Improving hemocompatibility of decellularized vascular tissue by structural modification of collagen fiber.

Decellularized vascular tissue has high potential as a tissue-engineered vascular graft because of its similarity to native vessels in terms of mechanical strength. However, exposed collagen on the tissue induces blood coagulation, and low hemocompatibility is a major obstacle to its vascular application. Here we report that freeze-drying and ethanol treatment effectively modify collagen fiber structure and drastically reduce blood coagulation on the graft surface without exogenous chemical modification. Decellularized carotid artery of ostrich was treated with freeze-drying and ethanol solution at concentrations ranging between 5 and 99.5 %. Collagen fiber distance in the graft was narrowed by freeze-drying, and the non-helical region increased by ethanol treatment. Although in vitro blood coagulation pattern was similar on the grafts, platelet adhesion on the grafts was largely suppressed by freeze-drying and ethanol treatments. Ex vivo blood circulation tests also indicated that the adsorption of platelets and Von Willebrand Factor was largely reduced to approximately 80 % by ethanol treatment. These results indicate that structural modification of collagen fibers in decellularized tissue reduces blood coagulation on the surface by inhibiting platelet adhesion.

Microwave-Assisted Incorporation of AgNP into Chitosan-Alginate Hydrogels for Antimicrobial Applications.

Herein, improving the antibacterial activity of a hydrogel system of sodium alginate (SA) and basic chitosan (CS) using sodium hydrogen carbonate by adding AgNPs was investigated. SA-coated AgNPs produced by ascorbic acid or microwave heating were evaluated for their antimicrobial activity. Unlike ascorbic acid, the microwave-assisted method produced uniform and stable SA-AgNPs with an optimal reaction time of 8 min. Transmission electron microscopy (TEM) confirmed the formation of SA-AgNPs with an average particle size of 9 ± 2 nm. Moreover, UV-vis spectroscopy confirmed the optimal conditions for SA-AgNP synthesis (0.5% SA, 50 mM AgNO3, and pH 9 at 80 °C). Fourier transform infrared (FTIR) spectroscopy confirmed that the -COO- group of SA electrostatically interacted with either the Ag+ or -NH3+ of CS. Adding glucono-δ-lactone (GDL) to the mixture of SA-AgNPs/CS resulted in a low pH (below the pKa of CS). An SA-AgNPs/CS gel was formed successfully and retained its shape. This hydrogel exhibited 25 ± 2 mm and 21 ± 1 mm inhibition zones against E. coli and B. subtilis and showed low cytotoxicity. Additionally, the SA-AgNP/CS gel showed higher mechanical strength than SA/CS gels, possibly due to the higher crosslink density. In this work, a novel antibacterial hydrogel system was synthesized via 8 min of microwave heating.

Immobilization of Arg-Gly-Asp peptides on silk fibroin via Gly-Ala-Gly-Ala-Gly-Ser sequences.

A simple method by which the functional peptide of Gly-Arg-Gly-Asp-Ser (GRGDS) is immobilized on the surface of silk fibroin (SF) films via Gly-Ala-Gly-Ala-Gly-Ser (GAGAGS) sequences is proposed. GAGAGS, a repeating amino acid sequence in the crystal region of Bombyx mori SF, performs a key role in interacting with and immobilizing SF molecules. Immobilization by this proposed method involves no chemical reaction, thereby preserving the original properties of the SF molecule. The density of GRGDS peptides existing on SF film was found to be higher in the GAGAGS-bound type than in the non-GAGAGS-bound type. Furthermore, results showed that the amount of immobilized (GAGAGS)GRGDS peptide increased as the ß-sheet crystallization was promoted in the SF film. Fibroblasts, which adhered to the surface of the SF film, showed more extensibility because of the (GAGAGS)GRGDS immobilization, which suggests that the cell adhesion activity of RGD is functioning effectively.

Dynamic in vitro hemocompatibility of oligoproline self-assembled monolayer surfaces.

