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Heterochronic Parabiosis Rejuvenates Aged Bones
PLASTIC AND MAXILLOFACIAL SURGERY I Recent studies have found that diabetics and the elderly are deficient in SDF-1, suggesting a possible mechanism for their cardiovascular complications. Utilizing newly developed murine models, we studied the effect of global (gKO) and endothelial cell-specific SDF-1 knockout (eKO) during wound healing and neovascularization.
Diminished Recruitment of Resident Skeletal Progenitor Cells in Diabetic Fracture Healing Ruth Tevlin, MB, BAO, BCh, MRCSI, Elly Seo, MA, Adrian McArdle, MB, BCh, MRCSI, Owen Marecic, BA, Taylor Wearda, BS, Kshemendra Senarath-Yapa, MBBChir, MRCS, Elizabeth R Zielins, MD, Charles K Chan, PhD, Irving L Weissman, MD, Michael T Longaker, MD, MBA, FACS Stanford University, Stanford, CA
METHODS: Humanized excisional wounds were created on the dorsum of gKO, eKO and wild type (WT) mice. An ischemic skin flap was created on the dorsum of eKO and WT mice. Wounds and flaps were photographed and assessed at regular intervals. Tissue was harvested for histology and quantitative reverse transcription polymerase chain reaction (qRT-PCR). The effects of SDF-1 on fibroblast behavior were assessed in vitro.
INTRODUCTION: Diabetes mellitus is associated with an increased incidence of non-union, delayed union and mal-union of fractures. The mechanism(s) leading to impaired fracture healing remain to be elucidated. Recruitment of tissue stem and progenitor cells is vital to restoring injured bones. Our laboratory has recently identified a clonal precursor of bone, cartilage and stroma (BCSP) that generates mature skeletal tissue through a series of functionally characterized cellular intermediates. We hypothesize that impaired recruitment of this BCSP and its downstream progenitors contributes to poor fracture healing in diabetes.
RESULTS: The eKO and gKO groups demonstrated similarly impaired wound healing (*p¼0.006). eKO wounds had reduced transcription, shown by qRT-PCR, and protein expression, on immunofluorescent staining, of SDF-1 (*p¼0.002; *p¼0.008), vascular endothelial growth factor (VEGF) (*p<0.05; *p<0.05) and fibroblast growth factor-2 (FGF-2) (*p<0.05; *p¼0.006) with decreased vascular density (*p<0.05). eKO mice also had reduced ischemic flap survival (39% vs 72%; *p¼0.008) associated with decreased vascular density (*p¼0.003). SDF-1 increased fibroblast proliferation (*p<0.001) and migration (*p¼0.002) in vitro.
METHODS: Closed, bi-cortical femoral fractures were created in age- and sex-matched diabetic (db/db) and wild-type mice. Fracture healing was assessed by radiography and mechanical testing. Fluorescence-activated cell sorting (FACS) was performed on fracture callus cells at multiple time-points post-fracture to determine the presence and quantity of activated BCSP [CD45(-) Tie2(-) CD51(+)CD105(+)Thy(-)6C3(-)] in diabetic and control femora. Microarray analysis was performed to identify differential gene expression in activated BCSP between the 2 groups.
CONCLUSIONS: Endothelial cell SDF-1 (eSDF-1) represents an important therapeutic target, as it plays a pivotal role in the vascular response, regulating the expression of cytokines responsible for neovascularization, modulating fibroblast behavior and increasing vascular density in ischemic tissue.
RESULTS: In vivo, diabetic mice displayed delayed callus development on radiographic assessment. Diabetic femora were weaker on mechanical assessment than the wild-type control (*p<0.05). FACS analysis demonstrated diminished recruitment of skeletal progenitor cells to the fracture callus of diabetic compared to wild-type mice. By microarray analysis of normal vs diabetic BCSP, we identified multiple genes that were differentially expressed in diabetic callus compared to wild-type control.
