title: Targeting of ALK2, a Receptor for Bone Morphogenetic Proteins, Using the Cre/lox System to Enhance Osseous Regeneration by Adipose-Derived Stem Cells.
abstract: Access to readily available autogenous tissue that regenerates bone would greatly improve clinical care. We believe the osteogenic phenotype caused by mutations in ALK2 can be harnessed in adipose-derived stem cells (ASCs) to improve bone tissue engineering. We set out to demonstrate that ALK2 may serve as a novel target to (a) improve in vitro ASC osteogenic differentiation and (b) enhance in vivo bone regeneration and calvarial healing. Transgenic mice were designed using the Cre/lox system to express constitutively active ALK2 (caALK2) with ubiquitously inducible Cre expression after tamoxifen exposure. ASCs from caALK2+/- and caALK2-/-(control) mice were exposed to tamoxifen and assessed for pro-osteogenic gene expression, bone morphogenetic protein (BMP) signaling, and osteogenic differentiation. Next, ASCs collected from these transgenic mice were analyzed in vivo using a calvarial defect model and analyzed by micro-computed tomography (micro-CT) and histology. ASCs from caALK2+/-mice had increased BMP signaling as demonstrated by upregulation of pSmad 1/5. ASCs from caALK2+/-mice had enhanced bone signaling and osteogenic differentiation compared with caALK2-/-mice (n=4, p<.05). Transcription of pro-osteogenic genes at day 7 was significantly higher in ASCs from caALK2-overexpressing mice (Alp, Runx2, Ocn, Opn) (n=4, p<.05). Using micro-CT and histomorphometry, we found that bone formation was significantly higher in mice treated with caALK2-expressing ASCs in vivo. Using a novel transgenic mouse model, we show that expression of constitutively active ALK2 receptor results in significantly increased ASC osteogenic differentiation. Furthermore, we demonstrate that this increased ASC differentiation can be harnessed to improve calvarial healing.
authors.affiliation: Department of Surgery, University of Michigan Medical School, Ann Arbor, Michigan, USA; Division of Advanced Prosthodontics, Biomaterials, and Hospital Dentistry, University of California, Los Angeles, Los Angeles, California, USA; Department of Biologic and Materials Sciences, University of Michigan Dental School, Ann Arbor, Michigan, USA; Department of Surgery, Massachusetts General Hospital, Boston, Massachusetts, USA.
authors.forename: Jonathan R
authors.initials: JR
authors.lastname: Peterson
authors.affiliation: Department of Surgery, University of Michigan Medical School, Ann Arbor, Michigan, USA; Division of Advanced Prosthodontics, Biomaterials, and Hospital Dentistry, University of California, Los Angeles, Los Angeles, California, USA; Department of Biologic and Materials Sciences, University of Michigan Dental School, Ann Arbor, Michigan, USA; Department of Surgery, Massachusetts General Hospital, Boston, Massachusetts, USA.
authors.forename: Oluwatobi
authors.initials: O
authors.lastname: Eboda
authors.affiliation: Department of Surgery, University of Michigan Medical School, Ann Arbor, Michigan, USA; Division of Advanced Prosthodontics, Biomaterials, and Hospital Dentistry, University of California, Los Angeles, Los Angeles, California, USA; Department of Biologic and Materials Sciences, University of Michigan Dental School, Ann Arbor, Michigan, USA; Department of Surgery, Massachusetts General Hospital, Boston, Massachusetts, USA.
authors.forename: Shailesh
authors.initials: S
authors.lastname: Agarwal
authors.affiliation: Department of Surgery, University of Michigan Medical School, Ann Arbor, Michigan, USA; Division of Advanced Prosthodontics, Biomaterials, and Hospital Dentistry, University of California, Los Angeles, Los Angeles, California, USA; Department of Biologic and Materials Sciences, University of Michigan Dental School, Ann Arbor, Michigan, USA; Department of Surgery, Massachusetts General Hospital, Boston, Massachusetts, USA.
authors.forename: Kavitha
authors.initials: K
authors.lastname: Ranganathan
authors.affiliation: Department of Surgery, University of Michigan Medical School, Ann Arbor, Michigan, USA; Division of Advanced Prosthodontics, Biomaterials, and Hospital Dentistry, University of California, Los Angeles, Los Angeles, California, USA; Department of Biologic and Materials Sciences, University of Michigan Dental School, Ann Arbor, Michigan, USA; Department of Surgery, Massachusetts General Hospital, Boston, Massachusetts, USA.
authors.forename: Steven R
authors.initials: SR
authors.lastname: Buchman
authors.affiliation: Department of Surgery, University of Michigan Medical School, Ann Arbor, Michigan, USA; Division of Advanced Prosthodontics, Biomaterials, and Hospital Dentistry, University of California, Los Angeles, Los Angeles, California, USA; Department of Biologic and Materials Sciences, University of Michigan Dental School, Ann Arbor, Michigan, USA; Department of Surgery, Massachusetts General Hospital, Boston, Massachusetts, USA.
authors.forename: Min
authors.initials: M
authors.lastname: Lee
authors.affiliation: Department of Surgery, University of Michigan Medical School, Ann Arbor, Michigan, USA; Division of Advanced Prosthodontics, Biomaterials, and Hospital Dentistry, University of California, Los Angeles, Los Angeles, California, USA; Department of Biologic and Materials Sciences, University of Michigan Dental School, Ann Arbor, Michigan, USA; Department of Surgery, Massachusetts General Hospital, Boston, Massachusetts, USA.
authors.forename: Stewart C
authors.initials: SC
authors.lastname: Wang
authors.affiliation: Department of Surgery, University of Michigan Medical School, Ann Arbor, Michigan, USA; Division of Advanced Prosthodontics, Biomaterials, and Hospital Dentistry, University of California, Los Angeles, Los Angeles, California, USA; Department of Biologic and Materials Sciences, University of Michigan Dental School, Ann Arbor, Michigan, USA; Department of Surgery, Massachusetts General Hospital, Boston, Massachusetts, USA.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Mishina
authors.affiliation: Department of Surgery, University of Michigan Medical School, Ann Arbor, Michigan, USA; Division of Advanced Prosthodontics, Biomaterials, and Hospital Dentistry, University of California, Los Angeles, Los Angeles, California, USA; Department of Biologic and Materials Sciences, University of Michigan Dental School, Ann Arbor, Michigan, USA; Department of Surgery, Massachusetts General Hospital, Boston, Massachusetts, USA benlevimd@gmail.com.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Levi
mesh_terms: D030201:Activin Receptors, Type I / Q000235:genetics / Q000378:metabolism; D000273:Adipose Tissue; D000818:Animals; D019485:Bone Morphogenetic Proteins / Q000235:genetics / Q000378:metabolism; D001861:Bone Regeneration / Q000235:genetics*; D016326:Extracellular Matrix Proteins / Q000235:genetics / Q000378:metabolism; D018390:Gene Targeting; D019426:Integrases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D008249:Protein-Lysine 6-Oxidase / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics*; D033581:Stem Cell Transplantation; D013234:Stem Cells / Q000166:cytology / Q000201:enzymology
chemical_list: D019485:Bone Morphogenetic Proteins; D016326:Extracellular Matrix Proteins; C077112:Lox protein, mouse; D008249:Protein-Lysine 6-Oxidase; D030201:Activin Receptors, Type I; C498943:Acvr1 protein, mouse; C045073:Cre recombinase; D019426:Integrases
keywords: ALK2; Bone tissue engineering; Calvarial defect; Mesenchymal stem cells