Electrospun curcumin gelatin blended nanofibrous mats accelerate wound healing by Dkk-1 ...

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TECHNISCHE UNIVERSITÄT MÜNCHEN
FAKULTÄT FÜR MEDIZIN

Klinik und Poliklinik für 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

Vollständiger Abdruck der von der Fakultät für Medizin der Technischen Universität
München 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

Prüfer 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 Fakultät für Medizin der
Technischen Universität München eingereicht und durch die Fakultät für 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/β-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 α, MCP-1 and
TSP-1 47
3.2.14. β-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 .......................................................... 50
3.2.21. Collagen content analysis ................................................. 50
3.2.22. Macrophage immunohistochemistry ................................ 51
3.2.23. Statistical analysis ............................................................. 51
4. RESULTS ................................................................................................ 52
4.1. Material characterization .......................................................... 52
4.1.1. Morphology of electrospun nanofibrous mat ................... 52
4.1.2. Electrospinning nanoformulates curcumin ...................... 53
4.1.3. Curcumin incorporation in nanofiber ................................ 54
4.1.4. Curcumin release profile .................................................... 55
4.1.5. Tensile mechanical properties of Cc/Glt NM .................... 56
4.2. Biocompatibility and cytotoxicity ............................................ 57
4.2.1. Cytotoxicity of electrospun NM in vitro ............................ 57
4.2.2. Fluorescence-based cytotoxicity in vitro ......................... 58
4.2.3. Cell visualization on the nanofibrous mat ........................ 60
4.3. Curcumin activates fibroblasts ................................................ 61
4.3.1. Fibroblasts in vitro wound healing .................................... 61
4.3.2. Fibroblasts cytokine paracrine profile array .................... 62
4.3.3. Quantified cytokine expression of Dkk-1, SDF-1α, MCP-1
and TSP-1 ......................................................................................... 63
4.4. Dkk-1 mediates curcumin induced fibroblasts mobilization . 64
4.4.1. β-catenin signaling in migrated fibroblasts ...................... 64
4.4.2. Dkk-1 mediates fibroblast migration ................................. 65
4.5. MCP-1 mediates curcumin induced anti-inflammation .......... 67
4.5.1. Curcumin inhibits PBMC chemotaxis ............................... 67
4.5.2. Curcumin inhibited macrophage (MV-4-11) chemotaxis is
mediated by MCP-1 ......................................................................... 68
4.6. Wound healing in vivo .............................................................. 69
4.6.1. Cc/Glt NM accelerates wound closure in vivo .................. 69
4.6.2. Cc/Glt NM enhances dermal regeneration ........................ 70
4.6.3. Cc/Glt NM enhances collagen deposition ......................... 72
4.6.4. Cc/Glt NM inhibits macrophage infiltration in vivo .......... 74
5. DISCUSSION .......................................................................................... 75
5.1. Electrospun Cc/Glt NM successfully nano-formulates
crucmumin and enhances its bioavailability for wound therapy ...... 77
5.2. Electrospun Cc/Glt NM is ideal for topical wound treatment 89
5.3. Delivered curcumin exterts synergistic signalling to
accelerate wound healing ..................................................................... 98
5.4. Electrospun Cc/Glt NM could be a benificial complement to
current wound therapy ........................................................................ 108
6. GENERAL CONCLUSIONS AND PERSPECTIVES ............................. 115
7. APPENDIX ............................................................................................ 116
8. ACKNOWLEDGEMENTS ..................................................................... 118
9. REFERENCES ...................................................................................... 126
LIST OF FIGURES AND TABLES
Figure 1: Structure of the human skin ........................ 错误！未定义书签。
Figure 2: "Brick and Motar" pattern of the stratum corneum ................. 8