The blood compatibility of self-assembled monolayers (SAMs) of oligoproline, a nonionic antifouling peptide, was investigated using the cone-and-plate assay imitating arterial blood flow conditions. End-capped oligoprolines composed of 6 and 9 proline residues (Pro6 and Pro9) and a Cys residue were synthesized for preparing SAMs (Pro-SAMs) on Au-sputtered glass. The surface of Pro-SAMs indicated hydrophilic property with a smooth topology. The adsorption of blood components and the adhesion of blood cells, including leukocytes and platelets, were strongly suppressed on Pro-SAMs. Moreover, Pro9-SAM did not trigger the activation of platelets (i.e., the conformational change of GPIIb/IIIa and P-selectin (CD62P) expression on platelets and the formation of aggregates). Our results demonstrate that Pro9-SAM completely inhibited acute thrombogenic responses and the activation of platelets under dynamic conditions.

Stable and direct coating of fibronectin-derived Leu-Asp-Val peptide on ePTFE using one-pot tyrosine oxidation for endothelial cell adhesion.

Expanded polytetrafluoroethylene (ePTFE) is widely used in clinical applications, such as in the manufacture of blood-contacting implantable devices, owing to its flexibility, biostability, and non-adhesiveness. Modification with peptides is an effective strategy to further improve the ePTFE function. However, the chemical stability of PTFE makes it difficult to modify with peptides. In this study, we reported a simple method for the dense and stable coating of biofunctional peptides on the ePTFE surface through the anchor sequence, Tyr-Lys-Tyr-Lys-Tyr-Lys (YK3). A peptide (YK3-LDV) incorporating the YK3 anchor and a ligand sequence for α4ß1 integrin, Leu-Asp-Val (LDV), was successfully coated on ePTFE grafts through one-pot oxidation. The peptide layer constructed via YK3-LDV coating on ePTFE was stable and resistant to extensive washing by aqueous solutions of highly concentrated salts and surfactants. YK3-LDV coating promoted the in vitro adhesion of endothelial cells to ePTFE. Furthermore, YK3-LDV coating accelerated the in vivo formation of neointima-like tissue in a rat model with an ePTFE patch implanted into the carotid artery.

Long-lasting hydrophilic surface generated on poly(dimethyl siloxane) with photoreactive zwitterionic polymers.

Poly(dimethylsiloxane) (PDMS) is known as one of the most established polymers for making elastomers. Therefore, it is commonly used for the fabrication of biomedical devices. Many PDMS surface modification processes have been proposed recently to increase PDMS reliability in medical fields. However, the modified surface's long-term stability is still limited. Hydrophobic recovery of PDMS is widely recognized as a factor that reduces the efficacy of PDMS surface modification. The photoreactive zwitterionic polymer effectively suppresses the hydrophobic recovery of PDMS, according to the current analysis. The photoreactive zwitterionic monomer, 2-[2-(Methacryloyloxy)ethyldimethylanmmonium] ethyl benzophenoxy phosphate (MBPP) was polymerized by conventional radical polymerization and coated on O2-plasma-treated PDMS specimens. The specimens were immersed in an aqueous solution of 2-methacryloyloxyethyl phosphorylcholine (MPC) and exposed under ultraviolet (UV) radiation for 3 h. Instead, of poly(MBPP) (PMBPP), benzophenone (BP) was also used as a conventional photoinitiator. The time-dependent change in the wettability and elemental composition of the specimen surface was monitored for nine weeks after photo-grafting of poly[2-methacryloyloxyethyl phosphorylcholine (MPC)] (PMPC). The advancing and receding contact angles (θA/θR) of the pristine PDMS specimen were 112°/71° and significantly decreased immediately after the grafting of PMPC regardless of types of photoinitiator. However, the hydrophobicity of the surface gradually recovered, and θA was changed from 12° to 81° for nine weeks of storage under air atmosphere when BP was used as a photoinitiator for graft polymerization of MPC. However, surface hydrophilicity (θA ≅ 20°) of the surface grafted with PMPC with PMBPP as an initiator was effectively preserved for nine weeks. This surface also showed excellent lubricity and non-fouling properties regardless of the storage periods. Therefore, zwitterionic photoreactive polymer, PMBPP, is then used as a macrophotoinitiator for the surface modification of PDMS.

