Adipose-Derived Stem Cells Enhance Graft Incorporation and Mineralization in a Murine Model of Irradiated Mandibular Nonvascularized Bone Grafting.

BACKGROUND: Nonvascularized bone grafting represents a practical method of mandibular reconstruction. However, the destructive effects of radiotherapy on native bone preclude the use of nonvascularized bone grafts in head and neck cancer patients. Adipose-derived stem cells have been shown to enhance bone healing and regeneration in numerous experimental models. The purpose of this study was to determine the impact of adipose-derived stem cells on nonvascularized bone graft incorporation in a murine model of irradiated mandibular reconstruction. METHODS: Thirty isogenic rats were randomly divided into 3 groups: nonvascularized bone graft (control), radiation with nonvascularized bone graft (XRT), and radiation with nonvascularized bone graft and adipose-derived stem cells (ASC). Excluding the control group, all rats received a human-equivalent dose of radiation. All groups underwent mandibular reconstruction of a critical-sized defect with a nonvascularized bone graft from the contralateral hemimandible. After a 60-day recovery period, graft incorporation and bone mineralization were compared between groups. RESULTS: Compared with the control group, the XRT group demonstrated significantly decreased graft incorporation (P = 0.011), bone mineral density (P = 0.005), and bone volume fraction (P = 0.001). Compared with the XRT group, the ASC group achieved a significantly increased graft incorporation (P = 0.006), bone mineral density (P = 0.005), and bone volume fraction (P = 0.013). No significant differences were identified between the control and ASC groups. CONCLUSIONS: Adipose-derived stem cells enhance nonvascularized bone graft incorporation in the setting of human-equivalent radiation.

Therapeutic Efficacy of Adipose-Derived Stem Cells Versus Bone Marrow Stromal Cells for Irradiated Mandibular Fracture Repair.

BACKGROUND: Mesenchymal stem cells have immense potential in applications of bone healing and regeneration. However, few studies have evaluated the therapeutic efficacy of adipose-derived stem cells (ASCs) and bone marrow stromal cells (BMSCs) in irradiated bone. The purpose of this study is to compare the ability of ASCs versus BMSCs to enhance healing outcomes in a murine model of irradiated mandibular fracture repair. METHODS: Forty-eight isogenic male Lewis rats underwent radiation therapy followed by mandibular osteotomy with intraoperative placement of either ASCs or BMSCs. Animals were killed on postoperative day 40. Mandibles were analyzed for union rate, biomechanical strength, vascularity, and mineralization. Groups were compared at P < 0.05 significance. RESULTS: The ASC and BMSC groups demonstrated 92% and 75% union rates. Compared with the BMSC group, the ASC group demonstrated a trending increase in maximum load ( P = 0.095) on biomechanical strength analysis and a significant increase in vessel number ( P = 0.001), vessel thickness ( P = 0.035), and vessel volume fraction ( P = 0.007) on micro-computed tomography angiography analysis. No significant differences in bone mineralization were identified on micro-computed tomography analysis. CONCLUSION: This study demonstrates the superior therapeutic efficacy of ASCs over BMSCs in irradiated fracture healing as evidenced by union rate, vascular morphometry, and a trend in biomechanical strength. We posit that the robust vascular response induced by ASCs better recapitulates the sequence and synchronicity of physiologic bone healing compared with BMSCs, thereby improving the reliability of irradiated fracture repair.

Therapeutic Interventions to Reduce Radiation Induced Dermal Injury in a Murine Model of Tissue Expander Based Breast Reconstruction.

