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  1. Article: Transient brown adipocyte-like cells derive from peripheral nerve progenitors in response to bone morphogenetic protein 2.

    Salisbury, Elizabeth A / Lazard, Zawaunyka W / Ubogu, Eroboghene E / Davis, Alan R / Olmsted-Davis, Elizabeth A

    Stem cells translational medicine

    2012  Volume 1, Issue 12, Page(s) 874–885

    Abstract: Perineurial-associated brown adipocyte-like cells were rapidly generated during bone morphogenetic protein 2 (BMP2)-induced sciatic nerve remodeling in the mouse. Two days after intramuscular injection of transduced mouse fibroblast cells expressing BMP2 ...

    Abstract Perineurial-associated brown adipocyte-like cells were rapidly generated during bone morphogenetic protein 2 (BMP2)-induced sciatic nerve remodeling in the mouse. Two days after intramuscular injection of transduced mouse fibroblast cells expressing BMP2 into wild-type mice, there was replication of beta-3 adrenergic receptor(+) (ADRB3(+)) cells within the sciatic nerve perineurium. Fluorescence-activated cell sorting and analysis of cells isolated from these nerves confirmed ADRB3(+) cell expansion and their expression of the neural migration marker HNK1. Similar analysis performed 4 days after BMP2 delivery revealed a significant decrease in ADRB3(+) cells from isolated sciatic nerves, with their concurrent appearance within the adjacent soft tissue, suggesting migration away from the nerve. These soft tissue-derived cells also expressed the brown adipose marker uncoupling protein 1 (UCP1). Quantification of ADRB3-specific RNA in total hind limb tissue revealed a 3-fold increase 2 days after delivery of BMP2, followed by a 70-fold increase in UCP1-specific RNA after 3 days. Expression levels then rapidly returned to baseline by 4 days. Interestingly, these ADRB3(+) UCP1(+) cells also expressed the neural guidance factor reelin. Reelin(+) cells demonstrated distinct patterns within the injected muscle, concentrated toward the area of BMP2 release. Blocking mast cell degranulation-induced nerve remodeling resulted in the complete abrogation of UCP1-specific RNA and protein expression within the hind limbs following BMP2 injection. The data collectively suggest that local BMP2 administration initiates a cascade of events leading to the expansion, migration, and differentiation of progenitors from the peripheral nerve perineurium to brown adipose-like cells in the mouse, a necessary prerequisite for associated nerve remodeling.
    MeSH term(s) Adenoviridae/genetics ; Adipocytes, Brown/cytology ; Adipocytes, Brown/physiology ; Age Factors ; Animals ; Bone Morphogenetic Protein 2/genetics ; Cell Adhesion Molecules, Neuronal/genetics ; Cell Differentiation/physiology ; Cell Division/physiology ; Cell Lineage/physiology ; Cell Movement/physiology ; Cells, Cultured ; Extracellular Matrix Proteins/genetics ; Fibroblasts/cytology ; Fibroblasts/physiology ; Humans ; Ion Channels/genetics ; Mast Cells/cytology ; Mast Cells/physiology ; Mice ; Mitochondrial Proteins/genetics ; Nerve Regeneration/physiology ; Nerve Tissue Proteins/genetics ; Norepinephrine/metabolism ; Peripheral Nerves/cytology ; Peripheral Nerves/physiology ; Receptors, Adrenergic, beta-3/genetics ; Serine Endopeptidases/genetics ; Stem Cell Transplantation/methods ; Stem Cells/cytology ; Stem Cells/physiology ; Transgenes/genetics ; Uncoupling Protein 1
    Chemical Substances BMP2 protein, human ; Bone Morphogenetic Protein 2 ; Cell Adhesion Molecules, Neuronal ; Extracellular Matrix Proteins ; Ion Channels ; Mitochondrial Proteins ; Nerve Tissue Proteins ; Receptors, Adrenergic, beta-3 ; UCP1 protein, human ; Ucp1 protein, mouse ; Uncoupling Protein 1 ; Serine Endopeptidases (EC 3.4.21.-) ; reelin protein (EC 3.4.21.-) ; Norepinephrine (X4W3ENH1CV)
    Language English
    Publishing date 2012-11-26
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
    ZDB-ID 2642270-0
    ISSN 2157-6580 ; 2157-6564
    ISSN (online) 2157-6580
    ISSN 2157-6564
    DOI 10.5966/sctm.2012-0090
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  2. Article ; Online: Far-red fluorescence gene reporter tomography for determination of placement and viability of cell-based gene therapies.

