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 .............................