BACKGROUND: Radiation therapy (XRT) induced dermal injury disrupts type I collagen architecture. This impairs cutaneous viscoelasticity, which may contribute to the high rate of complications in expander-based breast reconstruction with adjuvant XRT. The objective of this study was to further elucidate the mechanism of radiation-induced dermal injury and to determine if amifostine (AMF) or deferoxamine (DFO) mitigates type I collagen injury in an irradiated murine model of expander-based breast reconstruction. METHODS: Female Lewis rats (n = 20) were grouped: expander (control), expander-XRT (XRT), expander-XRT-AMF (AMF), and expander-XRT-DFO (DFO). Expanders were surgically placed. All XRT groups received 28 Gy of XRT. The AMF group received AMF 30 minutes before XRT, and the DFO group used a patch for delivery 5 days post-XRT. After a 20-day recovery period, skin was harvested. Atomic force microscopy and Raman spectroscopy were performed to evaluate type I collagen sheet organization and tissue compositional properties, respectively. RESULTS: Type I collagen fibril disorganization was significantly increased in the XRT group compared with the control (83.8% vs 22.4%; P = 0.001). Collagen/matrix ratios were greatly reduced in the XRT group compared with the control group (0.49 ± 0.09 vs 0.66 ± 0.09; P = 0.017). Prophylactic AMF demonstrated a marked reduction in type I collagen fibril disorganization on atomic force microscopy (15.9% vs 83.8%; P = 0.001). In fact, AMF normalized type I collagen organization in irradiated tissues to the level of the nonirradiated control (P = 0.122). Based on Raman spectroscopy, both AMF and DFO demonstrated significant differential protective effects on expanded-irradiated tissues. Collagen/matrix ratios were significantly preserved in the AMF group compared with the XRT group (0.49 ± 0.09 vs 0.69 ± 0.10; P = 0.010). ß-Sheet/α-helix ratios were significantly increased in the DFO group compared with the XRT group (1.76 ± 0.03 vs 1.86 ± 0.06; P = 0.038). CONCLUSIONS: Amifostine resulted in a significant improvement in type I collagen fibril organization and collagen synthesis, whereas DFO mitigated abnormal changes in collagen secondary structure in an irradiated murine model of expander-based breast reconstruction. These therapeutics offer the ability to retain the native microarchitecture of type I collagen after radiation. Amifostine and DFO may offer clinical utility to reduce radiation induced dermal injury, potentially decreasing the high complication rate of expander-based breast reconstruction with adjuvant XRT and improving surgical outcomes.

Noncultured Minimally Processed Adipose-Derived Stem Cells Improve Radiated Fracture Healing.

Adipose-derived stem cells mitigate deleterious effects of radiation on bone and enhance radiated fracture healing by replacing damaged cells and stimulating angiogenesis. However, adipose-derived stem cell harvest and delivery techniques must be refined to comply with the US Food and Drug Administration restrictions on implantation of cultured cells into human subjects prior to clinical translation. The purpose of this study is to demonstrate the preservation of efficacy of adipose-derived stem cells to remediate the injurious effects of radiation on fracture healing utilizing a novel harvest and delivery technique that avoids the need for cell culture. Forty-four Lewis rats were divided into 4 groups: fracture control (Fx), radiated fracture control (XFx), radiated fracture treated with cultured adipose-derived stem cells (ASC), and radiated fracture treated with noncultured minimally processed adipose-derived stem cells (MP-ASC). Excluding the Fx group, all rats received a fractionated human-equivalent dose of radiation. All groups underwent mandibular osteotomy with external fixation. Following sacrifice on postoperative day 40, union rate, mineralization, and biomechanical strength were compared between groups at P < 0.05 significance. Compared with Fx controls, the XFx group demonstrated decreased union rate (100% vs 20%), bone volume fraction (P = 0.003), and ultimate load (P < 0.001). Compared with XFx controls, the MP-ASC group tripled the union rate (20% vs 60%) and demonstrated statistically significant increases in both bone volume fraction (P = 0.005) and ultimate load (P = 0.025). Compared with the MP-ASC group, the ASC group showed increased union rate (60% vs 100%) and no significant difference in bone volume fraction (P = 0.936) and ultimate load (P = 0.202). Noncultured minimally processed adipose-derived stem cells demonstrate the capacity to improve irradiated fracture healing without the need for cell proliferation in culture. Further refinement of the cell harvest and delivery techniques demonstrated in this report will enhance the ability of noncultured minimally processed adipose-derived stem cells to improve union rate and bone quality, thereby optimizing clinical translation.

Amifostine Prophylaxis in Irradiated Breast Reconstruction: A Study of Oncologic Safety In Vitro.

