Pain hypersensitivity, sensorimotor impairment, and decreased muscle force in a novel rat model of radiation-induced peripheral neuropathy.

INTRODUCTION: Radiation-induced peripheral neuropathy is a rare, but serious complication often resulting in profound morbidity, life-long disability, and chronic debilitating pain. Unfortunately, this type of peripheral neuropathy is usually progressive, and almost always irreversible. To date, a standardized rat model of radiation-induced peripheral neuropathy has not been established. The purpose of the present study was to examine neuropathic pain, sensorimotor impairment, and muscle force parameters following the administration of a clinically relevant radiation dose in a rat model. METHODS: Ten rats were randomly assigned to one of two experimental groups: (1) radiation and (2) sham-radiated controls. Radiated animals were given a clinically relevant dose of 35 Gray (Gy) divided into five daily doses of 7 Gy/day. This regimen represents a human equivalent dose of 70 Gy, approximating the same dosage utilized for radiotherapy in oncologic patients. Sham-radiated controls were anesthetized and placed in the radiation apparatus but were not given radiation. All animals were tested for baseline values in both sensorimotor and pain behavioral tests. Sensorimotor testing consisted of the evaluation of walking tracks with the calculation of the Sciatic Functional Index (SFI). Pain-related behavioral measures consisted of mechanical allodynia (von Frey test), cold allodynia (Acetone test), and thermal allodynia (Hargreaves test). Animals were tested serially over an 8-week period. At the study endpoint, electrophysiological and muscle force assessments were completed, and histomorphometric analysis was performed on all sciatic nerves. RESULTS: Animals that underwent radiation treatment displayed significantly greater pain hypersensitivity to mechanical stimulation as compared to sham radiated controls from weeks 4 to 8 of testing. SFI values indicated sensorimotor impairments in the overground gait of radiated animals as compared to non-radiated animals. Furthermore, radiated animals displayed reduced twitch and tetanic muscle force when compared to sham radiated controls. CONCLUSIONS: A clinically relevant human equivalent dose of fractionated 35 Gy in rats established significant pain hypersensitivity, impairments in sensorimotor locomotion, and decreased muscle force capacity. This novel rodent model of radiation-induced peripheral neuropathy can be utilized to assess the potential efficacy of therapeutic treatments to either prevent or remediate this clinically debilitating condition.

Optimizing immunofluorescent staining of H vessels within an irradiated fracture callus in paraffin-embedded tissue samples.

H vessels are an essential link in angiogenic-osteogenic coupling and orchestrate the process of bone healing. H vessels are critically deficient in the setting of radiation-induced fractures, which have been reported to occur in up to 25% of patients undergoing radiotherapy. By increasing H-vessel proliferation, Deferoxamine (DFO) revitalizes the physiologic response to skeletal injury and accelerates irradiated fracture repair. H-vessel quantification is therefore an important outcome measure in histologic analysis of bone healing. However, an optimized protocol for staining H vessels in formalin-fixed paraffin-embedded (FFPE) tissue sections has not been reported. With this protocol, we describe a method of staining FFPE bone samples with minimal background fluorescence and high signal-to-noise ratio. We examined mandibular specimens in a rat model of bone healing from a range of fracture conditions, including healthy bone (Fx), irradiated bone (XFx), and irradiated bone with DFO treatment (XFx-DFO). Quantitative analysis revealed a significant increase of H vessels in the XFxDFO group compared to both the Fx and XFx groups. By optimizing immunofluorescent staining of H vessels in FFPE samples across a range of fracture conditions, we offer investigators an efficacious means of producing reliable imaging for quantitative analysis of H vessels in an irradiated fracture callus.

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.

Intraoperative Stromal Vascular Fraction Therapy Improves Histomorphometric and Vascular Outcomes in Irradiated Mandibular Fracture Repair.

BACKGROUND: Cell-based treatments have demonstrated the capacity to enhance reconstructive outcomes in recent decades but are hindered in clinical utility by regulatory hurdles surrounding cell culture. This investigation examines the ability of a noncultured stromal vascular fraction derived from lipoaspirate to enhance bone healing during fracture repair to further the development of translatable cell therapies that may improve outcomes in irradiated reconstruction. METHODS: Isogenic male Lewis rats were divided into three groups: fracture, irradiated fracture, and irradiated fracture with stromal vascular fraction treatment. Irradiated groups received a fractioned dose of 35 Gy before mandibular osteotomy. Stromal vascular fraction was harvested from the inguinal fat of isogenic donors, centrifuged, and placed intraoperatively into the osteotomy site. All mandibles were evaluated for bony union and vascularity using micro-computed tomography before histologic analysis. RESULTS: Union rates were significantly improved in the irradiated fracture with stromal vascular fraction treatment group (82 percent) compared to the irradiated fracture group (25 percent) and were not statistically different from the fracture group (100 percent). Stromal vascular fraction therapy significantly improved all metrics of bone vascularization compared to the irradiated fracture group and was not statistically different from fracture. Osteocyte proliferation and mature bone formation were significantly reduced in the irradiated fracture group. Bone cellularity and maturity were restored to nonirradiated levels in the irradiated fracture with stromal vascular fraction treatment group despite preoperative irradiation. CONCLUSIONS: Vascular and cellular depletion represent principal obstacles in the reconstruction of irradiated bone. This study demonstrates the efficacy of stromal vascular fraction therapy in remediating these damaging effects and provides a promising foundation for future studies aimed at developing noncultured, cell-based therapies for clinical implementation.

