Chondroitin-4-sulfate: a bioactive macromolecule to foster vascular healing around stent-grafts after endovascular aneurysm repair

Deficient healing after endovascular aneurysm repair with a stent-graft is thought to be related to pro-apoptotic environment in abdominal aortic aneurysms and inertness of graft materials. We developed a bioactive coating containing chondroitin-4-sulfate and assessed its potential to improve cell adhesion, viability and resistance to apoptosis on PET surfaces. Coatings of collagen type I and CS were prepared and characterized by DMMB, FT-IR, DSC, SEM and contact angle goniometry. Preliminary cell culture experiments with vascular smooth muscle cells showed increased adhesion and viability in serum-free medium on CS-coated surfaces compared to control PET films.     

HuBiogel incorporated fibro-porous hybrid nanomatrix graft for vascular tissue interfaces

Native extracellular matrix (ECM) possesses the biochemical cues to promote cell survival. However, decellularized, the ECM loses its cell supporting mechanical integrity. We report, here, a new biohybrid vascular graft fabricated from a blend of polycaprolactone (PCL), poliglecaprone (PGC), and incorporated with human biomatrix as functional materials for vascular tissue interfacing applications, thus harnessing the biochemical cues from the ECM and the mechanical integrity of the polymer blends. The fabricated fibro-porous tubular small diameter graft (i.d. = 4 mm) from electrospun polymer blend was coated with HuBiogel^tm, a cocktail of collagenous matrix derived from human placenta called . The compositional, morphological, and mechanical properties of graft were measured, analyzed, and compared with a non-coated tubular PCL/PGC graft using Fourier Transform infrared spectroscopy (FTIR), x-ray photoelectron spectroscopy (XPS), and scanning electron microscopy (SEM). BCA assay was used to calculate the protein content and coating-uniformity throughout the hybrid graft. Mechanical properties such as tensile strength (1.6 MPa), Young's modulus (2.4 MPa), burst pressure (>1900 mmHg), and suture retention strength (2.3 N) of hybrid graft were found to be comparable to native blood vessels. Protein coating has improved the hydrophilicity and the biocompatibility (cell viability and cell-attachment) enhanced with human umbilical vein endothelial cells (HUVECs) seeded in vitro onto the lumen layer of the graft over two weeks. The overall results promise this new biohybrid graft to be a potential candidate for vascular tissue interface and regeneration.     

具有内皮细胞选择性的细胞膜仿生支架材料的研究

利用AIBN引发自由基反应,由单体2-(甲基丙烯酰氧基)乙基-2-(三甲基氨基)乙基磷酸酯(MPC)、甲基丙烯酸十八酯(SMA)、对硝基苯氧羰基聚乙二醇甲基丙烯酸酯(MEONP)合成了一种新型类细胞膜仿生涂层材料.MPC可以阻抗非特异性吸附;MEONP可以结合抗体或多肽促进特异性识别.通过表面固定的方法引入多肽序列Arg-Glu-Asp-Val(REDV),使涂层具有内皮细胞选择性.核磁、紫外吸收、红外光谱表征证实聚合物的组成以及REDV多肽在表面的固定;并通过血浆复钙化实验表征涂层的血液相容性.细胞黏附与增殖实验反映REDV多肽构建的涂层表面具备良好的特异性识别并结合内皮细胞的能力.     

Alkali‐Mediated Miscibility of Gelatin/Polycaprolactone for Electrospinning Homogeneous Composite Nanofibers for Tissue Scaffolding

