Catherine N. Tchanque-Fossuo

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 reverses radiation induced hypovascularity during bone regeneration and repair in the murine mandible

BACKGROUND Deferoxamine (DFO) is an iron-chelating agent that has also been shown to increase angiogenesis. We hypothesize that the angiogenic properties of DFO will improve bone regeneration in distraction osteogenesis (DO) after x-ray radiation therapy (XRT) by restoring the vascularity around the distraction site. MATERIAL AND METHODS Three groups of Sprague-Dawley rats underwent distraction of the left mandible. Two groups received pre-operative fractionated XRT, and one of these groups was treated with DFO during distraction. After consolidation, the animals were perfused and imaged with micro-CT to calculate vascular radiomorphometrics. RESULTS Radiation inflicted a severe diminution in the vascular metrics of the distracted regenerate and consequently led to poor clinical outcome. The DFO treated group revealed improved DO bone regeneration with a substantial restoration and proliferation of vascularity. CONCLUSIONS This set of experiments quantitatively demonstrates the ability of DFO to temper the anti-angiogenic effect of XRT in mandibular DO. These exciting results suggest that DFO may be a viable treatment option aimed at mitigating the damaging effects of XRT on new bone formation.

An evidence-based approach to pressure sores

The Maintenance of Certification module series is designed to help the clinician structure his or her study in specific areas appropriate to his or her clinical practice. This article is prepared to accompany practice-based assessment of preoperative assessment, anesthesia, surgical treatment plan, perioperative management, and outcomes. In this format, the clinician is invited to compare his or her methods of patient assessment and treatment, outcomes, and complications, with authoritative, information-based references. This information base is then used for self-assessment and benchmarking in parts II and IV of the Maintenance of Certification process of the American Board of Plastic Surgery. This article is not intended to be an exhaustive treatise on the subject. Rather, it is designed to serve as a reference point for further in-depth study by review of the reference articles presented.

Raman spectroscopy demonstrates Amifostine induced preservation of bone mineralization patterns in the irradiated murine mandible

PURPOSE Adjuvant radiotherapy in the management of head and neck cancer remains severely debilitating. Fortunately, newly developed agents aimed at decreasing radiation-induced damage have shown great promise. Amifostine (AMF) is a compound, which confers radio-protection to the exposed normal tissues, such as bone. Our intent is to utilize Raman spectroscopy to demonstrate how AMF preserves the mineral composition of the murine mandible following human equivalent radiation. METHODS Sprague Dawley rats were randomized into 3 experimental groups: control (n=5), XRT (n=5), and AMF-XRT (n=5). Both XRT and AMF groups underwent bioequivalent radiation of 70Gy in 5 fractions to the left hemimandible. AMF-XRT received Amifostine prior to radiation. Fifty-six days post-radiation, the hemimandibles were harvested, and Raman spectra were taken in the region of interest spanning 2mm behind the last molar. Bone mineral and matrix-specific Raman bands were analyzed using one-way ANOVA, with statistical significance at p<0.05. RESULTS The full-width at half-maximum of the primary phosphate band (FWHM) and the ratio of carbonate/phosphate intensities demonstrated significant differences between AMF-XRT versus XRT (p<0.01) and XRT versus control (p<0.01). There was no difference between AMF-XRT and control (p>0.05) in both Raman metrics. Computer-aided spectral subtraction further confirmed these results where AMF-XRT was spectrally similar to the control. Interestingly, the collagen cross-link ratio did not differ between XRT and AMF-XRT (p<0.01) but was significantly different from the control (p<0.01). CONCLUSION Our novel findings demonstrate that AMF prophylaxis maintains and protects bone mineral quality in the setting of radiation. Raman spectroscopy is an emerging and exceptionally attractive clinical translational technology to investigate and monitor both the destructive effects of radiation and the therapeutic remediation of AMF on the structural, physical and chemical qualities of bone.

Deferoxamine expedites consolidation during mandibular distraction osteogenesis.

