基于贻贝仿生化学改善聚己内酯纤维多孔支架的 细胞相容性

在组织工程领域,支架的表面化学性能对调控细胞的生长行为起着关键的作用。为进一步改善聚己内酯(PCL)纤维支架的细胞相容性,开发了一种基于贻贝仿生化学在PCL纤维支架表面接枝生物相容性大分子的方法。该方法主要包含多巴胺在PCL纤维的表面涂覆和自聚合,以及生物活性大分子精氨酸-甘氨酸-天冬氨酸(RGD)和肝素的引入。傅里叶变换红外光谱测试结果表明RGD和PDA被成功地引入到PCL纤维支架表面。扫描电镜形貌检测和水接触角测试结果表明该改性手段不仅增大了纤维支架的表面粗糙度并且改善了支架的表面润湿性能。血管内皮细胞在改性的支架表面表现出了良好的细胞黏附性和细胞存活性。这种不涉及任何有毒溶剂的改性方法在组织工程支架领域具有广阔的应用前景。     

Electrospun nanofibers immobilized with collagen for neural stem cells culture

Fibrous mats via electrospinning have been widely applied in tissue engineering. In this work, nanofibers were prepared via electrospinning from polymer with different content of carboxyl groups. A natural material, collagen, was then immobilized onto the nanofiber surface by N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC)/N-Hydroxysuccinimide (NHS) activation process. It was found that the immobilization degree of collagen could be facilely modulated. The obtained collagen-modified nanofibers were used for neural stem cells culture, and unmodified nanofibers were used as a control. Results indicated that the modification of collagen could enhance the attachment and viability of the cultured neural stem cells.     

静电纺丝天然-人工合成聚合物复合纳米纤维及其对细胞黏附、增殖和迁移的影响

生物材料组成成分对细胞生物功能有不同的影响。利用静电纺丝技术制备了基于聚己内酯(PCL,polycaprolactone)的不同天然蛋白、多糖(丝素蛋白(SF,silk fibroin)、透明质酸(HA,hyaluronicacid))的混合组分纳米纤维,采用了扫描电镜和接触角对纳米纤维进行基础表征。同时,进一步考察了纳米纤维作为组织工程支架的可行性。研究结果表明SF组分能增加材料的可纺性,有利于细胞的前期黏附,并能够促进细胞增殖。HA组分可以改善材料的亲水性,增加细胞伪足并促进细胞迁移。重要的是,PCL/SF/HA纳米纤维能同时结合SF和HA的优点,有望在组织工程领域得到应用。     

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.     

Electrospun nanofibers composed of poly(ε-caprolactone) and polyethylenimine for tissue engineering applications

Poly(ε-caprolactone) (PCL) electrospun nanofibers have been reported as a scaffold for tissue engineering application. However, high hydrophobicity of PCL limits use of functional scaffold. In this study, PCL/polyethylenimine (PEI) blend electrospun nanofibers were prepared to overcome the limitation of PCL ones because the PEI as a cationic polymer can increase cell adhesion and can improve the electrospinnability of PCL. The structure, mechanical properties and biological activity of the PCL/PEI electrospun nanofibers were studied. The diameters of the PCL/PEI nanofibers ranged from 150.4 ± 33 to 220.4 ± 32 nm. The PCL/PEI nanofibers showed suitable mechanical properties with adequate porosity and increased hydrophilic behavior. The cell adhesion and cell proliferation of PCL nanofibers were increased by blending with PEI due to the hydrophilic properties of PEI.     

Nanofibrous membrane of collagen–polycaprolactone for cell growth and tissue regeneration

Nanofibrous substrates of synthetic polymers including polycaprolactone (PCL) have shown considerable potential in tissue regeneration. This paper reports the use of PCL/collagen nanofibers to improve the in vitro osteoblastic responses for the applications in bone regeneration area. Collagen and PCL were dissolved in a co-solvent, and the resulting solution was electrospun into a nanofibrous web. Nonwoven fibrous matrices were successfully produced at various compositional ratios (PCL/collagen = 1/3, 1 and 3 by weight). Although the PCL nanofiber was hydrophobic, the presence of collagen significantly improved the water affinity, such as the water contact angle and water uptake capacity. Tensile mechanical tests showed that the collagen–PCL nanofiber had a significantly higher extension rate (approximately 2.8-fold) than the PCL while maintaining the maximum tensile load in a similar range. The osteoblastic cells cultured on the collagen–PCL nanofibrous substrate showed better initial adhesion and a higher level of growth than those cultured on the PCL nanofiber. Furthermore, real-time RT-PCR revealed the expression of a series of bone-associated genes, including osteopontin, collagen type I and alkaline phosphatase. The expression of these genes was significantly higher on the collagen–PCL nanofiber than on the PCL nanofiber. When subcutaneously implanted in mouse the collagen–PCL membrane facilitated tissue cells to well penetrate into the nanofibrous structure at day 7, whilst no such cell penetration was noticed in the pure PCL nanofiber. Overall, the presence of collagen within the PCL nanofiber improves the water affinity, tensile extension rate, and the tissue cell responses, such as initial adhesion, growth, penetration and the expression of bone-associated genes. Therefore, the collagen–PCL nanofibrous membrane may have potential applications in the cell growth and bone tissue regeneration.     

