Fabrication, in vitro, in vivo evaluation of hybrid biopolymers for artificial soft and hard tissue aplication

SEM morphology of fibroblast cells attachment for 1 hour cultured a cross-linked chitosan fibrous membrane; b crosslinked chitosan membrane treated with glycine; c crosslinked chitosan m

Doctorate Thesis

Fabrication, In-vitro, In-vivo

Evaluation of Hybrid Biopolymers for Artificial Soft and Hard Tissue

Application

2012 08

College of Medicine, SoonChunHyang University

Biomedical Engineering & Materials,

Nguyen Thi Hiep

Fabrication, In-vitro, In-vivo

Evaluation of Hybrid Biopolymers for Artificial Soft and Hard Tissue

Application

Advisor: Professor Lee Byong-Taek

Dissertation submitted for the degree of Doctor of

Philosophy

2012 07

College of Medicine, SoonChunHyang University

Biomedical Engineering & Materials,

Nguyen Thi Hiep

Acknowledgements

First of all I would like to express my profound gratitude and earnest reverence to Dr Byong-Taek Lee, Professor, Department of Biomedical Engineering and Materials, School of Medicine, Soonchunhyang University, Cheonan, Korea for his enthusiastic guidance, constructive and creative suggestions and continuous supervision throughout the progress of this work

I earnestly acknowledge that without his dynamic and encouragement, I could never be completed my thesis and matured in my works as it is now I would like to express my heartiest gratitude and thanks to professor Young-Ki Min and professor Hun-Mo Yang, Department of Physiology, SoonChunHyang University; for their kind and sage advices and encouragements to continue the work and for their enormous support during my work While doing the work I received help and encouragement not only in my academic matter but also in my personal life from a number of people; I owe my thanks to my faithful husband, Mr Dao Van Hoa, always stands side-by-side and supports the spiritual atmosphere for me I thank all of my department members for their effort and guidance in my academic life I also wish to thank Mr S-W Kim, Mr DV Quang, Dr KP Rajat, and Ms Lee for their kind supports It is

my pleasure to express my gratitude to Mr D-W Jang, Mr DV Tuyen, Mr.TV Viet, Dr NTB Linh, Ms NT Phuong, Mr TQ Bao, Ms YH Kim, Dr KS Swapan, Ms H-J Hong, Mr M-S Kim, Ms S-H Lim, Mr M Dibakar, Mr DNh Subrato, Ms S So-Ra, Ms Rose-Ann F, Ms J-H Kim, Mr Alexandar S, Ms.Celine A, Mr Nathaiel C, Mr Andrew RP for their help I would like to

extend my thankful gratitude to Prof Kap-Ho Lee, School of Advanced Materials Engineering, Chungnam National University, and Dr S-J Lee, Department of Thoracic and Cardiovascular Surgery, Cheonan Hospital, Soonchunhyang University, Cheonan, 330-090, Korea, for their valuable assistance And, I am deeply indebted to my grand-fathers, my grand-mother,

my father, my mother, my father-in-law, my mother-in-law, my brothers, my sisters, my brothers-in-law, my sisters-in-law, my nieces and my nephew for their encouragement, patience and love without which it was impossible to achieve all that I have done

Cheonan, South Korea, July 2012

Nguyen Thi Hiep

Dedication

To my parents

1.3 Objectives of this dissertation 3

2.3 Study II: Fabrication of Cross-linked Nano-fibrous Chitosan Membranes

Using an Electro-spinning Process and Their Biocompatibility Evaluation 16

3.2 Study I: The effect of cross-linking on the microstructure, mechanical

properties and biocompatibility of electrospun

polycaprolactone-gelatin/PLGA-gelatin/PLGA-chitosan hybrid composite

PU/PCL for Artificial Blood Vessel Application

3.3.2 Materials and Methods 50 3.3.3 Results and Discussion 54

3.4 Study III: Hemocompatibility Testing of Conjugated Linoleic Acid

Loaded Electrospun Polycaptrolactone/Polyurethane Membrane 62

4.2 Study I: In-Vitro and In-Vivo Studies of rhBMP2-Coated PS/PCL Fibrous

4.2.2 Materials and Methods 74 4.2.3 Results and Discussion 78

4.3 Study II: Distribution of BCP in PCL/PLGA Porous Scaffold to Enhance

Osteoblastic Differentiation of human mesenchymal stem cell and

Osteogenesis in Rabbit Defect Model

4.4 Study III: Comparison of the effect osteoinductive (RGD, BMP2) and

osteoconductive (BCP) agents to bone remodeling of poly vinylalcohol/gelatin

List of Tables

Chapter 2

Study I: Nano Ag Loaded PVA Nano-Fibrous Mats for Skin Applications

Table 1 Zone of inhibition test from the disc diffusion test 13

Chapter 3

Study I: The effect of cross-linking on the microstructure, mechanical

properties and biocompatibility of electrospun

polycaprolactone-gelatin/PLGA-gelatin/PLGA-chitosan hybrid composite

Table 1 The parameters used for fabricating chitosan, PLGA, gelatin and PCL

Chapter 4

Study III: Comparison of the effect osteoinductive (RGD, BMP2) and

osteoconductive (BCP) agents to bone remodeling of poly vinylalcohol/gelatin

hydrogel matrices

Table 1 Characterization and properties of hydrogels

107

List of Figures

Chapter 1

Fig 1 (A) The tissue engineering cycle, using autologous cells 1: A small number

of cells are removed from the body 2: They are screened for phenotype and increased in number through proliferation 3: These cells are seeded onto porous scaffolds together with growth factors to enhance proliferation 4: The seeded scaffolds are placed in culture to further increase cell number 5: Finally, the regenerated tissue is implanted into the site of damage to integrate with the natural tissue (B) Example of tissue-engineered cartilage in the shape of a human ear

1

Fig 2 Different scaffold architectures provide cells with different topological cues.(A) A foamed or salt leached scaffold with large interconnected pores to permits cell ingrowth presents cells with a large, continuous surface area for attachment (B) Micro-fibrous scaffolds can be made to have greater porosity Where there is a large gap between fibres, focal adhesion placement guides cells to elongating along the fibre and this may restrict the contact with cells on different fibres (C) Nanofibre scaffolds have a larger surface area in relation to mass and can be fabricated with high porosity Focal adhesion size is limited by the fibre diameter, reducing a cell’s ability to develop a strong cytoskeleton such that cells may remain more rounded and stationary within the scaffold

2

Chapter 2

Fig 1 schematically process of tissue-engineered skin 4

Study I: Nano Ag Loaded PVA Nano-Fibrous Mats for Skin Applications

Fig 1 SEM micrographs of Ag NPs loaded electro-spun PVA mats with microwave 60 seconds (a), 90 seconds (b); c) and d) are EDS profile on the large area of a) and b)

8

Fig.2 UV-Vis absorption spectra of PVA/Ag solution with different microwave

Fig 3 XRD curves of AgNO3 loaded electrospun PVA mat and Ag NPs loaded

Fig 4 SEM micrographs Ag NPs loaded electrospun PVA mats with microwave

60 seconds at 80°C (a), 120 °C (c), and 150 °C (e); with 90 seconds at 150 °C (g); (b), (d) (f) and (h) are enlarge image of (a), (c) (e) and (h)

10

Fig.5 TEM and HRTEM image of Ag NPs loaded electrospun PVA nano-fiber with 60 seconds microwave irradiation and heat at 150 °C 10 Fig 6 FT-IR curves of PVA powder, Ag NPs loaded electro-spun PVA mat before

Fig 7 DSC curves of PVA powder (a), electro-spun PVA (b), Ag NPs loaded electro-spun PVA 7 before heat treatment (c) and after heat treatment (d) 12 Fig 8 Comparison of the inhibition zone test between a, e) pure PVA mat; b, f) PVA-Ag mat; c, g) PVA-Ag NPs-2 (120℃); d, h) PVA–Ag NPs-2 (150℃) The photographs of the disk sensitivity test for E coli (a, b, c, d) and S aureus (e, f, g, h)

Fig.1 SEM morphology of chitosan nano-fibrous membrane; (a) 2 wt%_ 2 wt %

AA, (b) 5 wt%_ 2 wt % AA, (c) 2 wt%_ 90 wt % AA and (a) 5 wt%_ 90 wt % AA 19 Fig.2 SEM morphology of chitosan nano-fibrous membranes depend on chitosan 20

concentration 2 wt % (a), 5 wt % (b), 7 wt % (c), and 9 wt % (d)

Fig.3 SEM morphology of chitosan nano-fibrous membranes; (a) uncrosslinked, (b) cross-linked (b), (c) cross-linked and treated glycerin, (d) washing in NaOH from sample (c)

20

Fig.4 FT- IR spectrum of chitosan bulk (a), chitosan nano-fibers membrane (b), cross-linked chitosan nano-fibers membrane (c), and cross-linked chitosan nano-fibers treated by glycine (d)

21

Fig.5 Cytotoxicity of uncrosslinked and crosslinked chitosan fibrous membranes depend on glycerin treatment and washing process 22 Fig.6 SEM morphology of fibroblast cells attachment for 1 hour cultured (a) cross-linked chitosan fibrous membrane; (b) crosslinked chitosan membrane treated with glycine; (c) crosslinked chitosan membrane treated by glycerin and washed (c)

