PURPOSE: The difficulty of harvest and relative scarcity of bone marrow stromal cells (BMSCs) has limited the widespread use and clinical application of this technology, thereby necessitating inquiry into other therapies including adipose-derived stromal cells (ASCs). The goal of this study was to compare the ability of ASCs and BMSCs to heal mandibular defects and understand the mechanism through which this occurs. We hypothesize that ASCs will enhance fracture healing by improving vasculogenesis, while BSMCs will directly affect osteogenesis. METHODS: Male Lewis rats were radiated (35Gy), and subsequently underwent mandibular osteotomy with external fixation with implantation of two million BMSCs (n=12) or ASCs (n=16) marked with Green fluorescent protein (GFP). After 40 days, union rates were evaluated using microCT. Confocal microscopy visualized the contribution of ASCs/BMSCs to the bone regenerate. Quantitative polymerase chain reaction of ASCs/BMSCs compared expression of osteogenic and vasculogenic genes. Coculture of ASCs (n=3) or BMSCs (n=3) with human umbilical vein endothelial cells (HUVECs) was performed in vitro in transwells to measure tubule formation as a marker of vasculogenesis. RESULTS: ASC-implantation resulted in higher union rates than BMSC-implantation (union rate: 94% vs. 66%). These cells contribute indirectly to fracture healing, as GFP was not visualized at the site. BMSCs expressed osteogenic genes including osteopontin to a significantly greater degree than did ASCs, while ASCs expressed greater levels of vascular endothelial growth factor. This translated to greater tubule formation among HUVECs co-cultured with ASCs than with BMSCs (64.3 ± 7.3 vs. 23.3 ± 2.6, p=0.0008), and increased vasculogenesis in vivo in mandibles after ASC implantation. CONCLUSIONS: ASCs heal fracture defects better than BMSCs. This effect is likely mediated by indirect modulation of vasculogenesis, rather than by a direct effect on osteogenesis. Clinicians interested in cell-based therapies for irradiated bone injury should consider ASCs as a promising option, given their abundance, ease of acquisition, and improved fracture healing.
Endometriosis of the appendix is an uncommon mimicker of acute appendicitis which makes for a diagnostic dilemma.We present a rare case of a menstruating woman presenting with classic symptoms of appendicitis, without the characteristic inflammatory changes seen on laparoscopy consistent with appendicitis. Instead, the appendix appeared unusually contracted on itself. Pathologic review of the appendix revealed microscopic findings of endometriosis.We theorize the growth and shedding of the endometrial tissue during menstruation caused compression of the neural plexi in the wall of the appendix leading to the presentation mimicking acute appendicitis.Given the potential for endometrial appendicitis, we propose appendectomy in reproductive age female patients with right lower quadrant pain, regardless of appendix appearance on laparoscopy.
PURPOSE: The ability of deferoxamine (DFO) to mitigate the deleterious effects of radiation on bone healing and regeneration for head and neck cancer (HNC) reconstruction is currently being investigated. However, there remains concern about the tumorigenic potential of DFO, due to the ability of DFO to induce angiogenesis and promote tissue vascularization. The purpose of this study is to investigate the effects of DFO on head and neck squamous cell carcinoma (HNSCC) in-vitro and in-vivo, to delineate the clinical safety of DFO administration to HNC patients. METHODS: MDA-1986 HNSCC cells were exposed to increasing doses of DFO (0, 25, and 50µM) and XRT (0, 5, and 10Gy) in triplicate and counted via hemocytometer to define the dose-dependent effects of each therapy. A 3-D sphere assay was performed to confirm the observed DFO dose response. Subsequently, an MTS assay was performed to comparatively analyze the following groups: control, XRT (5Gy), DFO (100µM), and XRT+DFO. Xenograft mouse models were then created in Nu/Nu mice using two million green-luciferase-tagged cells, which were injected SQ to 12 mice. Resulting tumors were allowed 14 days to proliferate. XRT mice received 3 fractionated doses of 3 Gy over the 10-day study. DFO mice received 5 doses of DFO via peritumoral injection. Tumor volumes were measured every third day during treatments. Statistical analysis was performed using ANOVA and paired-t-test, and p=0.05 was considered significant. RESULTS:In vitro, cell proliferation significantly decreased with increasing doses of XRT. Unexpectedly, DFO also displayed a significant