Surface modified electrospun nanofibrous scaffolds for nerve tissue engineering

The development of biodegradable polymeric scaffolds with surface properties that dominate interactions between the material and biological environment is of great interest in biomedical applications. In this regard, poly-ε-caprolactone (PCL) nanofibrous scaffolds were fabricated by an electrospinning process and surface modified by a simple plasma treatment process for enhancing the Schwann cell adhesion, proliferation and interactions with nanofibers necessary for nerve tissue formation. The hydrophilicity of surface modified PCL nanofibrous scaffolds (p-PCL) was evaluated by contact angle and x-ray photoelectron spectroscopy studies. Naturally derived polymers such as collagen are frequently used for the fabrication of biocomposite PCL/collagen scaffolds, though the feasibility of procuring large amounts of natural materials for clinical applications remains a concern, along with their cost and mechanical stability. The proliferation of Schwann cells on p-PCL nanofibrous scaffolds showed a 17% increase in cell proliferation compared to those on PCL/collagen nanofibrous scaffolds after 8 days of cell culture. Schwann cells were found to attach and proliferate on surface modified PCL nanofibrous scaffolds expressing bipolar elongations, retaining their normal morphology. The results of our study showed that plasma treated PCL nanofibrous scaffolds are a cost-effective material compared to PCL/collagen scaffolds, and can potentially serve as an ideal tissue engineered scaffold, especially for peripheral nerve regeneration.     

Heparin-functionalized collagen matrices with controlled release of basic fibroblast growth factor

Tissue engineering scaffolds with controlled long-term release of growth factors are constructed in an attempt to mimic the intelligent ability of the extracellular matrix (ECM) to release endogenous growth factors. In this study, collagen sponges (Collagen group) were modified by N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) crosslinking (EDC/NHS group) and heparin immobilization (EDC/NHS-H group), and subsequently seeded with human umbilical vein endothelial cells (HUVECs). Native and modified sponges were pre-adsorbed with basic fibroblast growth factor (bFGF) to evaluate the sustained release and bioactive maintenance of bFGF from the sponges. We found that modified collagen matrices permitted HUVECs to proliferate and migrate well and to distribute uniformly. The EDC/NHS-H group exhibited an excellent sustained-release profile and bioactive maintenance of the pre-adsorbed bFGF as compared with the Collagen and EDC/NHS groups. These results suggest that heparin-functionalized collagen matrices can support a controlled release of bFGF and thus, have potential as a tissue engineering scaffold.     

Electrospun nanofibers of collagen-chitosan and P(LLA-CL) for tissue engineering

Electrospun nanofibers could be used to mimic the nanofibrous structure of the extracellular matrix (ECM) in native tissue.In tissue engineering,the ECM could be used as tissue engineering scaffold to ...     

Preparation of electrospun nanofibers of star-shaped polycaprolactone and its blends with polyaniline

The electrospun nanofibers of synthetic star-shaped poly(?-caprolactone) (PCL) and its blends with polyaniline (PANI) were prepared. We utilized the advantages of star-shaped PCL and benefits of electrospinning method for obtainment of the uniform nanofibers with improved properties for tissue engineering. Biodegradable star-shaped PCL with four arms was synthesized by Sn(Oct)₂-catalyzed ring-opening polymerization (ROP) of ?-caprolactone (CL) from a pentaerythritol core. The chemical structure of star-shaped PCL was investigated by FTIR, and average molecular weight of polymer was determined by ¹HNMR (about 38000 g mol^?1). Thermal behavior of star-shaped PCL was also studied by Thermogravimetric Analysis (TGA). Moreover, the cyclic voltammetry (CV) measurement confirmed the preparation of electroactive nanofibers. The scanning electron microscopy (SEM) was also used to investigate the morphology of electrospun nanofibers produced from star-shaped PCL and its blends with PANI with different feed ratios. The presence of PANI resulted in fibers with diameters less than 100 nm and significant decrease of bead formation.     