BACKGROUND: Indications for adjuvant radiation therapy (XRT) in breast cancer have expanded. Although highly effective, XRT damages surrounding tissues and vasculature, often resulting in delayed or compromised breast reconstruction. Thus, effective yet safe methods of radiation injury prophylaxis would be desirable. Amifostine is a Food and Drug Administration-approved radioprotectant; however, concerns about its potential to also protect cancer remain. The purpose of this study was to evaluate the oncologic safety of amifostine (AMF) in vitro and determine its effect on human breast cancer cells in the setting of XRT. METHODS: One ER+/PR+/Her2- (MCF-7) and two ER-/PR-Her2- (MDA-MB-231, MDA-MB-468) breast cancer cell lines were investigated. Female fibroblasts were used as controls. Cells were treated with WR-1065, the active metabolite of AMF, 20 minutes before 0Gy, 10Gy, or 20Gy XRT. Live and dead cells were quantified; percent cell death was calculated. RESULTS: WR-1065 treatment significantly preserved viability and reduced healthy female fibroblasts death after XRT compared with untreated controls. All three breast cancer cells lines exhibited radiosensitivity with substantial cell death. Cancer cells retained their radiosensitivity despite WR-1065 pretreatment, achieving the same degree of cell death as untreated controls. CONCLUSIONS: This study demonstrated the proficiency of AMF to selectively protect healthy cells from XRT while breast cancer cells remained radiosensitive. These results support the oncologic safety of AMF in breast cancer in vitro. Further investigation is now warranted in vivo to ascertain the translational potential of using AMF as a radioprotectant to improve breast reconstruction after radiation treatment.

Stem Cells for Bone Regeneration: Current State and Future Directions.

Mesenchymal stem cells (MSCs) are capable of differentiating into osteoblasts, chondrocytes, and adipocytes, each of which is important for musculoskeletal tissue regeneration and repair. Reconstruction and healing of bony defects remains a major clinical challenge. Even as surgical practices advance, some severe cases of bone loss do not yield optimal recovery results. New techniques involving implantation of stem cells and tissue-engineered scaffolds are being developed to help improve bone and cartilage repair. The invasiveness and low yield of harvesting MSCs from the bone marrow (BMSCs) has led to the investigation of alternatives, including adipose-derived mesenchymal stem cells (ASCs). A review of the literature yielded several studies concerning the use of BMSCs and ASCs for the treatment of bone defects in both in vitro and in vivo models. Although both ASCs and BMSCs have demonstrated bone regenerative capabilities, BMSCs have outperformed ASCs in vitro. Despite these in vitro study findings, in vivo study results remain variable. Analysis of the literature seems to conclude there is no significant difference between bone regeneration using ASCs or BMSCs in vivo. Improved study design and standardization may enhance the application of these studies to patient care in the clinical setting.

Nonvascularized Bone Graft Reconstruction of the Irradiated Murine Mandible: An Analogue of Clinical Head and Neck Cancer Treatment.

Nonvascularized bone grafts (NBGs) represent a practical method of mandibular reconstruction that is precluded in head and neck cancer patients by the destructive effects of radiotherapy. Advances in tissue-engineering may restore NBGs as a viable surgical technique, but expeditious translation demands a small-animal model that approximates clinical practice. This study establishes a murine model of irradiated mandibular reconstruction using a segmental iliac crest NBG for the investigation of imperative bone healing strategies. Twenty-seven male isogenic Lewis rats were divided into 2 groups; control bone graft and irradiated bone graft (XBG). Additional Lewis rats served as graft donors. The XBG group was administered a fractionated dose of 35Gy. All rats underwent reconstruction of a segmental, critical-sized defect of the left hemi-mandible with a 5 mm NBG from the iliac crest, secured by a custom radiolucent plate. Following a 60-day recovery period, hemi-mandibles were evaluated for bony union, bone mineralization, and biomechanical strength (P < 0.05). Bony union rates were significantly reduced in the XBG group (42%) compared with controls (80%). Mandibles in the XBG group further demonstrated substantial radiation injury through significant reductions in all metrics of bone mineralization and biomechanical strength. These observations are consistent with the clinical sequelae of radiotherapy that limit NBGs to nonirradiated patients. This investigation provides a clinically relevant, quantitative model in which innovations in tissue engineering may be evaluated in the setting of radiotherapy to ultimately provide the advantages of NBGs to head and neck cancer patients and reconstructive surgeons.

Novel Formulation Strategy to Improve the Feasibility of Amifostine Administration.

