PURPOSE: Mandibular reconstruction secondary to oncologic resection remains limited to invasive free tissue transfer (FTT) by the detrimental effects of radiotherapy. Nonvascularized bone grafts (NBGs) represent a practical surgical alternative to FTT, but are precluded from irradiated reconstruction due to sequelae such as diminished capacity for revascularization and the destruction of osteocompetent cells. In order to overcome these principal barriers to irradiated bone healing, our laboratory has developed an implantable, hyaluronic acid (HA)-deferoxamine nanoparticle designed to stimulate angiogenesis during the critically-important weeks following surgery. This study examines the efficacy of this novel therapy in a murine model of irradiated mandibular reconstruction with the goal of ultimately reintroducing NBGs as a viable alternative to FTT in the irradiated setting. METHODS: Male Lewis rats (n = 33) were equally divided into three groups; control bone graft (CBG), irradiated bone graft (XBG), and irradiated bone graft with intraoperative HA-deferoxamine implantation (XHDBG). Irradiated groups received a fractionated dose of 35Gy over 5 days, comparable to 70Gy administered to head and neck cancer patients clinically. Following a 2-week recovery period, all rats underwent creation of a 5 mm critical-sized segmental defect in the left hemi-mandible and reconstruction with a NBG from the iliac crest of an isogenic donor. On post-operative day 60, all mandibles were perfused and evaluated for bony union upon dissection. Vascularity was evaluated throughout the bone graft and healing interfaces through microcomputed tomography prior to histologic analysis of osteocyte proliferation and mature bone volume. Statistical analysis was performed using ANOVA, with p values less than 0.05 considered significant. RESULTS: Bony union rates were improved by HA-deferoxamine treatment in the XHDBG group (82%) compared to the XBG group (64%) and were similar to union rates observed in the CBG group (91%). Radiotherapy resulted in decreased vessel number, vessel volume, vessel volume fraction, and vessel thickness in the XBG group compared to CBG. Implantation of HA-deferoxamine significantly increased all metrics of bone vascularity compared to the XBG group. No significant differences were observed between the XHDBG and CBG groups. Radiotherapy-induced cell depletion at the bone graft interfaces was evidenced through a significant reduction of osteocytes in the XBG group compared to CBG. Mature bone formation was also significantly decreased in XBG in comparison to CBG. Osteocyte proliferation and mature bone formation were significantly increased in the XHDBG group compared to XBG and were not statistically different from non-irradiated control levels. CONCLUSION: The results of this study demonstrate the ability of HA-deferoxamine implantation to significantly improve the vascularity and cellularity of NBGs in reconstruction of the irradiated mandible. Given the pre-existing status of deferoxamine on hospital formulary, this treatment represents a highly translatable method of enhanced bone healing in the setting of radiotherapy that may expand the utility of NBGs in mandibular reconstruction following tumor ablation. While further investigations are necessary, such translation would offer the practical benefits of NBGs to both surgeons and head and neck cancer patients including reduced donor site morbidity and technical demand in comparison to FTT procedures.