22

Fig.7 SEM morphology of fibroblast cells attachment for 1 day cultured (a) linked chitosan fibrous membrane; (b) crosslinked chitosan membrane treated with glycine; (c) crosslinked chitosan membrane treated by glycerin and washed (c)

Fig 5 SEM morphology of uncross-linked gelatin (a) and cross-linked gelatin mat

at the point of fracture after tensile strength (b) 31 Fig 6 DSC thermograms of gelatin powder, uncross-linked electro-spun gelatin mats and cross-linked electro-spun gelatin mats 31 Fig 7 Swelling behavior of uncross-linked electro-spun gelatin (a) and cross-linked electro-spun gelatin (b) after immersing in the SPF solution for various periods of time

32

Fig 8 SEM morphology of cross-linked electro-spun gelatin after immersing in the SPF solution for 30 min (a), 120 min (b), 720 min (c) and 1440 min (d) 33

Chapter 3

Fig 1 Rapid Engineering Autologous Blood Vessel 35

Study I: The effect of cross-linking on the microstructure, mechanical properties and biocompatibility of electrospun polycaprolactone- gelatin/PLGA-gelatin/PLGA-chitosan hybrid composite

Fig 1 Schematics of double-ejection electrospinning (A), triple-layer artificial blood vessel scaffold (B) and crosslinking process (C) 40 Fig 2 SEM morphology of the outer layer of the artificial blood vessel: chitosan (a), PLGA (b), PLGA-chitosan (c) and cro-PLGA-chitosan scaffolds (d) 41 Fig 3 SEM morphology of the intermediate layer of the artificial blood vessel: gelatin (a), PLGA (b), PLGA-gelatin (c) and cro-PLGA-gelatin scaffolds (d) 41 Fig 4 SEM morphology of the inner layer of the artificial blood vessel: gelatin (a), PCL (b), PCL-gelatin (c) and cro-PCL-gelatin scaffolds (d) 42 Fig 5 Artificial blood vessels with different inner diameters (a), cross-section (b), the three layers of the PCL-gelatin/PLGA-gelatin/PLGA-chitosan fibrous tube stained in different colors (c,d) and the bilayers of the PCL-gelatin/PLGA-chitosan fibrous tube (e)

42

Fig 6 Stress-strain curves and corresponding SEM morphology of PCL- 43

gelatin/PLGA-gelatin/PLGA-chitosan ABV scaffold: non-crosslinked (a1-2) and crosslinked (b1-2)

Fig 7 Burst strength of non-crosslinked and crosslinked gelatin/PLGA-chitosan scaffolds 43 Fig 8 Cytotoxicity of electrospun PCL-gelatin, cro-PCL-gelatin, gelatin-PLGA , cro-gelatin-PLGA, PLGA-chitosan and cro-PLGA-chitosan scaffolds 44 Fig 9 Optical absorbance of fibroblast cells seeded on PLGA-chitosan, cro-PLGA-chitosan and TCPS or endothelial cells seeded on PCL-gelatin, cro-PCL-gelatin and TCPS after 1, 3 and 5 days of incubation

Study II: A Hybrid Electrospun PU/PCL Scaffold Satisfied the Requirements

of Blood Vessel Prosthesis in terms of Mechanical Properties, Pore Size and Biocompatibility

Fig 1 SEM morphology of electro-spun PU (a), PCL (b) and hybrid PU/PCL (c)

Fig 2 Photograph of electrospun hybrid PU/PCL tube (a); SEM morphologys of inner surface (b), outer surface (c) and cross-section (d, e) of electrospun PU/PCL tube

54

Fig 3 FT-IR spectrum of electro-spun PU, PCL and PU/PCL membranes 55 Fig 4 Pore size distribution of PU (a), PCL (b) and PU-PCL (c) tubes 55 Fig 5 Photograph of water droplet and contact angle on the electrospun PU (a), PCL (b) and hybrid PU/PCL (c) membranes 56 Fig 6 Stress-strain curves of electro-spun PU (a), PCL (b) and hybrid PU/PCL (c)

Fig 7 Pressure strength of electro-spun PU, PCL and PU/PCL tubes 56 Fig 8 Cell viability of electrospun PU, PCL and hybrid PU/PCL membranes 57 Fig 9 Optical density of fibroblast and endothelial cells respond on electrospun

Fig 10 SEM images showing attachment and proliferation of fibroblast cells on TCPs (a1-d1) and electrospun PU/PCL (a2-d2) depending on the incubation times

1 hour (a), 1 day (b), 3 days (c) and 5 days (d)

57

Fig 11 SEM images showing attachment and proliferation of endothelial cells on TCPs (a1-d1) and electrospun hybrid PU/PCL membrane (a2-d2) depending on the incubation times, 1 hour (a), 1 day (b), 7 days (c) and 14 days (d)

58

Fig 12 Immunofluoresence images of fibroblast cells adhesion on TCPs (a1, b1) and electrospun hybrid PU/PCL membrane (a2-4, b2-4) for 1 day (a1-4) and 2 days (b1-4)

58

Fig 13 Immunofluoresence images of endothelial cells adhesion on TCPs (a1, b1) and electrospun hybrid PU/PCL membrane (a2-4, b2-4) for 1 day (a1-4) and 2 days (b1-4)

59

Fig.14 Immunofluoresence images of endothelial cells adhesion on inner surface and fibroblast cells on outer layer of PU/PCL tube for 3days (a1-2,), 10 days (b1-2) and 1 months (A,B)

adhered on electrospun PCL/PU (a1-2) and PCL/PU-CLA (b1-2) membranes

Fig 4 confocal morphology of platelets adhered on electrospun PCL/PU (a) and PCL/PU-CLA (b) membranes seeded endothelial cells for 14 days 67 Fig 5 Photographs of whole blood clotting on electrospun PCL/PU (a1, b1, c1) and PCL/PU-CLA (a2, b2, c2) for 15, 30 and 60 minutes (a, b and c) membranes 67 Fig.6 SEM morphology of whole fresh blood adhered on electrospun PCL/PU (a)

Fig 7 Absorbance of whole blood cells clotted on PCL/PU and PCL/PU-CLA 68 Fig 8 Absorbance of whole blood cells hemolyzed by PCL/PU and PCL/PU-CLA 68

Chapter 4

Fig 1 Schematically process of tissue-engineered bone 72

Fig 3 Fabrication Process for Bone Substitute 72

Study I: In-Vitro and In-Vivo Studies of rhBMP2-Coated PS/PCL Fibrous Scaffolds for Bone Regeneration

Fig 1 SEM images of (a1) PS fibrous scaffold, (b1) PCL fibrous scaffold, (c1) PS/PCL fibrous scaffold and (d) diagram of fiber diameter of PS/PCL fibrous scaffold, PS fibrous scaffold and PCL fibrous scaffold Arrows in (a,b and c) indicate the distance of fiber-fiber

79

Fig 4 SEM (a) and BSEM (b) morphology of PS/PCLBMP2; the inserted image is

at low magnification; and the profile of rhBMP2 released from PS/PCLBMP2 fibrous scaffold (c) The amount of rhBMP-2 released from fibrous scaffold was determined by ELISA Arrows in (a and b) indicate the rhBMP2 presence

81

Fig 7 SEM micrograph of osteoblast cell proliferation on control (a1,b1,c1), PCL (a2,b2,c2), PS (a3,b3,c3), PS/PCL (a4,b4,c4) and PS/PCL-BMP2 (a5,b5,c5) fibrous scaffold after 1 day (a1-5), 3 days (b1-5), 7 days (c1-5)

81

Fig 8 Osteoblast proliferation on the control, PS, PCL, PS/PCL and BMP2 fibrous scaffold using MTT assay; *p<0.05 82 Fig 9 Cell proliferation of mesenchymal stem cells on control (a), PS/PCL (b) and PS/PCL-BMP2 (c) fibrous scaffolds after 7 days cultured DAPI (Red color for staining nuclear); Phalloidin (Green color for membrane)

PS/PCL-83

Fig 10 X-ray irradiation profiles (A), Sigittal single images (B) and dimensional structures (C) of defect (control), PS/PCL, PS/PCL-BMP2 and native skull after 8 weeks implantation

Three-83

Fig 11 Quantitative analysis of new bone formation on PS/PCL and PS/PCLBMP2 compared to the control and native bone by analyzing micro-CT results

84

Fig 12 Hematoxylin & eosin staining of the control (a,b), PS/PCL fibrous scaffold (c,d), PS/PCL-BMP2 fibrous scaffold (e,f) and native skull (g,h) with their enlarged image at the center and the edge after 8 weeks implantation The asterisk indicated osteoid formation; NB=New Bone

85

Fig 13 Masson’s Trichrome staining of the control, the PS/PCL fibrous scaffold, PS/PCL-BMP2 fibrous scaffold at the center and the edge after 8 weeks implantation; NB: new bone; the blue color indicates collagen formation inside the new bone; the black arrows indicate the electrospun fibers; NB=New Bone

86

Study II: Distribution of BCP in PCL/PLGA Porous Scaffold to Enhance Osteoblastic Differentiation of Human Mesenchymal Stem Cell and Osteogenesis in Rabbit Defect Model