PURPOSE: Amifostine (AMF), a radioprotectant, is FDA-approved for intravenous administration in cancer patients receiving radiation therapy (XRT). Unfortunately, it remains clinically underutilized due to adverse side effects. The purpose of this study is to define the pharmacokinetic profile of an oral AMF formulation potentially capable of reducing side effects and increasing clinical feasibility. METHODS: Calvarial osteoblasts were radiated under three conditions: no drug, AMF, and WR-1065 (active metabolite). Osteogenic potential of cells was measured using alkaline phosphatase staining. Next, rats were given AMF intravenously or directly into the jejunum, and pharmacokinetic profiles were evaluated. Finally, rats were given AMF orally or subcutaneously, and blood samples were analyzed for pharmacokinetics. RESULTS: WR-1065 preserved osteogenic potential of calvarial osteoblasts after XRT to a greater degree than AMF. Direct jejunal AMF administration incurred a systemic bioavailability of 61.5%. Subcutaneously administrated AMF yielded higher systemic levels, a more rapid peak exposure (0.438 vs. 0.875 h), and greater total systemic exposure of WR-1065 (116,756 vs. 16,874 ng*hr/ml) compared to orally administered AMF. CONCLUSIONS: Orally administered AMF achieves a similar systemic bioavailability and decreased peak plasma level of WR-1065 compared to intravenously administered AMF, suggesting oral AMF formulations maintain radioprotective efficacy without causing onerous side effects, and are clinically feasible.

Histologic Improvements in Irradiated Bone Through Pharmaceutical Intervention in Mandibular Distraction Osteogenesis.

PURPOSE: Despite the relative surgical ease and reduced donor-site morbidity of distraction osteogenesis (DO) in comparison with free tissue transfer, DO is currently precluded as a reconstructive option for head and neck cancer (HNC) patients because of the destructive effects of radiotherapy (XRT). This study investigates the ability of a novel combined therapy (CT) of radioprotective amifostine (AMF) and angiogenic deferoxamine (DFO) to mitigate XRT-induced bone injury in a murine model of DO. MATERIALS AND METHODS: Thirty male Sprague-Dawley rats were divided into 5 groups: DO (primary control), XRT (secondary control), AMF, DFO, and CT. With the exclusion of the DO group, all rats were administered a fractionated, human-equivalent XRT dose of 35 Gy, comparable with 70 Gy administered to HNC patients clinically. All groups underwent mandibular osteotomy and distraction to 5.1 mm. After euthanasia administration on postoperative day 40, the mandibles were sectioned and stained with Gomori trichrome. Osteocyte number, bone volume, and osteoid volume were compared between all groups by analysis of variance (P < .05). RESULTS: All rats survived and were included in the final analysis. The XRT group exhibited substantial bone injury, evidenced by a decreased osteocyte number and bone volume, as well as an increase in immature osteoid volume, compared with DO controls. The AMF, DFO, and CT groups showed significant increases in osteocyte proliferation compared with the XRT group and were not statistically different from the DO group. Notably, the CT group showed remediation of XRT-induced impairment of bone maturation and exhibited significantly greater bone volume and reduced osteoid volume in comparison with all groups. CONCLUSIONS: Combined AMF and DFO treatment showed the capacity to remediate the deleterious effects of XRT, restore cellularity to nonirradiated levels, and surpass all groups in mature bone formation. Although further investigations of AMF and DFO are warranted, this study provides preliminary support for the potential use of DO in HNC patients through pharmaceutical facilitation of irradiated bone healing.

Topical Deferoxamine Alleviates Skin Injury and Normalizes Atomic Force Microscopy Patterns Following Radiation in a Murine Breast Reconstruction Model.