A well plate-based multiplexed platform for incorporation of organoids into an organ-on-a-chip system with a perfusable vasculature.

Owing to their high spatiotemporal precision and adaptability to different host cells, organ-on-a-chip systems are showing great promise in drug discovery, developmental biology studies and disease modeling. However, many current micro-engineered biomimetic systems are limited in technological application because of culture media mixing that does not allow direct incorporation of techniques from stem cell biology, such as organoids. Here, we describe a detailed alternative method to cultivate millimeter-scale functional vascularized tissues on a biofabricated platform, termed 'integrated vasculature for assessing dynamic events', that enables facile incorporation of organoid technology. Utilizing the 3D stamping technique with a synthetic polymeric elastomer, a scaffold termed 'AngioTube' is generated with a central microchannel that has the mechanical stability to support a perfusable vascular system and the self-assembly of various parenchymal tissues. We demonstrate an increase in user familiarity and content analysis by situating the scaffold on a footprint of a 96-well plate. Uniquely, the platform can be used for facile connection of two or more tissue compartments in series through a common vasculature. Built-in micropores enable the studies of cell invasion involved in both angiogenesis and metastasis. We describe how this protocol can be applied to create both vascularized cardiac and hepatic tissues, metastatic breast cancer tissue and personalized pancreatic cancer tissue through incorporation of patient-derived organoids. Platform assembly to populating the scaffold with cells of interest into perfusable functional vascularized tissue will require 12-14 d and an additional 4 d if pre-polymer and master molds are needed.

Surfaces immobilized with oligo-prolines prevent protein adsorption and cell adhesion.

In this study, oligo-prolines, (Pro)n (n = 6 and 9) inspired by the backbone structure of collagen, were evaluated as a novel non-ionic anti-fouling peptide. Two oligo-prolines with a cysteine residue were synthesized and immobilized on gold substrates via Au-thiol binding. The surfaces immobilized with oligo-prolines, and forming a polyproline-II conformation, indicated hydrophilic properties (water contact angle ≈ 25 degrees). The degree of adsorption of human serum albumin, human fibrinogen, and bovine serum components on these surfaces was quantified using a quartz crystal. The immobilization of oligo-prolines prevented the adsorption of proteins and serum components including small molecules, such as fatty acids. Pro9 specifically indicated good resistance to the adsorption of all components due to the highly-packed Pro9 chains on the surface. The adhesion of fibroblasts was drastically suppressed on the surfaces immobilized with oligo-prolines. Our findings suggest that oligo-proline-immobilized surfaces, specifically Pro9-s, are useful for the development of novel vascular devices that have ultra-low fouling properties.

Highly Durable Lubricity of Photo-Cross-Linked Zwitterionic Polymer Brushes Supported by Poly(ether ether ketone) Substrate.

