Tubular silk scaffolds for small diameter vascular grafts

Vascular surgeries such as coronary artery bypass require small diameter vascular grafts with properties that are not available at this time. Approaches using synthetic biomaterials have been not completely successful in producing non-thrombogenic grafts with inner diameters less than 6 mm, and there is a need for new biomaterials and graft designs. We propose silk fibroin as a microvascular graft material and describe tubular silk scaffolds that demonstrate improved properties over existing vascular graft materials. Silk tubes produced using an aqueous gel spinning technique were first assessed in vitro in terms of thrombogenicity (thrombin and fibrinogen adsorption, platelet adhesion) and vascular cell responses (endothelial and smooth muscle cell attachment and proliferation) in comparison with polytetrafluoroethylene (PTFE), a synthetic material most frequently used for vascular grafts. Silk tubes were then implanted into the abdominal aortas of Sprague-Dawley rats. At time points of 2 weeks and 4 weeks post implantation, tissue outcomes were assessed through gross observation (acute thrombosis, patency) and histological staining (H&E, Factor VIII, smooth muscle actin). Over the 4-week time period, we observed graft patency and endothelial cell lining of the lumen surfaces. These results demonstrate the feasibility of using silk fibroin as a vascular graft material and some advantages of silk tubes over the currently used synthetic grafts.     

Co-electrospun blends of PLGA, gelatin, and elastin as potential nonthrombogenic scaffolds for vascular tissue engineering

In search for novel biomimetic scaffolds for application in vascular tissue engineering, we evaluated a series of fibrous scaffolds prepared by coelectrospinning tertiary blends of poly(lactide-co-glycolide) (PLGA), gelatin, and elastin (PGE). By systematically varying the ratios of PLGA and gelatin, we could fine-tune fiber size and swelling upon hydration as well as the mechanical properties of the scaffolds. Of all PGE blends tested, PGE321 (PLGA, gelatin, elastin v/v/v ratios of 3:2:1) produced the smallest fiber size (317 ± 46 nm, 446 ± 69 nm once hydrated) and exhibited the highest Young's modulus (770 ± 131 kPa) and tensile strength (130 ± 7 kPa). All PGE scaffolds supported the attachment and metabolization of human endothelial cells (ECs) and bovine aortic smooth muscle cells (SMCs) with some variances in EC morphology and cytoskeletal spreading observed at 48 h postseeding, whereas no morphologic differences were observed at confluence (day 8). The rate of metabolization of ECs, but not of SMCs, was lower than that on tissue culture plastic and depended on the specific PGE composition. Importantly, PGE scaffolds were capable of guiding the organotypic distribution of ECs and SMCs on and within the scaffolds, respectively. Moreover, the EC monolayer generated on the PGE scaffold surface was nonthrombogenic and functional, as assessed by the basal and cytokine-inducible levels of mRNA expression and amidolytic activity of tissue factor, a key player in the extrinsic clotting cascade. Taken together, our data indicate the potential application of PGE scaffolds in vascular tissue engineering.     

Surface Modification and Characterisation of Silk Fibroin Fabric Produced by the Layer-by-Layer Self-Assembly of Multilayer Alginate/Regenerated Silk Fibroin

Silk-based medical products have a long history of use as a material for surgical sutures because of their desirable mechanical properties. However, silk fibroin fabric has been reported to be haemolytic when in direct contact with blood. The layer-by-layer self-assembly technique provides a method for surface modification to improve the biocompatibility of silk fibroin fabrics. Regenerated silk fibroin and alginate, which have excellent biocompatibility and low immunogenicity, are outstanding candidates for polyelectrolyte deposition. In this study, silk fabric was degummed and positively charged to create a silk fibroin fabric that could undergo self-assembly. The multilayer self-assembly of the silk fibroin fabric was achieved by alternating the polyelectrolyte deposition of a negatively charged alginate solution (pH = 8) and a positively charged regenerated silk fibroin solution (pH = 2). Finally, the negatively charged regenerated silk fibroin solution (pH = 8) was used to assemble the outermost layer of the fabric so that the surface would be negatively charged. A stable structural transition was induced using 75% ethanol. The thickness and morphology were characterised using atomic force microscopy. The properties of the self-assembled silk fibroin fabric, such as the bursting strength, thermal stability and flushing stability, indicated that the fabric was stable. In addition, the cytocompatibility and haemocompatibility of the self-assembled silk fibroin fabrics were evaluated. The results indicated that the biocompatibility of the self-assembled multilayers was acceptable and that it improved markedly. In particular, after the self-assembly, the fabric was able to prevent platelet adhesion. Furthermore, other non-haemolytic biomaterials can be created through self-assembly of more than 1.5 bilayers, and we propose that self-assembled silk fibroin fabric may be an attractive candidate for anticoagulation applications and for promoting endothelial cell adhesion for vascular prostheses.     

