF-beta1 and VEGF protein production. Decreases in total TGF-beta1 production were due to a decrease in TGF-beta1 production per cell. In contrast, decreased total VEGF production was secondary to decreases in cellular proliferation, because the cellular production of VEGF by irradiated osteoblasts was moderately increased when VEGF production was corrected for cell number. Additionally, in contrast to control cells (i.e., nonirradiated), conditioned media obtained from irradiated osteoblasts failed to stimulate the proliferation of bovine aortic endothelial cells. Finally, transfection of control and irradiated cells with a replication-deficient TGF-beta1 adenovirus before irradiation resulted in an increase in cellular production of TGF-beta1 protein and VEGF. Interestingly, this intervention did not alter the effects of irradiation on cellular proliferation, which implies that alterations in TGF-beta1 expression do not underlie the deficiencies noted in cellular proliferation. The authors hypothesize that ionizing radiation-induced alterations in the cytokine profiles and differentiation states of osteoblasts may provide insights into the cellular mechanisms underlying osteoradionecrosis and impaired fracture healing

A number of studies have demonstrated the critical role of angiogenesis for successful wound repair in the surgical patient. Vascular disruption from tissue injury due to trauma or surgery leads to a hypoxic zone in the healing wound. In this dynamic process, angiogenesis is vital for the delivery of oxygen, nutrients, and growth factors necessary to initiate the synthetic processes of wound healing. Fibroblasts, invading the wound early in the healing process, are involved in extracellular matrix (ECM) deposition as well as wound contraction. However, the exact mechanisms by which important genes are regulated remain unknown. In order to examine these processes, we studied the effects of hypoxia on fibroblasts for the expression of VEGF, type IalphaI collagen, and matrix-metalloproteinase-3, three genes essential for the regulation of angiogenesis, ECM deposition, and ECM degradation in wound healing. Primary cell cultures of normal human dermal fibroblasts (NHDFs) were placed in hypoxia for varying periods of time. Northern blot hybridization was performed with [alpha32P]dCTP-labeled cDNA probes for VEGF, type IalphaI collagen, and MMP-3. The results demonstrated a time-dependent VEGF mRNA upregulation (470% of baseline) under hypoxia. Type IalphaI collagen increased (170% of baseline) at 24 h, but was then abruptly downregulated to 3.8% of baseline at 48 h. MMP-3 was incrementally downregulated to 2.2% of baseline at 48 h. These experiments focused on the effect of hypoxia on genes thought to play a role in wound repair. VEGF upregulation in the hypoxic microenvironment of the early wound may serve to stimulate angiogenesis. Type IalphaI collagen, though upregulated early on, was abruptly downregulated at 48 h. This downregulation may reflect the in vivo requirement for angiogenesis to deliver oxygen for successful hydroxylation and collagen synthesis in the wound. MMP-3, also downregulated at 48 h, may also implicate the need for angiogenesis. These data support the theory that hypoxia-driven angiogenesis is critical for ECM formation and remodeling in successful soft tissue repair. Furthermore, they may represent the role of hypoxia as an important regulator to efficiently balance these complex processes in the healing wound