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  • ROLE OF MICRORNAS IN IMPROVED

    OSTEOGENICITY OF TOPOGRAPHICALLY

    MODIFIED TITANIUM IMPLANT

    SURFACES

    Nishant Chakravorty

    Bachelor of Medicine & Bachelor of Surgery (MBBS)

    Master of Medical Science & Technology (MMST)

    Submitted in fulfillment of the requirements for the degree of

    Doctor of Philosophy

    Institute of Health & Biomedical Innovation

    Science & Engineering Faculty

    Queensland University of Technology

    July, 2014

  • Role of MicroRNAs in Improved Osteogenicity of Topographically Modified Titanium Implant Surfaces i

    Keywords

    cell signaling, gene expression, microarray, microRNA, micro-roughened titanium

    surface, modSLA, SLA, osteogenesis, osteoprogenitor differentiation, titanium

    surface modification.

  • ii Role of MicroRNAs in Improved Osteogenicity of Topographically Modified Titanium Implant Surfaces

  • Role of MicroRNAs in Improved Osteogenicity of Topographically Modified Titanium Implant Surfaces iii

    Abstract

    Topographically modified micro-roughened titanium implants, like the clinically

    successful dental implants - sand-blasted, large grit, acid-etched (SLA) and its

    successor, the chemically modified hydrophilic SLA (modSLA) surfaces, are

    recognized to have improved osseointegration (in vivo) and osteogenic properties (in

    vitro). Micro-roughened surfaces are known to induce “contact osteogenesis”,

    wherein osseointegration occurs via direct deposition of new bone on the implant

    surface itself, making the in vitro osteogenic differentiation process an appropriate

    reflection of the in vivo osseointegration process. Micro-roughened surfaces, as such

    provide us with a unique model to explore the molecular mechanisms involved in the

    process of osteogenesis and osseointegration. Previous studies on modSLA and SLA

    surfaces have described the differential expression of several genes and pathways

    compared with smooth polished (SMO) surfaces, and have hinted on an early

    regulation of osteogenic differentiation on these surfaces; however, a structured

    approach exploring the molecular events was lacking.

    MicroRNA mediated RNA-interference mechanisms, are known to be vital

    regulators of cell differentiation by translational repression and gene silencing. This

    project hypothesized that an early modulation of molecular processes by microRNA-

    mediated mechanisms on micro-roughened surfaces, like the modSLA and SLA,

    leads to their improved osteogenic differentiation and osseointegration. A whole-

    genome transcriptome analysis of human osteoprogenitor cells, performed using

    Affymetrix Human Genome U133 Plus 2.0 arrays, following 3 and 24 hours

    exposure to SLA and SMO surfaces, confirmed an early pro-osteogenic response on

    micro-roughened titanium surfaces at 24 hours. An initial pro-angiogenic and

    immunomodulatory response, as seen on the SLA surfaces, was seen to condition

    them for the pro-osteogenic response. Subsequently, the microRNA expression

    profile of osteoprogenitor cells on modSLA and SLA surfaces at 24 hours revealed

    the differential expression of several microRNAs on these surfaces (compared with

    SMO). Relatively minor differences were observed between modSLA and SLA

    surfaces.

  • iv Role of MicroRNAs in Improved Osteogenicity of Topographically Modified Titanium Implant Surfaces

    The microRNAs downregulated on modSLA and SLA surfaces (compared with

    SMO), were seen to have several putative targets in the TGFβ/BMP and Wnt/Ca 2+

    pathways (based on bioinformatics-based target predictions using TargetScan);

    indicating a possible activation of these pro-osteogenic cell signaling pathways. To

    confirm whether signaling pathways are activated within 24 hours of interaction with

    these surfaces, the expression of key genes of TGFβ, Wnt, FGF, Notch, and

    Hedgehog pathways was investigated. The study concluded that TGFβ/BMP, Wnt

    (especially the Wnt/Ca 2+

    ), and Notch pathways show higher expression on SLA &

    modSLA (compared with SMO), as early as 24 hours. The study also demonstrated a

    relatively high expression of cell signaling genes at 24 hours compared to 72 hours,

    further highlighting the importance of the early interaction of cells with the micro-

    roughened surfaces.