Electrospinning collagen and hyaluronic acid nanofiber meshes

Collagen and hyaluronic acid (HA) are main components of the extracellular matrix and have been utilized in electrospinning; a technique that creates nanosized fibers for tissue scaffolds. A collagen/HA polymer solution was electrospun into a scaffold material for osteoporosis patients who have reduced bone strength. To synthesize nanofibers, a high voltage was applied to the polymer solution to draw out nanofibers that were collected on a ground plate as a uniform mesh. The meshes were then crosslinked to render them insoluble and conjugated with gold nanoparticles to promote biocompatibility. Characterization of the mesh was performed using scanning electron microscope, electron dispersive spectroscopy and fourier transform infrared spectroscopy. A WST-1 assay determined the potential biocompatibility. The results show that collagen/HA scaffolds were developed that were insoluble in aqueous solutions and promoted cellular attachment that could be used as a tissue engineered scaffold to promote cell growth.     

Stiffness Improvement of 45S5 Bioglass®‐Based Scaffolds Through Natural and Synthetic Biopolymer Coatings: An Ultrasonic Study

Due to its excellent bioactivity, 45S5 Bioglass^® is being highly considered in tissue engineering scaffold development. In order to enhance vascularization promoting tissue growth, these scaffolds typically have a highly interconnected porous structure with a porosity between 80 and >90%. Often, Bioglass^®‐based scaffolds of such a high porosity have insufficient stiffness. In order to increase the stiffness of Bioglass^®‐based scaffolds fabricated by the foam replica method, the herein investigated scaffolds were coated with a number of different biopolymers, including: collagen, gelatin, polycaprolactone (PCL), alginate and poly(l ‐lactic acid). The resulting stiffness gain was quantified by means of ultrasonic measurements. Accordingly, PCL and collagen coatings increased the scaffold stiffness, as compared to uncoated scaffolds, by 58 and 38%, respectively; while no remarkable stiffness increase was recorded for the other coatings. Additionally, scanning electron microscopy images of polymer coated scaffolds revealed that PCL coatings had not clogged the scaffold's micropores, which is deemed essential for cell seeding and to enable in‐growth of bone tissue. Thus, the application of PCL coatings represents a promising strategy for mechanical competence enhancement of Bioglass^®‐based scaffolds for bone tissue engineering.     

Mineralization of osteoblasts with electrospun collagen/hydroxyapatite nanofibers

Regeneration of fractured or diseased bones is the challenge faced by current technologies in tissue engineering. The major solid components of human bone consist of collagen and hydroxyapatite. Collagen (Col) and hydroxyapatite (HA) have potential in mimicking natural extracellular matrix and replacing diseased skeletal bones. More attention has been focused on HA because of its crystallographic structure similar to inorganic compound found in natural bone and extensively investigated due to its excellent biocompatibility, bioactivity and osteoconductivity properties. In the present study, electrospun nanofibrous scaffolds are fabricated with collagen (80 mg/ml) and Col/HA (1:1). The diameter of the collagen nanofibers is around 265 ± 0.64 nm and Col/HA nanofibers are 293 ± 1.45 nm. The crystalline HA (29 ± 7.5 nm) loaded into the collagen nanofibers are embedded within nanofibrous matrix of the scaffolds. Osteoblasts cultured on both scaffolds and show insignificant level of proliferation but mineralization was significantly (p < 0.001) increased to 56% in Col/HA nanofibrous scaffolds compared to collagen. Energy dispersive X-ray analysis (EDX) spectroscopy results proved the presence of higher level of calcium and phosphorous in Col/HA nanocomposites than collagen nanofibrous scaffolds grown osteoblasts. The results of the present study suggested that the designed electrospun nanofibrous scaffold (Col/HA) have potential biomaterial for bone tissue engineering.     