Fig 1 Schematic processing diagram of PCL/PLGA-BCP scaffold (1) Slurry of PCL/PLGA with or without BCP in DMF/THF/MC was stirred over night inside hole (2) Mixture slurry was casted in the mold for 2 days connecting to vacuum (3) The block typed scaffold were stirred in water for 2 hours

90

Fig.2 SEM morphology, EDS and micro-CT slices of PCL/PLGA (a, c, e and g), PCL/PLGA-BCP (b, d, f and h) scaffolds 94 Fig 3 XRD profile of PCL/PLGA, PCL/PLGA-BCP scaffolds and BCP powder 95 Fig 4 Diagram (a) of Photographs of contact angle of PCL/PLGA (b) and PCL/PLGA -BCP (c) scaffolds (with video supplement data) ANOVA was used to compare the contact angle degree of with and without BCP loaded PCL/PLGA porous scaffold * P < 0.01

95

Fig 5 Cell proliferation on the tissue culture plate, PCL/PLGA and BCP using MTT assay ANOVA was used to compare the cell proliferation among the 1, 5 and 15 weeks * P < 0.05, ** P < 0.01, *** P < 0.005 vs 1 week

97

Fig 9 Coronal (a) , Transaxial (b) and Sagittal (c) images of micro-CT slices and 3D images of the bone defect (3 mm in diameter, 4 mm depth), through the cortical bone surface into the cancellous bone in the medial epicondyles of bilateral femora

in rabbits

98

Fig 10 Micro-CT slices of PCL/PLGA and PCL/PLGA-BCP implanted for 2 months (a,b) and 8 months (c,d) 98 Fig 11 (A) New bone volume to total volume ratio (BV/TV, %) of each group evaluating the quantity of new bone (B) New Bone BMD (mg/cm3) of each group

to evaluate the strength of new bone ANOVA was used to compare the bone formation value after between BCP with and without loaded PCL/PLGA porous scaffold between 2 and 8 months implantation * P < 0.05 vs 2 months

99

Fig 12 Histological analysis, H&E staining (a,b) and MT staining (c,d), of PCL/PLGA (a,c) and PCL/PLGA-BCP (b,d) after 2 months implantation Arrows indicate the bone tissue formation surrounding the scaffold S character indicate the remain scaffolds

99

Figure 13 Histological analysis, H&E staining (a,b) and MT staining (c,d), of PCL/PLGA (a,c) and PCL/PLGA-BCP (b,d) after 8 months implantation S = scaffold, NB = new bone

100

Study III: Comparison of the effect osteoinductive (RGD, BMP2) and osteoconductive (BCP) agents to bone remodeling of poly vinylalcohol/gelatin hydrogel matrices

Fig 1 SEM morphology (a,b) and pose sires distribution diagram (c,d) of 107

uncrosslinked PVA/gelatin (a,c) and crosslinked PVA/Gelatin (b,d) hydrogel

Fig 2 FT-IR spectrum of uncrosslinked (a) and cross-linked (b) PVA-Gel

Fig 3 SEM morphology of H (a) and H-BCP (b); EDS profile of HBCP (c) Vonkossa- staining of HBCP (d) and slice (e) and 3-D micro-CT image of HBCP (f)

108

Fig 4 BMP-2 release from H and HBCP with various compositions Cumulative

Fig 5 A, Photographs of hydrogels immersed in media during cell culture process Scale bar 5 mm; B, Confocal images of MSC on crosslinked hydrogel taken at different magnifications for 1 week of culturing; (b) is enlarged from image R-zone and (c) is enlarged from Q-zone

109

Fig 6 SEM images of mesenchymal stem cell on control (a), H (b), HBCP (c), RGD (d), H-BMP2 (e) and HBCP-BMP2 (f) 110 Fig 7 Influence of growth factors on gene expression and matrix deposition of hBMSC cultured osteoinductive conditions on H, BCP, HRGD, HBMP2 and HBCPBMP2 compared to cover slips control Immunofluorescence microphotographs of BMP2 (red), F-actin (green), DAPI (blue) and their merger (yellow) deposition after differentiation of hBMSCs on cover slips over a period of

H-3 weeks Scale bar represents 50 μm

110

Fig 8 Influence of growth factors on gene expression and matrix deposition of hBMSC cultured osteoinductive conditions on H, BCP, HRGD, HBMP2 and HBCPBMP2 compared to cover slips control Immunofluorescence microphotographs of ABS (red), COL-I (green), DAPI (blue) and their merger (yellow)deposition after differentiation of hBMSCs on cover slips over a period of

3 weeks Scale bar represents 50 μm

111

Fig 9 Influence of growth factors on gene expression and matrix deposition of hBMSC cultured osteoinductive conditions on H, BCP, HRGD, HBMP2 and HBCPBMP2 compared to cover slips control Optical images and ARS staining of after differentiation of hBMSCs on culture plate over a period of 3 weeks Scale bar represents 50 μm

112

Fig 10 An alizarin Red S assay of cetylpyridinium extraction from scaffolds after

3 weeks differentiating in osteogenetic medium from hBMSC 112 Fig 11 3-D image (a), Z-axis (b), Y- axis (c) and X- axis (d) of mico-CT data of the bone defect (3 mm diameter, 3 mm depth) 113 Fig 12 Photographs of operation periods: rabbit’s distal epiphysis inserted crosslinked hydrogel (a), harvested after 1 week (a), 5 weeks (b) and 15 weeks (c) Dash cycles indicate the post-implantation areas

114

Fig 14 Bone volume to total volume (BV/TV) (a) and bone mineral density (b) of the implanted hydrogel at different surgery times (1 week, 5 weeks, 15 weeks), comparison in natural bone and the control ANOVA was used to compare the expression among the 1, 5 and 15 weeks * P < 0.05, ** P < 0.01 vs 1 week

115

Fig 15 H&E staining of the post-implantation after 1 week, 5 weeks, 15 weeks, using natural bone as positive control Scale bar 1 mm And the diagram show hydrogel degradation rate during implantation Dash squares indicated the post-implantation areas

116

Fig 16 Hematoxylin/eosin stained histological cross-sections of hydrogel 5 weeks after implantation (a); b,c and d are enlarged images of R, Q and P zones, 116

respectively, post-implantation Scale bar 100 µm Cap, Tr and BV mean capillaries, trabecular and blood vessel Dash square indicate the post-implantation Fig 17 Masson’s Trichrome stained histological cross-sections of hydrogel 5 weeks after implantation (a), b,c and d are enlarged images of R, Q and P zones post-implantation Scale bar 100 µm NB means new bone Dash square indicate the post-implantation

117

Fig 18 Hematoxylin/eosin stained histological cross-sections of hydrogel 15 weeks after implantation (a); b,c and d are enlarged images of Q, R and P zones post-implantation Scale bar 100 µm Cap, NB and BV mean capillaries, new bone and blood vessel Dash square indicate the post-implantation

117

Fig 19 Masson’s Trichrome stained histological cross-sections of hydrogel 15 weeks after implantation (a), b,c and d are enlarged images of Q, R and P zones post-implantation Scale bar 100 µm Cap, BV mean capillaries, and blood vessel Dash square indicate the post-implantation

117

Fig 20 Hematoxylin and Eosin staining of the defect of H, HBCP, RGD,

ABSTRACT

Fabrication, In-vitro, In-vivo Evaluation of Hybrid Biopolymers

for Artificial Soft and Hard Tissue Application

Nguyen Thi Hiep

Department of Biomedical Engineering and Materials,

College of Medicine, SoonChunHyang University,

Cheonan, Korea

(Supervised by Professor Byong-Taek Lee)

Tissue engineering has emerged as a promising alternative approach in the treatment of malfunctioning or lost organs The design strategies of fabricating the scaffold is needed

to investigate to create the functional scaffolds The scaffold should be akin the tissue about the structure, mechanical properties and biocompatible in invitro and invivo Therefore, the electrospun membranes might be good for applying in skin, blood vessel and skull using electrospinning method However, the porous structure is important for bone defect regeneration so that the hydrogel and slurry methods were employed to create the large pore-size scaffolds In addition to facilitating cell adhesion, promoting cell growth, and allowing the retention of differentiated cell functions, the scaffold should

be modified with osteoinductive/osteoconductive agents A number of scaffolds fabricated from various kinds of biodegradable materials such as polyvinyl alcohol, gelatin, chitosan, polyurethane, polycaprolactone, poly (lactic-glycolic acid), and of non-degradable material (polystyrene) have been developed In order to accelerate healing process, mineral (bicalcium phosphate), and growth factors (rhBMP2, RGD) were added Varies methods were employed to evaluate the mechanical properties as tensile strength, burst strength, compressive strength And, in order to characterize scaffolds, XRD, contact angle, FT-IR, SEM, BSEM, TEM, HTEM, the swelling ratio, the degradable ratios, the rhBMP release, hemostatic of platelet and whole blood adhesion were employed In vitro studies, the cytotoxicity and cell proliferation had done with MTT

assay method while SEM observation and confocal viewer methods were employed to observe cell attachment, spreading and proliferation using osteoblast cell (MG63), fibroblast cell (L-929), endothelial cell (CPAE), rabbit mesenchymal stem cell, human mesenchymal stem cell, human chondrocyte stem cell In vivo studies, the micro-CT or the histological stains (Hematoxyline & Eosin, Massion’s Trichrome) and immunology stain were employed to observe the bone ingrowths in the bone defect of the rabbits or the rats

