1 Enhanced patency and endothelialization of small- caliber vascular grafts fabricated by coimmobilization of heparin and cell adhesive peptides Lab 8 D 1 Zhang Yaxing 週報

2 Paper`s title and its translation in Japanese 2 Enhanced patency and endothelialization of small-caliber vascular grafts fabricated by coimmobilization of heparin and cell adhesive peptides ヘパリンと細胞接着ペプチドの共固定化による小口 径人工血管の開存性の向上と内皮化の促進

3 The reason why I chose this paper 3 ＜ Relevance to my own research ＞ Research theme ： Electrospun PU-PEG structures by Coimmobilization of Heparin and Cell Adhesive Peptides To understand the research trends of the world ， Identify my own research topic. ＜ Knowledge want to learn & Experimental methods ＞

4 Background 4 Cardiovascular are well known major causes of mortality over 40% in the worldwide.

5 Background 5 Tissue-engineered vascular grafts However, the increasing needs for vascular prostheses have not yet been satisfied with limited availability of autologous vessels and small-caliber (

6 Background 6

7 Introduction 7 According to Professor Robert Langer: Tissue Engineering is the field which applies principles of Biology and Engineering to the development of functional substitutes for damaged tissues(Langer et al. 1993).

8 Introduction 8 Scaffolds  Scaffolds are 3-dimensional materials constructed in order to provide structure to a developing tissue and to allow cells to adhere, proliferate, differentiate and most importantly, secrete extracellular matrix (ECM) (Leong MF. et al., 2009).  Many different materials have been investigated in order to construct scaffolds such as polymers (PLA, PGA, PCL, PEG), bioactive ceramics (HA, TCP) as well as natural polymers (Collagen, GAGs, Chitosan). Source:Figure 1. a) Electrospun artificial blood vessel. b) Closer view of the tube wall (image copied from Vaz. C. “Design of scaffolds for blood vessel tissue engineering using a multi-layering electrospinning technique.” Acta Biomaterialia (1) 2005: 575-582.)

9 Knowledge of scaffold 9 Scaffold construction requirements In order to engineer a successful scaffold, a plethora of requirements should be met, such as: 1. Architecture: Scaffolds should provide void volume for vascularization, new tissue formation and remodeling so as to facilitate host tissue integration upon implantation. The biomaterials should be processed to give a porous enough structure for efficient nutrient and metabolite transport without significantly compromising the mechanical stability of the scaffold. Moreover, the biomaterials should also be degradable upon implantation at a rate matching that of the new matrix production by the developing tissue( Berger J, et al., 2004). 2. Cyto and tissue compatibility: Scaffolds should provide support for either extraneously applied or endogenous cells to attach, grow and differentiate during both in vitro culture and in vivo implantation. The biomaterials used to fabricate the scaffolds need to be compatible with the cellular components of the engineered tissues and endogenous cells in host tissue ( Berger J, et al., 2004).

10 Knowledge of scaffold 10 Scaffold construction requirements 3. Bioactivity: Scaffolds may interact with the cellular components of the engineered tissues actively to facilitate and regulate their activities. The biomaterials may include biological cues such as cell-adhesive ligands to enhance attachment or physical cues such as topography to influence cell morphology and alignment. The scaffold may also serve as a delivery vehicle or reservoir for exogenous growth-stimulating signals such as growth factors to speed up regeneration (Discher DE, et al., 2005). 4. Mechanical property: Scaffolds provide mechanical and shape stability to the tissue defect. The intrinsic mechanical properties of the biomaterials used for scaffolding or their post-processing properties should match that of the host tissue. Exerting traction forces on a substrate, many mature cell types, such as epithelial cells, fibroblasts, muscle cells, and neurons, sense the stiffness of the substrate and show dissimilar morphology and adhesive characteristics (Engler AJ, et al., 2006).

11 Knowledge of scaffold 11 Scaffolds synthesis

12 Introduction of biomaterials 12

13 Introduction of biomaterials 13

14 My Experimental choose raw materials 14 Hydrophobic and negatively charged surfaces provide optimal conditions for cell attachment, as they facilitate the adhesion of cell-attachment proteins. MetarialdisadvantagesAdvantages Polylactic acid (PLA)The degradation product is lactic acid, that is easy to cause infection in the human body. Easily dissolved in organic solvents. Good mechanical properties and biocompatibility. Poly (caprolactone)(PCL)It can exist in the body for 2 years, it is difficult to degrade 1.biological suitability 2.non-toxic 3. product is water and carbon dioxide Polyethylene glycol (PEG) （ MW ＜ 20000 ） The mechanical properties are not very good 1.Antibacterial 2.non-toxic 3. Low molecular weight, easy to degrade Polyurethane (PU) Not soluble in water Non degradable The mechanical properties are very good