Electrospun natural‐synthetic composite nanofibers, which possess favorable biological and mechanical properties, have gained widespread attention in tissue engineering. However, the development of biomimetic nanofibers of hybrids remains a huge challenge due to phase separation of the polymer blends. Here, aqueous sodium hydroxide (NaOH) solution is proposed to modulate the miscibility of a representative natural‐synthetic hybrid of gelatin (GT) and polycaprolactone (PCL) for electrospinning homogeneous composite nanofibers. Alkali‐doped GT/PCL solutions and nanofibers examined at macroscopic, microscopic, and internal molecular levels demonstrate appropriate miscibility of GT and PCL after introducing the alkali dopant. Particularly, homogeneous GT/PCL nanofibers with smooth surface and uniform diameter are obtained when aqueous NaOH solution with a concentration of 10 m is used. The fibers become more hydrophilic and possess improved mechanical properties both in dry and wet conditions. Moreover, biocompatibility experiments show that stem cells adhere to and proliferate better on the alkali‐modified nanofibers than the untreated one. This study provides a facile and effective approach to solve the phase separation issue of the synthetic‐natural hybrid GT/PCL and establishes a correlation of compositionally and morphologically homogeneous composite nanofibers with respect to cell responses.     

Biofunctionalized Electrospun PCL‐PIBMD/SF Vascular Grafts with PEG and Cell‐Adhesive Peptides for Endothelialization

Artificial small‐caliber vascular grafts are still limited in clinical application because of thrombosis, restenosis, and occlusion. Herein, a small‐caliber vascular graft (diameter 2 mm) is fabricated from poly(ε‐caprolactone)‐b‐poly(isobutyl‐morpholine‐2,5‐dione) (PCL‐PIBMD) and silk fibroin (SF) by electrospinning technology and then biofunctionalized with low‐fouling poly(ethylene glycol) (PEG) and two cell‐adhesive peptide sequences (CREDVW and CAGW) with the purpose of enhancing antithrombogenic activity and endothelialization. The successful grafting of PEG and peptide sequences is confirmed by X‐ray photoelectron spectroscopy. The suitable surface wettability of the modified vascular graft is testified by water contact angle analysis. The surface hemocompatibility is verified by platelet adhesion assays and protein adsorption assays, and the results demonstrate that both platelet adhesion and protein adsorption on the biofunctionalized surface are significantly reduced. In vitro studies demonstrate that the biofunctionalized surface with suitable hydrophilicity and cell‐adhesive peptides can selectively promote the adhesion, spreading, and proliferation of human umbilical vein endothelial cells. More importantly, compared with control groups, this biofunctionalized small‐caliber vascular graft shows high long‐term patency and endothelialization after 10 weeks of implantation. The biofunctionalization with PEG and two cell‐adhesive peptide sequences is an effective method to improve the endothelialization and long‐term performance of synthetic vascular grafts.     

Controlling the Poly(ε‐caprolactone) Degradation to Maintain the Stemness and Function of Adipose‐Derived Mesenchymal Stem Cells in Vascular Regeneration Application

Biodegradable poly(ε‐caprolactone) (PCL) scaffolds with adipose‐derived mesenchymal stem cells (ADSCs) have been used in vascular regeneration studies. An evaluation method of the effect of PCL degradation products (DP) on the viability, stemness, and differentiation capacities of ADSCs is established. ADSCs are cultured in medium containing different concentrations of PCL DP before evaluating the effect of PCL DP on the cell apoptosis and proliferation, cell surface antigens, adipogenic and osteogenic differentiation capacities, and capacities to differentiate into endothelial cells and smooth muscle cells. The results demonstrate that PCL DP exceed 0.05 mg mL^?1 may change the stemness and differentiation capacities of ADSCs. Therefore, to control the proper concentration of PCL DP is essential for ADSCs in vascular regeneration application.     