Chapter 1

General Introduction

1.1 About Tissue Engineering

Organ and tissue loss or failure resulting from an injury or other type of damage is a major human health problem Tissue or organ transplantation is a standard therapy to treat these patients, but this is severely limited by donor shortage Other available therapies including surgical reconstruction, drug therapy, synthetic prostheses, and medical devices are not limited by supply, but they do have other problems For example, synthetic prostheses and medical devices are not able to replace all the functions of a damaged or lost organ or tissue The efforts to address these problems and limitations have elicited the development of new biomaterials and alternative therapies

Tissue engineering is the use of combination of cell, engineering, materials method and suitable biochemical and physio-chemical factors to improve or replace biological function [1] Tissue engineering has emerged as a promising alternative approach to treat the loss or malfunction of a tissue or organ without the limitations of current therapies Tissue engineering is a new development in biomedicine, involving a series of strategies using biologically based mechanisms to repair and heal damaged and diseased tissue The key elements include a specific living cell type (or several cell types), a material scaffold that forms a supporting structure for culturing the cells in vitro and surgical delivery in vivo to the patient, and, for the majority of mammalian cell types, a growth stimulus Similar to expand cells from a small biopsy, follow by the culturing of the cells in temporary three-dimensional scaffolds to form the new organ or tissue as shown in Figure 1 By using the patient's own cells, this approach has the advantages of autografts, but without the problems associated with adequate supply With this approach, porous three-dimensional temporary scaffolds play an important role in manipulating cell function and guidance of new organ formation Isolated and expanded cells adhere to the temporary scaffold in all three dimensions, proliferate, and secrete their own extracellular matrices, replacing the biodegrading scaffold

Fig 1 (A) The tissue engineering cycle, using autologous cells 1: A small number

of cells are

removed from the body 2: They are screened for phenotype and increased in number through proliferation 3: These cells are seeded onto porous scaffolds together with growth factors to enhance proliferation 4: The seeded scaffolds are placed in culture to further increase cell number 5: Finally, the regenerated tissue is implanted into the site

of damage to integrate with the natural tissue (B) Example of tissue-engineered cartilage

in the shape of a human ear

1.2 The Ideal of Biomaterials

A variety of different biomaterials are currently being used as scaffold for reconstruction

of soft (such as artery or skin) or hard (bone) defects Ideally, scaffolds for tissue engineering should meet several design criteria: (1) the surface should permit cell adhesion, promote cell growth, and allow the retention of differentiated cell functions; (2) the scaffolds should be biocompatible, neither the polymer nor its degradation by-products should provoke inflammation or toxicity in vivo; (3) the scaffold should be biodegradable and eventually eliminated; (4) the porosity should be high enough to provide sufficient space for cell adhesion, extracellular matrix regeneration, and minimal diffusional constraints during culture, and the pore structure should allow even spatial cell distribution throughout the scaffold to facilitate homogeneous tissue formation; (5) the material should be reproducibly processable into three-dimensional structure, and mechanically strong

To harness the regenerative capability of cells requires an intricate understanding

of the signals that stimulate the different cellular responses that lead to natural tissue genesis and regeneration Different types of scaffold provide cells with different cues These cues are presented both by the macroscale topology of the scaffold (see figure 2) as well as the nanoscale surface features and surface chemistry

Fig 2 Different scaffold architectures provide cells with different topological cues.(A) A foamed or salt leached scaffold with large interconnected pores to permits cell ingrowth presents cells with a large, continuous surface area for attachment (B) Micro-fibrous scaffolds can be made to have greater porosity Where there is a large gap between fibres, focal adhesion placement guides cells to elongating along the fibre and this may restrict the contact with cells on different fibres (C) Nanofibre scaffolds have a larger surface area in relation to mass and can be fabricated with high

porosity Focal adhesion size is limited by the fibre diameter, reducing a cell’s ability to develop a strong cytoskeleton such that cells may remain more rounded and stationary within the scaffold

1.3 Objective of this dissertation

The undertaken researches were focused to develop new hybrid biopolymers for skin,

blood vessel and bone applications with improved biocompatibility and performance As

a foundation work of the desired goal new hybrid polymers was applied for making

scaffold with improved biocompatibility and mechanical properties The main focus of

the works was using different methods (electrospining, hydrogel, solvent evaporation,

microwave irradiation etc) to the investigation of different structure of scaffolds to mimic

specific extraccellulose matrices of specific organs (skin, blood vessel or bone substitute)

Attaining high anti-organisms together with superior biocompatibility was target of an

attificial skin Attaining high burst strength together with superior endothelialization was

target of an attificial blood vessel And, Attaining high porosity/large pore sizes together

with superior biocompatability was target of a bone substitute Three different organs

includes skeleton structural, circulatory system and skin were investigated in this

dissertation

Chapter 2- Tissue Engineering of skin

2.1 Introduction

Skin loss can lead to death or to an unacceptable quality of life, and effort of tissue engineers to synthesize skin have focused on that vital clinical need The approaches used have varied from those in which an organoid that mimics skin is synthesized in vitro (cultured epidermal autograft, living skin equivalent, living dermal replacement) to those

in which a biologically active scaffold, optionally seeded with keratinocytes, is implanted (dermis regeneration template) at the desired anatomical site to achieve in-vivo synthesis (regeneration) In addition to traditional approaches such as split- or full-thickness skin grafts, tissue flaps and free-tissue transfers, skin bioengineering in vitro or in vivo has been developing over the past decades Currently, both engineering and life sciences toward the development of substitutes to restore and maintain skin structure and function These methods are valuable alternatives or complements to other techniques in reconstructive surgery Fig 1 shows a principle of fabrication of artificial skin composed

of polymer fiber-layer and functional cell-layer in order to mimic the structure of a natural skin

Fig 1 schematically process of tissue-engineered skin

2.2 Study I: Nano Ag Loaded PVA Nano-Fibrous Mats for Skin Applications

2.2.1 Introduction

Wound healing is a complex processes which include rebuilding the structure and functions of the skin However, bacterial overgrowth and infection disrupts this wound-healing process Therefore, an artificial matrix is fabricated to promote wound healing that has the ability to prevent microbial infection In this study, we fabricated silver nano-particles (Ag NPs) loaded electrospun PVA mats (PVA-Ag mats) through the combination of electro-spinning microwave and heat treatments Ag NPs resurface on the surface of the PVA fibers by a thermic process This mat has three five advantages for skin application: First, Silver (Ag) and its compounds have been known to have extraordinary bacterium-inhibitory and bactericidal properties [2-7] The silver ion exhibits broad-spectrum biocidal activity toward many different bacteria, fungi, and viruses[6, 8] and is believed to be the active component in silver-based antimicrobials

On the other hand, Ag has low toxicity toward mammalian cells [9] Because of these benefits, Ag has been widely studied as a coating material on particular medical devices, such as indwelling catheters, wound dressing, etc [10-12] However, Ag ions or its components has limited usefulness as an antimicrobial agent for several reasons which reported by Kim et al [5] These limitations can be overcome by the use of Ag NPs Besides, it is important to enhance the antimicrobial effect Second, PVA is a good host material for metal, due to its excellent thermo-stability and chemical resistance[13, 14] Moreover, PVA is non-toxic, has high mechanical stability and is degradable in human environments On the other hand, PVA contains alcohol groups that could reduce Ag+ to

Ag0 without the need for a reductant as reported by Pal et al [15, 16] Besides, using PVA as a reductant agent is cost-effective method Third, electro-pinning is the simplest method to fabricate a continuous fiber of small diameter (nano-fibers), high surface area, small pore size and good mechanical property [17, 18] Due to all of the advantages described above, PVA-Ag mat has been investigated for use in wound pressing [18] and antimicrobial applications [19] Fourth, microwave irradiation is interested in method for fabricated nano-metal in present As microwave is an environmentally friendly green method that is much faster, simpler and more economical than conventional methods [14, 16] such as chemical reduction NABH4 [20] Therefore, PVA-Ag was fabricated using four above advantages in this investigation However, when this approach was used Ag NPs were dispersed inside the electro-spun PVA fiber, which limited the antimicrobial

activity of the fiber Finally, to resurface Ag NPs on PVA nano-fibers, heat treatment process was applied This approach was different from the method used by Hong et al., where a microwave step was used instead of UV irradiation More over, we found that several more nano-particles were homogeneously dispersed on the surface when the heat treatment step was conducted at 150 0C The presence of Ag NPs was confirmed by UV-vis, XRD, SEM, and TEM Interactions between PVA and Ag NPs were examined by FT-IR The thermal-crystalline properties of the mat were assessed by DSC In addition, the mechanical properties showed that the mat had high mechanical stress, which is important for wound healing applications The antibacterial test showed that the mats had high antimicrobial activities against both Gram-positive and Gram-negative, which is important to decrease infection during the wound healing process By this investigation, PVA-Ag mat showed that it can have not only good mechanical property, but also the antimicrobial activity to further eliminate the risk of infection

2.2.2 Experimental and Methods

Polyvinyl alcohol (PVA, full hydrolyzed) and AgNO3 (99,998 per cent) were purchased Aldrich Co., USA)