Heterochronic Parabiosis Rejuvenates Aged Bones Adrian McArdle, MB, BCh, MRCSI, Ruth Tevlin, MB, BAO, BCh, MRCSI, Owen Marecic, BA, Taylor Wearda, BS, Kshemendra Senarath-Yapa, MBBChir, MRCS, Graham G Walmsley, BA, Michael P Snyder, PhD, Irving L Weissman, MD, Charles K Chan, PhD, Michael T Longaker, MD, MBA, FACS Stanford University, Stanford, CA
CONCLUSIONS: Fracture healing is impaired in our murine model of diabetes. Our findings illustrate a diminished recruitment of bone, cartilage and stromal progenitor cells in diabetic fracture healing.
INTRODUCTION: Aging results in a decline in the regenerative capacity of bone and is associated with increases in morbidity and mortality. We evaluated the role of skeletal stem cells and the niche microenvironment in promoting the aged bone phenotype.
Endothelial Cell Derived Stromal-Derived Factor-1 (SDF-1) Regulates Neovascularization and Fibroblast Physiology in Response to Ischemia Zeshaan N Maan, MBBS, MS, MRCS*, Alexander J Whittam, BA, Dominik Duscher, MD, Michael S Hu, MD, MPH, MS, Robert C Rennert, MD, Lauren H Fischer, MD, Graham G Walmsley, BA, Arnetha J Whitmore, BA, Michael T Longaker, MD, MBA, FACS, Geoffrey C Gurtner, MD, FACS Stanford University School of Medicine, Stanford, CA
METHODS: Differences in skeletal regeneration were compared in 6-week old (young) and 2-year old (aged) mice using a femoral fracture model. Heterochronic parabiosis between young and aged mice allowed for evaluation of the role of the skeletal niche in promoting an aged bone phenotype during normal homeostasis and in response to injury. The osteogenic potential of purified populations of skeletal stem cells from young, aged, and heterochronic paired mice was also assessed in vitro and in vivo. Transcriptional and metabolomic screens were used to identify differentially regulated pathways associated with skeletal aging.
INTRODUCTION: An impaired neovascular response underlies the significant cardiovascular complications seen in elderly and diabetic patients. SDF-1, a cytokine primarily known for its chemotactic function, is also thought to play a role in neovascularization.
ª 2014 by the American College of Surgeons Published by Elsevier Inc.
S82
http://dx.doi.org/10.1016/j.jamcollsurg.2014.07.193 ISSN 1072-7515/14
Vol. 219, No. 3S, September 2014
RESULTS: Aging is associated with cell intrinsic changes in the skeletal stem cell population and a decline in their regenerative potential. Heterochronic parabiosis rejuvenates aged bones by altering osteoclastogenesis. Transcriptional and metabolomic changes are associated with the age-related functional decline of skeletal stem cells. Manipulation of BMP and Wnt signaling pathways can rejuvenate aged bone by stimulating their innate regenerative capacity. CONCLUSIONS: Skeletal aging is a complex process, associated with cell intrinsic and cell extrinsic changes in the skeletal stem cell and the stem cell niche. We identified multiple differentially regulated transcriptional and metabolomic differences in aged skeletal stem cells and found that through the manipulation of BMP and Wnt signaling pathways, we were able to improve bone healing parameters in aged mice. Impaired Angiogenesis: A Critical Contributor to Problematic Fracture Healing in Diabetes Ruth Tevlin, MB, BAO, BCh, MRCSI, Adrian Mc Ardle, MB, BAO, BCh, MRCSI, Kshemendra Senarath-Yapa, MB, BChir, MRCS, Melanie Rodrigues, PhD, Zeshaan N Maan, MBBS, MS, MRCS, Shuli Li, MD, PhD, Charles K Chan, PhD, John Brunski, PhD, Geoffrey C Gurtner, MD, FACS Stanford University, Stanford, CA INTRODUCTION: Diabetes mellitus is associated with impaired fracture healing. The underlying mechanisms remain to be elucidated. We hypothesize that impaired angiogenesis contributes to poor fracture healing in diabetes. METHODS: Differences in fracture healing were compared in ageand sex-matched diabetic (db/db) and wild type mice using a bicortical femoral fracture model. Fracture healing was assessed by radiography and mechanical testing. Femora and sera were harvested weekly post-fracture for histologic, transcriptional, and protein analysis. RESULTS: Diabetic mice showed delayed callus development in vivo. Diabetic femora were weaker than wild-type femora on mechanical testing (*p<0.05). Diabetic fracture calluses demonstrated significantly less staining for endothelial markers using immunohistochemistry when comparing diabetic callus to control at 4 weeks post-fracture (**p<0.005). Throughout fracture healing, circulating levels of vascular endothelial growth factor (VEGF) were significantly higher in diabetic mice compared to wild type (**p<0.005). Protein analysis by western blot showed increased VEGF detected in the diabetic callus in the early phase of fracture healing, however levels of the VEGFR2 receptor were markedly lower than control (**p<0.005). Quantitative polymerase chain reaction showed no significant difference in VEGFR2 transcription between the two groups. In addition, angiogenesis immunoassay showed significantly reduced SDF-1 levels (**p<0.005) in the early stage of diabetic fracture healing compared to control. CONCLUSIONS: These findings illustrate a ligand-independent impairment of VEGF-VEGFR2 signaling, in the presence of inadequate angiogenesis in diabetic fracture healing. Furthermore,
Surgical Forum Abstracts
S83
reduction of SDF-1 at the early stage of diabetic fracture healing may result in poor recruitment of endothelial progenitor cells to the fracture. Extended Immunosupressive Therapy in a NonMyeloablative Conditioning Regimen: Achieving Tolerance in Vascularized Composite Allotransplantation (VCA) while Reducing Risk of Graft-vs-Host Disease (GVHD) Bruce J Swearingen, MD, Jeff Chang, MD, Tiffany Butts, BS, Scott Graves, PhD, Rainer Storb, MD, David W Mathes, MD, FACS Fred Hutchinson Cancer Research Center, Seattle, WA INTRODUCTION: Our hypothesis was that our non-myeloablative hematopoietic stem cell transplant protocol could be used to induce tolerance to a recipient VCA without the need for persistent donor cell chimerism. To mimic the clinical setting, we utilized a tapered cessation of immunosuppression. Our protocol was further modified by decreasing the amount of total body irradiation (TBI) in a second cohort. METHODS: 5 Haploidentical canine recipients received a nonmyeloablative conditioning regimen of 350cGy TBI, mobilized donor stem cells (PBMC) and VCA transplantation followed by a short course of immunosuppression with a tapered dose reduction. 3 dogs were then transplanted after a decreased irradiation dose of 300cGy. Peripheral blood chimerism was evaluated by polymerase chain reaction techniques weekly. VCA rejection was followed clinically and confirmed histologically after biopsy. RESULTS: All 8 tolerated the conditioning regimen. 3 of 5 dogs in the 350cGy group were followed long term. All 3 of developed full donor chimerism with 2 developing GVHD upon completion of immunosuppression. In the 300cGy cohort, 1 dog had transient chimerism <3 weeks and developed an acute rejection episode. One dog had poor stem cell engraftment but has not shown signs of VCA rejection while on immunosuppression. The last dog developed full donor chimerism without signs of GVHD while on immunosuppression. CONCLUSIONS: We demonstrated an extended immunosuppressive regimen led to tolerance of the VCA but at an increased risk of GVHD due to full donor chimerism at the higher irradiation dose. Decreasing the TBI led to variable engraftment and VCA tolerance outcomes despite the extended immunosuppressive regimen. Epidermal Aquaporin-3 Expression Is Increased in the Burn Wound after a Cutaneous Thermal Injury Jamil Matthews, MD, Raul Sebastian, MD, Parker Fillmore, MD, Leigh Ann Price, MD, Ramana Sidhaye, MD, Stephen Milner, DDS, MD, FACS Johns Hopkins University School of Medicine, Baltimore, MD INTRODUCTION: Aquaporins (AQP) are transmembrane proteins that transport water and small solutes across cell membranes. In the skin, AQPs play a role in skin hydration, cell proliferation, migration, immunity, and wound healing. However, the expression of
Diminished Recruitment of Resident Skeletal Progenitor Cells in Diabetic Fracture Healing Ruth Tevlin, MB, BAO, BCh, MRCSI, Elly Seo, MA, Adrian McArdle, MB, BCh, MRCSI, Owen Marecic, BA, Taylor Wearda, BS, Kshemendra Senarath-Yapa, MBBChir, MRCS, Elizabeth R Zielins, MD, Charles K Chan, PhD, Irving L Weissman, MD, Michael T Longaker, MD, MBA, FACS Stanford University, Stanford, CA
METHODS: Humanized excisional wounds were created on the dorsum of gKO, eKO and wild type (WT) mice. An ischemic skin flap was created on the dorsum of eKO and WT mice. Wounds and flaps were photographed and assessed at regular intervals. Tissue was harvested for histology and quantitative reverse transcription polymerase chain reaction (qRT-PCR). The effects of SDF-1 on fibroblast behavior were assessed in vitro.