Electrospun PCL/PLA/HA based nanofibers as scaffold for osteoblast-like cells

Polycaprolactone (PCL), poly (lactic acid) (PLA) and hydroxyapatite (HA) are frequently used as materials for tissue engineering. In this study, PCL/PLA/HA nanofiber mats with different weight ratio were prepared using electrospinning. Their structure and morphology were studied by FTIR and FESEM. FTIR results demonstrated that the HA particles were successfully incorporated into the PCL/PLA nanofibers. The FESEM images showed that the surface of fibers became coarser with the introduction of HA nanoparticles into PCL/PLA system. Furthermore, the addition of HA led to the decreasing of fiber diameter. The average diameters of PCL/PLA/HA nanofiber were in the range of 300-600 nm, while that of PCL/PLA was 776 +/- 15.4 nm. The effect of nanofiber composition on the osteoblast-like MC3T3-E1 cell adhesion and proliferation were investigated as the preliminary biological evaluation of the scaffold. The MC3T3-E1 cell could be attached actively on all the scaffolds. The MTT assay revealed that PCL/PLA/HA scaffold shows significantly higher cell proliferation than PCL/PLA scaffolds. After 15 days of culture, mineral particles on the surface of the cells was appeared on PCL/PLA/HA nanofibers while normal cell spreading morphology on PCL/PLA nanofibers. These results manifested that electrospun PCL/PLA/HA scaffolds could enhance bone regeneration, showing their marvelous prospect as scaffolds for bone tissue engineering.     

Preparation and characterization of electrospun poly(ε-caprolactone)-poly(l-lactic acid) nanofiber tubes

Recently, attempts have been made to develop nanofiber tubes suitable for nerve regeneration made of biodegradable nanofibers. Among all polymeric nanofibers, poly(ε-caprolactone) (PCL) is distinctively known for better mechanical stability and poly(l-lactic acid) (PLLA) for relatively faster biodegradability. Our purpose of study is to investigate their blending compatibility and the ability to form nanofiber tubes via electrospinning. We electrospun the PCL–PLLA nanofiber tubular using different blend ratios of PCL–PLLA. The electrospun nanofibers were continuously deposited over high speed rotating mandrel to fabricate nanofiber tubes having inner diameter of 2 mm and the wall thickness of 55–65 μm. The diameters of nanofibers were between 715 and 860 nm. The morphologies of PCL–PLLA nanofiber tubes were examined under scanning electron microscope, and showed better structural stability and formability than the neat PLLA nanofibers. Fourier transform infrared spectroscopy study revealed that the PCL–PLLA blend nanofiber exhibited characteristic peaks of both PCL and PLLA and was composition-dependent. Raman and X-ray diffraction studies showed that the increasing PCL ratio in the PCL–PLLA blend increased crystallinity of PCL–PLLA blends. Differential scanning calorimetry revealed recrystallization peaks in PCL–PLLA blends ratios of 1:2 and 1:1. Based on characterization, the electrospun PCL–PLLA nanofiber tubes is considered to be a better candidate for further in vivo or in vitro investigation, and resolve biocompatibility issues in tissue engineering.     

Fabrication of Nanofiber Reinforced Protein Structures For Tissue Engineering

Extracellular matrices and degradable nanofibers are two very promising materials in the field of tissue engineering; however both of these structures face limitations as tissue engineering scaffolds. Extracellular matrices, such as collagen, gelatin, and laminin, have excellent biocompatibility and allow cell in growth and survival, but structural weakness makes them difficult to handle and greatly limits their uses. Degradable nanofibers support cell attachment and can provide structural support and directional guidance, but individual degradable nanofibers are fragile and have a tendency to form dense fiber bundles which limit cell penetration into the spaces between the nanofibers, especially in the case of aligned nanofibers. To overcome these difficulties, degradable loose nanofibers were embedded in protein matrix in an attempt to fabricate a hybrid scaffold with improved properties, such as improved strength, guidance, spacing among nanofibers, etc. Polycaprolactone (PCL) was used as a model material for degradable nanofibers. Gelatin was employed as a model protein for matrix structure formation. Thin hybrid films (average thickness = 2.78 um) were fabricated by wetting the loose aligned undirectional nanofiber arrays or loose aligned bi-directional nanofiber grids with a gelatin aqueous solution, which also allows for live cell loading into the nanofiber-protein composite if cell are premixed with protein solution or on the surface of the films. Gelatin film alone without nanofiber reinforcement is difficult to handle due to the weakness of the thin membrane. Gelatin films with a fiber density as low as 3% v/v were structurally robust enough for handling, and manipulation into complex shapes. Mechanical testing confirmed that the addition of nanofibers enhanced the strength of gelatin films, in both dry and hydrated state. In vitro testing confirmed that nanofiber reinforced films were biocompatible and provided cells with directional guidance. Results demonstrate the promise of gelatin/PCL nanofiber composites as a tissue scaffolding material.     