BACKGROUND: Breast cancer is most commonly managed with a combination of tumor ablation, radiation, and/or chemotherapy. Despite the oncologic benefit of these treatments, the detrimental effect of radiation on surrounding tissue challenges the attainment of ideal breast reconstruction outcomes. The purpose of this study was to determine the ability of topical deferoxamine (DFO) to reduce cutaneous ulceration and collagen disorganization following radiotherapy in a murine model of expander-based breast reconstruction. METHODS: Female Sprague-Dawley rats (n = 15) were divided into 3 groups: control (expander), XRT (expander + radiation), and DFO (expander + radiation + deferoxamine [DFO]). Expanders were placed in a submusculocutaneous plane in the right upper back and ultimately filled to 15 mL. Radiation was administered via a fractionated dose of 28 Gy. Deferoxamine was delivered topically for 10 days following radiation. After a 20-day recovery period, skin ulceration and dermal type I collagen organization were analyzed. RESULTS: Compared with control, the XRT group demonstrated a significant increase in skin ulceration (3.7% vs 43.3%, P = 0.00) and collagen fibril disorganization (26.3% vs 81.8%, P = 0.00). Compared with the XRT group, treatment with topical DFO resulted in a significant reduction in ulceration (43.3% vs 7.0%, P = 0.00) and fibril disorganization (81.8% vs 15.3%, P = 0.00). There were no statistical differences between the control and DFO groups in skin ulceration or collagen disorganization. CONCLUSIONS: This study suggests topical DFO is capable of reducing skin ulceration and type I collagen fibril disorganization following radiotherapy. This novel application of DFO has potential to enhance expander-based breast reconstruction outcomes and improve quality of life for women suffering the devastating effects of breast cancer.

Radioprotection With Amifostine Enhances Bone Strength and Regeneration and Bony Union in a Rat Model of Mandibular Distraction Osteogenesis.

BACKGROUND: Using distraction osteogenesis (DO) to regenerate robust endogenous bone could greatly enhance postoncologic reconstruction of head and neck cancer. However, radiation (XRT) corrosive effects still preclude DO's immense potential. We posit that adjunctive pretreatment with the radioprotectant amifostine (AMF) can optimize wound healing and allow for successful DO with quantifiable enhancements in bony union and strength despite previous surgical bed irradiation. METHODS: Two groups of murine left hemimandibles were exposed to a human equivalent radiation dosage fractionated over 5 daily doses of 7 Gy. AMF-XRT-DO (n = 30) received AMF before radiation, whereas XRT-DO (n = 22) was untreated. All animals underwent left hemimandibular osteotomy and external fixator placement, followed by distraction to a 5.1-mm gap. Left hemimandibles were harvested and mechanically tested for parameters of strength, yield, and breaking load. RESULTS: Radiation-related complications such as severe alopecia were significantly increased in XRT-DO compared with the AMF-treated group (P = 0.001), whereas infection and death were comparable (P = 0.318). Upon dissection, bony defects were grossly visible in XRT-DO distraction gap compared with AMF-XRT-DO, which exhibited significantly more complete unions (P = 0.004). Those results were significantly increased in the specimens prophylactically treated with AMF (yield: 39.41 N vs 21.78 N, P = 0.023; breaking load: 61.74 N vs 34.77 N, P = 0.044; respectively). CONCLUSIONS: Our study revealed that AMF enhances biomechanical strength, regeneration, and bony union after radiation in a murine model of DO. The use of prophylactic AMF in combination with DO offers the promise of an alternative reconstructive option for patients afflicted with head and neck cancer.

Significant Differences in the Bone of an Isogenic Inbred Versus Nonisogenic Outbred Murine Mandible: A Study in Rigor and Reproducibility.

Inattention to differences between animal strains is a potential cause of irreproducibility of basic science investigations. Accordingly, the authors' laboratory sought to ensure that cross-comparisons of results generated from studies of mandibular physiology utilizing the Sprague Dawley and Lewis rat strains are valid. The authors specifically investigated baseline histomorphometrics, bone mineral density, and biomechanical strength of the unaltered endogenous mandibles of the inbred, isogenic Lewis rat, and the outbred, nonisogenic Sprague Dawley rat to determine if they are indeed equal. The authors hypothesized that little difference would be found within these metrics.The authors' study utilized 20 male Lewis and Sprague Dawley rats, which underwent no manipulation other than final dissection and analysis. Ten rats from each strain underwent bone mineral density and biomechanical strength analysis. The remaining rats underwent histological analysis. Descriptive and bivariate statistics were computed and the P value was set at 0.05.Lewis rats had a significantly greater number of empty lacunae. Sprague Dawley rats exhibited a significantly greater ratio of bone volume-to-total volume, bone mineral density, tissue mineral density, bone volume fraction, and total mineral content. No differences were found during biomechanical testing.This study demonstrates that differences exist between the Lewis and Sprague Dawley rat within unaltered baseline mandibular tissue. However, these differences appear to have limited functional impact, as demonstrated by similar biomechanical strength metrics. Other specific differences not addressed in this manuscript may exist. However, the authors believe that researchers may confidently cross-compare results between the 2 strains, while taking into account the differences found within this study.