Electrospun PLGA�Csilk fibroin�Ccollagen nanofibrous scaffolds for nerve tissue engineering

Electrospun nanofibrous scaffolds varying different materials are fabricated for tissue engineering. PLGA, silk fibroin, and collagen-derived scaffolds have been proved on good biocompatibility with neurons. However, no systematic studies have been performed to examine the PLGA-silk fibroin-collagen (PLGA-SF-COL) biocomposite fiber matrices for nerve tissue engineering. In this study, different weight ratio PLGA-SF-COL (50:25:25, 30:35:35) scaffolds were produced via electrospinning. The physical and mechanical properties were tested. The average fiber diameter ranged from 280 + 26 to 168 + 21 nm with high porosity and hydrophilicity; the tensile strength was 1.76 ± 0.32 and 1.25 ± 0.20 Mpa, respectively. The results demonstrated that electrospinning polymer blending is a simple and effective approach for fabricating novel biocomposite nanofibrous scaffolds. The properties of the scaffolds can be strongly influenced by the concentration of collagen and silk fibroin in the biocomposite. To assay the cytocompatibility, Schwann cells were seeded on the scaffolds; cell attachment, growth morphology, and proliferation were studied. SEM and MTT results confirmed that PLGA-SF-COL scaffolds particularly the one that contains 50% PLGA, 25% silk fibroin, and 25% collagen is more suitable for nerve tissue engineering compared to PLGA nanofibrous scaffolds.     

A high molecular weight silk fibroin scaffold that resists degradation and promotes cell proliferation

The regulation of the biodegradation rate of 3D-regenerated silk fibroin scaffolds and the avoidance of premature collapse are important concerns for their effective applications in tissue engineering. In this study, bromelain, which is specific to sericin, was used to remove sericin from silk, and high molecular weight silk fibroin was obtained after the fibroin fibers were dissolved. Afterwards, a 3D scaffold was prepared via freeze-drying. The Sodium dodecyl sulfate–polyacrylamide gel electrophoresis results showed that the average molecular weight of the regenerated silk fibroin prepared by using the bromelain-degumming method was approximately 142.2 kDa, which was significantly higher than that of the control groups prepared by using the urea- and Na₂CO₃-degumming methods. The results of enzyme degradation in vitro showed that the biodegradation rate and internal three-dimensional structure collapse of the bromelain-degumming fibroin scaffolds were significantly slower than those of the two control scaffolds. The proliferation activity of human umbilical vein vascular endothelial cells inoculated in bromelain-degumming fibroin scaffolds was significantly higher than that of the control scaffolds. This study provides a novel preparation method for 3D-regenerated silk fibroin scaffolds that can effectively resist biodegradation, continuously guide cell growth, have good biocompatibility, and have the potential to be used for the regeneration of various connective tissues.     

Preparation, characterization and biocompatibility of electrospinning heparin-modified silk fibroin nanofibers

In this study, the electrospun silk fibroin nanofibrous scaffolds were modified with heparin by grafting after plasma treatment and blending electrospinning. Morphology, microstructure, chemical composition and grafting efficiency of the heparin-modified silk fibroin nanofibrous scaffolds were characterized to evaluate the effect of modification by means of scanning electron microscopy (SEM), Fourier transform infrared spectra (FTIR) and X-ray photoelectron spectrometer (XPS). The results showed that the heparin was successfully introduced to the silk fibroin nanofibrous scaffolds by both the two kinds of modification, and there was a hydrogen bonding between the silk fibroin and heparin. Moreover, the hydrophilicity, O-containing groups and negative charge density of the heparin-modified scaffolds were enhanced. In vitro coagulation time tests showed that the activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT) of the heparin-modified scaffolds were much higher than those of the pure silk fibroin scaffolds. L929 fibroblasts and EVCs spread and proliferated better on the heparin-modified scaffolds than on the pure silk fibroin scaffolds. Macrophages, neutrophils and lymphocytes were not observed in the heparin-modified scaffolds, which indicated that the modified scaffolds could induce minor inflammation in vivo. The results indicated that the electrospun heparin-modified silk fibroin nanofibrous scaffolds could be considered as ideal candidates for tissue engineering scaffolds.     