含悬挂羧基手臂聚乳酸材料的内皮细胞黏附及其细胞活性

为了考察内皮化材料表面的细胞活性, 在前期工作的基础上, 分别在聚乳酸(PLA)、乳酸-苹果酸共聚物(PLMA), 以及含悬挂羟基或羧基的乳酸-苹果酸共聚物膜(PLMAHE,PLMACA)表面种植人脐静脉内皮细胞(HUVEC), 成功地制备了内皮化表面. 通过测定内皮化材料表面内皮细胞释放的内皮型一氧化氮合酶(eNOS)以及一氧化氮的释放量, 间接考察了内皮细胞的抗凝血活性; 另外, 通过内皮化表面的血小板黏附实验, 直接观察了血小板在内皮细胞上的黏附情况. 实验结果表明, 含羧基材料表面的内皮细胞活性比PLA和PLMAHE的高; 相对其它材料PLMACA能更有效地保留黏附于其表面内皮细胞的活性, 其单位内皮细胞的eNOS以及NO的释放量分别为(41.8±8.1) μmol/104 cells和(0.76±0.16) U/104 cells. 电镜照片(SEM)显示, 各种材料表面的内皮细胞均能有效地减少血小板的黏附与聚集; 在内皮细胞脱落的区域, PLMACA仍能较好地实现其抑制血小板黏附的功能, 有望成为新型血管修复(替代)材料.     

Immobilization of anti-CD31 antibody on electrospun poly(ɛ-caprolactone) scaffolds through hydrophobins for specific adhesion of endothelial cells

Hydrophilicity improvement and bioactive surface design of poly(?-caprolactone) (PCL) grafts are of key importance for their application in tissue engineering. Herein, we develop a convenient approach for achieving stable hydrophilic surfaces by modifying electrospun PCL grafts with a class II hydrophobin (HFBI) coating. Static water contact angles (WCA) demonstrated the conversion of the PCL grafts from hydrophobic to hydrophilic after the introduction of amphiphilic HFBI. ATR-FTIR and XPS confirmed the presence of self-assembled HFBI films on the surface of the PCL nanofibers. The biocompatibility of the HFBI-modified PCL grafts was evaluated by cell proliferation in vitro, and by arteriovenous shunt (AV shunt) experiments ex vivo. Anti-CD31 antibody, which is specific for endothelial cells (ECs), was subsequently immobilized on the HFBI-coated PCL scaffolds through protein-protein interactions. This bioactive PCL graft was found to promote the attachment and retention of endothelial cells. These results suggest that this stepwise strategy for introducing cell-specific binding molecules into PCL scaffolds may have potential for development of vascular grafts that can endothelialize rapidly in vivo.     

Incorporation of nanohydroxyapatite and vitamin D3 into electrospun PCL/Gelatin scaffolds: The influence on the physical and chemical properties and cell behavior for bone tissue engineering

Scaffold, an essential element of tissue engineering, should provide proper physical and chemical properties and evolve suitable cell behavior for tissue regeneration. Polycaprolactone/Gelatin (PCL/Gel)‐based nanocomposite scaffolds containing hydroxyapatite nanoparticles (nHA) and vitamin D3 (Vit D3) were fabricated using the electrospinning method. Structural and mechanical properties of the scaffold were determined by scanning electron microscopy (SEM) and tensile measurement. In this study, smooth and bead‐free morphology with a uniform fiber diameter and optimal porosity level with appropriate pore size was observed for PCL/Gel/nHA nanocomposite scaffold. The results indicated that adding nHA to PCL/Gel caused an increase of the mechanical properties of scaffold. In addition, chemical interactions between PCL, gelatin, and nHA molecules were shown with XRD and FT‐IR in the composite scaffolds. MG‐63 cell line has been cultured on the fabricated composite scaffolds; the results of viability and adhesion of cells on the scaffolds have been confirmed using MTT and SEM analysis methods. Here in this study, the culture of the osteoblast cells on the scaffolds showed that the addition of Vit D3 to PCL/Gel/nHA scaffold caused further attachment and proliferation of the cells. Moreover, DAPI staining results showed that the presence and viability of the cells were greater in PCL/Gel/nHA/Vit D3 scaffold than in PCL/Gel/nHA and PCL/Gel scaffolds. The results also approved increasing cell proliferation and alkaline phosphatase (ALP) activity for MG‐63 cells cultured on PCL/Gel/nHA/Vit D3 scaffold. The results indicated superior properties of hydroxyapatite nanoparticles and vitamin D3 incorporated in PCL/Gel scaffold for use in bone tissue engineering.     

