Salman Ahsan

Localized deferoxamine injection augments vascularity and improves bony union in pathologic fracture healing after radiotherapy

BACKGROUND Medically based efforts and alternative treatment strategies to prevent or remediate the corrosive effects of radiotherapy on pathologic fracture healing have failed to produce clear and convincing evidence of success. Establishing an effective pharmacologic option to prevent or treat the development of non-unions in this setting could have immense therapeutic potential. Experimental studies have shown that deferoxamine (DFO), an iron-chelating agent, bolsters vascularity and subsequently enhances normal fracture healing when injected locally into a fracture callus in long bone animal models. Since radiotherapy is known to impede angiogenesis, we hypothesized that the pharmacologic addition of DFO would serve to mitigate the effects of radiotherapy on new vessel formation in vitro and in vivo. MATERIALS AND METHODS In vitro investigation of angiogenesis was conducted utilizing HUVEC cells in Matrigel. Endothelial tubule formation assays were divided into four groups: Control, Radiated, Radiated+Low-Dose DFO and Radiated+High-Dose DFO. Tubule formation was quantified microscopically and video recorded for the four groups simultaneously during the experiment. In vivo, three groups of Sprague-Dawley rats underwent external fixator placement and fracture osteotomy of the left mandible. Two groups received pre-operative fractionated radiotherapy, and one of these groups was treated with DFO after fracture repair. After 40 days, the animals were perfused and imaged with micro-CT to calculate vascular radiomorphometrics. RESULTS In vitro, endothelial tubule formation assays demonstrated that DFO mitigated the deleterious effects of radiation on angiogenesis. Further, high-dose DFO cultures appeared to organize within 2h of incubation and achieved a robust network that was visibly superior to all other experimental groups in an accelerated fashion. In vivo, animals subjected to a human equivalent dose of radiotherapy (HEDR) and left mandibular fracture demonstrated quantifiably diminished μCT metrics of vascular density, as well as a 75% incidence of associated non-unions. The addition of DFO in this setting markedly improved vascularity as demonstrated with 3D angiographic modeling. In addition, we observed an increased incidence of bony unions in the DFO treated group when compared to radiated fractures without treatment (67% vs. 25% respectively). CONCLUSION Our data suggest that selectively targeting angiogenesis with localized DFO injections is sufficient to remediate the associated severe vascular diminution resulting from a HEDR. Perhaps the most consequential and clinically relevant finding was the ability to reduce the incidence of non-unions in a model where fracture healing was not routinely observed.

Deferoxamine restores Callus size, mineralization, and mechanical strength in fracture healing after radiotherapy

Background: Therapeutic augmentation of fracture-site angiogenesis with deferoxamine has proven to increase vascularity, callus size, and mineralization in long-bone fracture models. The authors posit that the addition of deferoxamine would enhance pathologic fracture healing in the setting of radiotherapy in a model where nonunions are the most common outcome. Methods: Thirty-five Sprague-Dawley rats were divided into three groups. Fracture, irradiated fracture, and irradiated fracture plus deferoxamine. The irradiated fracture and irradiated fracture plus deferoxamine groups received a human equivalent dose of radiotherapy [7 Gy/day for 5 days, (35 Gy)] 2 weeks before mandibular osteotomy and external fixation. The irradiated fracture plus deferoxamine group received injections of deferoxamine into the fracture callus after surgery. After a 40-day healing period, mandibles were dissected, clinically assessed for bony union, imaged with micro–computed tomography, and tension tested to failure. Results: Compared with irradiated fractures, metrics of callus size, mineralization, and strength in deferoxamine-treated mandibles were significantly increased. These metrics were restored to a level demonstrating no statistical difference from control fractures. In addition, the authors observed an increased rate of achieving bony unions in the irradiated fracture plus deferoxamine–treated group when compared with irradiated fracture (67 percent and 20 percent, respectively). Conclusions: The authors’ data demonstrate nearly total restoration of callus size, mineralization, and biomechanical strength, and a threefold increase in the rate of union with the use of deferoxamine. The authors’ results suggest that the administration of deferoxamine may have the potential for clinical translation as a new treatment paradigm for radiation-induced pathologic fractures.

Deferoxamine administration delivers translational optimization of distraction osteogenesis in the irradiated mandible

Background: The authors’ laboratory has previously demonstrated that deferoxamine promotes angiogenesis and bone repair in the setting of radiation therapy coupled with distraction osteogenesis. However, clinically relevant effects of deferoxamine administration on union rate and micro–computed tomographic and biomechanical parameters are unknown. The authors posit that administration of deferoxamine will increase union rate, mineralization, and strength of the regenerate in an irradiated distraction osteogenesis model. Methods: Sprague-Dawley rats were randomized into three groups: distraction osteogenesis–control, distraction osteogenesis–radiation therapy, and distraction osteogenesis–radiation therapy–deferoxamine. All animals underwent an osteotomy and distraction osteogenesis across a 5.1-mm distraction gap. Irradiated animals received 35-Gy human-equivalent radiation therapy 2 weeks before surgery, and deferoxamine was injected postoperatively in the regenerate site of treatment animals. Animals were killed on postoperative day 40, and mandibles were harvested to determine rates of bony union and micro–computed tomographic and biomechanical parameters. Results: Compared with irradiated mandibles, deferoxamine-treated mandibles exhibited a higher union rate (11 percent versus 92 percent, respectively). Across micro–computed tomographic and biomechanical parameters, significant diminutions were observed with administration of radiation therapy, whereas deferoxamine therapy resulted in significant restoration to levels of controls, with select metrics exhibiting significant increases even beyond controls. Conclusions: The authors’ data confirm that deferoxamine restores clinically relevant metrics of bony union and micro–computed tomographic and biomechanical parameters in a model of irradiated distraction osteogenesis in the murine mandible. Their findings support a potential use for deferoxamine in treatment protocols to allow predictable and reliable use of distraction osteogenesis as a viable reconstructive option in patients with head and neck cancer.