For improving lubricity, the formation of zwitterionic polymer brushes on material surfaces is one of the most promising approaches. In this study, the photoreactive zwitterionic monomer 2-[2-(methacryloyloxy)ethyldimethylanmmonium] ethyl benzophenoxy phosphate (MBPP) was synthesized to improve the stability of zwitterionic polymer brushes. Although MBPP contains a benzophenone moiety in this molecule, it is water-soluble because of the zwitterionic linker. As a substrate, poly(ether ether ketone) (PEEK) was selected because it has recently been used to replace metals in orthopedic implants. Furthermore, PEEK is photosensitive, and UV graft polymerization of (meth)acrylic monomers on the surface can be performed without using any photoinitiators. Aqueous solutions containing various molar ratios of 2-(methacryloyloxy)ethyl phosphorylcholine (MPC) and MBPP were prepared, and the PEEK specimens were immersed in these solutions. UV light was used to irradiate the solutions for 180 min, and the formation of grafting layers of zwitterionic polymers on PEEK specimens was confirmed using contact angle measurement and X-ray photoelectron spectroscopy. The surface friction of PEEK was effectively reduced via the photopolymerization of zwitterionic monomers. However, the surface lubricity of poly(MPC) (PMPC)-grafted surface deteriorated during continuous friction because of the removal of PMPC from the surface. Nevertheless, the stability of polymer brushes was effectively improved by adding only 0.5-0.75 mol % of MBPP in the monomer solution. Moreover, the reduction of wear on the surface was determined using confocal laser microscopy. The excellent lubrication phenomenon was attributed to preserving the hydration state of grafted polymers under compressive stress. Moreover, bacterial adhesion on substrates was tested and observed on a neat PEEK and scratched regions of uncross-linked PMPC-grafted PEEK. Note that bacterial adhesion was completely suppressed on the surface of PEEK modified with cross-linked PMPC brushes with MBPP. Thus, we conclude that the surface modification of PEEK with MPC and MBPP can provide ideal surface properties for orthopedic devices.

Short-term evaluation of thromboresistance of a poly(ether ether ketone) (PEEK) mechanical heart valve with poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC)-grafted surface in a porcine aortic valve replacement model.

Improved thromboresistance of mechanical valves is desired to decrease the risk of thromboembolism and thrombosis and reduce the dosage of anticoagulation with a vitamin K antagonist (e.g., warfarin). For several mechanical valves, design-related features are responsible for their improved thromboresistance. However, it remains unclear whether material-related features provide a practical level of thromboresistance to mechanical valves. Here, we studied the effect of a bileaflet valve made of poly(ether ether ketone) (PEEK) with a poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC)-grafted surface (PEEK-g-PMPC). PMPC is a well-known thromboresistant polymeric material. A short-term (<26 h) porcine aortic valve replacement model using neither an anticoagulant nor an antiplatelet agent showed that the PEEK-g-PMPC valve opened and closed normally with an allowable transvalvular gradient. Unlike an untreated PEEK valve, no thrombus formed on the PEEK-g-PMPC valves on gross anatomy examination in addition to the absence of traveled thrombi in the kidney and lung tissues. Material (PEEK-g-PMPC)-related thromboresistance appeared to decrease the risk of thromboembolism and thrombosis for patients with mechanical valves. However, thromboresistance of the PEEK-g-PMPC valve requires improvement because fibrous fouling was still observed on the leaflet. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1052-1063, 2019.

Surface modification of porous alpha-tricalcium phosphate granules with heparin enhanced their early osteogenic capability in a rat calvarial defect model.

Heparin binds to and modulates various growth factors, potentially augmenting the bone-forming capability of biomaterials. Here, α-tricalcium phosphate (α-TCP) granules were modified with peptide containing the marine mussel-derived adhesive sequence, which reacts with α-TCP surface, and cationic sequence, which binds to heparin (α-Ph). α-Ph retained the α-TCP phase and intergranule spaces after the surface modification. The existence of heparin on α-Ph granules was confirmed using X-ray photoelectron spectroscopy. Granules of α-TCP and α-Ph were implanted into critical-size defects in rat calvaria for 4 weeks. Micro-computed tomography, histological evaluation, and Alcian blue staining revealed that α-Ph induced superior bone formation compared with α-TCP. Newly formed bone on α-Ph was preferentially in contact with the Alcian blue-stained surfaces of granules. These results suggested that heparinization enhanced the early osteogenic capacity of α-TCP, possibly by modulating the secretion of Alcian blue-stained extracellular matrixes.