Photolithographically Patterned Cell-Repellent PEG-b-PTHPMA Diblock Copolymer for Guided Cell Adhesion and Growth

Surfaces for guided cell adhesion and growth are indispensable in several diagnostic and therapeutic applications. Towards this direction, four diblock copolymers comprising polyethylene glycol (PEG) and poly(2-tetrahydropyranyl methacrylate) (PTHPMA) are synthesized employing PEG macroinitiators of different chain lengths. The copolymer with a 5000 Da PEG block and a PEG-PTHPMA comonomers weight ratio of 43–57 provides a film with the highest stability in the culture medium and the strongest cell repellent properties. This copolymer is used to develop a positive photolithographic material and create stripe patterns onto silicon substrates. The highest selectivity regarding smooth muscle cell adhesion and growth and the highest fidelity of adhered cells for up to 3 days in culture is achieved for stripe patterns with widths between 25 and 27.5 µm. Smooth muscle cells cultured on such patterned substrates exhibit a decrease in their proliferation rate and nucleus area and an increase in their major axis length, compared to the cells cultured onto non-patterned substrates. These alterations are indicative of the adoption of a contractile rather than a synthetic phenotype of the smooth muscle cells grown onto the patterned substrates and demonstrate the potential of the novel photolithographic material and patterning method for guided cell adhesion and growth.     

Thermoresponsive poly(N-isopropyl acrylamide)-grafted polycaprolactone films with surface immobilization of collagen

Thermoresponsive poly(N-isopropylacrylamide) (P(NIPAAm))-grafted polycaprolactone (PCL) films with a suitable amount of immobilized cell-adhesive collagen were prepared to improve cell adhesion and proliferation above the lower critical solution temperature (LCST, 32°C) of P(NIPAAm) without destroying cell detachment properties at lower temperatures. Covalently tethered P(NIPAAm) brushes on PCL film surfaces were first prepared via surface-initiated atom transfer radical polymerization (ATRP). The alkyl bromide end groups of the grafted P(NIPAAm) brushes were used in nucleophilic substitution reactions for the direct coupling of collagen to produce the collagen-immobilized thermoresponsive PCL surface. At 37°C, the cell attachments on the collagen-immobilized thermoresponsive PCL surface were enhanced substantially. The attached cells could be recovered simply by lowering culture temperature. The P(NIPAAm)-grafted PCL films with immobilized collagen are potentially useful as adhesion modifiers for advanced cell culture and tissue engineering applications.     

Fabrication of random and aligned electrospun nanofibers containing graphene oxide for skeletal muscle cells scaffold

Graphene oxide (GO)‐based materials have been explored in biomedical applications as active engineered materials for diagnosis and therapy. Although a large number of studies have been carried out in the last years, aspects involving the orientation and elongation of cells on GO immobilized on polymeric nanofibers are still scarce. We investigated the interactions between skeletal muscle cells and GO immobilized on random and aligned electrospun nanofibers of poly(caprolactone) (PCL), a biocompatible and biodegradable polymer. Oxygen plasma was employed to modify the nanofiber polymer surface to enhance the interactions between the PCL fibers and GO. Scanning electron microscopy and confocal microscopy revealed the morphology and orientation of skeletal muscle cells (C2C12 cells) on random and aligned GO/PCL nanofibers. The approach employed here is useful to investigate the interaction of skeletal muscle cells with biocompatible polymer nanofibers modified with GO intended for cell scaffolds and tissue engineering.     