    Lu, Yujie / Darne, Chinmay D / Tan, I-Chih / Zhu, Banghe / Hall, Mary A / Lazard, Zawaunyka W / Davis, Alan R / Simpson, Lashan / Sevick-Muraca, Eva M / Olmsted-Davis, Elizabeth A

    Optics express

    2013  Volume 21, Issue 20, Page(s) 24129–24138

    Abstract: Non-invasive injectable cellular therapeutic strategies based on sustained delivery of physiological levels of BMP-2 for spinal fusion are emerging as promising alternatives, which could provide sufficient fusion without the associated surgical risks. ... ...

    Abstract Non-invasive injectable cellular therapeutic strategies based on sustained delivery of physiological levels of BMP-2 for spinal fusion are emerging as promising alternatives, which could provide sufficient fusion without the associated surgical risks. However, these injectable therapies are dependent on bone formation occurring only at the specific target region. In this study, we developed and deployed fluorescence gene reporter tomography (FGRT) to provide information on in vivo cell localization and viability. This information is sought to confirm the ideal placement of the materials with respect to the area where early bone reaction is required, ultimately providing three dimensional data about the future fusion. However, because almost all conventional fluorescence gene reporters require visible excitation wavelengths, current in vivo imaging of fluorescent proteins is limited by high tissue absorption and confounding autofluorescence. We previously administered fibroblasts engineered to produce BMP-2, but is difficult to determine 3-D information of placement prior to bone formation. Herein we used the far-red fluorescence gene reporter, IFP1.4 to report the position and viability of fibroblasts and developed 3-D tomography to provide placement information. A custom small animal, far-red fluorescence tomography system integrated into a commercial CT scanner was used to assess IFP1.4 fluorescence and to demark 3-D placement of encapsulated fibroblasts with respect to the vertebrae and early bone formation as assessed from CT. The results from three experiments showed that the placement of the materials within the spine could be detected. This work shows that in vivo fluorescence gene reporter tomography of cell-based gene therapy is feasible and could help guide cell-based therapies in preclinical models.
    MeSH term(s) Animals ; Bone Morphogenetic Protein 2/genetics ; Bone Morphogenetic Protein 2/therapeutic use ; Cell Survival ; Fluorescence ; Genes, Reporter ; Genetic Therapy ; Humans ; Image Processing, Computer-Assisted ; Mice ; Optical Imaging ; Spinal Fusion ; Tomography, X-Ray Computed/methods
    Chemical Substances Bone Morphogenetic Protein 2
    Language English
    Publishing date 2013-10-09
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
    ZDB-ID 1491859-6
    ISSN 1094-4087 ; 1094-4087
    ISSN (online) 1094-4087
    ISSN 1094-4087
    DOI 10.1364/OE.21.024129
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  3. Article ; Online: Cell-based gene therapy for repair of critical size defects in the rat fibula.

    Lazard, Zawaunyka W / Heggeness, Michael H / Hipp, John A / Sonnet, Corinne / Fuentes, Angie S / Nistal, Rita P / Davis, Alan R / Olabisi, Ronke M / West, Jennifer L / Olmsted-Davis, Elizabeth A

    Journal of cellular biochemistry

    2011  Volume 112, Issue 6, Page(s) 1563–1571

    Abstract: More than a decade has passed since the first experiments using adenovirus-transduced cells expressing bone morphogenetic protein 2 were performed for the synthesis of bone. Since this time, the field of bone gene therapy has tackled many issues ... ...