Bone marrow stem cells assuage radiation-induced damage in a murine model of distraction osteogenesis: A histomorphometric evaluation.

The purpose of this study is to determine if intraoperatively placed bone marrow stem cells (BMSCs) will permit successful osteocyte and mature bone regeneration in an isogenic murine model of distraction osteogenesis (DO) following radiation therapy (XRT). Lewis rats were split into three groups, DO only (Control), XRT followed by DO (xDO) and XRT followed by DO with intraoperatively placed BMSCs (xDO-BMSC). Coronal sections from the distraction site were obtained, stained and analyzed via statistical analysis with analysis of variance (ANOVA) and subsequent Tukey or Games-Howell post-hoc tests. Comparison of the xDO-BMSC and xDO groups demonstrated significantly improved osteocyte count (87.15 ± 10.19 vs. 67.88 ± 15.38, P = 0.00), and empty lacunae number (2.18 ± 0.79 vs 12.34 ± 6.61, P = 0.00). Quantitative analysis revealed a significant decrease in immature osteoid volume relative to total volume (P = 0.00) and improved the ratio of mature woven bone to immature osteoid (P = 0.02) in the xDO-BMSC compared with the xDO group. No significant differences were found between the Control and xDO-BMSC groups. In an isogenic murine model of DO, BMSC therapy assuaged XRT-induced cellular depletion, resulting in a significant improvement in histological and histomorphometric outcomes.

A Comparison of Vascularity, Bone Mineral Density Distribution, and Histomorphometrics in an Isogenic Versus an Outbred Murine Model of Mandibular Distraction Osteogenesis.

PURPOSE: The vascularity, bone mineral density distribution, and histomorphometric data between the inbred, isogenic Lewis rat and the outbred, nonisogenic Sprague Dawley rat within mandibular distraction osteogenesis (MDO) were evaluated to allow future researchers to compare the results generated from these 2 animals. We hypothesized that little difference would be found between the 2 strains within these metrics. MATERIALS AND METHODS: We implemented a comparative study between the Lewis and Sprague Dawley rat strains within MDO. The sample was composed of 17 male Lewis and 17 male Sprague Dawley rats that underwent surgical external fixation and distraction. The rats' hemimandibles were distracted to a total distance of 5.1 mm. After 28 days of consolidation, 9 rats from each group underwent bone mineral density distribution analysis. The remaining rats from each group were analyzed for the vascular and histologic metrics. Descriptive and bivariate statistics were computed, and the P value was set at .05. RESULTS: We demonstrated successful MDO in all the rats, with no significant difference found in the histologic or bone mineral density distribution metrics. No significant differences were found in any of the vascular metrics, with the exception of vascular separation, which was not normalized to the mandibular volume (P = .048). CONCLUSIONS: The results of the present study have demonstrated that little dissimilarity exists between the isogenic Lewis and outbred Sprague Dawley models of MDO. Thus, researchers can confidently compare the gross results between the 2 strains, with consideration of the very small differences between the 2 models. For studies that require an isogenic strain, the Lewis rat is an apt surrogate for the Sprague Dawley strain.

Amifostine Treatment Mitigates the Damaging Effects of Radiation on Distraction Osteogenesis in the Murine Mandible.

According to the American Society of Clinical Oncology, in 2012, more than 53,000 new cases of head and neck cancers (HNCs) were reported in the United States alone and nearly 12,000 deaths occurred relating to HNC. Although radiotherapy (XRT) has increased survival, the adverse effects can be unrelenting and their management is rarely remedial. Current treatment dictates surgical mandibular reconstruction using free tissue transfer. These complex operations entail extended hospitalizations and attendant complications often lead to delays in initiation of adjuvant therapy, jeopardizing prognosis as well as quality of life. The creation of new bone by distraction osteogenesis (DO) generates a replacement of deficient tissue from local substrate and could have immense potential therapeutic ramifications. Radiotherapy drastically impairs bone healing, precluding its use as a reconstructive method for HNC. We posit that the deleterious effects of XRT on bone formation could be pharmacologically mitigated. To test this hypothesis, we used a rodent model of DO and treated with amifostine, a radioprotectant, to assuage the XRT-induced injury on new bone formation. Amifostine had a profound salutary effect on bone regeneration, allowing the successful implementation of DO as a reconstructive technique. The optimization of bone regeneration in the irradiated mandible has immense potential for translation from the bench to the bedside, providing improved therapeutic options for patients subjected to XRT.