Effect of honeycomb-patterned surface topography on the adhesion and signal transduction of porcine aortic endothelial cells

Surface topography has vital roles in cellular response. Here, to investigate the mechanism behind cellular response to surface topography, we prepared honeycomb (HC)-patterned films from poly(epsilon-caprolactone) (PCL) with micropatterned surface topography by casting a polymer solution of water-immiscible solvent under high humidity. We characterized the adsorption of fibronectin (Fn) on the film using atomic force microscopy (AFM) and confocal laser scanning microscopy (CLSM). The response of porcine aortic endothelial cells (PAECs) to adsorbed Fn molecules onto HC-patterned films was observed by immunofluorescence labeling of vinculin and the actin fiber of PAECs cultured for 1 and 72 h in serum-free medium. The expression of focal adhesion kinase autophosphorylated at the tyrosine residue (pFAK) at 1 h culture was determined using an immunoprecipitation method. Fn adsorbed selectively around the pore edges to form ring-shaped aggregates. The immunostaining results revealed that PAECs adhered to the HC-patterned films at focal contact points localized around pore peripheries. These points correspond to adsorption sites of Fn. The expression of pFAK after 1 h on the HC-patterned film was 3 times higher than that on a corresponding flat film, indicating that the signaling mediated by the binding between Fn and the integrin receptor was more highly activated on the HC-patterned film. These results suggest that the cellular response to HC-patterned films (e.g., adhesion pattern and phosphorylation of FAK) originates from the regularly aligned adsorption pattern of Fn determined by the pore structure of the film.     

Thermo-responsive gelatin-functionalized PCL film surfaces for improvement of cell adhesion and intelligent recovery of gene-transfected cells

Efficient local gene transfection on a tissue scaffold is dependent on good cell-adhesion characteristics. In this work, the thermo-responsive gelatin-functionalized polycaprolactone (PCL) films were proposed for improvement of cell adhesion and intelligent recovery of gene-transfected cells. Functional copolymer brushes (PCL-g-P(NIPAAm-co-MAAS)) were first prepared via surface-initiated ATRP of N-isopropylacrylamide (NIPAAm) and methacrylic acid sodium salt (MAAS) from the initiator-funcationalized PCL surfaces. The pendant carboxyl end-groups of the PCL-g-P(NIPAAm-co-MAAS) surface were subsequently coupled with gelatin via carbodiimide chemistry to produce the thermo-responsive gelatin-functionalized PCL surface. The thermo-responsive gelatin-functionalized PCL film surface can improve cell adhesion and proliferation above the LCST of P(NIPAAm) without destroying cell detachment properties at lower temperatures. The dense transfected cells can be recovered simply by lowering culture temperature. The thermo-responsive gelatin-functionalized PCL films are potentially useful as intelligent adhesion modifiers for directing cellular functions within tissue scaffolds.     

Stimulants from gardeniae fructus for cultured endothelial cell proliferation.

Bioassay-guided fractionation of Gardeniae Fructus extract (GFE), which stimulates the proliferation of cultured endothelial cells, led to the isolation of glycerol and D-mannitol. Both compounds significantly increased the incorporation of [3H]thymidine and [14C]leucine into the acid-insoluble fraction of bovine aortic endothelial cell layers in culture. This clearly indicated that glycerol and D-mannitol are active components of GFE on endothelial cell proliferation. On the other hand, they did not change the number of cultured vascular smooth muscle cells from bovine aorta. Glycerol and D-mannitol may be beneficial drugs for vascular disorders.     

Morphological regimes of poly(ε-caprolactone)/octaisobutyl polyhedral oligosilsesquioxane composite films in relation to film composition and thickness

The morphological evolution of poly(ε-caprolactone) (PCL)/octaisobutyl polyhedral oligomeric silsesquioxane (IBUPOSS) films was analyzed using scanning electron microscopy (SEM) and polarized optical microscopy (POM). The morphologies of the blend films with PCL/IBUPOSS mass ratios of 95:5 to 50:50 were discussed according to decomposition mechanism in relation to film composition and thickness. In addition to the morphological regime for films with lower IBUPOSS loadings, in which the growth of PCL spherulites was nearly independent on the presence of fine IBUPOSS aggregates, two new morphological regimes were observed for the films with higher IBUPOSS loadings: (1) thicker blend films exhibited a rich dynamics, giving rise to a trilayer structure and (2) the decomposition of thinner films was induced by the kinetically controlled growth of IBUPOSS aggregates. By varying the thickness and the composition of the blend films, the current study provides important new insight into the rich phase behavior of nanoparticle-filled polymer films.     