INTRODUCTION: Diabetes mellitus is associated with an increased incidence of non-union, delayed union and mal-union of fractures. The mechanism(s) leading to impaired fracture healing remain to be elucidated. Recruitment of tissue stem and progenitor cells is vital to restoring injured bones. Our laboratory has recently identified a clonal precursor of bone, cartilage and stroma (BCSP) that generates mature skeletal tissue through a series of functionally characterized cellular intermediates. We hypothesize that impaired recruitment of this BCSP and its downstream progenitors contributes to poor fracture healing in diabetes.
RESULTS: The eKO and gKO groups demonstrated similarly impaired wound healing (*p¼0.006). eKO wounds had reduced transcription, shown by qRT-PCR, and protein expression, on immunofluorescent staining, of SDF-1 (*p¼0.002; *p¼0.008), vascular endothelial growth factor (VEGF) (*p<0.05; *p<0.05) and fibroblast growth factor-2 (FGF-2) (*p<0.05; *p¼0.006) with decreased vascular density (*p<0.05). eKO mice also had reduced ischemic flap survival (39% vs 72%; *p¼0.008) associated with decreased vascular density (*p¼0.003). SDF-1 increased fibroblast proliferation (*p<0.001) and migration (*p¼0.002) in vitro.
METHODS: Closed, bi-cortical femoral fractures were created in age- and sex-matched diabetic (db/db) and wild-type mice. Fracture healing was assessed by radiography and mechanical testing. Fluorescence-activated cell sorting (FACS) was performed on fracture callus cells at multiple time-points post-fracture to determine the presence and quantity of activated BCSP [CD45(-) Tie2(-) CD51(+)CD105(+)Thy(-)6C3(-)] in diabetic and control femora. Microarray analysis was performed to identify differential gene expression in activated BCSP between the 2 groups.
CONCLUSIONS: Endothelial cell SDF-1 (eSDF-1) represents an important therapeutic target, as it plays a pivotal role in the vascular response, regulating the expression of cytokines responsible for neovascularization, modulating fibroblast behavior and increasing vascular density in ischemic tissue.
RESULTS: In vivo, diabetic mice displayed delayed callus development on radiographic assessment. Diabetic femora were weaker on mechanical assessment than the wild-type control (*p<0.05). FACS analysis demonstrated diminished recruitment of skeletal progenitor cells to the fracture callus of diabetic compared to wild-type mice. By microarray analysis of normal vs diabetic BCSP, we identified multiple genes that were differentially expressed in diabetic callus compared to wild-type control.