    Abstract More than a decade has passed since the first experiments using adenovirus-transduced cells expressing bone morphogenetic protein 2 were performed for the synthesis of bone. Since this time, the field of bone gene therapy has tackled many issues surrounding safety and efficacy of this type of strategy. We present studies examining the parameters of the timing of bone healing, and remodeling when heterotopic ossification (HO) is used for bone fracture repair using an adenovirus gene therapy approach. We use a rat fibula defect, which surprisingly does not heal even when a simple fracture is introduced. In this model, the bone quickly resorbs most likely due to the non-weight bearing nature of this bone in rodents. Using our gene therapy system robust HO can be introduced at the targeted location of the defect resulting in bone repair. The HO and resultant bone healing appeared to be dose dependent, based on the number of AdBMP2-transduced cells delivered. Interestingly, the HO undergoes substantial remodeling, and assumes the size and shape of the missing segment of bone. However, in some instances we observed some additional bone associated with the repair, signifying that perhaps the forces on the newly forming bone are inadequate to dictate shape. In all cases, the HO appeared to fuse into the adjacent long bone. The data collectively indicates that the use of BMP2 gene therapy strategies may vary depending on the location and nature of the defect. Therefore, additional parameters should be considered when implementing such strategies.
    MeSH term(s) Adenoviridae/genetics ; Animals ; Bone Morphogenetic Protein 2/genetics ; Bone Morphogenetic Protein 2/metabolism ; Bone and Bones/abnormalities ; Cell Line ; Cell- and Tissue-Based Therapy/methods ; Fibula/abnormalities ; Genetic Therapy/methods ; Humans ; Mice ; Osteogenesis/physiology ; Rats ; Wound Healing/physiology
    Chemical Substances Bone Morphogenetic Protein 2
    Language English
    Publishing date 2011-02-23
    Publishing country United States
    Document type Journal Article ; Research Support, U.S. Gov't, Non-P.H.S.
    ZDB-ID 392402-6
    ISSN 1097-4644 ; 0730-2312
    ISSN (online) 1097-4644
    ISSN 0730-2312
    DOI 10.1002/jcb.23068
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  4. Article ; Online: Characterization of chemical, radiochemical and optical properties of a dual-labeled MMP-9 targeting peptide.

    Azhdarinia, Ali / Wilganowski, Nathaniel / Robinson, Holly / Ghosh, Pradip / Kwon, Sunkuk / Lazard, Zawaunyka W / Davis, Alan R / Olmsted-Davis, Elizabeth / Sevick-Muraca, Eva M

    Bioorganic & medicinal chemistry

    2011  Volume 19, Issue 12, Page(s) 3769–3776

    Abstract: Optical imaging possesses similar sensitivity to nuclear imaging and has led to the emergence of multimodal approaches with dual-labeled nuclear/near-infrared (NIR) agents. The growing impact of (68)Ga (t(1/2)=68 min) labeled peptides on preclinical and ... ...