Changes in Skin Vascularity in a Murine Model for Postmastectomy Radiation.

BACKGROUND: Postmastectomy radiation causes persistent injury to the breast microvasculature, and the prevailing assumption is that longer delays before breast reconstruction allow for recovery of blood supply. This study uses a murine model to examine the effects of radiation on skin vascularity to help determine when radiation-induced effects on the microvasculature begin to stabilize. STUDY DESIGN: Isogenic Lewis rats were divided into 2 groups: radiation therapy (XRT) (n = 24) and control (n = 24). The XRT rats received a breast cancer therapy human dose-equivalent of radiation to the groin, whereas control rats received no radiation. Animals were sacrificed at 4, 8, 12, and 16 weeks after completion of radiation. The vasculature was injected with Microfil, and groin skin was harvested for radiomorphometric analysis by microcomputed tomography. One-way analysis of variance with post hoc Tukey tests was used to determine significance between groups. RESULTS: Augmentation in vascularity was observed in the XRT group at 4 weeks after radiation compared to the control group (P = 0.045). Vessel number was decreased at 12 weeks (P = 0.002) and at 16 weeks (P = 0.001) in the XRT rats compared to control rats. Vessel separation in the XRT group was higher than that in the control group at 12 weeks (P = 0.009) and 16 weeks (P = 0.001). There was no change in vessel number and separation between weeks 12 and 16. CONCLUSIONS: A period of augmented skin vascularity is seen after radiation injury followed by decreased vascularity which demonstrates stabilization at approximately 12 weeks in this murine model. This model can be used to further study breast flap vascularity and the optimization of the timing of delayed breast reconstruction.

Clinical Use of Deferoxamine in Distraction Osteogenesis of Irradiated Bone.

The deleterious effects of radiotherapy, including hypovascularity and hypocellularity, have made distraction of irradiated bones challenging. Animal studies, however, have demonstrated adjunctive measures such as the administration of deferoxamine to significantly improve bone regeneration across irradiated distraction gaps. In this report, the authors demonstrate, for the first time, enhanced bone formation following deferoxamine application in a patient following distraction of a previously irradiated maxilla. Computed tomography imaging of the pterygomaxillary buttress on the side of administration revealed significantly increased bone area and density relative to the contralateral buttress. This is the first presentation of clinical deferoxamine use to promote bone formation following irradiated bone distraction and highlights the promise for this adjunctive measure to make outcomes after distraction of irradiated bone more reliable.

Raman spectroscopy delineates radiation-induced injury and partial rescue by amifostine in bone: a murine mandibular model.

Despite its therapeutic role in head and neck cancer, radiation administration degrades the biomechanical properties of bone and can lead to pathologic fracture and osteoradionecrosis. Our laboratories have previously demonstrated that prophylactic amifostine administration preserves the biomechanical properties of irradiated bone and that Raman spectroscopy accurately evaluates bone composition ex vivo. As such, we hypothesize that Raman spectroscopy can offer insight into the temporal and mechanical effects of both irradiation and amifostine administration on bone to potentially predict and even prevent radiation-induced injury. Male Sprague-Dawley rats (350-400 g) were randomized into control, radiation exposure (XRT), and amifostine pre-treatment/radiation exposure groups (AMF-XRT). Irradiated animals received fractionated 70 Gy radiation to the left hemi-mandible, while AMF-XRT animals received amifostine just prior to radiation. Hemi-mandibles were harvested at 18 weeks after radiation, analyzed via Raman spectroscopy, and compared with specimens previously harvested at 8 weeks after radiation. Mineral (ρ958) and collagen (ρ1665) depolarization ratios were significantly lower in XRT specimens than in AMF-XRT and control specimens at both 8 and 18 weeks. amifostine administration resulted in a full return of mineral and collagen depolarization ratios to normal levels at 18 weeks. Raman spectroscopy demonstrates radiation-induced damage to the chemical composition and ultrastructure of bone while amifostine prophylaxis results in a recovery towards normal, native mineral and collagen composition and orientation. These findings have the potential to impact on clinical evaluations and interventions by preventing or detecting radiation-induced injury in patients requiring radiotherapy as part of a treatment regimen.