Preparation of Ag NPs loaded electrospun PVA mats using combination of microwave and electro-spinning methods

We fabricated PVA-Ag mats from a PVA solution containing Ag NPs This suspension was synthesized using a PVA solution that contained AgNO3 irradiated microwave Firstly, 12 wt % PVA in distilled water was prepared at 80 ºC Then, an aqueous solution

of 0.1ml 1M AgNO3 in distilled water was added and irradiated for 60 or 90 seconds in a microwave oven (LG Electronics Co., Korea) At that time, the color of the PVA solution started to change gradually from an achromatic color to faint yellow to brownish red at 60 and 90 seconds of irradiation, respectively, along with a reduction of Ag+ to Ag0 Then, the PVA containing Ag0 solutions were placed in a plastic syringe and connected through a metal syringe needle The solutions were electro-spun directly to a high voltage power supply (NNC–30 kilovolts–2mA portable type, Korea) A grounded steel cylinder,

10 centimeters away from the tip of the syringe needle was used to collect the nano-fiber mats The flow rates (ml/hr) of the PVA solutions were controlled by a syringe pump (lure-lock type, Korea) To resurface Ag NPs on PVA fibers, heat treatment process was used at 80oC, 120oC and 150oC

Characterization of PVA nano-fibrous mat loading Ag NPs

The detailed microstructure of the PVA nano-fiber mats loading Ag NPs was observed by scanning electron microscopy (SEM) (SM-65F, JEOL, Japan) and transmission electron microscopy (TEM) (JEM2010, JEOL, Japan) Ag NPs were conformed by high-resolution transmission electron microscopy (HRTEM) The average diameter of the electro-spun nano-fibers was determined by analyzing the SEM images using a custom code image analysis program

Absorption spectra of the Ag NPs dispersed in a PVA solution after microwave irradiatation was measured in the wavelength ranging from 300 to 800 nm using a UV-vis spectrophotometer (U2101 PC)

Differential scanning calorimetry (DSC) measurements of the PVA electro-spun, Ag NPs loaded PVA mat before heat treatment (PVA-Ag NPs-1) and PVA loading Ag NPs after heat treatment (PVA-Ag NPs-2) were performed using a DSC (METTLER TOLEDO KOREA-DSC822e) instrument in the temperature range from 0-250 ºC under a nitrogen atmosphere with a scanning speed of 5 ºC/min

X-ray diffractions (Rigaku, D/MAX-2500V) of Ag+ loaded PVA electro-spun and PVA-

Ag NPs-2 mat were acquired using Cu Kα radiation The diffraction angle was ranged from 5 to 1200 2θ

Fourier transform-infrared (FT-IR) spectroscopy analysis was performed on a Spectrum

GX PerkinElmer, USA The infrared spectra of the PVA electro-spun, PVA-Ag NPs-1 and PVA Ag NPs-2 were measured over a wavelength range of 4000-500 cm-1

Mechanical properties

The dimensions of PVA, PVA-Ag NPs-1 and PVA-Ag NPs-2 (wide 1 mm and length 27 mm) were measure by digital micrometer and thickness (100 µm) was measure by SEM before measuring the tensile strength Mechanical characterization was achieved by application of tensile test loads to the specimens prepared from the electro-spun ultra fine non-woven fiber mats Preparation of specimens and tensile strength testing were reported from our previous researches [21, 22]

Antibacterial tests were then carried out by the disc diffusion method [23] using 3.0 x 106

cfu/ml of Staphylococcus aureus and 4.0 x 106 cfu/ml of Escherichia coli The surface of

a Mueller-Hinton Argar (MHA, Becton-Dickinson) plate was inoculated with the bacteria

strains for testing using a sterile cotton swab Then, samples (1 mm x 1 mm) were spread onto a MHA plate After incubation at 37oC for 24 hours, the agar plates were examined for inhibition of bacterial growth In these experiments, the PVA mat was used as a control The antibacterial activity was evaluated by measuring the zone of inhibition against the test organisms

2.2.3 Results and Discussion

Characterization of PVA nano-fibrous mat loading Ag NPs

Figure 1 shows the SEM micrograph of electrospun PVA mat loading Ag NPs, which were fabricated under microwave irradiation for 60 seconds (Figure 1a) (sample A) and

90 seconds (Figure 1b) (sample B) Sample A showed homogenous nano-fibrous mats with fiber diameters ranging between 100-200 nm In contrast, Sample B showed non-homogenous nano-fibrous mats with diameters ranging between 100-500 nm For explanation of the more increase microwave irradiation time the more increase fiber diameter; water evaporation is the reason of increasing polymer solution concentration after increasing irradiation time due to the fiber diameter was increased [24] Figure 1c) and Figure 1d) are the EDS profiles of large square areas from sample A and sample B

As can be seen in these profiles, Ag as well as O and C elements of PVA were detected The percent of Ag was determined relative to only C, H, O and Ag The percent Ag in sample A was 9.93 % Ag This was higher than the percent Ag in sample B (5.73 %)

Fig 1 SEM micrographs of Ag NPs loaded electro-spun PVA mats with microwave 60 seconds (a), 90 seconds (b); c) and d) are EDS profile on the large area of (a) and (b)

To confirm that Ag NPs were present in the PVA solution after microwave treatment, UV-vis absorption spectra of PVA containing the Ag NPs solution treated with different microwave irradiation times were obtained (Figure 2) A maximum absorption located at 416 nm, which is a characteristic peak for Ag NPs, was observed in the PVA/Ag NPs solution The peak was found to be more intense when microwave irradiation was conducted for 90 seconds than when conducted for 60 seconds

Fig 2 UV-Vis absorption spectra of PVA/Ag solution with different microwave irradiation time

To confirm successful formation of Ag NPs through microwave irradiation, XRD profiles of AgNO3 loaded electro-spun PVA without and with microwave-irradiation were acquired (Figure 3) Typically, the XRD pattern

of the PVA nano-fibrous mat loading

Ag NPs had diffraction peaks at a 2θ of 38.2° and this peak could be indexed as (111), which is similar to the results reported by Galya et al who fabricated a PVA/Ag film for antibacterial applications [25] The sharp crystalline structure of electro-spun PVA with strong peaks at 2θ = 20.9º, and shoulders could be indexed as 2θ = 19.4º, 20.9, and 23.6º, which also displayed an XRD profile of both electro spun mats Similar findings on the XRD profiles were previously reported [26, 27] The peak 2θ = 9.2 of crystalline PVA-

Ag+ was high in the XRD profiles of electro-spun mats that were not irradiated, while no peak at 2θ = 38.2, which is characteristic of Ag NPs, was observed

In contrast, a peak at 2θ = 38.2 was observed for electro-spun mats that were irradiated [28] This result demonstrates that Ag and Ag+ added to PVA altered the

microwave-crystalline structure of electro-spun PVA The Ag NPs peak was remarkably weak due to the small number of Ag molecules This result was similar to the results we obtained in a previous study where we examined PMMA and Ag substrates [29] These combined results described above indicate that the Ag NPs were successfully dispersed in PVA nano-fibers using the microwave and electro-spinning method

Fig 3 XRD curves of AgNO 3 loaded electrospun PVA mat and Ag NPs loaded electrospun PVA mat

Electro-spun PVA fibers loading Ag NPs, which were subjected to microwave irradiation, were placed in an oven for heat treatment Electrospun PVA fiber loading Ag NPs mats

were then heated at the following temperatures, 80 ºC, 120 ºC and 150 ºC for 24 hours

Fig 4 SEM micrographs Ag NPs loaded electrospun PVA mats with microwave 60 seconds at 80°C (a), 120 °C (c), and 150 °C (e); with 90 seconds at 150 °C (g); (b), (d) (f) and (h) are enlarge image of (a), (c) (e) and (h)

Figure 4 (a, c, e) shows the SEM micrographs of PVA loading Ag NPs after

60 seconds of microwave-irradiation and heat treatment at different temperatures while Figure 4g shows a SEM micrograph

of electro-spun PVA loading Ag NPs mats heated at 150 ºC after 90 seconds of microwave irradiation Figure 4 (b, d, f, h) are three-fold enlarged images of Figure 4 (a, c, e, g) at X90 000 The heat treatment process was found to affect the morphology

of fiber, as reflected in the SEM images For instance, as shown in the comparison of Figure 4a and Figure 4c, under similar experiment conditions (60 seconds microwave irradiation, 24 hours heating), Ag NPs appeared on the fibers surface when the temperature was changed from 80ºC to 120 ºC Moreover, in the case of 150 ºC (Figure 4e), the Ag NPs that were deposited on the PVA fibers surface were larger in size than at 120ºC Figure 4g was added to compare the effect of microwave-irradiation on this

process A smaller number of Ag NPs were observed in Figure 4g compare with Figure 4e even though the samples were fabricated under similar conditions with the only difference being elimination of microwave-irradiation The combined results of these experiments indicate that the most optimal conditions for Ag NP resurfacing were 60 seconds of microwave irradiation and heat treatment at 150ºC Variations in the resurfacing process were shown in more detail in the enlarged images

Fig.5 TEM and HRTEM image of Ag NPs loaded electrospun PVA nano-fiber with 60 seconds microwave irradiation and heat at 150 °C

