Electrospun curcumin gelatin blended nanofibrous mats ... · PDF fileABSTRACT Background:...

Click here to load reader

  • date post

    30-Aug-2019
  • Category

    Documents

  • view

    0
  • download

    0

Embed Size (px)

Transcript of Electrospun curcumin gelatin blended nanofibrous mats ... · PDF fileABSTRACT Background:...

  • TECHNISCHE UNIVERSITT MNCHEN FAKULTT FR MEDIZIN

    Klinik und Poliklinik fr Plastische und Handchirurgie Klinikum rechts der Isar

    Electrospun curcumin gelatin blended nanofibrous mats accelerate wound healing by Dkk-1 mediated fibroblast mobilization and MCP-1 mediated anti-inflammation

    Xinyi Dai Vollstndiger Abdruck der von der Fakultt fr Medizin der Technischen Universitt Mnchen zur Erlangung des akademischen Grades eines

    Doctor of Philosophy (Ph.D.) genehmigten Dissertation.

    Betreuer: Univ.-Prof. Dr. A. F. Schilling Vorsitzender: Univ.-Prof. Dr. C. Zimmer

    Prfer der Dissertation:

    1. Univ.-Prof. Dr. H.-G. Machens

    2. Priv.-Doz. Dr. R. H. H. Burgkart

    Die Dissertation wurde am 26.09.2016 bei der Fakultt fr Medizin der

    Technischen Universitt Mnchen eingereicht und durch die Fakultt fr Medizin

    am 14.12.2016 angenommen.

  • ABSTRACT

    Background: Acute wounds occur as a result of trauma as well as surgery. It

    is estimated that 300 million acute wounds are treated globally each year [1].

    In the United States alone, approximately 11 million people are affected and 3

    million are hospitalized annually. These numbers continue to rise, making

    wound management a great challenge to society and medical professionals,

    especially plastic surgeons [2,3]. Traditional reconstructive surgical

    intervention such as skin flap transplantation still faces the problem of donor

    tissue insufficiency, post-operative flap compromise, host immunological

    rejection or even loss of function [4,5,6,7]. Therefore, wound therapy via

    noninvasive approaches is of major importance and a variety of compounds

    have been discussed to improve it [8].

    Turmeric, the product of Curcuma longa, is one of these compounds and has a

    very long history of being used for treatment of wounds in many Asian

    countries. Curcumin, the principal curcuminoid of turmeric, has been recently

    identified as a powerful modulator of processes involved in healing. However,

    the inherent limitations of the compound itself, such as hydrophobicity,

    instability, poor absorption and rapid systemic elimination, pose big hurdles for

    translation to wider clinical application. Accumulating evidence indicates that

    nano-formulation is capable of improving solubility of previously unsoluble

    compounds. Electrospinning, known for many years in the textile industry and

    organic polymer science, has recently emerged as a novel technique for

  • generating nanoscale biomimetic scaffolds for tissue engineering. The

    simplicity of the electrospinning process itself and the possibility to incorporate

    therapeutic compounds into the nonwoven nanofiber meshes during spinning

    allow the development of controlled drug delivery systems with this method.

    For wound healing applications, such a system should ideally be able to

    mimick the structure and biological function of extracellular matrix (ECM)

    proteins, to provide structural and mechanical integrity as well as support for

    cellular processes.

    The aim of the present PhD thesis was it to engineer curcumin/gelatin blended

    nanofibrous mats by electrospinning to adequately enhance the bioavailability

    of the hydrophobic curcumin for wound treatment. For this purpose these

    electrospun curcumin/gelatin blended nanofibrous mats were evaluated as

    both drug-release system and biomimetic scaffold/dressing for topical

    application. The potential mechanisms being involved were also scrutinized.

    Materials and methods: We prepared curcumin/gelatin blended nanofibrous

    mats by electrospinning and scrutinized their properties including material

    characterization (SEM, XRD, FITR), curcumin release profile, tensile

    mechanical properties, cytotoxicity/biocompatibility (LDH, WST, LIVE/DEAD).