    Abstract Optical imaging possesses similar sensitivity to nuclear imaging and has led to the emergence of multimodal approaches with dual-labeled nuclear/near-infrared (NIR) agents. The growing impact of (68)Ga (t(1/2)=68 min) labeled peptides on preclinical and clinical research offers a promising opportunity to merge the high spatial resolution of NIR imaging with the clinically-accepted positron emission tomography (PET). Previously, dual-labeled agents have been prepared with longer-lived radiometals and showed no detrimental effects on optical properties as a result of radiolabeling. In this study, we selected a peptide (M(2)) that targets MMP-2/9 and is dual-labeled with IRDye 800 CW and (68)Ga. Since (68)Ga chelation typically requires low pH (3.5-4) and elevated heating temperatures (95 °C), we sought to evaluate the impact of (68)Ga labeling on the optical properties of M(2). An efficient method for preparation of (68)Ga-M(2) was developed and reaction conditions were optimized. Stability studies in PBS, DTPA, and serum were performed and high levels of intact agent were evident under each condition. The addition of multiple reporters to a targeting agent adds further complexity to the characterization and validation and thus requires not only testing to ensure the agent is stable chemically and radiochemically, but also optically. Therefore, fluorescence properties were evaluated using a spectrofluorometer as well as by fluorescence detection via HPLC. It was determined that (68)Ga-labeling conditions did not impair the fluorescent properties of the agent. The agent was then used for in vivo imaging in a mouse model of heterotopic ossification (HO) with activated MMP-9 expression as an early biomarker which precedes mineralization. Although (68)Ga-complexation greatly reduced binding affinity of the peptide and negated tracer uptake on PET, NIR imaging showed consistent fluorescent signal that correlated to MMP-9 expression. This attests to the feasibility of using (68)Ga/NIR for dual-labeling of other peptides or small molecules for multimodality molecular imaging.
    MeSH term(s) Animals ; Chromatography, High Pressure Liquid ; Drug Delivery Systems ; Fluorescent Dyes/chemistry ; Gallium Radioisotopes/chemistry ; Humans ; Matrix Metalloproteinase 9/chemistry ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Molecular Structure ; Peptides/chemistry
    Chemical Substances Fluorescent Dyes ; Gallium Radioisotopes ; Peptides ; Matrix Metalloproteinase 9 (EC 3.4.24.35)
    Language English
    Publishing date 2011-05-06
    Publishing country England
    Document type Journal Article ; Research Support, U.S. Gov't, Non-P.H.S.
    ZDB-ID 1161284-8
    ISSN 1464-3391 ; 0968-0896
    ISSN (online) 1464-3391
    ISSN 0968-0896
    DOI 10.1016/j.bmc.2011.04.054
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  5. Article ; Online: Hydrogel microsphere encapsulation of a cell-based gene therapy system increases cell survival of injected cells, transgene expression, and bone volume in a model of heterotopic ossification.

    Olabisi, Ronke M / Lazard, Zawaunyka W / Franco, Christy L / Hall, Mary A / Kwon, Sun Kuk / Sevick-Muraca, Eva M / Hipp, John A / Davis, Alan R / Olmsted-Davis, Elizabeth A / West, Jennifer L

    Tissue engineering. Part A

    2010  Volume 16, Issue 12, Page(s) 3727–3736

    Abstract: Bone morphogenetic proteins (BMPs) are well known for their osteoinductive activity, yet harnessing this capacity remains a high-priority research focus. We present a novel technology that delivers high BMP-2 levels at targeted locations for rapid ... ...

    Abstract Bone morphogenetic proteins (BMPs) are well known for their osteoinductive activity, yet harnessing this capacity remains a high-priority research focus. We present a novel technology that delivers high BMP-2 levels at targeted locations for rapid endochondral bone formation, enhancing our preexisting cell-based gene therapy system by microencapsulating adenovirus-transduced cells in nondegradable poly(ethylene glycol) diacrylate (PEGDA) hydrogels before intramuscular delivery. This study evaluates the in vitro and in vivo viability, gene expression, and bone formation from transgenic fibroblasts encapsulated in PEGDA microspheres. Fluorescent viability and cytotoxicity assays demonstrated >95% viability in microencapsulated cells. ELISA and alkaline phosphatase assays established that BMP-2 secretion and specific activity from microencapsulated AdBMP2-transduced fibroblasts were not statistically different from monolayer. Longitudinal transgene expression studies of AdDsRed-transduced fibroblasts, followed through live animal optical fluorescent imaging, showed that microencapsulated cells expressed longer than unencapsulated cells. When comparable numbers of microencapsulated AdBMP2-transduced cells were intramuscularly injected into mice, microcomputed tomography evaluation demonstrated that the resultant heterotopic bone formation was approximately twice the volume of unencapsulated cells. The data suggest that microencapsulation protects cells and prolongs and spatially distributes transgene expression. Thus, incorporation of PEGDA hydrogels significantly advances current gene therapy bone repair approaches.
    MeSH term(s) Alkaline Phosphatase/metabolism ; Animals ; Bone Morphogenetic Protein 2/genetics ; Bone Morphogenetic Protein 2/metabolism ; Cell Line ; Cell Survival/genetics ; Cell Survival/physiology ; Enzyme-Linked Immunosorbent Assay ; Female ; Fibroblasts/cytology ; Fibroblasts/metabolism ; Humans ; Hydrogel, Polyethylene Glycol Dimethacrylate/chemistry ; Mice ; Mice, SCID ; Microspheres ; Tissue Engineering/methods ; Transgenes/genetics ; Transgenes/physiology ; X-Ray Microtomography
    Chemical Substances BMP2 protein, human ; Bone Morphogenetic Protein 2 ; Hydrogel, Polyethylene Glycol Dimethacrylate (25852-47-5) ; Alkaline Phosphatase (EC 3.1.3.1)
    Language English
    Publishing date 2010-09-01
    Publishing country United States
    Document type Journal Article ; Research Support, U.S. Gov't, Non-P.H.S.
    ZDB-ID 2420582-5
    ISSN 1937-335X ; 1937-3341
    ISSN (online) 1937-335X
    ISSN 1937-3341
    DOI 10.1089/ten.TEA.2010.0234
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  6. Article ; Online: Vessel formation is induced prior to the appearance of cartilage in BMP-2-mediated heterotopic ossification.