Heterochronic Parabiosis Rejuvenates Aged Bones Adrian McArdle, MB, BCh, MRCSI, Ruth Tevlin, MB, BAO, BCh, MRCSI, Owen Marecic, BA, Taylor Wearda, BS, Kshemendra Senarath-Yapa, MBBChir, MRCS, Graham G Walmsley, BA, Michael P Snyder, PhD, Irving L Weissman, MD, Charles K Chan, PhD, Michael T Longaker, MD, MBA, FACS Stanford University, Stanford, CA
CONCLUSIONS: Fracture healing is impaired in our murine model of diabetes. Our findings illustrate a diminished recruitment of bone, cartilage and stromal progenitor cells in diabetic fracture healing.
INTRODUCTION: Aging results in a decline in the regenerative capacity of bone and is associated with increases in morbidity and mortality. We evaluated the role of skeletal stem cells and the niche microenvironment in promoting the aged bone phenotype.
Endothelial Cell Derived Stromal-Derived Factor-1 (SDF-1) Regulates Neovascularization and Fibroblast Physiology in Response to Ischemia Zeshaan N Maan, MBBS, MS, MRCS*, Alexander J Whittam, BA, Dominik Duscher, MD, Michael S Hu, MD, MPH, MS, Robert C Rennert, MD, Lauren H Fischer, MD, Graham G Walmsley, BA, Arnetha J Whitmore, BA, Michael T Longaker, MD, MBA, FACS, Geoffrey C Gurtner, MD, FACS Stanford University School of Medicine, Stanford, CA
METHODS: Differences in skeletal regeneration were compared in 6-week old (young) and 2-year old (aged) mice using a femoral fracture model. Heterochronic parabiosis between young and aged mice allowed for evaluation of the role of the skeletal niche in promoting an aged bone phenotype during normal homeostasis and in response to injury. The osteogenic potential of purified populations of skeletal stem cells from young, aged, and heterochronic paired mice was also assessed in vitro and in vivo. Transcriptional and metabolomic screens were used to identify differentially regulated pathways associated with skeletal aging.
INTRODUCTION: An impaired neovascular response underlies the significant cardiovascular complications seen in elderly and diabetic patients. SDF-1, a cytokine primarily known for its chemotactic function, is also thought to play a role in neovascularization.
ª 2014 by the American College of Surgeons Published by Elsevier Inc.
S82
http://dx.doi.org/10.1016/j.jamcollsurg.2014.07.193 ISSN 1072-7515/14
Vol. 219, No. 3S, September 2014
RESULTS: Aging is associated with cell intrinsic changes in the skeletal stem cell population and a decline in their regenerative potential. Heterochronic parabiosis rejuvenates aged bones by altering osteoclastogenesis. Transcriptional and metabolomic changes are associated with the age-related functional decline of skeletal stem cells. Manipulation of BMP and Wnt signaling pathways can rejuvenate aged bone by stimulating their innate regenerative capacity. CONCLUSIONS: Skeletal aging is a complex process, associated with cell intrinsic and cell extrinsic changes in the skeletal stem cell and the stem cell niche. We identified multiple differentially regulated transcriptional and metabolomic differences in aged skeletal stem cells and found that through the manipulation of BMP and Wnt signaling pathways, we were able to improve bone healing parameters in aged mice. Impaired Angiogenesis: A Critical Contributor to Problematic Fracture Healing in Diabetes Ruth Tevlin, MB, BAO, BCh, MRCSI, Adrian Mc Ardle, MB, BAO, BCh, MRCSI, Kshemendra Senarath-Yapa, MB, BChir, MRCS, Melanie Rodrigues, PhD, Zeshaan N Maan, MBBS, MS, MRCS, Shuli Li, MD, PhD, Charles K Chan, PhD, John Brunski, PhD, Geoffrey C Gurtner, MD, FACS Stanford University, Stanford, CA INTRODUCTION: Diabetes mellitus is associated with impaired fracture healing. The underlying mechanisms remain to be elucidated. We hypothesize that impaired angiogenesis contributes to poor fracture healing in diabetes. METHODS: Differences in fracture healing were compared in ageand sex-matched diabetic (db/db) and wild type mice using a bicortical femoral fracture model. Fracture healing was assessed by radiography and mechanical testing. Femora and sera were harvested weekly