Mechanical and biological performances of new scaffolds made of collagen hydrogels and fibroin microfibers for vascular tissue engineering

A microstructured composite material made of collagen hydrogel (matrix) and silk fibroin microfibers (randomly oriented reinforcing fibers) is investigated in order to conjugate the mechanical resistance of fibroin with the suitable biological performance of collagen to design new scaffolds for vascular tissue engineering. Results show that fibroin microfibers and collagen fibrils have suitable interfacial adhesion, and the scaffold exhibits improved mechanical properties if compared with a pure collagen hydrogel. Furthermore, the overall biological performance is improved.     

Vascular tissue engineering: Fabrication and characterization of acetylsalicylic acid-loaded electrospun scaffolds coated with amniotic membrane lysate

As the incidence of small-diameter vascular graft (SDVG) occlusion is considerably high, a great amount of research is focused on constructing a more biocompatible graft. The absence of a biocompatible surface in the lumen of the engineered grafts that can support confluent lining with endothelial cells (ECs) can cause thrombosis and graft failure. Blood clot formation is mainly because of the lack of an integrated endothelium. The most effective approach to combat this problem would be using natural extracellular matrix constituents as a mimic of endothelial basement membrane along with applying anticoagulant agents to provide local antithrombotic effects. In this study, we fabricated aligned and random electrospun poly-L-lactic acid (PLLA) scaffolds containing acetylsalicylic acid (ASA) as the anticoagulation agent and surface coated them with amniotic membrane (AM) lysate. Vascular scaffolds were structurally and mechanically characterized and assessed for cyto- and hemocompatibility and their ability to support endothelial differentiation was examined. All the scaffolds showed appropriate tensile strength as expected for vascular grafts. Lack of cytotoxicity, cellular attachment, growth, and infiltration were proved using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and scanning electron microscopy. The blood compatibilities of different scaffolds examined by in vitro hemolysis and blood coagulation assays elucidated the excellent hemocompatibility of our novel AM-coated ASA-loaded nanofibers. Drug-loaded scaffolds showed a sustained release profile of ASA in 7 days. AM-coated electrospun PLLA fibers showed enhanced cytocompatibility for human umbilical vein ECs, making a confluent endothelial-like lining. In addition, AM lysate-coated ASA-PLLA-aligned scaffold proved to support endothelial differentiation of Wharton's jelly-derived mesenchymal stem cells. Our results together indicated that AM lysate-coated ASA releasing scaffolds have promising potentials for development of a biocompatible SDVG.     

Human cytomegalovirus infection of endothelial cells triggers platelet adhesion and aggregation

Human cytomegalovirus (HCMV) is an opportunistic pathogen that has been implicated in the pathogenesis of vascular diseases. HCMV infection of endothelial cells may lead to vascular damage in vitro, and acute-phase HCMV infection has been associated with thrombosis. We hypothesized that viral infection of endothelial cells activates coagulation cascades and contributes to thrombus formation and acute vascular catastrophes in patients with atherosclerotic disease. To assess the effects of HCMV on thrombogenesis, we examined the adhesion and aggregation of blood platelets to uninfected and HCMV-infected endothelial cells. At 7 days after infection, platelet adherence and aggregation were greater in infected than in uninfected cultures (2,000 platelets/100 cells and 225 +/- 15 [mean +/- standard error of the mean] aggregates/five microscopic fields versus 100 platelets/100 cells and no aggregates). von Willebrand factor (vWF), ICAM-1, and VCAM-1 but not collagen IV, E-selectin, P-selectin, CD13, and CD31 were expressed at higher levels on infected cells than on uninfected cells. Platelet aggregation was inhibited by blocking of platelet GPIb (with blocking antibodies) or GPIIb/IIIa (with ReoPro) or by blocking of vWF (with polyclonal antibodies to vWF). Furthermore, blocking of vWF, platelet GPIb, and ICAM-1 but not of the endothelial cell marker CD13, alpha(5)beta(3)-integrin, or HCMV glycoprotein B reduced platelet adherence to infected cells by 75% +/- 5%, 74% +/- 5%, or 18% +/- 5%, respectively. The increased thrombogenicity was dependent on active virus replication and could be inhibited by foscarnet and ganciclovir; these results suggest that a late viral gene may be mediating this phenomenon, which may contribute to vascular catastrophes in patients with atherosclerotic disease.     