Periodontal regeneration induced by porous alpha-tricalcium phosphate with immobilized basic fibroblast growth factor in a canine model of 2-wall periodontal defects.

We evaluated the effect of porous alpha-tricalcium phosphate (α-TCP) with immobilized basic fibroblast growth factor (bFGF) on periodontal regeneration in a canine model of 2-wall periodontal defects. Identical bone defects were made in the canine mandible; six defects in each animal were filled with porous α-TCP with bFGF bound via heparin (bFGF group), and the remaining defects were filled with unmodified porous α-TCP (control group). Micro-computed tomography and histological evaluation were performed at 2, 4, and 8 weeks post-implantation. The bone mineral content of the bFGF group was higher than that of the control group at 2 and 4 weeks (p < 0.05). Histological evaluation at 2 weeks post-implantation revealed degradation of the porous α-TCP, and bone had formed on the surface of α-TCP particles in the bFGF group. Some of these collagen fibers connected the newly formed cementum with the alveolar bone, revealing the formation of new periodontal ligaments with Sharpey's fibers. At 8 weeks, continuous cortical bone with a Haversian structure covered the top of the bone defects in the bFGF group. These findings indicate that porous α-TCP with immobilized bFGF could promote periodontal regeneration at the early regeneration phase in a canine model of 2-wall periodontal defects.

Direct surface modification of metallic biomaterials via tyrosine oxidation aiming to accelerate the re-endothelialization of vascular stents.

Rapid in-situ re-endothelialization of coronary stents is one of the most effective approaches to inhibit late thrombosis and restenosis. Strut surfaces allowing excellent adhesion and migration of endothelial cells and endothelial progenitor cells may accelerate in-situ re-endothelialization. Here, a well-known endothelial cell adhesive peptide, Arg-Glu-Asp-Val (REDV), was directly immobilized onto metallic surfaces by means of single-step tyrosine oxidation with copper chloride (II) and hydrogen peroxide, which we recently reported as a new biomaterial modification technique. REDV immobilization on a 316L stainless steel plate improved endothelial cell adhesion and effectively suppressed platelet adhesion in vitro. In addition, a Co-Cr stent immobilized with Ac-Tyr-Gly-Gly-Gly-Arg-Glu-Asp-Val (Y-REDV) was implanted into a rabbit abdominal aorta. On 7 days postimplantation, 80% of the strut surface of the Y-REDV-immobilized stent was covered by a thin neointimal layer and was similar in appearance to native endothelium. Restenosis and late thrombosis were not observed in the Y-REDV-immobilized stent for 42 days. These findings suggest that direct immobilization of Y-REDV peptide onto metallic biomaterials by tyrosine oxidation is effective for promoting in-situ re-endothelialization in vascular stents. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 491-499, 2018.

Individual evaluation of cardiac marker expression and self-beating during cardiac differentiation of P19CL6 cells on different culture substrates.

Cell-based therapies using self-beating cardiomyocytes have been attracting great attention for use in cardiac regeneration, although an effective procedure to improve cardiac differentiation and self-beating induction is required. The purpose of this study is to clarify the effect of the culture substrate on cardiac maturation by separately evaluating the cardiac differentiation step and the beating induction step in vitro. To this end, the well-studied cardiomyocyte-like progenitor cell line P19CL6 and neonatal cardiomyocytes (NCMs) were selected and cultured on substrates coated with collagen type I (Col-I), gelatin (Gel), fibronectin (FN), or poly-l-lysine (PLL). It was found that the cardiac differentiation step, which was assessed using cardiac marker gene expression (GATA-binding protein 4 (GATA4), myocyte-specific enhancer factor 2D (MEF2D), and hyperpolarization-activated cyclic nucleotide-gated potassium channel 4 (HCN4)) in the P19CL6 embryonal carcinoma cells, was greatly enhanced on Col-I, Gel, and PLL. In contrast, the spontaneous beating step, which was directly assessed by counting the beating colonies and measuring contractile protein gene expression (α-myosin heavy chain (α-MHC), troponin C type 1 (TnC1), and troponin T type 2 (TnT2)) in the rat NCMs, was enhanced on the FN and PLL surfaces. In the present study, for the first time, it was found that PLL enhances both the cardiac differentiation and the beating induction steps of cardiac maturation, which can aid in preparing beating cardiomyocytes for regenerative medicine. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1166-1174, 2017.