Figure 5 shows TEM and HRTEM images of PVA electro-spun fiber loading Ag NPs fabricated using 60 second microwave irradiation and then 150 ºC heat treament TEM

images show that Ag NPs loaded electrospun PVA nano fibers was aggregated from a smaller size (5-15 nm) to larger size (around 100nm) (Figure 5) The Ag NPs were spherical dispersed on the surface of the fibers Figure 5a shows resurfacing and aggregation of Ag NPs on the PVA fiber Aggregation of Ag NPs was shown more clearly in the HRTEM image (Figure 5b), the large spherical structures resulted from the aggregation of smaller Ag NPs (5~10 nm)

Fig 6 FT-IR curves of PVA powder,

Ag NPs loaded electro-spun PVA mat before and after heat at 150 °C

To examine the interaction between PVA and Ag, the FT-IR spectrum of PVA powder, PVA-Ag NPs-1 and PVA-Ag NPs-2 were acquired (Figure 6) A similar approach was used by Mbhele et al [30] The main characteristic peaks of PVA were assigned as follows: skeletal vibrations (814 and 916 cm-1); -CH2-wagging (1376 cm-1); and -C-H- and -O-H- bending (1328 cm-1) The peak at 1096 cm-1 was the attributed to the C-O stretching vibrations of the remaining non-hydrolysed vinyl acetate group of PVA [31, 32] The peak at 1420 cm-1 is the result of the coupling of the O-H in plane vibration with the C-H wagging vibration [33] The FT-IR spectrum of PVA-Ag NPs-1 and PVA-Ag NPs-2 are shown in Figure 6 The decrease in the ratio of 1420 cm-1 relative

to the presence of Ag NPs, indicates decoupling between the corresponding vibrations due to interactions between Ag NPs and the –OH groups of the PVA chains This finding was reported in previous studies [33-35] This group also reported that the band peaking

at 1141 cm-1, which occurs due to symmetric C-C stretching, corresponds to the crystalline regions in PVA [33] The peak at 1255 cm-1 was reported to be associated with C-O-C vibrations and indicates cross-linking of some PVA radicals [36]

Thermal characterization of the PVA powder (a), PVA electro-spun (b), PVA-Ag mats without (c) and with (d) heat treatment were carried out in the temperature range of 0 -

450 ºC at a heating rate of 5 ºC per minute in nitrogen Based on the thermogram

presented in Figure 7, glass transition temperatures, Tg, (determined by the ASTM midpoint technique using STARe software) were observed at 75.20 ºC and 73.39 ºC for PVA powder and the electro-spun PVA mat, respectively Meanwhile, the melting point,

Tm, of PVA and the electro-spun PVA mat were observed at 226.84 ºC and 225.02 ºC, respectively In the case of loading Ag NPs before and Ag NPs loaded electro-spun mat

after heat treatment, no Tg was observed, but Tm were observed at 211.84ºC and 197.54

ºC, respectively

Fig 7 DSC curves of PVA powder (a), electro-spun PVA (b), Ag NPs loaded electro- spun PVA 7 before heat treatment (c) and after heat treatment (d)

Antibacterial activities

To test the antibacterial activity of the sample, the disc diffusion method was used to assess

activity against S aureus and E.coli The zone

inhibition was given in Table 1 The test was repeated four times, for each sample Figure 8 shows the inhibition microbial activity of pure PVA, PVA-Ag NPs-1, PVA-Ag NPs-2 (120 ºC) and PVA-Ag NPs-2 (150 ºC) electro-spun fibrous mats Results showed that a pure PVA, no zones of inhibition were observed Figure 8(b, f), Figure 8(c, g) and Figure 8(d, h) showed high the antibacterial

activity against S aureus and E.coli on all Ag NPs loaded electro-spun PVA fibrous mats,

respectively There was a slight increase in the zones of inhibition when the mat was heated at 120 ºC, which increased for the mat heated at 150 ºC The zone created by the PVA loading Ag NPs nano-fibrous mat placed on the bacteria-inoculated surface killed all S aureus bacteria under and around the samples The PVA Ag NPs-2 mat showed the strongest anti-microbial activities against gram-positive S aureus and gram-negative E.coli and the gram-positive S aureus was more sensitive then gram-negative E coli

Fig.8 Comparison

of the inhibition zone test between a, e) pure PVA mat; b, f) PVA-Ag mat; c, g) PVA-Ag NPs-2

(120℃); d, h) PVA–

Ag NPs-2 (150℃) The photographs of the disk sensitivity test for E coli (a, b, c, d) and S aureus (e, f, g, h) Table 1 showed that PVA-Ag NPs-2 (150 ºC) mat was highest against S aureus and E.coli with increasing zone inhibition However, the gram-positive bacteria (S aureus) was more sensitive than the gram-negative bacteria (E coli) on all Ag NPs loaded electrospun PVA mats with larger zone inhibition

Table 1 Zone of inhibition test from the disc diffusion test

Mechanical properties

Fig 9 Tensile strength curves of spun PVA, Ag NPs loaded electro-spun PVA before and after heat treatment

electro-For skin applications, mechanical properties are also important Therefore, the tensile strength of electro-spun PVA mat, PVA-Ag NPs-1 and PVA-Ag NPs-2 (150 oC) with heat treatment at 150 oC were also investigated (Figure 9) The stress-strain curve of the PVA nano-fibrous mat changed after introduction of the Ag nano-particles Contrary to the pure PVA fibrous mat, where the stress increased after loading the Ag NPs, the nano-composite fibrous mats were stronger and more brittle than the pure PVA fibrous mat Without heat treatment, the stress increased from 9 MPa to 35 MPa, while with heat treatment, the stress increased to 47 MPa; however, the strain decreased from

23 % to 15 % for both cases These results correspond to those of reported by Mbhele at el.[30], who examined PVA-Ag NPs film (59 MPa for 0.73 wt% Ag) prepared by the evaporation method using NaBH4 as a reducing agent However, the percentage of Ag in this study was higher (9.93 wt% Ag was dispersed into the sample A), which caused a dispersed interruption of Ag NPs on the PVA fibers and resulted in a brittle PVA-Ag nano-fibrous composite mat compared PVA mat The stress-strain curves indicate that heat treatment had the following effect: the sample A after heat treatment showed increased tensile stress and brittleness compared with un-treated, but the tensile strain was unchanged for both cases (PVA-Ag NPs-1 or PVA-Ag NPs-2) Even though PVA-

Ag NPs-1 and PVA-Ag NPs-2 mats showed more brittle than PVA mat, its still have

potential for skin applications because those mat showed higher than tensile strength of mammalian (flank skin) [37]

Discussion

In this study, we used two steps to fabricate Ag NPs loaded electrospun PVA nano fibrous mats for skin applications First, the Ag NPs were dispersed on the electro-spun PVA nano-fibers through microwave and electro-spinning methods Then, the Ag NPs were resurfaced on the surface of the PVA nano-fibers using a heat treatment process In the case of loading Ag NPs, Ag NPs were dispersed and stabilized in the PVA solution before electro-spinning due to the reduction of the alcohol group [16] and the chelating ability of PVA [28, 38] Since PVA prevents aggregation and precipitation of the particles, the Ag+ was stabilized by PVA chelation and reduction through microwave-irradiation [28, 38-40] Therefore, PVA has been frequently used not only as a particle stabilizer but also as a reductant agent [41] The reduction of alcohol groups in PVA through microwave-irradiation is similar to the reduction of alcohol groups in PEG [39, 40] The reduction during the microwave-irradiation process was visualized by the naked-eye because the color of the PVA solution started to change gradually from an achromatic color to a faint yellow to brownish red after 60 and 90 seconds of irradiation, which was reported in previous studies [34] The color of the PVA solution was stable over the spinning time and the nanoparticles showed almost no tendency to aggregate because the

Ag NPs were capped by PVA chains The presence of dispersed Ag NPs in the spinning solution after microwave-irradiation, results in change solution color, was confirmed by UV-vis absorption (Figure 2) [16, 42, 43] This suspension was spun, resulting in the homogeneous dispersion of Ag NPs in the PVA nano-fibers Because dispersion of the Ag NPs was homogenous, the surface of the PVA fiber loading Ag NPs was smooth in both cases (Figure 1) However, the EDS profiles indicate that the PVA nano-fibrous mat contained different amounts of Ag (Figure 1c and Figure 1d) Therefore, even though the same amount of AgNO3 was added, the amount that was loaded varied (when irradiation time is different) This phenomenon occurred because of the effect of microwave-irradiation on the Ag NPs size Since microwave-irradiation time from 60 second to 90 second increased, larger Ag NPs were produced Bigger Ag NPs were heavy for PVA to carry and possible to precipitate during microwave and electro-spinning process XRD profiles confirmed that Ag NPs loaded electrospun PVA mats successfully through microwave irradiation (Figure 3) whereas the interaction between PVA and Ag with and without heat treatment was confirmed by FT-IR (Figure 6) and DSC (Figure 7) The results showed that the addition of Ag not only affected the structure of the PVA mat but also changed the crystalline structure and melting point of the mats

electro-In this study, an Ag NPs resurfacing process was investigated by varying the temperature

at the same microwave-irradiation time The effect of temperature is shown in Figure 4 When the temperature was changed from 80ºC, 120ºC and 150ºC, the number of Ag NPs

on the PVA surface also increased Based on the report by Hong et al [18], we believed this occurred because Ag and Ag+ might remain in a cluster while diffusing and then aggregate on the surface of PVA fibers during heat treatment Moreover, Luo et al [41] explained that PVA could be cross-linked and their chains could be cross-linked by Ag and Ag+ during hydrothermal treatment at 160ºC This report also described a potential mechanism by which Ag could be reduced by PVA and then aggregate with each other Combination of Hong et al and Luo et al reports, we can ensure that the Ag and Ag+diffusing from inside fiber and aggregate on the surface of PVA fibers base on temperature of oven However, by comparing the SEM images in Figure 4f and Figure 4h (enlarge of Figure 4e and Figure 4g), indicate that increasing the microwave-irradiated time from 60 to 90 second did not increase the number of Ag NPs This results are according to reported EDS profiles As shown in Figure 8, the antibacterial activity, as measured by the diameter of the growth inhibition zone, depended on the test sample used [44] In fact, 60 seconds of microwave irradiation followed by heating at 150ºC for