    The project further explored the role of two miRNAs, miR-26a and miR-17, that

    were observed to be downregulated on the modSLA and SLA surfaces (compared

    with SMO) and had putative targets in TGFβ/BMP and Wnt/Ca 2+

    pathways, during

    the process of osteogenic differentiation. Both of the miRNAs were found to have

    inhibitory effects on the TGFβ/BMP and Wnt/Ca 2+

    pathways; and over-expressing

    these miRNAs was seen to delay the process of osteogenesis. Empirical evidences

    also suggested a coordinated modulation of these two pathways, with the activation

    of the TGFβ/BMP having a stimulatory effect on the Wnt/Ca 2+

    pathway. To explore

    the plausibility of using miRNA modulators to regulate osteogenesis on implant

    surfaces, inhibitors of miR-26a (i-miR-26a) and miR-17 (i-miR-17) were used on

    smooth polished titanium surfaces. i-miR-26a and i-miR-17 transfected cells were

    seen to have superior osteogenic differentiation on SMO surfaces.

    Taken together, the findings of the project strongly suggest a highly regulated

    microRNA-mediated control of molecular mechanisms during the process of

    osteogenesis, that may be responsible for the superior osseointegration and

    osteogenic differentiation properties on micro-roughened titanium implant surfaces.

    The conclusions derived from the project, indicate the possibility of using microRNA

    modulators to enhance osseointegration and bone formation in clinically demanding

    circumstances.

  • Role of MicroRNAs in Improved Osteogenicity of Topographically Modified Titanium Implant Surfaces v

    List of Publications

    Published or accepted manuscripts relevant to the work performed in this PhD:

    1. Nishant Chakravorty, Anjali Jaiprakash, Saso Ivanovski & Yin Xiao.

    “Implant surface modifications and osseointegration.” In Li, Qing & Mai,

    Yiu-Wing (Ed.) Biomaterials for Implants and Scaffolds (Springer Series in

    Biomaterials Science and Engineering). Springer Science+Business Media

    New York (accepted Nov, 2012)

    2. Nishant Chakravorty, Stephen Hamlet, Anjali Jaiprakash, Ross Crawford,

    Adekunle Oloyede, Mohammed Alfarsi, Yin Xiao & Saso Ivanovski. “Pro-

    osteogenic topographical cues promote early activation of osteoprogenitor

    differentiation via enhanced TGFβ, Wnt, and Notch signaling.” Clinical and

    Oral Implants Research. 2014;25(4):475-86. (doi: 10.1111/clr.12178)

    3. Nishant Chakravorty, Saso Ivanovski, Indira Prasadam, Ross Crawford,

    Adekunle Oloyede & Yin Xiao. “The microRNA expression signature on

    modified titanium implant surfaces influences genetic mechanisms leading to

    osteogenic differentiation.” Acta Biomaterialia. 2012;8(9):3516-23 (doi:

    10.1016/j.actbio.2012.05.008)

    Manuscripts in preparation relevant to the work performed in this PhD:

    1. Nishant Chakravorty, Saso Ivanovski & Yin Xiao. “Osteogenic

    differentiation on micro-roughened titanium surfaces: A review of current

    concepts and knowledge.”

    2. Saso Ivanovski*, Nishant Chakravorty*, Jamie Harle, Stephen Hamlet, Yin

    Xiao, Peter Brett & Maurizio Tonetti. “Genome-wide transcriptional analysis

    of early interactions of osteoprogenitor cells with micro-rough titanium

    implants.” ( * Co-first authors)

    3. Nishant Chakravorty, Anjali Jaiprakash, Ross Crawford, Adekunle

    Oloyede, Saso Ivanovski & Yin Xiao. “The miR-26a and miR-17 mediated

    cell signaling cross-talks guide osteogenic differentiation and

    osseointegration.”

  • vi Role of MicroRNAs in Improved Osteogenicity of Topographically Modified Titanium Implant Surfaces

    Other publications

    1. Yinghong Zhou, Nishant Chakravorty, Yin Xiao & Wenyi Gu.

    “Mesenchymal stem cells and nano-structured surfaces.” In Turksen, Kurshad

    (Ed.) Stem Cell Nanotechnology: Methods and Protocols. Springer Protocols,

    Humana Press Inc., New York. (Methods in Molecular Biology.

    2013;1058:133-48) (doi: 10.1007/7651_2013_30)

    2. Indira Prasadam, Nishant Chakravorty, Ross Crawford & Yin Xiao.

    “Mesenchymal stem cell concepts in osteoarthritis therapy: Current status of

    theory, technology, and applications.” In Xiao, Yin (Ed.) Mesenchymal Stem

    Cells. Nova Science Publishers, Inc., Hauppauge, New York, 2012

    Research Grant

    1. Yin Xiao, Nishant Chakravorty & Saso Ivanovski. "Role of micro-RNAs in

    improved osteogenicity of modified titanium implant surfaces". Funded by

    Australian Dental Research Foundation, 2012 ($10,101.60)

  • Role of MicroRNAs in Improved Osteogenicity of Topographically Modified Titanium Implant Surfaces vii

    Table of Contents

    Keywords .............

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