Accelerated tissue integration into porous materials by immobilizing basic fibroblast growth factor using a biologically safe three-step reaction.

Soft tissue integration into a porous structure is important to prevent bacterial infection of percutaneous devices and improve tissue regeneration using porous scaffolds. Here, basic fibroblast growth factor (bFGF) was immobilized on porous polymer materials using a mild and biologically safe three-step reaction: (1) modification with a novel surface-modification peptide (penta-lysine-mussel adhesive sequence, which reacts with various matrices), (2) electrostatic binding of heparin with introduced penta-lysine, and (3) biologically specific binding of bFGF to heparin. Porous polyethylene specimens (PPSs) (D = 6.0 mm, H = 2.0 mm) with a good size for tissue integration were selected as a base material, immobilized with bFGF, and subcutaneously implanted into mice. Half of the unmodified PPSs extruded out of the body on day 112 postimplantation; however, the three-step reaction completely prevented sample rejection. Tissue integration was greatly accelerated by immobilizing bFGF. Direct physical coating of bFGF on PPS resulted in greater immobilization but lesser tissue integration than that after the three-step bFGF immobilization, indicating that heparin binds and enhances bFGF efficacy. This three-step bFGF immobilization reaction will be applicable to various polymeric, metallic, and ceramic materials and is a simple strategy to integrate tissue on porous medical devices or scaffolds for tissue regeneration.

Single-step immobilization of cell adhesive peptides on a variety of biomaterial substrates via tyrosine oxidation with copper catalyst and hydrogen peroxide.

Immobilization of biologically active peptides which were isolated from extracellular matrix proteins is a powerful strategy for the design and functionalization of biomaterial substrates. However, the method of peptide immobilization was restricted, that is, peptide is often immobilized through the reactive groups inherent in substrates with multistep reactions. Here, we report a single-step immobilization of fibronectin-derived cell adhesive peptide (Arg-Glu-Asp-Val; REDV) onto polymer materials by use of tyrosine oxidation with copper catalyst and hydrogen peroxide. REDV peptide was successfully immobilized on tissue culture polystyrene, poly(ethylene terephthalate), poly(vinyl chloride), expanded-poly(tetrafluoroethylene), and poly(l-lactic acid), resulting in enhanced adhesion of human umbilical vein endothelial cells. This method is a single-step reaction under very mild conditions and is available for the biological functionalization of various medical devices.

Directing stem cell differentiation by changing the molecular mobility of supramolecular surfaces.

Polymer surfaces with a wide range of hydrated surface mobility are developed by a simple deposition method with supramolecular block copolymers. The morphologies of adhering stem cells are greatly dependent on the surface mobility of polymers, and this induces significant changes in the cytoskeletal signaling pathway to direct the downstream stem cell differentiation.

Mobility of the Arg-Gly-Asp ligand on the outermost surface of biomaterials suppresses integrin-mediated mechanotransduction and subsequent cell functions.

