Characterization of smooth muscle cells on poly(ε-caprolactone) films

Bioresorbable vascular grafts can be used for direct implantation. Over time, the grafts will degrade and be replaced by natural tissue. In this study, the potential application of biaxially drawn poly(ε-caprolactone) (PCL) films for the design of vascular grafts was examined. PCL films were first modified to enhance cell physiological response to the surface. Two methods of surface modification were studied: surface hydrolysis by immersion in sodium hydroxide, and immobilization of collagen onto PCL film surface. Tensile tests indicate that immersion in sodium hydroxide results in a significant drop in ultimate tensile strength, whereas collagen-immobilized films remained uncompromised. Human coronary artery smooth muscle cells were cultured on the different surfaces, and it was demonstrated that collagen-immobilized films elicited the most favorable response from the cultured cells. This indicates the potential for collagen-immobilized PCL films for vascular tissue engineering applications.     

静电纺丝制备壳聚糖/聚己内酯血管支架及表征

结合壳聚糖(CS)和聚己内酯(PCL)二者的优点, 以静电纺丝的方法制备了CS/PCL血管支架。采用SEM和电子万能试验机检测了该支架的结构和力学性能, 将内皮祖细胞(EPCs)与该支架膜复合培养, 评估了该血管支架维持细胞黏附、 繁殖和分化的能力。SEM结果显示: 通过静电纺丝可以得到多孔、 类似于天然细胞外基质的直径约400nm的纤维微结构; 当CS与PCL质量比为0.5时, 静电纺丝所制备的CS/PCL血管支架弹性最大形变达到31.64%, 应力-应变曲线显示其弹性变形能力较强; EPCs在CS/PCL血管支架黏附率可达95.1%, 荧光显微镜观察结果也显示了CS/PCL血管支架利于细胞黏附、 生长。      

The synergistic effect of aligned nanofibers and hyaluronic acid modification on endothelial cell behavior for vascular tissue engineering

The endothelialization of tissue-engineered vascular grafts (TEVGs) is considered to be an effective strategy to prevent the coagulation and restenosis of small-diameter vascular grafts. In this study, we fabricated well aligned nanofibrous scaffolds with PCL using a high speed rotating collector, modified those surfaces with hyaluronic acid (HA) and studied the synergistic effect of the scaffolds on the endothelial cells behavior in vitro. The well-aligned oriented architecture was observed by SEM images in the nanofibrous scaffolds. The contact angle measurements and FTIR-ATR evidenced that HA was successfully modified on the PCL nanofibrous scaffolds and hydrophilicity of the scaffolds was increased after HA coating. The results of adhesion and morphology of human umbilical vein endothelial cells (HUVECs) showed that the HA-coating aligned PCL (HA-aPCL) nanofibrous scaffolds could highly promote attachment and guide HUVECs bipolar spread with the parallel aligned nanofibers. Furthermore, HUVECs on the HA-aPCL formed a confluent monoendothelial cell layer and exhibited superior protein expression levels of von Willebrand factor (vWF). This study suggested that the combination of aligned nanostructure and HA modification was more capable of promoting the regeneration of functional endothelium for vascular tissue engineering than individual use.     

Zwitterionic PMCP‐Modified Polycaprolactone Surface for Tissue Engineering: Antifouling,Cell Adhesion Promotion,and Osteogenic Differentiation Properties

Biodegradable polycaprolactone (PCL) has been widely applied as a scaffold material in tissue engineering. However, the PCL surface is hydrophobic and adsorbs nonspecific proteins. Some traditional antifouling modifications using hydrophilic moieties have been successful but inhibit cell adhesion, which is not ideal for tissue engineering. The PCL surface is modified with bioinspired zwitterionic poly[2‐(methacryloyloxy)ethyl choline phosphate] (PMCP) via surface‐initiated atom transfer radical polymerization to improve cell adhesion through the unique interaction between choline phosphate (CP, on PMCP) and phosphate choline (PC, on cell membranes). The hydrophilicity of the PCL surface is significantly enhanced after surface modification. The PCL‐PMCP surface reduces nonspecific protein adsorption (e.g., up to 91.7% for bovine serum albumin) due to the zwitterionic property of PMCP. The adhesion and proliferation of bone marrow mesenchymal stem cells on the modified surface is remarkably improved, and osteogenic differentiation signs are detected, even without adding any osteogenesis‐inducing supplements. Moreover, the PCL‐PMCP films are more stable at the early stage of degradation. Therefore, the PMCP‐functionalized PCL surface promotes cell adhesion and osteogenic differentiation, with an antifouling background, and exhibits great potential in tissue engineering.     

