Immunological tolerance to composite tissue allograft transplantation in a DLA-identical canine...
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METHODS: hASCs were isolated from human lipoaspirate (n5) and transfected with a Noggin shRNA construct. Knockdown was confirmed on a gene and protein level. Angiogenesis was assessed by tubule formation, and qRT-PCR. Cells were seeded on a scaffold with or without BMP-2 and implanted into a 4mm critical size calvarial defect. In vivo angiogenic signaling was assessed by in situ hybridization, immunofluoresence and immunohistochemistry. RESULTS: By ELISA, hASCs with noggin suppression secreted sig- nificantly higher amounts of VEGF protein (*p0.05). hASCs with noggin knockdown expressed higher levels of angionegic gene mark- ers by qRT-PCR (VE-cadherein, VEGFA, VEGFB and HIF1A), and displayed enhanced vascular tubule formation in vitro. This en- hanced angiogenesis was observed in vivo, as well, as calvarial defects seeded with noggin shRNA hASCs exhibited a significantly higher number of vessels than controls, by immunohistochemistry (*p0.05). Additionally, BMP-2 releasing scaffolds significantly en- hanced VEGF protein levels in hASCs with noggin knockdown. CONCLUSIONS: When suppressing noggin, and supplementing BMP-2, hASCs demonstrate significant increases in angiogenesis both in vitro and in vivo. By creating a hASC population with the robust ability to undergo angiogenesis, we can optimize the the po- tential utility of cell-based therapies in tissue regeneration. Fibromodulin, a TGF-beta modulator, inhibits scar formation and increases tensile strength Zhong Zheng PhD, Janette N Zara MD, MS, Virginia T Nguyen BSc, Aaron W James MD, Yi Liu DDS DNB, Khoi Le BS, Sandra Yoon BSc, Harnek Singh BS, Chia Soo MD, FACS University of California-Los Angeles, Los Angeles, CA INTRODUCTION: Scar formation is a common sequelae of adult cutaneous injury. Fibromodulin (FMOD), a proteoglycan and mod- ulator of TGF-B signaling, has been previously shown to be impor- tant in fetal scarless wound healing. In this study, we sought to define the extent to which fibromodulin would inhibit scar formation and affect tensile strength in adult rat and pig skin. METHODS: One cm primary closure wounds were created in 3 month old Sprague Dawley rats, and 1.5 cm primary closure wounds were created in Yorkshire pigs. Rat wounds were injected with 100 uL recombinant FMOD or PBS control, while pig wounds were in- jected with 200 uL recombinant FMOD or PBS control. Rat wounds were harvested at 2 weeks post-operation, while pig wounds were harvested at 2, 4, and 8 weeks. Wounds were analyzed by histology and INSTRON tensiometry. RESULTS: Fibromodulin reduced scar size by approximately 50% in both rat and pig models based on quantification of scar area in high power histological images. Moreover, fibromodulin signifi- cantly increased wound tensile strength in both animal models as measured by tensiometry. CONCLUSIONS: Fibromodulin not only reduces scar-size across multiple species, but also increases wound breaking strength. Thus, fibromodulin may be of benefit as both a novel antifibrotic agent, as well as one to reduce the likelihood of wound dehiscence. Erasing Langer’s lines: Establishing a molecular basis for the fibrotic response to tension Victor W Wong MD, Satoshi Akaishi MD, PhD, Kristine C Rustad, Michael Sorkin MD, Jason P Glotzbach MD, Michael Januszyk MD, Michael T Longaker MD, MBA, FACS, Geoffrey C Gurtner MD, FACS Stanford University, Stanford, CA INTRODUCTION: Skin tension lines have directed surgical plan- ning since first described by Langer exactly 150 years ago. Al- though the causal relationship between tension and fibrosis re- mains poorly understood, more recent studies have implicated a pathogenic role for dysregulated cell-matrix interactions. We hy- pothesize that fibroblast-specific focal adhesion kinase (FAK), a key component of intracellular mechanotransduction, critically regulates the fibroproliferative response to tension following in- jury. METHODS: Mechanical activation of FAK and downstream Akt, p38, JNK, and ERK signaling were assessed by immunoblotting. Strain and/or PDGF were used to activate FAK in vitro, and small molecule inhibitors of FAK, Akt, p38, JNK, and ERK were utilized to define pathways linking mechanical force with collagen and chemokine production. Wildtype mice were treated with FAK inhib- itor during application of a mechanical load model and scar forma- tion was followed