Mechanotransduction in the regulation of cellular responses has been previously studied using elastic hydrogels. Because cells interact only with the surface of biomaterials, we are focusing on the molecular mobility at the outermost surface of biomaterials. In this study, surfaces with the mobile Arg-Gly-Asp-Ser (RGDS) peptide have been constructed. Cell culture substrates were coated with ABA-type block copolymers composed of poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate) segments (A) and a polyrotaxane (PRX) unit with RGDS bound to α-cyclodextrin (B). Adhesion, morphological changes and actin filament formation of human umbilical vein endothelial cells were reduced on the surfaces containing mobile PRX-RGDS in comparison to the immobile RGDS surfaces constructed from random copolymers with RGDS side groups (Prop-andom-RGDS). In the neurite outgrowth assay using rat adrenal pheochromocytoma cells (PC12), only ∼20% of adherent PC12 cells had neurites on PRX-RGDS surfaces, but more than 50% did on the Random-RGDS surface. The beating colony of dimethyl-sulfoxide-treated mouse embryonic carcinoma cells (P19CL6) were found 10 and 14 days after induction on PRX-RGDS and Random-RGDS surfaces, respectively. After 22 days, the beating colony disappeared on PRX-RGDS surfaces, but many colonies remained on Random-RGDS surfaces. These data suggest that the molecular mobility of the cell-binding ligand on the outermost surface of materials effectively suppresses the actin filament formation and differentiation of these functional cell lines, and may be used as a culture substrate for immature stem cells or progenitor cells.

Water absorbing and quick degradable PLLA/PEG multiblock copolymers reduce the encapsulation and inflammatory cytokine production.

Biomaterials that contact with soft tissues such as postoperative adhesion prevention membrane or tissue-regenerative scaffolds should possess specific features such as hydrophilicity, mild to no immunogenicity, and quick degradability. The inflammation reaction to multiblock copolymers of poly(L-lactic acid) (PLLA) and poly(ethylene glycol), named as Multi, which we developed as a good adhesion prevention materials with a very high degradation rate were investigated and compared with usual PLLA, non-degradable polyethylene (PE), and acellular collagenous tissue (COL). Tissue encapsulation, inflammatory cell recruitment, and expression of four cytokines (IL-1ß, IL-6, IL-10, and TGFß) affecting the promotion or inhibition of inflammation and wound healing were evaluated. The thick encapsulation for PE might have related to high expression of TGFß, and it was largely reduced in the cases of PLLA and Multi. The cytokine expression pattern in PE was dominantly alternatively activated macrophage (M2) type, while expression patterns to Multi were classically activated macrophage (M1)-type dominant, as with the COL specimen. Thus, multi is a tissue compatible material in spite of the large degradability. By introducing low molecular weight PEG into PLLA as multiblock-type sequence, we successfully prepared biocompatible PLLA derivatives with high molecular weight, large degradation rate, and mild tissue responses.

Three-layer microfibrous peripheral nerve guide conduit composed of elastin-laminin mimetic artificial protein and poly(L-lactic acid).

We developed a microfibrous poly(L-lactic acid) (PLLA) nerve conduit with a three-layered structure to simultaneously enhance nerve regeneration and prevent adhesion of surrounding tissue. The inner layer was composed of PLLA microfiber containing 25% elastin-laminin mimetic protein (AG73-(VPGIG)30) that promotes neurite outgrowth. The thickest middle layer was constructed of pure PLLA microfibers that impart the large mechanical strength to the conduit. A 10% poly(ethylene glycol) was added to the outer layer to prevent the adhesion with the surrounding tissue. The AG73-(VPGIG)30 compositing of an elastin-like repetitive sequence (VPGIG)30 and a laminin-derived sequence (RKRLQVQLSIRT: AG73) was biosynthesized using Escherichia coli. The PLLA microfibrous conduits were fabricated using an electrospinning procedure. AG73-(VPGIG)30 was successfully mixed in the PLLA microfibers, and the PLLA/AG73-(VPGIG)30 microfibers were stable under physiological conditions. The PLLA/AG73-(VPGIG)30 microfibers enhanced adhesion and neurite outgrowth of PC12 cells. The electrospun microfibrous conduit with a three-layered structure was implanted for bridging a 2.0-cm gap in the tibial nerve of a rabbit. Two months after implantation, no adhesion of surrounding tissue was observed, and the action potential was slightly improved in the nerve conduit with the PLLA/AG73-(VPGIG)30 inner layer.