post-fracture for histologic, transcriptional, and protein analysis. RESULTS: Diabetic mice showed delayed callus development in vivo. Diabetic femora were weaker than wild-type femora on mechanical testing (*p<0.05). Diabetic fracture calluses demonstrated significantly less staining for endothelial markers using immunohistochemistry when comparing diabetic callus to control at 4 weeks post-fracture (**p<0.005). Throughout fracture healing, circulating levels of vascular endothelial growth factor (VEGF) were significantly higher in diabetic mice compared to wild type (**p<0.005). Protein analysis by western blot showed increased VEGF detected in the diabetic callus in the early phase of fracture healing, however levels of the VEGFR2 receptor were markedly lower than control (**p<0.005). Quantitative polymerase chain reaction showed no significant difference in VEGFR2 transcription between the two groups. In addition, angiogenesis immunoassay showed significantly reduced SDF-1 levels (**p<0.005) in the early stage of diabetic fracture healing compared to control. CONCLUSIONS: These findings illustrate a ligand-independent impairment of VEGF-VEGFR2 signaling, in the presence of inadequate angiogenesis in diabetic fracture healing. Furthermore,
Surgical Forum Abstracts
S83
reduction of SDF-1 at the early stage of diabetic fracture healing may result in poor recruitment of endothelial progenitor cells to the fracture. Extended Immunosupressive Therapy in a NonMyeloablative Conditioning Regimen: Achieving Tolerance in Vascularized Composite Allotransplantation (VCA) while Reducing Risk of Graft-vs-Host Disease (GVHD) Bruce J Swearingen, MD, Jeff Chang, MD, Tiffany Butts, BS, Scott Graves, PhD, Rainer Storb, MD, David W Mathes, MD, FACS Fred Hutchinson Cancer Research Center, Seattle, WA INTRODUCTION: Our hypothesis was that our non-myeloablative hematopoietic stem cell transplant protocol could be used to induce tolerance to a recipient VCA without the need for persistent donor cell chimerism. To mimic the clinical setting, we utilized a tapered cessation of immunosuppression. Our protocol was further modified by decreasing the amount of total body irradiation (TBI) in a second cohort. METHODS: 5 Haploidentical canine recipients received a nonmyeloablative conditioning regimen of 350cGy TBI, mobilized donor stem cells (PBMC) and VCA transplantation followed by a short course of immunosuppression with a tapered dose reduction. 3 dogs were then transplanted after a decreased irradiation dose of 300cGy. Peripheral blood chimerism was evaluated by polymerase chain reaction techniques weekly. VCA rejection was followed clinically and confirmed histologically after biopsy. RESULTS: All 8 tolerated the conditioning regimen. 3 of 5 dogs in the 350cGy group were followed long term. All 3 of developed full donor chimerism with 2 developing GVHD upon completion of immunosuppression. In the 300cGy cohort, 1 dog had transient chimerism <3 weeks and developed an acute rejection episode. One dog had poor stem cell engraftment but has not shown signs of VCA rejection while on immunosuppression. The last dog developed full donor chimerism without signs of GVHD while on immunosuppression. CONCLUSIONS: We demonstrated an extended immunosuppressive regimen led to tolerance of the VCA but at an increased risk of GVHD due to full donor chimerism at the higher irradiation dose. Decreasing the TBI led to variable engraftment and VCA tolerance outcomes despite the extended immunosuppressive regimen. Epidermal Aquaporin-3 Expression Is Increased in the Burn Wound after a Cutaneous Thermal Injury Jamil Matthews, MD, Raul Sebastian, MD, Parker Fillmore, MD, Leigh Ann Price, MD, Ramana Sidhaye, MD, Stephen Milner, DDS, MD, FACS Johns Hopkins University School of Medicine, Baltimore, MD INTRODUCTION: Aquaporins (AQP) are transmembrane proteins that transport water and small solutes across cell membranes. In the skin, AQPs play a role in skin hydration, cell proliferation, migration, immunity, and wound healing. However, the expression of