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.

Implantable hyaluronic acid-deferoxamine conjugate prevents nonunions through stimulation of neovascularization.

Approximately 6.3 million fractures occur in the U.S. annually, with 5-10% resulting in debilitating nonunions. A major limitation to achieving successful bony union is impaired neovascularization. To augment fracture healing, we designed an implantable drug delivery technology containing the angiogenic stimulant, deferoxamine (DFO). DFO activates new blood vessel formation through iron chelation and upregulation of the HIF-1α pathway. However, due to its short half-life and rapid clearance, maintaining DFO at the callus site during peak fracture angiogenesis has remained challenging. To overcome these limitations, we composed an implantable formulation of DFO conjugated to hyaluronic acid (HA). This compound immobilizes DFO within the fracture callus throughout the angiogenic window, making it a high-capacity iron sponge that amplifies blood vessel formation and prevents nonunions. We investigated implanted HA-DFO's capacity to facilitate fracture healing in the irradiated rat mandible, a model whereby nonunions routinely develop secondary to obliteration of vascularity. HA-DFO implantation significantly improved radiomorphometrics and metrics of biomechanical strength. In addition, HA-DFO treated mandibles exhibited a remarkable 91% bone union rate, representing a 3.5-fold improvement over non-treated/irradiated controls (20% bone union rate). Collectively, our work proposes a unique methodology for the targeted delivery of DFO to fracture sites in order to facilitate neovascularization. If these findings are successfully translated into clinical practice, millions of patients will benefit from the prevention of nonunions.

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.

The Role of Deferoxamine in Irradiated Breast Reconstruction: A Study of Oncologic Safety.

BACKGROUND: Radiotherapy plays an essential role in the oncologic management of breast cancer. However, patients who undergo radiotherapy experience significantly more wound complications during the reconstructive process. Deferoxamine has immense potential to up-regulate angiogenesis and improve reconstructive outcomes. The purpose of this study was to determine the impact of deferoxamine on breast cancer cell proliferation in vitro, to delineate oncologic safety concerns regarding the use of deferoxamine as a regenerative therapeutic. METHODS: The dose-dependent effect of radiation and deferoxamine on two triple-negative breast cancer cell lines (MDA-MB-231 and MDA-MB-468) was determined by means of MTS (percentage cell viability) and tumorsphere (sphere number) analysis. Radiation therapy and deferoxamine were delivered both individually and in combination, and all experiments were completed in triplicate. Intracellular iron, nuclear factor-κB localization, and apoptosis/necrosis assays were performed to delineate mechanism. Analysis of variance statistical analysis was performed using SPSS (p < 0.05). RESULTS: For both cell lines, percentage viability and sphere number significantly decreased following exposure to 10 Gy of radiation. Surprisingly, the administration of 25 µM deferoxamine also significantly decreased each metric. The administration of deferoxamine (100 µM) in combination with radiation (10 Gy) resulted in significantly reduced percentage viability and sphere number compared with the administration of radiation alone. Deferoxamine treatment decreased intracellular iron, suppressed nuclear factor-κB activation, and induced apoptosis. CONCLUSION: Radiation and deferoxamine significantly decrease breast cancer proliferation when delivered independently and in combination, suggesting deferoxamine may be safely used to facilitate improved reconstructive outcomes among triple-negative breast cancer survivors. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.

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.

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.

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.

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.

Role of parathyroid hormone in regeneration of irradiated bone in a murine model of mandibular distraction osteogenesis.

BACKGROUND: The purpose of this study was to measure the histologic and histomorphometric effects of parathyroid hormone (PTH) treatment on irradiated bone undergoing distraction osteogenesis (DO). METHODS: Thirty-four rats were divided into 3 groups. The control group underwent DO and the radiation control group underwent radiotherapy (RT) before DO. The PTH group underwent RT and received PTH during DO. Quantitative histology and histomorphometry were performed. RESULTS: RT resulted in a depletion of osteocytes and increase in empty lacunae. Treatment with PTH resulted in an increase in osteocyte counts and decrease in empty lacunae (p < .05), restoring osteocytes to levels seen in nonradiated bone (p = .121). RT decreased bone volume to tissue volume (BV-TV) ratio and increased osteoid volume to tissue volume (OV-TV) ratio, signifying increased immature bone formation. PTH treatment restored OV-TV ratio to that observed in nonradiated bone. CONCLUSION: PTH treatment of irradiated bone enhanced bone regeneration and restored osteocyte counts and OV-TV ratio to levels comparable to nonradiated bone. © 2016 Wiley Periodicals, Inc. Head Neck 39: 464-470, 2017.

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.

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.