BACKGROUND A limitation of mandibular distraction osteogenesis (DO) is the length of time required for consolidation. This drawback subjects patients to possible pin-site infections, as well as a prolonged return to activities of normal daily living. Developing innovative techniques to abridge consolidation periods could be immensely effective in preventing these problematic morbidities. Deferoxamine (DFO) is an angiogenic activator that triggers the HIF-1α pathway through localized iron depletion. We previously established the effectiveness of DFO in enhancing regenerate vascularity at a full consolidation period (28 days) in a murine mandibular DO model. To investigate whether this augmentation in vascularity would function to accelerate consolidation, we progressively shortened consolidation periods prior to μCT imaging and biomechanical testing (BMT). MATERIALS AND METHODS Three time points (14d, 21d and 28d) were selected and six groups of Sprague-Dawley rats (n = 60) were equally divided into control (C) and experimental (E) groups for each time period. Each group underwent external fixator placement, mandibular osteotomy, and a 5.1 mm distraction. During distraction, the experimental groups were treated with DFO injections into the regenerate gap. After consolidation, mandibles were imaged and tension tested to failure. ANOVA was conducted between groups, and p < 0.05 was considered statistically significant. RESULTS At 14 days of consolidation the experimental group demonstrated significant increases in bone volume fraction (BVF), bone mineral density (BMD) and ultimate load (UL) in comparison to non-treated controls. The benefit of treatment was further substantiated by a striking 100% increase in the number of bony unions at this early time-period (C:4/10 vs. E:8/10). Furthermore, metrics of BVF, BMD, Yield and UL at 14 days with treatment demonstrated comparable metrics to those of the fully consolidated 28d control group. CONCLUSION Based on these findings, we contend that augmentation of vascular density through localized DFO injection delivers an efficient means for accelerating bone regeneration without significantly impacting bone quality or strength.

Amifostine Remediates the Degenerative Effects of Radiation on the Mineralization Capacity of the Murine Mandible

Background: Radiotherapy, a cornerstone of head and neck cancer treatment, causes substantial morbidity to normal adjoining bone. The authors assessed the radioprotective effect of amifostine therapy on the mineralization of the mandible using micro–computed tomography. They hypothesized that amifostine would safeguard the mandible from radiation-induced disruption of the mineralization process and the associated failure of new bone creation. Methods: Male Sprague-Dawley rats were randomized into three groups: control (n = 8), radiation therapy (n = 5), and amifostine (n = 8). Animals in the radiation therapy and amifostine groups underwent human bioequivalent radiation of 70 Gy in five fractions to the left hemimandible. Fifty-six days after irradiation, the hemimandibles were harvested for radiomorphometric analyses. Results: Amifostine-treated animals exhibited less alopecia, mucositis, and weight loss in addition to increased cortical density in comparison with those treated with radiation therapy. Bone and tissue mineral densities showed statistically significant improvement in amifostine versus radiation therapy, and no difference was observed between amifostine and control groups. Detailed micro–computed tomographic analysis further demonstrated significant differences in the mineralization profile when comparing radiation therapy and amifostine. Amifostine maintained regions of lower mineralization consistent with the preservation of normal remodeling. Conclusions: The authors have successfully demonstrated the ability of amifostine pretreatment to protect the natural mineralization profile of bone. This reflects the capacity of amifostine prophylaxis to safeguard the normal surrounding mandible from the impediments of collateral damage imposed by irradiation. Further study can correlate these findings with the potential use of amifostine to prevent the devastating associated morbidities of radiotherapy such as pathologic fractures and osteoradionecrosis.

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.

Stem Cell Therapy Remediates Reconstruction of the Craniofacial Skeleton After Radiation Therapy

This study utilized transplanted bone marrow stromal cells (BMSCs) as a cellular replacement therapy to remedy radiation-induced injury and restore impaired new bone formation during distraction osteogenesis (DO). BMSC therapy brought about the successful generation of new bone and significantly improved both the rate and quality of a bony union of irradiated, distracted [X-ray radiation therapy (XRT)/DO] murine mandibles to the level of nonirradiated DO animals. The bone mineral density and bone volume fraction were also significantly improved by the BMSC replacement therapy showing no difference when compared to nonirradiated animals. Finally, a biomechanical analysis examining the yield, failure load, and ultimate load also demonstrated a significantly improved structural integrity in BMSC-treated XRT/DO mandibles over XRT/DO alone. These results indicate that administration of BMSCs intraoperatively to a radiated distraction gap can function as an adequate stimulant to rescue the ability for irradiated bone to undergo DO and produce a healed regenerate of a vastly superior quality and strength. We believe that the fundamental information on the optimization of bone regeneration in the irradiated mandible provided by this work has immense potential to be translated from the bench to the bedside to lead to improved therapeutic options for patients suffering from the disastrous sequelae of radiation therapy.