for up to 21 days. RESULTS: Load-induced collagen production and MCP-1 secre- tion were tightly linked to fibroblast FAK-ERK signaling. Strain and PDGF synergistically induced MCP-1 secretion via FAK in vitro. In vivo, FAK inhibitor treatment significantly abrogated fibrosis and scar formation by almost 40% (p0.05) at three weeks. Scar atten- uation was associated with blunted ERK and MCP-1 signaling and decreased recruitment of macrophages. CONCLUSIONS: Fibroblast-driven mechanotransduction path- ways are critically regulated by FAK-ERK signaling during scar formation. Mechanical tension converts the fibroblast into an inflammatory cell that recruits other pro-fibrotic cells (macro- phages) via MCP-1 signaling, perpetuating a vicious cycle of fi- broproliferation. Offloading of wound tension can be achieved with FAK inhibition, in effect “erasing” the pathologic outcome of crossing Langer’s lines. Immunological tolerance to composite tissue allograft transplantation in a DLA-identical canine model Jeff Chang MD, MS, Billanna Hwang BS, Scott Graves PhD, Tiffany Miwongtum BS, Richard A Nash MD, Rainer Storb MD, David W Mathes MD, FACS University of Washington, Seattle, WA and Fred Hutchinson Cancer Research Center, Seattle, WA INTRODUCTION: The successful transplantation of composite tis- sue allografts (CTA) such as hand or face is a clinical reality. However, survival of these transplants is dependent on chronic immunosup- pression. Our hypothesis was that tolerance to the CTA in a large S99 Vol. 213, No. 3S, September 2011 Surgical Forum Abstracts
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Transcript of Immunological tolerance to composite tissue allograft transplantation in a DLA-identical canine...
METHODS: hASCs were isolated from human lipoaspirate (n�5)and transfected with a Noggin shRNA construct. Knockdown wasconfirmed on a gene and protein level. Angiogenesis was assessed bytubule formation, and qRT-PCR. Cells were seeded on a scaffoldwith or without BMP-2 and implanted into a 4mm critical sizecalvarial defect. In vivo angiogenic signaling was assessed by in situhybridization, immunofluoresence and immunohistochemistry.
RESULTS: By ELISA, hASCs with noggin suppression secreted sig-nificantly higher amounts of VEGF protein (*p�0.05). hASCs withnoggin knockdown expressed higher levels of angionegic gene mark-ers by qRT-PCR (VE-cadherein, VEGFA, VEGFB and HIF1A), anddisplayed enhanced vascular tubule formation in vitro. This en-hanced angiogenesis was observed in vivo, as well, as calvarial defectsseeded with noggin shRNA hASCs exhibited a significantly highernumber of vessels than controls, by immunohistochemistry(*p�0.05). Additionally, BMP-2 releasing scaffolds significantly en-hanced VEGF protein levels in hASCs with noggin knockdown.
CONCLUSIONS: When suppressing noggin, and supplementingBMP-2, hASCs demonstrate significant increases in angiogenesisboth in vitro and in vivo. By creating a hASC population with therobust ability to undergo angiogenesis, we can optimize the the po-tential utility of cell-based therapies in tissue regeneration.
Fibromodulin, a TGF-beta modulator, inhibits scarformation and increases tensile strengthZhong Zheng PhD, Janette N Zara MD, MS, Virginia T Nguyen BSc,Aaron W James MD, Yi Liu DDS DNB, Khoi Le BS, Sandra Yoon BSc,Harnek Singh BS, Chia Soo MD, FACSUniversity of California-Los Angeles, Los Angeles, CA
INTRODUCTION: Scar formation is a common sequelae of adultcutaneous injury. Fibromodulin (FMOD), a proteoglycan and mod-ulator of TGF-B signaling, has been previously shown to be impor-tant in fetal scarless wound healing. In this study, we sought to definethe extent to which fibromodulin would inhibit scar formation andaffect tensile strength in adult rat and pig skin.
METHODS: One cm primary closure wounds were created in 3month old Sprague Dawley rats, and 1.5 cm primary closure woundswere created in Yorkshire pigs. Rat wounds were injected with 100 uLrecombinant FMOD or PBS control, while pig wounds were in-jected with 200 uL recombinant FMOD or PBS control. Rat woundswere harvested at 2 weeks post-operation, while pig wounds wereharvested at 2, 4, and 8 weeks. Wounds were analyzed by histologyand INSTRON tensiometry.
RESULTS: Fibromodulin reduced scar size by approximately 50%in both rat and pig models based on quantification of scar area inhigh power histological images. Moreover, fibromodulin signifi-cantly increased wound tensile strength in both animal models asmeasured by tensiometry.