A novel polyurethane modified with biomacromolecules for small-diameter vascular graft applications

There is a growing demand for small-caliber tissue-engineered vascular grafts to replace damaged vessels. Fabricated scaffolds are unable to precisely mimic the mechanical properties of native vessels, provide long-term patency and support cell adhesion and growth, in particular support endothelialization. In this study, a new biodegradable poly(ether ester urethane) urea (PEEUU) was synthesized. The synthesized polyurethane was then functionalized by introducing free amino groups through aminolysis for further surface modification by immobilization of biomacromolecules on the surface of vascular grafts. The modified surfaces were then characterized using attenuated total reflectance-Fourier transform infrared spectroscopy, water contact angle measurement and atomic force microscopy. The mechanical properties of the fabricated scaffolds were analyzed, revealing mechanical properties close to that of the natural vessels. Surface modifications led to improved cell–scaffold interactions, showing appropriate cell attachment and function on the scaffolds. A confluent layer of endothelial cells was formed on biomacromolecule-immobilized PEEUU vascular grafts. The preliminary results of this study demonstrated that the new polyurethane modified with biomacromolecules can be considered as a candidate material for vascular tissue engineering application with capability to support endothelialization of fabricated vascular grafts.     

A novel strategy to graft RGD peptide on biomaterials surfaces for endothelization of small-diamater vascular grafts and tissue engineering blood vessel

To improve the performance of small-diamater vascular grafts, endothelization of biomaterials surfaces and tissue engineering are more promising strategies to fabricate small-diamater vascular grafts. In this study, a Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) peptide was grafted on the surfaces of poly(carbonate urethane)s (PCUs), with photoactive 4-benzoylbenzoic acid (BBA) by UV irradiation. The photoactive peptides (BBM-GRGDSP) were synthesized with classical active ester of peptide synthesis. The modified surfaces of PCU with the photoactive RGD peptides were characterized by water contact angle measurement and X-ray Photoelectron Spectroscopy (XPS), which results suggested that the peptides were successfully grafted on the PCU surfaces. The effect of these modified surfaces on endothelial cells (ECs) adhesion and proliferation was examined over 72 h. PCU surfaces coupled with the synthetic photoactive RGD peptides, as characterized with phase contrast microscope and the metabolic activity (MTT) assay enhanced ECs proliferation and spreading with increasing concentration of RGD peptides grafted on their surfaces. Increased retention of ECs was also observed on the polymers surfaces under flow shear stress conditions. The results demonstrated that GRGDSP peptides grafted on the surfaces of polymers with photoactive 4-benzoylbenzoic acids could be an efficient method of fabrication for artificial small-diamater blood vessels. The modified polymer is expected to be used for small-diamater vascular grafts and functional tissue engineered blood vessels to improve ECs adhesion and retention on the polymer surfaces under flow shear stress conditions.     

Synthesis and characterization of biodegradable poly (ethylene glycol) and poly (caprolactone diol) end capped poly (propylene fumarate) cross linked amphiphilic hydrogel as tissue engineering scaffold material

Biodegradable poly (caprolactone diol-co-propylene fumarate-co-ethylene glycol) amphiphilic polymer with poly (ethylene glycol) and poly (caprolactone diol) chain ends (PCL–PPF–PEG) was prepared. PCL–PPF–PEG undergoes fast setting with acrylamide (aqueous solution) by free radical polymerization and produces a crosslinked hydrogel. The cross linked and freeze-dried amphiphilic material has porous and interconnected network. It undergoes higher degree of swelling and water absorption to form hydrogel with hydrophilic and hydrophobic domains at the surface and appreciable tensile strength. The present hydrogel is compatible with L929 fibroblast cells. PCL–PPF–PEG/acrylamide hydrogel is a candidate scaffold material for tissue engineering applications.     