Purpose: Adipose-derived stems cells (ASC) have demonstrated promise across many areas of regenerative medicine, including bone tissue engineering. One major barrier to clinical translation and broad application of these therapeutics is current protocols which require laboratory cell culture and enzymatic digestion to produce stem cell rich product. More optimal methodologies would circumvent the current protocols, and instead, employ mechanically-based techniques to produce ASC rich stromal vascular fraction (SVF) intraoperatively for immediate clinical application. Therefore, the purpose of this study was to develop a clinically translatable technique for intra-operative harvest, isolation, and implantation of SVF with the primary aim of enhancing bone healing at irradiated fracture sites. Methods: Male Lewis rats (n=29) were divided into groups: Fracture (Fx), Radiation with Fracture (XRT), and Radiation with Fracture and SVF implantation (SVF). Experimental groups received 35Gy of radiation. All groups underwent mandibular osteotomy and external fixation. Inguinal fat pads were minced and serially processed using Tulip Sizing Transfers (2.4mm, 1.4mm, 1.2mm). Serial filtration (800 micron, 400 micron) and centrifugation was performed. The resultant oil and aqueous layers were discarded and the cell pellet was collected for immediate implantation at the osteotomy site. Animals were sacrificed on post-operative day 40. Gross pathology and MicroCT analysis were utilized to determine union rates and the quality of the bone formed at the osteotomy site. Biomechanical strength testing was performed until failure to evaluate yield and ultimate load of new bone at the osteotomy site. Results: Immediate implantation of SVF increased union rates compared to XRT alone (79% vs. 20%). Additionally, MicroCT analysis demonstrated high quality new bone formation in irradiated fractures treated with SVF compared to the control based on bone mineral density (666.2 ± 32.0 vs. 312.2 ± 51.7; p=0.000) and bone volume fraction (0.744 ± 0.072 vs. 0.350 ± 0.041; p=0.000). In fact, implantation of SVF into irradiated fracture sites resulted in bone quality similar to the bone formed at non-irradiated fracture sites, as there was no significant difference found between groups (BMD: 666.2 ± 32.0 vs. 710.3 ± 38.0; p=0.390, BVF: 0.744 ± 0.072 vs. 0.803 ± 0.04; p=0.300). Radiation significantly diminished the biomechanical properties of bone, including yield (23.6 ± 28.2 vs. 81.9 ± 31.3; p=0.002) and ultimate load (33.7 ± 30.9 vs. 87.3 ± 26.7; p=0.005). SVF implantation improved yield (52.0 ± 28.6 vs. 81.9 ± 31.3; p=0.161) and ultimate load (33.7 ± 30.9 vs. 87.3 ± 26.7; p=0.145) of the irradiated bone to the level of the non-irradiated control, as there was no significant difference between groups. Conclusions: Use of the stromal vascular fraction for bone tissue engineering demonstrates great potential, including applications in irradiated fracture healing. In this study, we developed a novel approach that eliminates laboratory dependent techniques and instead, utilizes mechanical methods that would enable intraoperative SVF harvest, isolation, and immediate implantation. While further studies are required to optimize this approach, the results of this study are incredibly promising for the long-awaited translation of cell-based therapeutics into the clinical arena.
The ability to examine bone vascularity using micro-computed tomography following vessel perfusion with Microfil® and to subsequently perform histologic bone analysis in the same specimen would provide an efficient method by which the vascular and cellular environment of bone can be examined simultaneously. The purpose of this report is to determine if the administration of Microfil precludes accurate histologic assessment of bone quality via osteocyte count and empty lacunae count. Sprague–Dawley rats (n = 6) underwent perfusion with Microfil. Left hemi-mandibles were harvested, decalcified, and underwent vascular analysis via micro-computed tomography prior to sectioning and staining with Gomori's trichrome. Quantitative histomorphometric evaluation was performed. Ninety-five percent confidence intervals (CIs) were used to determine statistical differences from an established set of controls (n = 12). Histologic analyses were successfully performed on specimens that had been perfused. Quantitative measures of bone cellularity of perfused versus control specimens revealed no statistical difference in osteocyte count per high-power field (95·33 versus 94·66; 95% CI: −7·64 to 6·30) or empty lacunae per high-power field (2·73 versus 1·89; 95% CI: −1·81 to 0·13). A statistical validation is reported that allows histologic analysis of cell counts in specimens which had been perfused with Microfil.
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.
AMERICAN SOCIETY OF PLASTIC SURGEONS PLASTIC & RECONSTRUCTIVE SURGERY PRS GLOBAL OPEN ASPS EDUCATION NETWORK AMERICAN SOCIETY OF PLASTIC SURGEONS PLASTIC & RECONSTRUCTIVE SURGERY PRS GLOBAL OPEN ASPS EDUCATION NETWORK
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.