24 hours was found to be optimal for preparing the Ag NPs loaded electro-spun PVA electro-spun mat for skin because of biggest zone inhibition area and high tensile strength

2.2.4 Conclusion

In summary, the PVA-Ag NPs mat, which can be used to promote in wound healing, was fabricated from a suspension of PVA and Ag NPs after microwave-irradiation for 60 seconds The PVA-Ag mat was then subjected to heat treatment at 150ºC to resurface the

Ag NPs on the surface of the PVA fibers to increase the antibacterial properties of the mat The presence of Ag NPs was confirmed by SEM, TEM, XRD, EDS, FT-IR and DSC confirming that the mat was successfully fabricated using this approach The fabricated electro-spinning method produced a mat that has high tensile stress, which is beneficial for skin applications The Ag NPs loaded electrospun PVA displayed excellent

antimicrobial activity against gram-positive S aureus and gram-negative E.coli In this

study, we developed a novel method to fabricate Ag NPs loaded electrospun PVA mats, which not only has high tensile stress but also excellent anti-bacterial activities, using a combination of three simple methods; electrospinning, microwave and low aging temperature methods

2.3 Study II: Fabrication of Cross-linked Nano-fibrous Chitosan Membranes Using an Electro-spinning Process and Their Biocompatibility Evaluation

2.3.1 Introduction

Chitosan is a polysaccharide that is obtained by deacetylating chitin[45] Chitosan is used widely in biomaterials field because chitosan is formed by joining thousands of monosaccharides which are important in living organisms [46] This polysaccharide is nontoxic, has antibacterial properties, biodegradability and can be further enhanced by artificial culturing [47] Therefore, chitosan is considered in skin applications recently because it has several beneficial characteristics such as biocompatibility, biodegradability, high efficient antibacterial action [48, 49] The electro-spinning method is one of the simplest among all methods for the preparation of fibrous membranes as extra-cellular matrices Electro-spinning offers a new direction for biopolymers since this approach allows one to decrease the diameter of the pore size from micrometers to nanometers; thereby, increasing the surface area to volume or mass ratio and the mechanical properties of the polymer [50] The methods for fabricating chitosan electro-spun membranes can be divided into two categories: pure CS electro-spun membranes that can

be fabricated by dissolving chitosan in a pure acid, and chitosan mixed electro-spun membranes fabricated by blending with polyvinyl alcohol [51], poly (ethylene oxide) [52], polycaprolactone [53], etc Chitosan electro-spun membranes are of interest in biomedical applications because they can provide a highly porous scaffold to support and guide tissue growth [54] The highly porous nature of the nano-fiber scaffolds allows cell migration and growth as well as the transport of nutrients and metabolic waste Since the electros-spun pure chitosan fibers are dissolved in water, cross-linking the chitosan fibers

is an essential step for achieving insolubility in water [55] Glutaraldehyde is often used

as a crosslinker to improve the limited properties of chitosan, including their mechanical properties [56] In addition, the biomineralization properties of the crosslinked chitosan materials might be influenced by the degree of glutaraldehyde crosslinking because it affects the structure and tailored surface of chitosan materials [57] The crosslinked chitosan materials are soft and rubbery in the swollen state, resembling living tissue when immersed, and some also possess excellent biocompatibility [58] In this work, the fabrication of electro-spun chitosan membranes used the similar solvents, acetic acid (AA) and trifluoroacetic acid (TFA), as reported by De Vrieze et al [59] and Ohkawa K

et al [60], respectively The aim of this work, we investigated washed an electros-pun

chitosan membrane to supplement a high antibacterial substrate for skin application The washing procedure includes washing glutaraldehyde, acid and organic salt created during the cross-linking step The glutaraldehyde cross-linked and uncrosslinked chitosan electro-spun nano fibrous membranes were analyzed by scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR) The biocompatibility of the cross-linked membrane indicated non-toxicity according to a MTT assay and fibroblasts cell well attachment through SEM images This substrate will be coated by the hydrogel

to supplement the other factors as growth factor, drug delivery system (DDS) etc… on the next investigations

2.3.2 Materials and Methods

Chitosan (from crab shells, minimum 85 % deacetylation) and glycine were from Aldrich USA Trifluoroacetic acid (TFA, CF3COOH, 99.0%) and acetic acid (CH3COOH, glacial, 99.0 %) were purchased from Duksan Pure Chemical Co., Korea Glutaraldehyde was obtained from DeaJung Co., Korea Sodium hydroxide (NaOH) was supplied by SamChun Pure Chemical Co., Korea Fetal bovine serum (FBS), P.S (penicillin/streptomycin (antibiotics)), Dulbecco’s phosphate buffered saline (D-PBS) without calcium or magnesiumand, MTT solution and trypsin-EDTA were purchased from GIBCO (Carlsbad, CA) The L-929 cell line was obtained from the ATCC Cell Line (CCL-1TM, NCTC clone 929 [L cell, L-929, derivative of Strain L], Korea) HDMS (Hexamethyldisilazane, Sigma, USA), DMSO (Dimethylsulfoxide 99, 0%, Samchun Pure Chemical Co., LTD, Korea), ethanol (Merck, Germany) were used as received

Sigma-For Electro-Spinning Setting and Membranes Treatment

2 wt % and 5 wt % chitosan dissolved in different acetic acid concentrations (2 and 90

wt %) and heat at 50 °C In separate solutions, 2 wt %, 5 wt %, 7 wt % and 10 wt % of chitosan were dissolved in TFA for 24 hrs and connected directly to the nozzle For Electro-Spinning Setting, the chitosan solutions were placed in a plastic syringe (lure-lock type, 12 ml) fitted to a needle with a tip in diameter 0.25 mm The flow rate of the chitosan solutions (0.5 ml/h) was controlled using a syringe pump (lure-lock type, Korea) The electro-spinning voltage (25 kV) was supplied directly by a high DC voltage power supply (NNC–30 kilovolts–2mA portable type, Korea) A grounded steel plate located 15 centimeters away from the tip of the syringe needle was used to collect the nano-fiber membranes For Cross linking Process and Washing Procedure, the chitosan electro-spun nano-fibrous membranes were placed on aluminum foil, which were then covered with a beaker 25 % gutaraldehyde was added to the beaker, and the membranes were incubated

at 37 ºC for 1 hour The electro-spun cross-linked membranes were dipped in 0.1 M

glycine for 15 minutes followed by 1.0 M NaOH for 3 hours Cross-linked samples were dissociated in a 1.0 M NaOH solution and washed 3 times with distilled water

Characterization of Chitosan Membranes

The structure of the chitosan fibrous membranes with AA and TFA was observed by SEM (SM-65F, JEOL, Japan) The uncross-linked and cross-linked chitosan electro-spun membranes were characterized by attenuated reflectance Fourier transform spectroscopy (Spectrum GX, PerkinElmer, USA).The infrared spectra of the samples were measured over a wavelength range of 4000-500 cm-1

In-Vitro Study- Cell Line and Maintenance

The cell culture studies were carried out using L-929 mouse fibroblasts The cells were subcultured in flasks using RPMI media, supplemented with 10 % FBS and 1 % P.S and maintained at 37ºC in a humidified CO2 (5%) atmosphere (incubator, ASTEC, Japan) The cells were dissociated with trypsin-EDTA and centrifuged and resuspended in the culture medium The culture medium was replaced every 2 days Gamma sterilized (SPL Life Science, Becton Dickinson Labware) 12-well tissue culture test plates was prepared before the cell culture experiments The culture plates were then sprayed with 70 % ethanol before being placed under UV light for 30 min to sterilize them Chitosan fibrous membranes (washed and unwashed) had a circular shape, 20 mm in diameter

In-Vitro Study- MTT Assay/ Cytotoxicity Assay

The cellular viability and cytotoxicity of chitosan membranes were determined using MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) assays For cytotoxicity testing, the fibroblasts cells were seeded in 96-well tissue culture plates at

1000 cells/well in 100 μL RPMI Then tissue culture plate was incubated overnight The extraction solutions were prepared following ISO 10933-5 Diluted extract solutions (100 μL) of every fibrous membrane at various concentrations (100 %, 75 %, 50 %, 25 % and 0 %) were then added The L 929 fibroblast cells were treated for 3 days and then