Investigation of the graft length impact on the interfacial toughness in a cellulose/poly(ε-caprolactone) bilayer laminate

Interfacial adhesion between immiscible cellulose-polymer interfaces is a crucial property for fibrous biocomposites. To tailor the interfacial adhesion, the grafting of polymers from cellulose films was studied using ring-opening polymerization of ε-caprolactone. The poly(ε-caprolactone) (PCL) grafted cellulose was analyzed with FTIR, AFM and via water CA measurements. The graft length was varied by the addition of a free initiator, enabling tailoring of the interfacial toughness. Films of microfibrillated cellulose were grafted with PCL and hot-pressed together with a PCL-film to form a bilayer laminate. Interfacial peeling toughness correlates very strongly with PCL degree of polymerization (DP). PCL grafts form physical entanglements in the PCL matrix and promote significant plastic deformation in the PCL bulk, thus increasing interfacial peeling energy.     

Evaluation of electrospun fibrous scaffolds of poly(dl-lactide) and poly(ethylene glycol) for skin tissue engineering

This study is derived from the innate concerns of electrospun poly(DL-lactide) (PDLLA) fibers as tissue engineering scaffolds: hydrophobic surface, surface erosion and dimensional shrinkage, which are not favorable to trigger the initial adhesion and further growth and population of cells. Blending electrospinning of PDLLA and poly(ethylene glycol) (PEG) with different PEG contents was evaluated for optimal tissue engineering scaffolds. The surface hydrophilicity was improved, and the degradation patterns of PDLLA/PEG mats changed from surface erosion to bulk degradation with the increase in PEG contents. The dimensional shrinkage was alleviated through the formation of crystal regions of PEG in the fiber matrix. The PDLLA/PEG fibrous mats were slightly weakened with the increase in the PEG contents, but a significant decrease in the tensile strength could be found for those with PEG contents of over 40%. Human dermal fibroblasts (HDFs) interacted and integrated well with the surrounding fibers containing 20 and 30% PEG, which provided significantly better environment for biological activities of HDFs than electrospun PDLLA mats. It indicated that electrospun mats containing 30% PEG exhibited the most balanced properties, including moderately hydrophilic surface, minimal dimensional changes, adaptable bulk biodegradation pattern and enhancement of cell penetration and growth within fibrous mats.     

Chemical activation and changes in surface morphology of poly(ε-caprolactone) modulate VEGF responsiveness of human endothelial cells

The high degree of clinical routine in percutaneous transluminal coronary angioplasty (PTCA) with and without stenting has not changed the fact that a large number of coronary heart disease patients are still affected by post-operative complications such as restenosis and thrombosis. Because re-endothelialization is the crucial aspect of wound healing after cardiovascular implant surgery, there is a need for modern biomaterials to aid endothelial cells in their adhesion and functional recovery post-stenting. This study systematically examines the potential of numerous chemical polymer modifications with regard to endothelialization. Poly(ε-caprolactone) (PCL) and its chemically activated forms are investigated in detail, as well as the impact of polymer surface morphology and precoating with matrix protein. Human umbilical vein endothelial cells (HUVECs) are used to characterize endothelial cell responses in terms of in vitro viability and adhesion. As a potential component in drug eluting implants, VEGF is applied as stimulus to boost endothelial cell proliferation on the polymer. In conclusion, plasma chemical activation of PCL combined with VEGF stimulation best enhances in vitro endothelialization. Examining the impact of morphological, chemical and biological modifications of PCL, this study makes an important new contribution towards the existing body of work on polymer endothelialization.     

Synthesis of multiblock thermoplastic elastomers based on biodegradable poly (lactic acid) and polycaprolactone

A new biodegradable (AB)_n type of multiblock copolymers derived from poly (ε-caprolactone) (PCL) and poly (lactic acid) (PLA) was prepared via the method of the chain extending reaction among PCL oligomers, PLA oligomers and hexamethylene diisocyanate (HDI). Fourier transform infrared spectra (FTIR), ¹H NMR, thermal gravity analysis (TGA) and derivative thermograms (DTG) were used to characterize the copolymers and the results showed that PCL and PLA were coupled by the reaction between –NCO groups and terminal –OH and –COOH groups of PCL and PLA, respectively. The material displayed enhanced mechanical properties: Young's modulus was as low as 2.7 ± 0.7 MPa and elongation at break value was above 790% at the composition of PCL/PLA = 80/20 (w/w). Moreover, according to SEM micrographs interfacial adhesion of the composites was improved. Thermal degradation temperature of the composites was higher than PLA but was lower than PCL, which is an advantage for industry process.     

