Matthew E. Dudziak

The effects of ionizing radiation on osteoblast-like cells in vitro.

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-&bgr;1 (TGF-&bgr;1), 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-&bgr;1 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-&bgr;1 and VEGF protein production. Decreases in total TGF-&bgr;1 production were due to a decrease in TGF-&bgr;1 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-&bgr;1 adenovirus before irradiation resulted in an increase in cellular production of TGF-&bgr;1 protein and VEGF. Interestingly, this intervention did not alter the effects of irradiation on cellular proliferation, which implies that alterations in TGF-&bgr;1 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.

Hypoxia regulates VEGF expression and cellular proliferation by osteoblasts in vitro.

Numerous studies have demonstrated the critical role of angiogenesis for successful osteogenesis during endochondral ossification and fracture repair. Vascular endothelial growth factor (VEGF), a potent endothelial cell-specific cytokine, has been shown to be mitogenic and chemotactic for endothelial cells in vitro and angiogenic in many in vivo models. Based on previous work that (1) VEGF is up-regulated during membranous fracture healing, (2) the fracture site contains a hypoxic gradient, (3) VEGF is up-regulated in a variety of cells in response to hypoxia, and (4) VEGF is expressed by isolated osteoblasts in vitro stimulated by other fracture cytokines, the hypothesis that hypoxia may regulate the expression of VEGF by osteoblasts was formulated. This hypothesis was tested in a series of in vitro studies in which VEGF mRNA and protein expression was assessed after exposure of osteoblast-like cells to hypoxic stimuli. In addition, the effects of a hypoxic microenvironment on osteoblast proliferation and differentiation in vitro was analyzed. These results demonstrate that hypoxia does, indeed, regulate expression of VEGF in osteoblast-like cells in a dose-dependent fashion. In addition, it is demonstrated that hypoxia results in decreased cellular proliferation, decreased expression of proliferating cell nuclear antigen, and increased alkaline phosphatase (a marker of osteoblast differentiation). Taken together, these data suggest that osteoblasts, through the expression of VEGF, may be in part responsible for angiogenesis and the resultant increased blood flow to fractured bone segments. In addition, these data provide evidence that osteoblasts have oxygen-sensing mechanisms and that decreased oxygen tension can regulate gene expression, cellular proliferation, and cellular differentiation.

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.

Transforming growth factor-β1 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-β1 (TGF-β1) modulates bone differentiation, matrix formation, and cytokine expression. The purpose of this study was to investigate the relationship between TGF-β1 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-β1 (2.5 ng/ml). The stability of VEGF mRNA in MC3T3-E1 cells was not increased after TGF-β1 treatment. Actinomycin D inhibited the TGF-β1-induced peak in VEGF mRNA, whereas cycloheximide did not. Blockade of TGF-β1 signal transduction via a dominant-negative receptor II adenovirus significantly decreased TGF-β1 induction of VEGF mRNA. Additionally, TGF-β1 induced a dose-dependent increase in VEGF protein expression by MC3T3-E1 cells ( P < 0.01). Dexamethasone similarly inhibited VEGF protein expression. Both TGF-β1 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-β1-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.

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.

Adenovirus‐Mediated Gene Therapy of Osteoblasts In Vitro and In Vivo

Modulation of biological pathways governing osteogenesis may accelerate osseous regeneration and reduce the incidence of complications associated with fracture healing. Transforming growth factor β1 (TGF‐β1) is a potent growth factor implicated in the regulation of osteogenesis and fracture repair. The use of recombinant proteins, however, has significant disadvantages and has limited the clinical utility of these molecules. Targeted gene therapy using adenovirus vectors is a technique that may circumvent difficulties associated with growth factor delivery. In this study, we investigate the efficacy of replication‐deficient adenoviruses containing the human TGF‐β1 and the bacterial lacZ genes in transfecting osteoblasts in vitro and osseous tissues in vivo. We demonstrate that adenovirus‐mediated gene therapy efficiently transfects osteoblasts in vitro with the TGF‐β1 virus causing a marked up‐regulation in TGF‐β1 mRNA expression even 7 days after transfection. Increased TGF‐β1 mRNA expression was efficiently translated into protein production and resulted in approximately a 46‐fold increase in TGF‐β1 synthesis as compared with control cells (vehicle‐ or B‐galactosidase–transfected). Moreover, virally produced TGF‐β1 was functionally active and regulated the expression of collagen IαI (5‐fold increase) and the vascular endothelial growth factor (2.5‐fold increase). Using an adenovirus vector encoding the Escherichia coli LacZ gene, we demonstrated that adenovirus‐mediated gene transfer efficiently transfects osteoblasts and osteocytes in vivo and that transfection can be performed by a simple percutaneous injection. Finally, we show that delivery of the hTGF‐β1 gene to osseous tissues in vivo results in significant changes in the epiphyseal plate primarily as a result of increased thickness of the provisional calcification zone.

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.)

Regional differentiation of rat cranial suture-derived dural cells is dependent on association with fusing and patent cranial sutures.

A significant body of literature supports a role for the dura mater underlying cranial sutures in the regulation of sutural fate. These studies have implicated regional differentiation of the dura mater based on association with fusing and patent rat cranial sutures. The purpose of these experiments was to isolate and characterize dural cells associated with fusing (posterior frontal) and patent (sagittal) rat cranial sutures. Six-day-old rats were killed, and the dura mater underlying the posterior frontal and sagittal sutures was harvested. Dural cells were briefly trypsinized and allowed to reach confluence. Two litters (10 animals per litter) were used for each set of experiments. Cells were harvested after the first and fifth passages for analysis of vimentin and desmoplakin expression (characteristic of human meningeal cells), cellular proliferation, density at confluence (a measure of cellular contact inhibition), and alkaline phosphatase production. In addition, bone nodule formation and collagen I production were analyzed in first passage cells. The results indicate that suture-derived dural cells can be established and that these cells coexpress vimentin and desmoplakin. In addition, it is demonstrated that first-passage sagittal suture-derived dural cells proliferate significantly faster and have decreased cellular contact inhibition than posterior frontal suture-derived cells (p < 0.01). Finally, it is shown that suture-derived dural cells have osteoblast-like properties, including alkaline phosphatase production, collagen I expression, and bone nodule formation in vitro. The possible mechanisms by which regional differentiation of suture-derived dural cells occur are discussed.

Gene expression of insulin-like growth factors I and II in rat membranous osteotomy healing.

Poorly healing mandibular osteotomies can be a difficult problem in reconstructive surgery. Many therapies have been attempted to augment the healing of mandibular fractures, defects, or osteotomies, but these methods have substantial drawbacks or have been ineffective. The difficulty in treating poorly healing bony defects has led to the exploration of gene therapy as a possible approach to supplement or accelerate mandibular fracture healing. To understand at what point the introduction of a suitable gene candidate might be of benefit in mandibular healing, it is imperative to examine the temporal expression of bone growth factors in a model of membranous bone healing. Insulinlike growth factors (IGFs) I and II are two such bone growth factor candidates because of their known potent in vitro as well as in vivo effects on bone formation. In this study the authors demonstrate the temporal pattern of IGF I and IGF II gene expression during mandibular osteotomy healing using a rat model. Their data reveal that IGF I and IGF II were elevated 7 days after a mandibular osteotomy that was held in external fixation. The upregulation of IGF I and IGF II during mandibular bone healing underscores the importance of these growth factors in bone repair. Gene therapy utilizing recombinant viral constructs containing IGFs I and II may be of benefit during mandibular bone healing in an effort to augment clinical scenarios of poor or retarded bony repair.