CONCLUSIONS: Fibromodulin not only reduces scar-size acrossmultiple species, but also increases wound breaking strength. Thus,fibromodulin may be of benefit as both a novel antifibrotic agent, aswell as one to reduce the likelihood of wound dehiscence.
Erasing Langer’s lines: Establishing a molecular basis forthe fibrotic response to tensionVictor W Wong MD, Satoshi Akaishi MD, PhD, Kristine C Rustad,Michael Sorkin MD, Jason P Glotzbach MD, Michael Januszyk MD,Michael T Longaker MD, MBA, FACS,Geoffrey C Gurtner MD, FACSStanford University, Stanford, CA
INTRODUCTION: Skin tension lines have directed surgical plan-ning since first described by Langer exactly 150 years ago. Al-though the causal relationship between tension and fibrosis re-mains poorly understood, more recent studies have implicated apathogenic role for dysregulated cell-matrix interactions. We hy-pothesize that fibroblast-specific focal adhesion kinase (FAK), akey component of intracellular mechanotransduction, criticallyregulates the fibroproliferative response to tension following in-jury.
METHODS: Mechanical activation of FAK and downstream Akt,p38, JNK, and ERK signaling were assessed by immunoblotting.Strain and/or PDGF were used to activate FAK in vitro, and smallmolecule inhibitors of FAK, Akt, p38, JNK, and ERK were utilizedto define pathways linking mechanical force with collagen andchemokine production. Wildtype mice were treated with FAK inhib-itor during application of a mechanical load model and scar forma-tion was followed for up to 21 days.
RESULTS: Load-induced collagen production and MCP-1 secre-tion were tightly linked to fibroblast FAK-ERK signaling. Strain andPDGF synergistically induced MCP-1 secretion via FAK in vitro. Invivo, FAK inhibitor treatment significantly abrogated fibrosis andscar formation by almost 40% (p�0.05) at three weeks. Scar atten-uation was associated with blunted ERK and MCP-1 signaling anddecreased recruitment of macrophages.
CONCLUSIONS: Fibroblast-driven mechanotransduction path-ways are critically regulated by FAK-ERK signaling during scarformation. Mechanical tension converts the fibroblast into aninflammatory cell that recruits other pro-fibrotic cells (macro-phages) via MCP-1 signaling, perpetuating a vicious cycle of fi-broproliferation. Offloading of wound tension can be achievedwith FAK inhibition, in effect “erasing” the pathologic outcomeof crossing Langer’s lines.
Immunological tolerance to composite tissue allografttransplantation in a DLA-identical canine modelJeff Chang MD, MS, Billanna Hwang BS, Scott Graves PhD,Tiffany Miwongtum BS, Richard A Nash MD, Rainer Storb MD,David W Mathes MD, FACSUniversity of Washington, Seattle, WA and Fred HutchinsonCancer Research Center, Seattle, WA
INTRODUCTION: The successful transplantation of composite tis-sue allografts (CTA) such as hand or face is a clinical reality. However,survival of these transplants is dependent on chronic immunosup-pression. Our hypothesis was that tolerance to the CTA in a large
S99Vol. 213, No. 3S, September 2011 Surgical Forum Abstracts
animal model could be induced via the simultaneous transplantationof hematopoietic stem cells (HSC) and CTA.
METHODS: 4 dog transplants were performed across a DLA matched,minor mismatch barrier. Recipients received 200 cGy of radiation andunderwent a CTA transplant (myocutaneous rectus flap) with intraop-erative injection of HSC followed by a short course of immunosuppres-sion. 2 dogs underwent the same protocol without any HSC infusion.All were followed for donor cell chimerism. The CTAs were evaluatedwith biopsies and followed clinically. T-regulatory cells were assessed byFOXP3 expression from the transplanted skin and muscle. Tolerantanimals underwent donor and third-party skin grafts.
RESULTS: All animals receiving CTA and HSC demonstrate long-term tolerance to their CTA (�1 year) and accepted secondary donorskin grafts independent of donor chimerism. 3 dogs had stable donorchimerism while the fourth dog lost chimerism at 10 weeks post-transplant. The 2 dogs transplanted without HSC rejected theirtransplants after the cessation of immunosuppression. In the CTA oftolerant dogs, FOXP3 expression was elevated.
CONCLUSIONS: The simultaneous transplant of HSC and CTAleads to tolerance. Tolerance induction appears to be dependent onthe administration of HSC but not its long-term engraftment.T-regulatory cells appear to play a role in this tolerance.
S100 Surgical Forum Abstracts J Am Coll Surg