    Dilling, Christine Fouletier / Wada, Aya M / Lazard, Zawaunyka W / Salisbury, Elizabeth A / Gannon, Francis H / Vadakkan, Tegy J / Gao, Liang / Hirschi, Karen / Dickinson, Mary E / Davis, Alan R / Olmsted-Davis, Elizabeth A

    Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

    2009  Volume 25, Issue 5, Page(s) 1147–1156

    Abstract: Heterotopic ossification (HO), or endochondral bone formation at nonskeletal sites, often results from traumatic injury and can lead to devastating consequences. Alternatively, the ability to harness this phenomenon would greatly enhance current ... ...

    Abstract Heterotopic ossification (HO), or endochondral bone formation at nonskeletal sites, often results from traumatic injury and can lead to devastating consequences. Alternatively, the ability to harness this phenomenon would greatly enhance current orthopedic tools for treating segmental bone defects. Thus, understanding the earliest events in this process potentially would allow us to design more targeted therapies to either block or enhance this process. Using a murine model of HO induced by delivery of adenovirus-transduced cells expressing bone morphogenetic protein 2 (BMP-2), we show here that one of the earliest stages in this process is the establishment of new vessels prior to the appearance of cartilage. As early as 48 hours after induction of HO, we observed the appearance of brown adipocytes expressing vascular endothelial growth factors (VEGFs) simultaneous with endothelial progenitor replication. This was determined by using a murine model that possesses the VEGF receptor 2 (Flk1) promoter containing an endothelial cell enhancer driving the expression of nuclear-localized yellow fluorescent protein (YFP). Expression of this marker has been shown previously to correlate with the establishment of new vasculature, and the nuclear localization of YFP expression allowed us to quantify changes in endothelial cell numbers. We found a significant increase in Flk1-H2B::YFP cells in BMP-2-treated animals compared with controls. The increase in endothelial progenitors occurred 3 days prior to the appearance of early cartilage. The data collectively suggest that vascular remodeling and growth may be essential to modify the microenvironment and enable engraftment of the necessary progenitors to form endochondral bone.
    MeSH term(s) Adipocytes, Brown/metabolism ; Animals ; Bone Morphogenetic Protein 2/pharmacology ; Cartilage/blood supply ; Ki-67 Antigen/biosynthesis ; Mice ; Ossification, Heterotopic/metabolism ; RNA, Messenger/metabolism ; Vascular Endothelial Growth Factor A/biosynthesis ; Vascular Endothelial Growth Factor Receptor-2/biosynthesis ; von Willebrand Factor/biosynthesis
    Chemical Substances Bone Morphogenetic Protein 2 ; Ki-67 Antigen ; RNA, Messenger ; Vascular Endothelial Growth Factor A ; vascular endothelial growth factor A, rat ; von Willebrand Factor ; Vascular Endothelial Growth Factor Receptor-2 (EC 2.7.10.1)
    Language English
    Publishing date 2009-10-14
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, U.S. Gov't, Non-P.H.S.
    ZDB-ID 632783-7
    ISSN 1523-4681 ; 0884-0431
    ISSN (online) 1523-4681
    ISSN 0884-0431
    DOI 10.1359/jbmr.091031
    Database MEDical Literature Analysis and Retrieval System OnLINE

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