An Anti-CD34 Antibody-Functionalized Clinical-Grade POSS-PCU Nanocomposite Polymer for Cardiovascular Stent Coating Applications: A Preliminary Assessment of Endothelial Progenitor Cell Capture and Hemocompatibility

In situ endothelialization of cardiovascular implants has emerged in recent years as an attractive means of targeting the persistent problems of thrombosis and intimal hyperplasia. This study aimed to investigate the efficacy of immobilizing anti-CD34 antibodies onto a POSS-PCU nanocomposite polymer surface to sequester endothelial progenitor cells (EPCs) from human blood, and to characterize the surface properties and hemocompatibility of this surface. Amine-functionalized fumed silica was used to covalently conjugate anti-CD34 to the polymer surface. Water contact angle, fluorescence microscopy, and scanning electron microscopy were used for surface characterization. Peripheral blood mononuclear cells (PBMCs) were seeded on modified and pristine POSS-PCU polymer films. After 7 days, adhered cells were immunostained for the expression of EPC and endothelial cell markers, and assessed for the formation of EPC colonies. Hemocompatibility was assessed by thromboelastography, and platelet activation and adhesion assays. The number of EPC colonies formed on anti-CD34-coated POSS-PCU surfaces was not significantly higher than that of POSS-PCU (5.0±1.0 vs. 1.7±0.6, p>0.05). However, antibody conjugation significantly improved hemocompatibility, as seen from the prolonged reaction and clotting times, decreased angle and maximum amplitude (p<0.05), as well as decreased platelet adhesion (76.8±7.8 vs. 8.4±0.7, p<0.05) and activation. Here, we demonstrate that POSS-PCU surface immobilized anti-CD34 antibodies selectively captured CD34⁺ cells from peripheral blood, although only a minority of these were EPCs. Nevertheless, antibody conjugation significantly improves the hemocompatibility of POSS-PCU, and should therefore continue to be explored in combination with other strategies to improve the specificity of EPC capture to promote in situ endothelialization.     

小鼠胚胎干细胞结合丝素材料向神经细胞分化的实验研究

以小鼠胚胎干细胞（ES）为种子细胞,使用改良的4-/4+ RA方案,诱导小鼠ES细胞在丝素材料上向神经细胞分化,探讨丝素材料对其生长、黏附、分化等情况的影响。将小鼠ES细胞悬浮培养4 d得到的拟胚体（EBs）分别接种到经丝素膜和明胶包被的培养皿上进行诱导,比较不同材料上EBs的贴壁率及向神经元分化的比率。结果表明EBs在明胶和柞蚕丝素蛋白膜（TSF）上贴壁较快,平均贴壁率为90.3%和84.4%,在桑蚕丝素蛋白膜（SF）上贴壁较慢,贴壁率低,仅为38.5%,同时三者神经元的分化比率均能达到40%以上,无明显差异。通过以上实验,我们得出,TSF有望成为小鼠ES细胞向神经细胞分化的支架材料。     

A comparison of Thai silk fibroin-based and chitosan-based materials on in vitro biocompatibility for bone substitutes

The novel hybrid scaffolds fabricated from silk fibroin, gelatin, low deacetylation degree chitosan and hydroxyapatite were investigated for their in vitro biocompatibility and osteoconductivity to mouse pre-osteoblast cell line (MC3T3-E1) and rat bone marrow-derived stem cells (MSC). We found that gelatin-conjugated silk fibroin films and scaffolds dominantly promoted cell adhesion and proliferation. Film and scaffold prepared from gelatin-conjugated silk fibroin with hydroxyapatite grown crystals effectively enhanced osteogenic differentiation of both cell types, as evaluated by alkaline phosphatase activity and calcium content. However the blend of hydroxyapatite/low deacetylation degree chitosan hybrid materials did not support cell growth. Furthermore, the blended hydroxyapatite in the bulk scaffold was found to be less effective for osteogenic differentiation than the scaffold with hydroxyapatite grown crystals. The comparative study between MC3T3-E1 and MSC showed that both cell types had similar trend of proliferation and osteogenic differentiation on the same material. Also, higher proliferative rate of MC3T3-E1 than MSC was observed.     