Bone Regeneration in Distraction Osteogenesis Demonstrates Significantly Increased Vascularity in Comparison to Fracture Repair in the Mandible

Background Tissue analysis of bone regenerate has suggested an intense vascular response after mandibular distraction osteogenesis (DO). Quantifying and three-dimensionally imaging this vascular response could be of immense clinical import in efforts to advance the utility of bone regeneration and repair. Conventional quantification of vascular responses has heretofore focused on inexact, cumbersome measurements of blood flow and histologic vessel counting. Using micro–computed tomography after vessel perfusion, we posit that quantitative vascular metrics will be significantly higher in mandibular DO compared with those observed in fracture repair (FxR) after bony union. Methods Sprague-Dawley rats underwent mandibular osteotomy and external fixator placement. A DO group (n = 9) underwent a 5.1-mm distraction, whereas a FxR group (n = 12) had a 2.1-mm fixed gap set. Forty days after surgery, Microfil was perfused into the vasculature, and imaging ensued. Vascular radiomorphometrics were calculated for the regions of interest. Independent-samples t-test was performed for comparison, with statistical significance set at P ⩽ 0.05. Results Stereological analysis demonstrated statistically significant increases in the distracted vasculature compared with fracture repair: vessel volume fraction (5.4% versus 2.8%, P = 0.030) and vessel number (0.86 versus 0.50 mm−1, P = 0.014). Conclusions We report robust and quantifiable increases in vascular density in DO compared with FxR. Our findings support a significant distinction between the regenerative processes of mandibular DO from the reparative mechanisms controlling fracture healing. A better understanding of the differences between the 2 types of bone formation may enable clinicians to selectively optimize therapeutic outcomes in the future.

Quantification and characterization of radiation-induced changes to mandibular vascularity using micro-computed tomography.

ObjectivePerhaps the most vexing and exigent problem confronting head and neck cancer reconstruction is overcoming the impediments of collateral damage imposed by radiation therapy (XRT) on normal surrounding tissue. Radiation therapy is detrimental to bone and soft tissue repair resulting in an unacceptably high incidence of devastating wound healing complications as well as the associated morbidity of late pathologic fractures, reduced bone healing, and osteoradionecrosis. The consequences of XRT on bone vasculature, long known to be affected by radiation, have been poorly understood. The purpose of this study was to analyze the degree by which irradiation degrades existing bone vascularity using a powerful micro–computed tomography technique to attain highly precise quantitative metrics of the vascular tree. MethodsFourteen 400-g male Sprague-Dawley rats underwent 35 Gy of fractionated XRT at 7 Gy/d. The animals were euthanized after 28 days, and the left ventricle was fixed and injected with Microfil (MV-122; Flow Tech, Carver, Mass) contrast. Left hemimandibles were dissected and scanned using high-resolution micro–computed tomography (18-&mgr;m voxels). The vessel number, thickness, separation, connectivity, and vessel volume fraction were analyzed for the region of interest, defined to be the volume behind the third molar spanning a total distance of 5.1 mm. ResultsStereologic analysis and subsequent analysis of variance test demonstrated a significant and quantifiable diminution in the irradiated vasculature when compared with control animals. The vessel volume fraction (0.016 vs 0.032, P ⩽ 0.003) and vessel thickness (0.042 vs 0.067 mm, P ⩽ 0.001) were markedly reduced. Interestingly, further analysis demonstrated no significant differences between vessel separation and vessel number. ConclusionsThe results of our study specifically quantify the corrosive affects of XRT on the vasculature of the mandible. The data from this novel technique go even further and imply retention of blood vessels but a degradation of their quality and size. Further experiments can now be directed at therapeutic interventions to reverse this process and better understand the underlying mechanism of XRT-induced bone injury.