Fabrication and characterization of PCL/gelatin composite nanofibrous scaffold for tissue engineering applications by electrospinning method

In the present study, composite nanofibrous tissue engineering-scaffold consisting of polycaprolactone and gelatin, was fabricated by electrospinning method, using a new cost-effective solvent mixture: chloroform/methanol for polycaprolactone (PCL) and acetic acid for gelatin. The morphology of the nanofibrous scaffold was investigated by using field emission scanning electron microscopy (FE-SEM) which clearly indicates that the morphology of nanofibers was influenced by the weight ratio of PCL to gelatin in the solution. Uniform fibers were produced only when the weight ratio of PCL/gelatin is sufficiently high (10:1). The scaffold was further characterized by Fourier transform infrared (FT-IR) spectroscopy, thermogravimetric (TG) analysis, and X-ray diffraction (XRD). FT-IR and TG analysis indicated some interactions between PCL and gelatin molecules within the scaffold, while XRD results demonstrated crystalline nature of PCL/gelatin composite scaffold. Cytotoxicity effect of scaffold on L929 mouse fibroblast cells was evaluated by MTT assay and cell proliferation on the scaffold was confirmed by DNA quantification. Positive results of MTT assay and DNA quantification L929 mouse fibroblast cells indicated that the scaffold made from the combination of natural polymer (gelatin) and synthetic polymer (PCL) may serve as a good candidate for tissue engineering applications.     

A comparative study of the chondrogenic potential between synthetic and natural scaffolds in an in vivo bioreactor

Abstract

The clinical demand for cartilage tissue engineering is potentially large for reconstruction defects resulting from congenital deformities or degenerative disease due to limited donor sites for autologous tissue and donor site morbidities. Cartilage tissue engineering has been successfully applied to the medical field: a scaffold pre-cultured with chondrocytes was used prior to implantation in an animal model. We have developed a surgical approach in which tissues are engineered by implantation with a vascular pedicle as an in vivo bioreactor in bone and adipose tissue engineering. Collagen type II, chitosan, poly(lactic-co-glycolic acid) (PLGA) and polycaprolactone (PCL) were four commonly applied scaffolds in cartilage tissue engineering. To expand the application of the same animal model in cartilage tissue engineering, these four scaffolds were selected and compared for their ability to generate cartilage with chondrocytes in the same model with an in vivo bioreactor. Gene expression and immunohistochemistry staining methods were used to evaluate the chondrogenesis and osteogenesis of specimens. The result showed that the PLGA and PCL scaffolds exhibited better chondrogenesis than chitosan and type II collagen in the in vivo bioreactor. Among these four scaffolds, the PCL scaffold presented the most significant result of chondrogenesis embedded around the vascular pedicle in the long-term culture incubation phase.     

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.     

Functional synergy of anti-mir221 and nanohydroxyapatite scaffold in bone tissue engineering of rat skull

An appropriate cell source, effective cell modification, and proper supportive matrices are the main bases of tissue engineering. The effectiveness of anti-mir221 or hydroxyapatite (HA) in improving the osteogenic differentiation of mesenchymal stem cells (MSCs) has been reported previously. Herein, simultaneous application of these osteogenic inducers was investigated in vivo. The Poly-caprolactone (PCL)/HA nanofibers were characterized using contact angle measurement, tensile test, Fourier transform infrared spectroscopy, and electron microscopy. Rat MSCs were isolated, characterized and transfected with anti-mir221. The rats were divided into 4 groups and an 8 mm defect were created in the mid-calvaria of each rat by trephine bur. Group 1 received (PCL)/HA nanofibers, group 2 received (PCL)/HA nanofibers plus autologous MSCs, group 3 received (PCL)/HA nanofibers plus MSCs transfected with anti-mir221, and group 4 rats were left empty as an additional control group. Histomorphometric and radiomorphometric evaluation after 4 and 8 weeks revealed more new bone formation in the cell-treated groups compared to the scaffold alone group. There was evidence for a combination of increased osteoclasts and osteoblast vascular lake containing red blood cells in the anti-mir221 transfected group. New bone penetration into the scaffolds empirically demonstrated the capability of this combination for efficient osteointegration. Altogether, the co-application of HA and anti-mir221 transfected cells can enhance bone healing of the rat skull.