A Histomorphometric Analysis of Radiation Damage in an Isogenic Murine Model of Distraction Osteogenesis.

PURPOSE: The devastation radiation therapy (XRT) causes to endogenous tissue in patients with head and neck cancer can be a prohibitive obstacle in reconstruction of the mandible, demanding a better understanding of XRT-induced damage and options for reconstruction. This study investigated the cellular damage caused by radiation in an isogenic murine model of mandibular distraction osteogenesis (DO). The authors posited that radiation would result in fewer osteocytes, with increased empty lacunae and immature osteoid. MATERIALS AND METHODS: Twenty Lewis rats were randomly assigned to a DO group (n = 10) or a XRT/DO group (n = 10). These groups underwent an osteotomy and mandibular DO across a 5.1-mm gap. XRT was administered to the XRT/DO group at a fractionated human equivalent dose of 35 Gy before surgery. Animals were sacrificed on postoperative day 40 and mandibles were harvested and sectioned for histologic analysis. RESULTS: Bone that underwent radiation showed a significantly decreased osteocyte count and complementary increase in empty lacunae compared with non-XRT bone (P = .019 and P = .000). In addition, XRT bone exhibited increased immature osteoid and decreased mature woven bone compared with nonradiated bone (P = .001 and P = .003, respectively). Furthermore, analysis of the ratio of immature osteoid to woven bone volume exhibited a significant increase in the XRT bone, further showing the devastating damage from XRT (P = .001). CONCLUSION: These results clearly show the cellular diminution that occurs as a result of radiation. This foundational study provides the groundwork on which to investigate cellular therapies in an immuno-privileged model of mandibular DO.

Burn injury enhances bone formation in heterotopic ossification model.

OBJECTIVE: To demonstrate the pro-osteogenic effect of burn injury on heterotopic bone formation using a novel burn ossicle in vivo model. BACKGROUND: Heterotopic ossification (HO), or the abnormal formation of bone in soft tissue, is a troubling sequela of burn and trauma injuries. The exact mechanism by which burn injury influences bone formation is unknown. The aim of this study was to develop a mouse model to study the effect of burn injury on heterotopic bone formation. We hypothesized that burn injury would enhance early vascularization and subsequent bone formation of subcutaneously implanted mesenchymal stem cells. METHODS: Mouse adipose-derived stem cells were harvested from C57/BL6 mice, transfected with a BMP-2 adenovirus, seeded on collagen scaffolds (ossicles), and implanted subcutaneously in the flank region of 8 adult mice. Burn and sham groups were created with exposure of 30% surface area on the dorsum to 60°C water or 30°C water for 18 seconds, respectively (n = 4/group). Heterotopic bone volume was analyzed in vivo by micro-computed tomography for 3 months. Histological analysis of vasculogenesis was performed with platelet endothelial cell adhesion molecule staining. Osteogenic histological analysis was performed by Safranin O, Picrosirius red, and aniline blue staining. Qualitative analysis of heterotopic bone composition was completed with ex vivo Raman spectroscopy. RESULTS: Subcutaneously implanted ossicles formed heterotopic bone. Ossicles from mice with burn injuries developed significantly more bone than sham control mice, analyzed by micro-computed tomography at 1, 2, and 3 months (P < 0.05), and had enhanced early and late endochondral ossification as demonstrated by Safranin O, Picrosirius red, and aniline blue staining. In addition, burn injury enhanced vascularization of the ossicles (P < 0.05). All ossicles demonstrated chemical composition characteristic of bone as demonstrated by Raman spectroscopy. CONCLUSIONS: Burn injury increases the predilection to osteogenic differentiation of ectopically implanted ossicles. Early differences in vascularity correlated with later bone development. Understanding the role of burn injury on heterotopic bone formation is an important first step toward the development of treatment strategies aimed to prevent unwanted and detrimental heterotopic bone formation.