15 Synthesis triblock copolymers 15 Reference: Novel pH-sensitive polyacetal-based block copolymers for controlled drug delivery The EtG solution in toluene (50%, w/w) was purified by distillation. Triethylamine (TEA)

16 Synthesis triblock copolymers 16 Reference: Novel pH-sensitive polyacetal-based block copolymers for controlled drug delivery

17 Schematic route of immobilization of heparin or the peptides 17 Heparin GRGDSYIGRD

18 Introduction of RGD 18 RGD-peptides are implicated in cellular attachment via integrins, and can be used to coat synthetic scaffolds in tissue engineering to enhance cellular attachment by mimicking in vivo conditions.

19 Knowledge of growth factors 19  Growth factors are protein molecules made by the body. They function to regulate cell devision and cell survival. GF can also be produced via genetic engineering in the lab and used in biological therapy.  GF bind to receptors on the cell surface, with the result of activating cellular proliferation and/or differentiation. GFs are very important, stimulating cellular devision in numerous different cell types.

20 Schematic route of immobilization of heparin or the peptides 20 Allophanate reaction The isocyanate end-group activated PEG was grafted onto the surface of the PU grafts via an allophanate reaction, in which isocyanate groups of end-functionalized PEG is coupled to amine groups of urethane linkage on the surface of PU.

21 Paper`s title and its translation in Japanese 21

22 Results 22 The grafts were approximately 15 mm in length, with an outer diameter of 4.3 mm, inner diameter of 2.5 mm, and thicknesses of 350 and 450 μm in the outer and inner layers, respectively (Figure 1A-C). The loose inner layer comprised randomly distributed electrospun fibers with diameters ranging from 4−8 μm, which showed a closely packed internal structure (Figure1.D,E). The fine outer layer consisted of fibers with diameters of about 1.5−3 μm, which showed morphology similar to that of the inner layer (Figure.1F).

23 Results 23 Cell-adhesive peptides were conjugated with a PEG chain anchored onto the surface of PU grafts via EDC/NHS chemistry. Amino acid analysis revealed that the surface densities of G and Y peptides on the PU-PEG surface were 0.26 and 0.31 nmol/cm 2, respectively.

24 Results 24  X-ray photoelectron spectroscopy was used to characterize the presence of anchored biomolecules on the surface of the PU grafts (Figure 3).  The presence of heparin could be confirmed by the signals of sulfur element (S 2s and S 2p) at the wide scan spectrum (Figure 3A).  In the narrow scan spectra, the spectrum of heparin (Figure 3B) showed different patterns from that of the two peptides (Figure 3C,D). Figure 3. X-ray photoelectron spectra of the modified PU vascular grafts. (A) Wide scan spectrum of the heparin-PU graft. Narrow scan spectra (O 1s) of the PU grafts immobilized with (B) heparin, (C) G peptide, and (D) Y peptide.

25 Results 25 Figure 4. (A) Water contact angles and (B) mechanical properties of the modified PU vascular grafts. Graph B presents the stress-strain curves of (a) the casted PU film, and (b) 20-wt % and (c) 15-wt % electrospun PU grafts

26 Results 26 Figure 5. (A) Human platelet adhesion and (B) human fibrinogen adsorption of the modified PU vascular grafts

27 Results 27 Figure 6. HUVEC (A) attachment and (B) proliferation for 1, 3, and 5 days on the modified PU vascular grafts

28 Results 28 Figure 7. In vivo implantation of modified PU vascular grafts using a rabbit abdominal aorta artery model. Photographs showing (A) natural abdominal aorta artery, (B)artery incision, (C)anastomosis of a vascular graft, and (D)completed implantation. (E)Angiographic image of an implanted vascular graft. (F)Photograph of an explanted vascular graft

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30 Results 30 Figure 8. Neointimal smooth muscle regeneration on day 26 after implantation. SMCs were stained with α-SMA antibody. (A) Immunohistochemical images of cross sections at the proximal, mid, and distal PU and modified PU vascular grafts. All images are at a magnification of 100×. (B) Stenosis rates of the PU and PU-PEG- Hep/G+Y grafts explanted after 26 days of implantation.

31 Results 31 Figure 9. Endothelialization analysis of PU-PEG-Hep/G+Y graft by immunohistochemical and histological staining. Cross-sections of (A-F) a PU-PEG-Hep/G+Y graft and (G-I) a natural rabbit artery stained with (A, B, G) hematoxylin and eosin, (C, D, H) antivon-Willebrand factor, and (E, F, I) anti-α-smooth muscle actin. All images are at a magnification of 400×

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