Surface-modified hyaluronic acid hydrogels to capture endothelial progenitor cells

A major challenge to the effective treatment of injured cardiovascular tissues is the promotion of endothelialization of damaged tissues and implanted devices. For this reason, there is a need for new biomaterials that promote endothelialization to enhance vascular repair. The goal of this work was to develop antibody-modified polysaccharide-based hydrogels that could selectively capture endothelial progenitor cells (EPCs). We showed that CD34 antibody immobilization on hyaluronic acid (HA) hydrogels provides a suitable surface to capture EPCs. The effect of CD34 antibody immobilization on EPC adhesion was found to be dependent on antibody concentration. The highest level of EPC attachment was found to be 52.2 cells per mm(2) on 1% HA gels modified with 25 μg mL(-1) antibody concentration. Macrophages did not exhibit significant attachment on these modified hydrogel surfaces compared to the EPCs, demonstrating the selectivity of the system. Hydrogels containing only HA, with or without immobilized CD34, did not allow for spreading of EPCs 48 h after cell seeding, even though the cells were adhered to the hydrogel surface. To promote spreading of EPCs, 2% (w/v) gelatin methacrylate (GelMA) containing HA hydrogels were synthesized and shown to improve cell spreading and elongation. This strategy could potentially be useful to enhance the biocompatibility of implants such as artificial heart valves or in other tissue engineering applications where formation of vascular structures is required.     

Combinatorial coating of adhesive polypeptide and anti-CD34 antibody for improved endothelial cell adhesion and proliferation

Improved attachment, adhesion and proliferation of the surrounding mature endothelial cells (ECs) and circulating endothelial progenitor cells (EPCs) is of primary importance to realize the in situ rapid re-endothelialization of cardiovascular stents. To achieve this, a combinatorial coating of synthesized mussel adhesive polypeptide mimics as well as anti-CD34 antibody was constructed onto the devices through a novel adsorption method in this study. To immobilize the polypeptide and target antibody effectively, polycaprolactone (PCL) was first spin-coated onto the substrate as intermediate. The immobilization of polypeptide and antibody was confirmed by the changes of water contact angles and the attachment, growth of ECs and EPCs on the substrates, respectively. The results showed that after adhesive polypeptide or/and antibody immobilization, the hydrophilicity of coated PCL substrate (PCLS) was obviously improved. The amount of the immobilized antibody, determined by enzymelinked immunoassay (ELISA) method, was enhanced with the increase of antibody concentrations in the range from 5 to 25 μg/ml. The coatings after BSA blocking prevented the unspecific protein adsorption as monitored by fluorescent microscopy. The results of in vitro cell culture showed that compared with the PCLS, polypeptide/anti-CD34 antibody coating could effectively enhance the attachment, growth and adhesion of ECs and EPCs, in particular EPCs. A platelet adhesion experiment revealed that the blood compatibility of the PCLS after polypeptide/anti-CD34 antibody coating was also obviously improved. The results showed that the surface modification with adhesive polypeptide and anti-CD34 antibody will be a promising coating technique for the surface modification of the intravascular prostheses for rapid re-endothelialization.     

Poly(ε-caprolactone)-based nanocomposites: Influence of compatibilization on properties of poly(ε-caprolactone)–silica nanocomposites

In the present paper, results about preparation and characterization of poly(ε-caprolactone) (PCL) based nanocomposites filled with silica nanoparticles are reported. In order to promote polymer/inorganic nanofiller compatibility and to increase the interfacial adhesion between the two components, silica nanoparticles surface has been functionalised by grafting a M_w = 10,000 Da PCL onto it. Successively, PCL based nanocomposites have been prepared by extrusion process. The relationships among size, amount of the nanofiller, organic coating and the final properties have been investigated. The morphological analysis has revealed that the silica functionalization can provide a useful method of preparation of the nanocomposites with the achievement of a fine, a good dispersion and a strong adhesion level. Thermal characterization has shown an improved thermal stability due to the presence of the silica nanoparticles, especially in the case of modified nanofillers. Finally mechanical tests revealed an increase of the Young’s modulus in the PCL based nanocomposites.     