Prophylactic amifostine preserves the biomechanical properties of irradiated bone in the murine mandible.

Background: The authors have previously demonstrated that amifostine prophylaxis mitigates the pernicious effects of radiation in settings of fracture repair and distraction osteogenesis. Expanding on these studies, the authors examined the biomechanical properties of uninjured bone exposed to both radiation and amifostine. The authors hypothesize that radiation will degrade the biomechanical properties of native bone, and further hypothesize that prophylactic amifostine will preserve biomechanical properties to levels of normal bone and protect against radiation-induced morbidities. Methods: Rats were randomized into control, irradiated, and amifostine pretreatment plus radiation (amifostine-pretreated) groups. Irradiated animals received a fractionated dosing schedule of 35 Gy, with amifostine-pretreated animals receiving amifostine before irradiation. Hemimandibles were harvested at 8 and 18 weeks for biomechanical testing and micro–computed tomographic analysis. Results: At 8 weeks, irradiated specimens displayed elevations above controls for all biomechanical properties. At 18 weeks, the biomechanical properties of irradiated specimens degraded in comparison with controls; at both time points, amifostine-pretreated specimens were maintained at levels comparable to controls. There was a significant decrease in tissue mineral density from 8- to 18-week irradiated specimens, whereas no such change existed for control and amifostine-pretreated specimens. Conclusions: The authors’ findings demonstrate paradoxical and transient elevations in the initial biomechanical properties of irradiated specimens that were not sustained through the later study time point. Amifostine pretreatment, however, provided uninterrupted preservation of the biomechanical properties of normal, native bone at both time points. This supports the contention that amifostine is capable of providing continuous protection to bone against the untoward effects of radiation therapy.

Abstract 53: prophylactic amifostine preserves the biomechanical properties of irradiated bone in the murine mandible.

ConClusion: An expanded krox20 expression domain at the embryonic stage of fgfr1b mRNA injected embryos suggests that this mutation has activating character in zebrafish, indicating a conserved role of fgfr1 in cranial suture development among vertebrates. The effects of genetic manipulation on adult suture phenotype will be assessed through histological analysis, skeletal morphology, and cell proliferation will be studied in our transgenic fgfr1b embryos. This zebrafish model of CS will advance our understanding of the role of Fgfr1b in cranial suture morphogenesis and the etiology of the disorder. In the future, it can be used for genetic and chemical screens to search for genetic modifiers and therapeutic agents that alter CS. 53 Prophylactic amifostine Preserves the Biomechanical Properties of irradiated Bone in the Murine Mandible

Abstract 62: A NOVEL COMBINATION OF AMIFOSTINE PROPHYLAXIS AND ANGIOGENIC DEFEROXAMINE PROTECTS AND RESTORES BIOMECHANICAL PROPERTIES OF BONE IN PATHOLOGIC FRACTURE HEALING AFTER RADIOTHERAPY

Purpose: Pathologic fractures and associated non-unions after radiotherapy for Head and Neck cancer pose a complex management dilemma for reconstructive surgeons. We have previously demonstrated a partial remediation of biomechanical strength and bony union formation with Deferoxamine (DFO), an angiogenic factor, in a murine model of fracture healing following radiotherapy. The purpose of this study was to investigate the synergistic potential of Amifostine (AMF), a cytoprotectant, in combination with DFO to produce a more robust biomechanical construct and predictable rate of bony union.

Abstract 211: DEFEROXAMINE RESTORES BIOMECHANICAL PROPERTIES AND POTENTIATES UNION OF IRRADIATED BONE IN DISTRACTION OSTEOGENESIS

Background: Our laboratory has shown the iron-chelating agent Deferoxamine (DFO) promotes angiogenesis and bone repair in the setting of radiation therapy (XRT) coupled with Distraction Osteogenesis (DO). While these studies demonstrate DFO improves vascular and histologic metrics for irradiated bone, the clinically impactful effect of DFO on union formation and biomechanics has never been examined. We hypothesize DFO administration will increase bony union and strengthen Biomechanical Properties (BPs) of the regenerate in an irradiated DO model.

Abstract 42P: Deferoxamine Decreases Non-Unions and Restores Bone Quality and Biomechanical Strength in Pathologic Fracture Healing After Radiotherapy

Objective: Adjuvant radiotherapy for head and neck cancer patient management continues to be paralleled with costly and devastating bone related pathologies. Most detrimental is the development of non-unions secondary to pathologic fractures. A pharmacologic means to prevent these pathologies would be highly desirable. Deferoxamine (DFO), an angiogenic therapy, augments vascularity and subsequently enhances fracture healing when injected into a fracture callus. We posit that the untoward effects of radiotherapy on bone quality, mechanical strength and non-union formation can be mitigated with this powerful angiogenic therapy.