dose-dependent antitumorigenic potency to HNSCC cells when analyzed via hemocytometer. The proceeding DFO-dose response sphere assay confirmed the abovementioned toxicity of DFO. The MTS assay exhibited a significant diminution of cell proliferation in all treatment groups compared to control. Specifically, the addition of DFO reduced cell proliferation to a significantly greater degree than XRT treatment alone, and the combination therapy decreased tumor proliferation significantly more than either single therapy. In vivo, buccal xenografts revealed an increase in control tumor volume by experimental day 6. However, the XRT and DFO groups did not experience a significant increase in tumor volume at any point during the 10-day treatment regimen. CONCLUSIONS:In vitro and in vivo studies reveal DFO exhibits an antitumorigenic effect that is equal to, if not more pronounced than, the potent effects of radiotherapy on HNSCC cell proliferation and tumor formation. Such findings provide preliminary evidence that DFO may be safely utilized in select HNC patient populations to promote new bone formation during head and neck reconstruction following radiotherapy. Moreover, the strong iron-chelating capacity of DFO may offer a promising chemotherapeutic approach to the oncologic management of HNC. Further studies examining the effect of DFO on HNSCC cell subtypes is warranted due to the heterogeneous nature of cancer cell biology. A. Donneys: None. J.V. Lynn: None. K. Urlaub: None. K. Kovatch: None. H.S. Uygur: None. J. Hoxie: None. L. Buchman: None. K. Ranganathan: None. A. Snider: None. N.S. Nelson: None. C. Subramanian: None. M.S. Cohen: None. S.R. Buchman: None.
INTRODUCTION: Over 60,000 new cases of head and neck cancer were diagnosed in the U.S. in 2016. Radiation is commonly required to reduce recurrence rates and improve survival; however, complications after radiation, including poor bone healing and osteoradionecrosis, contribute to the significant morbidity associated with this disease process. The current standard of treatment of such complications is limited to free tissue transfer. Given the significant morbidity associated with these procedures, it is important to examine the utility of cell-based therapies as a potential translational treatment to promote bone regeneration for irradiated patients. Adipose-derived stem cells (ASCs) and bone marrow derived stem cells (BMSCs) represent translational therapies that can improve osteogenesis. We recently demonstrated that ASCs are superior to BMSCs in enhancing bone healing using a segmental defect model in the rat mandible. We hypothesize that differing mechanisms of action between the two cell types contribute to the superiority of ASC’s to enhance bone healing. METHODS: BMSCs and ASCs were harvested from male Lewis rats (n=3), plated at a density of 200,000 cells/well, and treated with osteogenic differentiation medium. Alkaline phosphatase stain was performed to evaluate osteogenic potential. Vascular endothelial growth factor (VEGF) was also measured and compared. Finally, ASCs and BMSCs were cocultured with human umbilical vein endothelial cells using a transwell system to study the paracrine effect of these two cell types on vasculogenesis. Student’s t-tests were used to compare the osteogenic and vasculogenic potential of the two groups. RESULTS: ASCs had significantly less osteogenic potential than BMSCs (11.8 ± 0.9 vs. 16.3 ± 0.4; p<0.05). Conversely, ASCs were significantly more vasculogenic than BMSCs based on VEGF release (3,573 ± 87.4 vs. 1607.0 ± 45.0; p<0.001). These findings translated to significantly greater tubule formation in transwells treated with ASCs compared to BMSCs on video microscopy. The properties of ASCs that resulted in enhanced vasculogenesis are associated with enhanced bone formation in vivo and improved healing in our segmental defect model. CONCLUSION: ASCs and BMSCs enhance bone formation via different mechanisms. While to enhance bone healing as described in this study, the mechanism of a vasculogenic intermediate may hold greater promise in creating a translational therapeutic that more proficiently promotes bone healing and remediates the ravages of radiation injury.
Endocarditis is an uncommon complication of disseminated candidiasis among premature infants, but has been recently reported to be almost uniformly fatal. The lone previously documented survivor required extensive surgical resection as well as prolonged systemic antifungal therapy. The present report details a premature infant who recovered from Candida endocarditis with medical therapy alone.