    To further explore curcumin action through such nanoformulation, fibroblast

    cell based cytokine paracrine profile and subsequent fibroblast migration and

    monocyte/macrophage cell chemotaxis assays were performed. The healing

    capacity of the medicated nanofibrous mats was assessed both in vitro and in

  • vivo.

    Principle findings: 1) curcumin was successfully formulated into gelatin

    based nanofibers as amorphous nanosolid dispersion and could be

    control-released to enhance its bioavailability. 2) The resulting medicated

    nanofibrous mats showed agreeable biocompatibility without cytotoxic effects

    that facilitated cell-based dermal regeneration in vitro and accelerated wound

    healing in vivo. 3) The underlying mechanisms of the accelerated wound

    healing by topically applied curcumin/gelatin nanofibrous mats were found to

    be a combination of curcumin's ability of in situ fibroblasts mobilization, which

    is partially mediated by Dkk-1 regulated Wnt/-catenin signaling, and of its

    immunomodulatory action, specifically, the persistent inhibition of the

    inflammatory chemotaxis through decreased expression of MCP-1 by

    fibroblasts.

    Conclusions: These results demonstrate the feasibility and effectiveness of

    bioactive curcumin in a biomimetic nanofibrous structure on accelerated

    wound healing. These results open an avenue to translate this ancient

    medicine for modern wound therapy, which is promising for future non-invasive

    approaches to cure wounds faster through medicated wound dressings.

  • TABLE OF CONTENTS

    1. INTRODUCTION ....................................................................................... 1

    1.1. The human skin ........................................................................... 1

    1.1.1. Anatomy ................................................................................ 1

    1.1.2. Function ................................................................................. 6

    1.2. Acute wound.............................................................................. 13

    1.2.1. Acute wound classification ................................................ 13

    1.2.2. Skin regeneration and wound healing .............................. 17

    1.3. Curcumin from turmeric: a promising candidate for wound

    therapy.................................................................................................... 26

    1.4. Electrospinning: an encouraging approach for regenerative

    medicine ................................................................................................. 32

    1.4.1. Electrospinning history and fundamentals ...................... 32

    1.4.2. Electrospinning for advanced wound dressing ............... 37

    1.4.3. Electrospinning for drug delivery ...................................... 38

    2. HYPOTHESIS ......................................................................................... 38

    3. MATERIALS AND METHODS ................................................................ 39

    3.1. Materials .................................................................................... 39

    3.1.1. Reagents .............................................................................. 39

    3.1.2. Cell culture mediums .......................................................... 40

    3.1.3. Buffers ................................................................................. 40

    3.1.4. Cell lines .............................................................................. 40

    3.1.5. Commercial kits .................................................................. 40

    3.1.6. Antibodies ........................................................................... 40

    3.1.7. Devices ................................................................................ 41

    3.2. Methods ..................................................................................... 41

    3.2.1. Electrospinning of Curcumin/Gelatin blended nanofibrous

    mat 41

    3.2.2. Scanning electron microscopy (SEM) ............................... 42

    3.2.3. X-ray diffraction (XRD) spectroscopy ............................... 42

    3.2.4. Fourier transform infrared (FTIR) spectrometer............... 42

    3.2.5. Curcumin release profile .................................................... 43

    3.2.6. Tensile mechanical properties........................................... 43

    3.2.7. Cell isolation and culture ................................................... 44

    3.2.8. LDH and WST-1 assay ........................................................ 45

    3.2.9. Live and dead assay ........................................................... 45

    3.2.10. Cell visualization on the scaffold ...................................... 46

    3.2.11. Fibroblasts in vitro wound healing assay ........................ 46

    3.2.12. Fibroblasts cytokine profile array ..................................... 46

    3.2.13. Quantified ELISA assay for Dkk-1, SDF-1 , MCP-1 and

    TSP-1 47

    3.2.14. -catenin immunofluorescence and fibroblast migration

    47

  • 3.2.15. Dkk-1 mediated fibroblast migration ................................ 48

    3.2.16. MCP-1 mediated PBMC and macrophage chemotaxis.... 49

    3.2.17. Animal housing conditions ............................................... 49

    3.2.18. Surgical procedure for acute wound animal model ........ 49

    3.2.19. Wound closure analysis .................................................... 50

    3.2.20. Histological analysis .............................