Dynamic light scattering of native silk fibroin solution extracted from different parts of the middle division of the silk gland of the Bombyx mori silkworm

Dynamic light scattering (DLS) measurements were performed on aqueous solutions of native silk fibroin extracted from three parts, the posterior (MP), the middle (MM), and the anterior parts (MA), of the middle division (M) of the silk gland of the Bombyx mori silkworm to study the dynamics and aggregation properties of silk fibroin. In the MP part, fibroin molecules are present as aggregates (or clusters) being composed of several large protein complexes or elementary unit (EU), which are further associated to make a large assembly connected via divalent metallic ions. In the MM part, such clusters of EU take more compact structure, and finally in the MA part, clusters disappear, but EUs are more or less aligned to keep the assembly, and the EU takes the conformation of wormlike cylinder capped with hemispheres at both ends. The overall conformational change in solution structure was interpreted as being due to the change in ionic environment in the solution. DLS study was also performed on regenerated silk fibroin solutions, which revealed that fibroin is present as a single molecule dominantly and their association behavior seems completely different from that of native samples and does not depend on types and concentration of added metallic ions.     

Structural evolution of regenerated silk fibroin under shear: combined wide- and small-angle x-ray scattering experiments using synchrotron radiation

The structural evolution of regenerated Bombyx mori silk fibroin during shearing with a Couette cell has been studied in situ by synchrotron radiation small- and wide-angle x-ray scattering techniques. An elongation of fibroin molecules was observed with increasing shear rate, followed by an aggregation phase. The aggregates were found to be amorphous with beta-conformation according to infrared spectroscopy. Scanning x-ray microdiffraction with a 5 microm beam on aggregated material, which had solidified in air, showed silk II reflections and a material with equatorial reflections close to the silk I structure reflections, but with strong differences in reflection intensities. This silk I type material shows up to two low-angle peaks suggesting the presence of water molecules that might be intercalated between hydrogen-bonded sheets.     

Surface modification of poly(L-lactic acid) membrane via layer-by-layer assembly of silver nanoparticle-embedded polyelectrolyte multilayer

The improvement of hydrophilicity, antibacterial activity, hemocompatibility, and cytocompatibility of poly(L-lactic acid) (PLLA) membrane was developed via polyelectrolyte multilayer (PEM) immobilization. Colloidal silver nanoparticles were prepared by using dextran sulfate (DS) as a stabilizer to precede chemical reduction by dextrose. The polysaccharide PEMs, including chitosan (CH) and dextran sulfate (DS)-stabilized silver nanosized colloid (DSS), were successfully deposited on the aminolyzed PLLA membrane in a layer-by-layer (LBL) self-assembly manner. The obtained results showed that the contact angle of PLLA membranes decreased with PEMs grafting layers and reached a steady value after four bilayers of coating, hence suggesting that full coverage was achieved. The PLLA-PEM membranes with DSS as the outermost layer could resist platelet adhesion and human plasma fibrinogen (HPF) adsorption, while prolonging the blood coagulation time. The PLLA-PEM membranes could possess antibacterial activity against Methicilin-resistant Staphylococus aureus (MRSA). In addition, the proliferation and viability of human endothelial cells (ECs) on PLLA-PEM membranes could be significantly improved. Overall results demonstrated that such a fast, easy processing and shape-independent method for an antithrombogenic coating can be used for applications in hemodialysis devices.     

Electrospinning Bombyx mori silk with poly(ethylene oxide)

Electrospinning for the formation of nanoscale diameter fibers has been explored for high-performance filters and biomaterial scaffolds for vascular grafts or wound dressings. Fibers with nanoscale diameters provide benefits due to high surface area. In the present study we explore electrospinning for protein-based biomaterials to fabricate scaffolds and membranes from regenerated silkworm silk, Bombyx mori, solutions. To improve processability of the protein solution, poly(ethylene oxide) (PEO) with molecular weight of 900,000 was blended with the silk fibroin. A variety of compositions of the silk/PEO aqueous blends were successfully electrospun. The morphology of the fibers was characterized using high-resolution scanning electron microscopy. Fiber diameters were uniform and less than 800 nm. The composition was estimated by X-ray photoelectron spectroscopy to characterize silk/PEO surface content. Aqueous-based electrospining of silk and silk/PEO blends provides potentially useful options for the fabrication of biomaterial scaffolds based on this unique fibrous protein.     