PURPOSE: Bone regeneration is impaired after radiotherapy-induced vascular injury. Therapeutic enhancement of vascular response offers a solution for regenerating damaged vessels with resultant improvement in bone healing. There is evidence that tissue regeneration is stimulated with the use of cytokine-rich adipose derived stem cells (ASCs). We posit that use of ASCs will substantially enhance the quality of vascularity in an irradiated mandibular fracture model. METHODS: Lewis rats (n=37) were divided into 3 groups: mandible fracture (Fx), radiotherapy followed by fracture (XFx), and radiotherapy followed by fracture and adiposederived stem cell treatment (XFxASC). All experimental groups underwent external fixator placement with surgical creation of a mandibular osteotomy. After a 40-day recovery period, animals were perfused and micro-CT vascular radiomorphometrics were obtained. The measured vessel metrics included volume fraction, number, thickness, and separation were assessed and statistically analyzed using ANOVA. RESULTS: A statistically significant three-fold increase in vessel volume fraction was seen in both Fx (p=0.001) and XFxASC treatment (p= 0.048) groups when compared to the XFx group. No statistically significant difference was seen in vessel volume fraction between control and ASC groups (p=0.28). Vessel thickness was also significantly higher in Fx (p=0.031) and XFxASC (p= 0.006) groups when compared to XFx groups. While not statistically significant, there were numerically distinctive improvements for vessel number and vessel separation when comparing groups. The XFx group had the expected lowest vessel number at a value of 0.15 mm-1, the Fx group had the highest value of 0.49 mm-1, and the XFxASC group exhibited an intermediate vessel number of 0.27 mm-1. Vessel separation also demonstrated an expected trend with XFx displaying the most sparse vessel characteristics at a vessel separation of 44.9 mm and Fx group with increased vascular presence with a separation of 2.34 mm. Treatment with XFxASC group improved vessel separation from XFx with a value of 0.778 mm, a vascularity improvement that exceeded the Fx group. CONCLUSION: Use of ASCs in fracture defects following radiotherapy improves the clinically relevant vascular metrics of vessel volume fraction and vessel thickness. Favorable trends were also noted with the metrics of vessel number and vessel separation. Our results support further study for the potential use of ASCs in restoring vascularity in irradiated bone.
INTRODUCTION: The goal of this proposal is to therapeutically reverse the damaging effects of radiotherapy on bone formation and healing to enable non-vascularized grafting in irradiated bone. Utilizing a rodent model of mandibular bone grafting, we quantified metrics of diminished graft take and bone healing in response to radiation treatment. Subsequently, we utilized implantable deferoxamine (DFO)-an angiogenic stimulant, to reverse these radiation-induced detriments. We hypothesized that the addition of our proposed therapy, would evidence quantifiable degrees of remediation on the process of tissue regeneration, graft incorporation and bone healing. METHODS: Male Lewis rats received a human equivalent dose of radiotherapy (7Gy/d x 5d) to left hemi-mandibles. After recovery, a circular trephine burr (6mm) was utilized to create a critical size defect just posterior to the third molar, and a bone graft was harvested from the right hemi-mandible of the same animal and secured with a custom PLA resorbable plate. Three groups (n=8/group) of animals were investigated: Control, (irradiated) XRT and irradiated + implantable deferoxamine (DFO). Mandibles were imaged at 14, 40 and 60 days with in vivo µCT, and a 60-day healing period was allowed prior to further outcomes testing. Bony union was judged clinically by 3 blinded reviewers on a scale from 0 to 4, representing the approximate percentage of robust union formation along the circular graft-recipient site interface (e.g. 1=25%, 4=100%). Statistical comparisons were conducted with ANOVA (p<0.05). RESULTS: We observed a significant diminution of bone graft healing after radiotherapy. At 60 days, the bone volume fraction (BVF) of the XRT group decreased by 20% (p=0.001), and exhibited lower bony union scores when compared to control (p=0.005). With the addition of DFO, these findings were largely remediated. At 60 days, the BVF improved upon the XRT BVF by 12% (p=0.025), and was not different than control (p=0.282). In addition, the bony union scores of the implanted DFO group significantly improved from XRT levels (p=0.05), and were not different than control (p=0.200). CONCLUSION: Implantable DFO strongly remediates the effects of radiation on non-vascularized bone graft incorporation and healing as measured by micro-densitometry and bony union analysis. These observations are promising with regards to the potential utility of this therapy to enhance bone graft incorporation in the irradiated mandible for head and neck cancer survivors.