Electrospun PELCL membranes loaded with QK peptide for enhancement of vascular endothelial cell growth

One of the major challenges in tissue engineering of small-diameter vascular grafts is to inhibit intimal hyperplasia and keep long-term patency after implantation. Rapid endothelialization of the grafts could be an effective approach. In this study, QK, a peptide mimicking vascular endothelial growth factor, was selected as the bioactive substrate and loaded in electrospun membranes for enhancement of vascular endothelial cell growth. In detail, QK peptide was firstly introduced with poly(ethylene glycol) diacrylate into a thiolated chitosan solution that could transfer into hydrogel. Then, suspensions or emulsions of poly(ethylene glycol)-b-poly(l-lactide-co-ε-caprolactone) (PELCL) containing QK peptide (with or without chitosan hydrogel) were electrospun into fibrous membranes. For comparison, the electrospun PELCL membrane without QK was also fabricated. Results of release behaviors showed that the electrospun membranes, especially that contained chitosan hydrogel prepared by suspension electrospinning, could successfully encapsulate QK peptide and maintain its secondary structure after released. In vitro cell culture studies exhibited that the release of QK peptide could accelerate the proliferation of vascular endothelial cells in the 9 days. It was suggested that the electrospun PELCL membranes loaded with QK peptide might have potential applications in vascular tissue engineering.     

Electrospun poly(ε-caprolactone)/Ca-deficient hydroxyapatite nanohybrids: Microstructure,mechanical properties and cell response by murine embryonic stem cells

Nanohybrid scaffolds mimicking extracellular matrix are promising experimental models to study stem cell behaviour, in terms of adhesion and proliferation. In the present study, the structural characterization of a novel electrospun nanohybrid and the analysis of cell response by a highly sensitive cell type, embryonic stem (ES) cells, are investigated. Ca-deficient hydroxyapatite nanocrystals (d-HAp) were synthesized by precipitation. Fibrous PCL/d-HAp nanohybrids were obtained by electrospinning, d-HAp content ranging between 2 and 55 wt.%. Electrospun mats showed a non-woven architecture, average fiber size was 1.5 ±0.5 μm, porosity 80–90%, and specific surface area 16 m² g^? 1. Up to 6.4 wt.% d-HAp content, the nanohybrids displayed comparable microstructural, mechanical and dynamo-mechanical properties. Murine ES cell response to neat PCL and to nanohybrid PCL/d-HAp (6.4 wt.%) mats was evaluated by analyzing morphological, metabolic and functional markers. Cells growing on either scaffold proliferated and maintained pluripotency markers at essentially the same rate as cells growing on standard tissue culture plates with no detectable signs of cytotoxicity, despite a lower cell adhesion at the beginning of culture. These results indicate that electrospun PCL scaffolds may provide adequate supports for murine ES cell proliferation in a pluripotent state, and that the presence of d-HAp within the mat does not interfere with their growth.     

Nanofibrous modification on ultra-thin poly(e-caprolactone) membrane via electrospinning

Poly(e-caprolactone) (PCL) is a favorable material for tissue engineering. PCL was successfully fabricated into less than 10 μm thin membranes using a 2-roll-heated-mill and biaxial stretching process. However, PCL is known for its poor cellular adhesion and surface modifications are needed for any tissue engineering applications. This paper reports on a novel surface modification technique of the PCL membrane by coating with electrospun nanofibers. The purpose was to mimic the architecture of the natural extracellular matrix and create nanotopography for enhanced cellular attachment. The surfaces were characterized by scanning electron microscopy (SEM), water contact angle and atomic force microscopy. The results showed that uniform nanofibrous topology were successfully achieved on the surface of the PCL membrane, with increased roughness (more than 17 times) and surface area. This nanofibrous topology induced capillary effects after sodium hydroxide (NaOH) treatment, causing the water contact angle to drop to almost zero. Scratch tests revealed a strong interaction of PCL nanofiber coating on the PCL membrane. AlamarBlue assay indicated that 3T3 fibroblast cells proliferated well on the nanofibrous membrane. Confocal Laser Scanning Microscope revealed better cell attachment onto the nanofibrous membranes than the untreated membranes. Results from SEM showed that the cells' spindle-shaped morphology on the NaOH-treated fibrous surface was evident while they remained in isolated spherical shaped entities in the non-treated fibrous surfaces.     