Control of surface topography in biomimetic calcium phosphate coatings

The behavior of cells responsible for bone formation, osseointegration, and bone bonding in vivo are governed by both the surface chemistry and topography of scaffold matrices. Bone-like apatite coatings represent a promising method to improve the osteoconductivity and bonding of synthetic scaffold materials to mineralized tissues for regenerative procedures in orthopedics and dentistry. Polycaprolactone (PCL) films were coated with calcium phosphates (CaP) by incubation in simulated body fluid (SBF). We investigated the effect of SBF ion concentration and soaking time on the surface properties of the resulting apatite coatings. CaP coatings were examined by scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transform infrared spectrometry (FTIR), and energy dispersive X-ray spectrometry (EDX). Young's modulus (E(s)) was determined by nanoindentation, and surface roughness was assessed by atomic force microscopy (AFM) and mechanical stylus profilometry. CaP such as carbonate-substituted apatite were deposited onto PCL films. SEM and AFM images of the apatite coatings revealed an increase in topographical complexity and surface roughness with increasing ion concentration of SBF solutions. Young's moduli (E(s)) of various CaP coatings were not significantly different, regardless of the CaP phase or surface roughness. Thus, SBF with high ion concentrations may be used to coat synthetic polymers with CaP layers of different surface topography and roughness to improve the osteoconductivity and bone-bonding ability of the scaffold.     

Three‐Layered PCL Grafts Promoted Vascular Regeneration in a Rabbit Carotid Artery Model

In this study, a three layered poly (ε‐caprolactone) (PCL) graft (tPCL) was fabricated by electrospinning PCL and electrospraying poly (ethylene oxide) (PEO), which has a thin dense inner layer, a loose middle layer, and a dense outer layer. Regular PCL grafts (rPCL) with only a dense layer were used as control. In vivo evaluation was performed in rabbit carotid artery. Enhanced cell infiltration, rapid regeneration of endothelium and smooth muscle layers, and increased elastin deposition were observed within the tPCL graft wall. After 3 months, tPCL grafts showed faster PCL degradation than the rPCL grafts. Infiltrated macrophages in the tPCL grafts secreted higher level of monocyte chemoattractant protein‐1 (MCP‐1) and vascular endothelial growth factor (VEGF) which enhanced vascular regeneration. In conclusion, the tPCL graft may be a useful vascular prosthesis and worth for further investigation.

     

Biofunctionalization of titanium with PEG and anti-CD34 for hemocompatibility and stimulated endothelialization

Thrombosis and restenosis are the main causes of failure of cardiovascular and other blood-contacting biomedical devices. It is recognized that rapid endothelialization is a promising method for preventing these complications. Convincing evidence in vivo has further emerged that the vascular homing of endothelial progenitor cells (EPCs) contributes to rapid endothelial regeneration. This study deals with improving the hemocompatibility and enhancing EPC colonization of titanium by covalently bonding PEG(600) or PEG(4000), then end-grafting of an anti-CD34 antibody. For this, a chemically hydroxylated titanium surface was aminosilanized, which was further used for covalent grafting of polyethylene glycol and the antibody. The grafting efficiency was verified in each step. In vitro platelet adhesion analysis confirmed superior hemocompatibility of the modified surface over the control. Affinity of EPC to the surface and inhibition of smooth muscle cell adhesion, two prerequisites for endothelialization, are demonstrated in in vitro cell culture. While the coating selectively stimulates EPC adhesion, its antifouling properties prevent formation of an extracellular matrix and proliferation of the cells. Additional affinity for matrix proteins in the coating is considered for further studies. Potent inhibitory effect on macrophage activation and the relative stability of the coating render this technique applicable.