20 μL of filtered MTT solution (5 mg/mL in PBS) was added For cell viability testing,

10 000 cells per well were seed on the electrospun membranes After 1, 3 and 5 day (s), the chitosan electrospun membranes were changed to a new plate, then MTT solution was added on the membranes After incubation at 37 °C for 3.5 hours, MTT solution was removed from the well and DMSO was added to dissolve any insoluble formazan crystals The absorbance was measured at 595 nm wave-length using an ELISA reader (Turner Biosystems CE, Promega Corporation, USA)

In-Vitro Study- Adhesion, Growth Behavior of Cells

For cell attachment and cell proliferation analysis, the fibroblasts cells L-929 (1x

104cells/cm2) were seeded on chitosan cross-linked and cross-linked-washed nanofibrous

membranes After culturing for 1 day, the cellular constructs were harvested, rinsed twice with PBS to remove the non-adherent cells, fixed with 2% glutaraldehyde for 30 min and washed twice with DPBS (15 min/ time) Subsequently, the samples were dehydrated with a series of ethanol Finally, the samples were washed twice with HDMS All samples were air-dried overnight The dry cellular constructs were sputtered with gold using a SPI-module Sputter Coater at 7 mA for 6 minutes and examined by SEM observation

2.3.3 Results and Discussion

Fabrication of Chitosan Nanofibrous Membranes

Acetic acid (AA) and TFA were used as solvents to fabricate the chitosan fibers After examining the electro-spinning conditions, the following variables were fixed: voltage 22

kV, distance 15 cm, rate of polymer solution 0.5 ml/h, and 25 gauge needle In the case of

AA, it was difficult to dissolve a large amount of chitosan Therefore, the highest chitosan concentration was 5 wt% In this experiment, different chitosan concentrations were made with different AA concentrations Fig.1a shows electrospun chitosan from 2 wt % chitosan in 2 % AA The image shows that only a polymer dense film was collected (no beads or fibers) The fibers contained many beads when the chitosan concentration was increased 5 wt % (Fig.1b) Unfortunately, the same was observed when the AA concentration was increased to 90 % and 2 wt % chitosan (Fig.1c) This was more serious when the chitosan concentration increased to 5 wt % (Fig.1d) Contrary to AA solvent, good quality fibers were obtained when TFA was used Fig.2 shows the SEM morphology of the chitosan electro-spun fibrous membranes with various concentrations Clearly, beads with a small number of fibers had formed at the lowest

concentration (2 wt %), as shown in Fig.2a Beads and a small proportion of fibers were observed from the solution with a slightly higher concentration (5 wt %), as shown in Fig.2b However, a small proportion of fibers were observed under the same electro-spinning and concentration conditions but with 7 wt % chitosan, as shown in Fig.2c

Fig.1 SEM morphology of chitosan nano-fibrous membrane; (a) 2 wt%_ 2 wt % AA, (b)

5 wt%_ 2 wt % AA, (c) 2 wt%_ 90 wt % AA and (a) 5 wt%_ 90 wt % AA

Ultrafine chitosan fibers were observed when the chitosan concentration was increased from 5 to 7 wt %, as shown in Fig.2c Beads appeared instead of ultrafine chitosan fibers with further increases in the chitosan concentration (9 wt %) (Fig 2d) Interestingly, all chitosan fibers were cylindrical, continuous and randomly oriented at 7 wt% chitosan

with 100 – 1000 nm fiber diameter When the chitosan concentration was varied slightly, the as-spun fibers were not uniform or cylindrical and there was an increase in the number of beads From these results, TFA was used as the solvent for fabricating the chitosan membranes during the electro-spinning process

Fig 2 SEM morphology of chitosan nano-fibrous membranes depend on chitosan

concentration 2 wt % (a), 5 wt % (b), 7 wt % (c), and 9 wt % (d)

Chitosan Electrospun Nanofibers Membrane Treatments

Fig 3 SEM morphology of chitosan nano-fibrous membranes; (a) uncrosslinked, (b) cross-linked (b), (c) cross-linked and treated glycerin, (d) washing in NaOH from sample (c)

The fibrous membranes were then linked with 25 % glutaraldehyde in the chamber, as described in the experimental section The activity of the aldehyde group

cross-in the cross-lcross-inked chitosan was quenched by 0.1 M glyccross-ine and washed with distilled water Fig.3a presents the best chitosan fibrous membrane mentioned in Fig.2c Fig.3b shows a change in morphology of the chitosan fibrous membrane from random, individual fibers and soft membranes to a tie membrane with a random circular morphology because cross-linking bonds the individual fibers with each other in all directions Fig.3d displays the change in the morphology of the chitosan fibrous membranes from Fig.3b after using glycine to quench the activity of the aldehyde group

of glutaraldehyde, as this group inhibits fibroblast cell division Fig.3c shows a sample that had been washed with a 1M NaOH solution Fig.3d shows the morphology of the final product of the chitosan fibrous membranes after washing in water The fibers can be seen more clearly than in Fig.3c because of the removal of some organic salt resin through the washing step The individual fibers are interconnected, making a stable

chitosan membrane that would be viable for biomedical applications SEM images showed that the morphology of the CS fibrous membranes changed after each treatment period Moreover, after the cross-linking and washing steps, the fiber membranes retained their integrity as long, randomly oriented, cylindrical fibers However, it is unclear if the

GA vapor coated or penetrated into the fibers from the SEM images

Characterization of Chitosan Membranes

To determine if the chitosan electro-spun fibers were successfully crosslinked, we analyzed the FT-IR spectrum of the bulk chitosan samples (a) and the electro-spun membranes before (b) and after cross-linking with glutaraldehyde (c) and chitosan-glutaraldehyde with glycine treatment (d) (Fig.4) Fig.4a showed the transmission spectrum of the bulk chitosan with 75 % deacetylation The peaks were similar to the

78 % deacetylation reported elsewhere [24] In Fig.4b, the FT-IR spectrum showed new peaks that could be used to determine the level of deacetylation of chitosan [7] Figs 4c and 4d show the spectra of the cross-linked chitosan electrospun and cross-linked chitosan electrospun and washed the spectra showed C=N stretching or amide I bands at

1655 and 1630 cm-1, an amide II band at 1560 cm-1, a bridge oxygen stretching band at

1160 cm-1, the CH2 group of glutaraldehyde 700-740 cm-1, and the C-O stretching bands

at 1070 and 1030 cm-1 All fibers were analyzed directly As a result of the cross-linking reaction, there were significant differences in the FTIR spectra between the as-spun and

cross-linked electrospun fibers The FTIR spectra of the cross-linked chitosan fibers showed a distinct change in the carbonyl-amide FTIR showed that the chitosan fibrous membranes were successfully cross-linked; all cross-linked samples were insoluble in water

Fig 4 FT- IR spectrum of chitosan bulk (a), chitosan nano-fibers membrane (b), cross-linked chitosan nano-fibers membrane (c), and cross-linked chitosan nano-fibers treated by glycine (d)

Cytotoxicity Assay

The cell viability in the presence of uncrosslinked chitosan, cross-linked chitosan, crosslinked chitosan treated with glycine, and cross-linked chitosan, crosslinked chitosan treated with glycine and then washed in water were examined using a MTT assay Fig.5 shows the relative cell viability of the chitosan fibrous membranes in accordance with the diluted extracted solution The results highlight the importance of the washing step L-

929 fibroblast cells were left to grow in the media for 1 day before adding the diluted extract solution, which resulted in significant variations in the toxicity profiles of the singular chitosan membranes The results showed high toxicity on the chitosan electro-spun membrane even though the chitosan bulk is biocompatible [45], chitosan electro-

spun cross-linked membrane, and chitosan electro-spun cross-linked membrane treated with glycine In contrast, the chitosan electro-spun cross-linked membrane was washed thoroughly, and showed either low

cytotoxicity or high biocompatibility

Fig 5 Cytotoxicity of uncrosslinked and crosslinked chitosan fibrous membranes depend on glycerin treatment and washing process

The cell viability profiles of the cross-linked chitosan membranes and those treated with glycine but not washed by DI water was considerably lower than those of the chitosan cross-linked, treated by glycine and washed in DI water The cross-linked electro-spun chitosan was insoluble in water but the chitosan electro-spun membranes displayed the lowest biocompatibility due to the presence of toxic agents, such as aldehyde and acid groups [61] The chitosan electro-spun membrane showed low biocompatibility and water solubility, even though chitosan is biocompatible and insoluble This was attributed

to the residual TFA in the latter case The MTT assay showed that carefully washed chitosan fibrous membranes can be used in biomaterial applications The washing step is

an important step because TFA is a strong acid Although all samples were placed in a vacuum oven at low pressure for several days, some TFA still remained in the samples,

as showed toxic

Cells Attachment and Cells Proliferation: The results of the MTT assay were confirmed

by the SEM images of the fibroblast cell L-929 attached to each membrane The one cell morphology was tested to continually check the biocompatibility of all chitosan membranes

Fig 6 SEM morphology of fibroblast cells attachment for 1 hour cultured (a) cross-linked chitosan fibrous membrane; (b) crosslinked chitosan membrane treated with glycine; (c) crosslinked chitosan membrane treated by glycerin and washed (c)