A qualitative in vitro evaluation of the degradable materials poly(caprolactone), poly(hydroxybutyrate) and a poly(hydroxybutyrate)-(hydroxyvalerate) copolymer

A qualitative in vitro evaluation of poly(caprolactone) (PCL), poly(hydroxybutyrate) (PHB) and a poly(hydroxybutyrate)-(hydroxyvalerate) (PHB-PHV) copolymer was carried out using primary human osteoblasts (HOB) and a human osteosarcoma (HOS) cell line. The cells were grown on films of these polymers and cultured for 2 and 4 days with cells grown on Thermanox as a control. The cells on each of the polymers exhibited different cellular morphologies with different rates of cell proliferation. Results from a preliminary degradation study demonstrated that biodegradable materials can be partially degraded using enzymes such as papain and trypsin. Of the solutions tested, papain caused the greatest degradation, with phosphate-buffered saline (PBS) a physiological buffer having very little effect over a six week period. The bone cells were grown on partially degraded polymers and no differences in the performance of HOS and HOB cells on the materials were observed.     

Tetracycline hydrochloride (TCH)-loaded drug carrier based on PLA:PCL nanofibre mats: experimental characterisation and release kinetics modelling

The experimental characterisation of electrospun poly(lactic acid) (PLA):poly(ε-caprolactone) (PCL) as drug carriers, at five blend ratios from 1:0, 3:1, 1:1, 1:3 and 0:1, was holistically investigated in terms of their morphological structures, crystallinity levels and thermal properties. A widely used antibiotic tetracycline hydrochloride (TCH) was loaded to prepared fibrous mats at TCH concentrations of 1 and 5 wt%. The additional TCH into PLA:PCL better facilitates the reduction of fibre diameter than polymer blends. Increasing the TCH concentration from 1 to 5 wt% was found to result in only a modest decrease in the crystallinity level, but a significant increase in the crystallisation temperature (T _c) for PLA within PLA:PCL blends. The infrared spectra of fibre mats confirm the successful TCH encapsulation into fibrous networks. The first order and Zeng models for drug release kinetics were in better agreement with experimental release data, indicating the release acceleration of TCH with increasing its concentration. In a typical case of PLA:PCL (1:1) loaded with 5 wt% TCH, the fibre mats apparently demonstrate more wrinkled and floppy structures and increased fibre diameters and decreased inter-fibrous spaces after 7-day in vitro fibre degradation, as opposed to those obtained after 3-h degradation.     

The role of crystallinity on differential attachment/proliferation of osteoblasts and fibroblasts on poly(caprolactone-co-glycolide) polymeric surfaces

The objective of the present study is to systematically evaluate the role of polymer crystallinity on fibroblast and osteoblast adhesion and proliferation using a series of poly(caprolactone-co-glycolide) (PCL/PGA) polymers. PCL/PGA polymers were selected since they reflect both highly crystalline and amorphous materials. PCL/PGA polymeric materials were fabricated by compression molding into thin films. Five compositions, from PCL or PGA to intermediate copolymeric compositions of PCL/PGA in ratios of 25:75, 35:65 and 45:55, were studied. Pure PCL and PGA represented the crystalline materials while the copolymers were amorphous. The polymers/copolymers were characterized using DSC to assess crystallinity, contact angle measurement for hydrophobicity, and AFM for nanotopography. The PCL/PGA films demonstrated similar hydrophobicity and nanotopography whereas they differed significantly in crystallinity. Cell adhesion to and proliferation on PCL/PGA films and proliferation studies were performed using osteoblasts and NIH-3T3 fibroblasts. It was observed that highly crystalline and rigid PCL and PGA surfaces were significantly more efficient in supporting fibroblast growth, whereas amorphous/flexible PCL/PGA 35:65 was significantly more efficient in supporting growth of osteoblasts. This study demonstrated that while chemical composition, hydrophobicity and surface roughness of PCL/PGA polymers were held constant, crystallinity and rigidity of PCL/PGA played major roles in determining cell responses.