Figure 3: Acute wounds ........................................................................... 14
Figure 4: Molecular structures of turmeric-derived curcuminoids....... 28
Figure 5: Electrospinning process .......................................................... 33
Figure 6: Bending instability .................................................................... 34
Figure 7: Morphology ............................................................................... 52
Figure 8: Curcumin nanoformulation ...................................................... 53
Figure 9: Curcumin incorporation in gelatin nanofiber ......................... 54
Figure 10: Curcumin release in vitro ....................................................... 55
Figure 11: Mechanical property of Cc/Glt NM ......................................... 57
Figure 12: Cytotoxicity of electrospun NM in vitro ................................ 58
Figure 13: Fluorescence-based cytotoxicity in vitro ............................. 59
Figure 14: Cell visualization on the nanofibrous mat ............................ 61
Figure 15: Fibroblast in vitro wound healing ......................................... 62
Figure 16: Paracrine profile of fibroblasts .............................................. 63
Figure 17: Quantified cytokine expression............................................. 64
Figure 18: β-catenin signaling in migrated cell ...................................... 65
Figure 19: Dkk-1 mediates fibroblast migration ..................................... 66
Figure 20: Curcumin inhibits PBMC chemotaxis ................................... 67
Figure 21: Curcumin inhibited macrophage (MV-4-11) chemotaxis is
mediated by MCP-1............................................................................ 68
Figure 22: Topical application of Cc/Glt NM accelerates wound
closure in vivo ................................................................................... 69
Figure 23: Topical application of Cc/Glt NM enhances dermal
regeneration ....................................................................................... 71
Figure 24: Topical application of Cc/Glt NM enhances collagen
deposition .......................................................................................... 73
Figure 25: Topical application of Cc/Glt NM inhibits macrophage
infiltration in vivo ............................................................................... 74
Figure 26: Scheme of curcumin nanoformulation through
electrospinning .................................................................................. 87
Figure 27: Scheme of potential mechanism of curcumin induced
accelerated wound healing ............................................................. 104
LIST OF ABBREVIATIONS
AKT or PKB Protein kinase B
Bcl-2 B-cell lymphoma 2
bFGF /FGF-2 Basic fibroblast growth factor
BSA Bovine serum albumin
CAT Catalase
Cc/Glt NM Curcumin/Gelatin nanofibrous mats
CCL5 Chemokine (C-C motif) ligand 5
CE Collision energy
CM-Cc/Glt Conditioned medium of curcumin/gelatin nanofibrous mat
NM
CM-Glt NM Conditioned medium of gelatin nanofibrous mat
CXP Collision cell exit potential
Dkk-1 Dickkopf-related protein-1
DMEM Dulbecco's Modified Eagle's Medium
DMSO Dimethyl sulfoxide
DNA Deoxyribonucleic acid
Dox Doxorubicin hydrochloride
DP Declustering potential
E. coli Escherichia coli
ECM Extracellular matrix
EEP Ethyl ethylene phosphate
EGF Epidermal growth factor
EHD Electrohydrodynamic
FDA US Food and Drug Administrationthe
FTIR Fourier transform infrared
Glt NM Gelatin nanofibrous mats
GPx Glutathione peroxidase
GRAS Generally recognized as safe
HB-EGF Heparin binding epidermal growth factor
HBO Hyperbaric oxygen
HGF Hepatocyte growth factor
HHC Hexahydrocurcumin
HO Heme oxygenase
HPLC High-performance liquid chromatography
HS-27 Human fibroblast cell line
I.P. Intraperitoneal injection
IGF Insulin-like growth factor
IKK I kappa B kinase
IL-1 Interleukin-1
IL-6 Interleukin-6
IL-8 Interleukin-8
KGF Keratinocyte growth factor
LD50 Lethal 50% dose values
LDH Lactate dehydrogenase
LPS Lipopolysaccharide
MCP-1 Monocyte chemoattractant protein 1
MMPs Matrix metalloproteinases
MNPs Magnetic nanoparticles
MRSA Methicillin-resistant Staphylococcus aureus
MV-4-11 Human Macrophage cell line
NAC N-acetyl-l-cysteine
NFDs Nanofiber-based wound dressings
NF-κB Nuclear factor kappa-light-chain-enhancer of activated B cells
NGF Nerve growth factor
NM Nanofibrous mat
NO Nitric oxide