Analysis of the effect of disturbed flow on monocytic adhesion to endothelial cells

The preferential adhesion of monocytes to vascular endothelial cells (ECs) at regions near branches and curvatures of the arterial tree, where flow is disturbed, suggests that hemodynamic conditions play significant roles in monocyte adhesion. The present study aims to elucidate the effects of disturbed flow on monocyte adhesion to ECs and the adhesive properties of ECs. We applied, for the first time, the micron-resolution particle image velocimetry (μPIV) technique to analyze the characteristics of the disturbed flow produced in our vertical-step flow (VSF) chamber. The results demonstrated the existence of a higher near-wall concentration and a longer residence time of the monocytic analog THP-1 cells near the step and the reattachment point. THP-1 cells showed prominent adhesion to ECs pretreated with TNF in the regions near the step and the reattachment point, but they showed virtually no adhesion to un-stimulated ECs. Pre-incubation of the TNF-treated ECs with antibodies against intercellular adhesion molecule-1 (ICAM-1), vascular adhesion molecule-1 (VCAM-1), and E-selectin inhibited the THP-1 adhesion; the maximal inhibition was observed with a combination of these antibodies. Pre-exposure of ECs to disturbed flow in VSF for 24 h led to significant increases in their surface expressions of ICAM-1 and E-selectin, but not VCAM-1, and in the adhesion of THP-1 cells. Our findings demonstrate the importance of complex flow environment in modulating the adhesive properties of vascular endothelium and consequently monocyte adhesion in regions of prevalence of atherosclerotic lesions.     

Freeze-gelled silk fibroin protein scaffolds for potential applications in soft tissue engineering

Recently tissue engineering has escalated much interest in biomedical and biotechnological applications. In this regard, exploration of new and suitable biomaterials is needed. Silk fibroin protein is used as one of the most preferable biomaterials for fabrication of scaffolds and several new techniques are being adopted to fabricate silk scaffolds with greater ease, efficiency and perfection. In this study, a freeze gelation technique is used for fabrication of silk fibroin protein 3D scaffolds, which is both time and energy efficient as compared to the conventional freeze drying technique. The fabricated silk fibroin freeze-gelled scaffolds are evaluated micro structurally for morphology with scanning electron microscopy which reveals relatively homogeneous pore structure and good interconnectivity. The pore sizes and porosity of these scaffolds ranges between 60-110 μm and 90-95%, respectively. Mechanical test shows that the compressive strength of the scaffolds is in the range of 20-40 kPa. The applicability to cell culture of the freeze gelled scaffolds has been examined with human keratinocytes HaCat cells which show the good cell viability and proliferation of cells after 5 days of culture suggesting the cytocompatibility. The freeze-gelled 3D scaffolds show comparable results with the conventionally prepared freeze dried 3D scaffolds. Thus, this technique may be used as an alternative method for 3D scaffolds preparation and may also be utilized for tissue engineering applications.     

Impact of pre-incubation time of silk fibroin scaffolds in culture medium on cell proliferation and attachment

Cell behaviours such as proliferation and attachment can be affected by the length of pre-incubation period of the scaffolds in the culture medium for long term. The aim of this study was to investigate the long term pre-incubation of 3D silk fibroin scaffolds in complete culture medium on cell attachment and proliferation. After the preparation of silk fibroin scaffolds by the technique of freeze drying, the scaffolds were pre-incubated in complete culture medium for 2 d, 6 d or 10 d before apical papilla stem cells (SCAP) seeding. Modifications of the scaffold surface and wettability were examined by FE-SEM and water contact angle, respectively. Results showed a decrease both in roughness and water contact angle as pre-incubation time increases. DNA measurement after 18 h and 10 d cell seeding showed a significant increase of DNA concentration which represents better attachment and proliferation with pre-incubation time increase. Qualitative examination, live&dead assay or H&E staining method after 30 h and 10 d cell seeding respectively, indicated that pre-incubation of scaffolds has time dependent effect on cell proliferation and attachment. This suggests that improvement of cell attachment and proliferation may be mediated by differences in the amount of wettability (decreased water contact angle) after exposure of scaffold to culture medium for long term which, in turn, causes more protein adsorption in the surface of silk fibroin scaffold (decreased roughness).     

Scaffold-Free Tubular Tissues Created by a Bio-3D Printer Undergo Remodeling and Endothelialization when Implanted in Rat Aortae

BackgroundSmall caliber vascular prostheses are not clinically available because synthetic vascular prostheses lack endothelial cells which modulate platelet activation, leukocyte adhesion, thrombosis, and the regulation of vasomotor tone by the production of vasoactive substances. We developed a novel method to create scaffold-free tubular tissue from multicellular spheroids (MCS) using a “Bio-3D printer”-based system. This system enables the creation of pre-designed three-dimensional structures using a computer controlled robotics system. With this system, we created a tubular structure and studied its biological features.ConclusionsThe scaffold-free tubular tissues made of MCS using a Bio-3D printer underwent remodeling and endothelialization. Further studies are warranted to elucidate the underlying mechanism of endothelialization and its function, as well as the long-term results.