Growth of outgrowth endothelial cells on aligned PLLA nanofibrous scaffolds

Tissue engineering holds great promise in providing vascular grafts as substitutes for damaged small-diameter blood vessels. Two of the key factors in vascular tissue engineering are biocompatible scaffolds that mimic the effects of extracellular matrix and the source of seeding cells. Synthetic poly-l-lactic acid (PLLA) nanofibers has been shown to be excellent scaffolds for tissue engineering. Outgrowth endothelial cells (OECs) isolated from human peripheral blood could also be expanded in vitro and stably maintain the differentiated phenotypes and could be used as the seeding cells for engineering autologous vascular crafts. Here we tested the possibility of combining these two together. We found that PLLA nanofibers are not only biocompatible with OECs originally isolated from rabbit peripheral blood, the aligned PLLA fibers actually promoted and guided their sustained proliferation. These results suggest that aligned PLLA could be excellent both as the scaffolds and as a promoter of cell growth during vascular tissue engineering.     

Biocompatibility evaluation of protein-incorporated electrospun polyurethane-based scaffolds with smooth muscle cells for vascular tissue engineering

Nanotechnology has enabled the engineering of a variety of materials to meet the current challenges and needs in vascular tissue regeneration. In this study, four different kinds of native proteins namely collagen, gelatin, fibrinogen, and bovine serum albumin were incorporated with polyurethane (PU) and electropsun to obtain composite PU/protein nanofibers. SEM studies showed that the fiber diameters of PU/protein scaffolds ranged from 245 to 273 nm, mimicking the nanoscale dimensions of native ECM. Human aortic smooth muscle cells (SMCs) were cultured on the electrospun nanofibers, and the ability of the cells to proliferate on different scaffolds was evaluated via a cell proliferation assay. Cell proliferation on PU/Coll nanofibers was found the highest compared to other electrospun scaffolds and it was 42 % higher than the proliferation on PU/Fib nanofibers after 12 days of cell culture. The cell–biomaterial interaction studies by SEM confirmed that SMCs adhered to PU/Coll and PU/Gel nanofibers, with high cell substrate coverage, and both the scaffolds promoted cell alignment. The functionality of the cells was further demonstrated by immunocytochemical analysis, where the SMCs on PU/Coll and PU/Gel nanofibers expressed higher density of SMC proteins such as alpha smooth muscle actin and smooth muscle myosin heavy chain. Cells expressed biological markers of SMCs including aligned spindle-like morphology on both PU/Coll and PU/Gel with actin filament organizations, better than PU/Fib and PU/BSA scaffolds. Our studies demonstrate the potential of randomly oriented elastomeric composite scaffolds for engineering of vascular tissues causing cell alignment.     

Long-term viability of coronary artery smooth muscle cells on poly(L-lactide-co-epsilon-caprolactone) nanofibrous scaffold indicates its potential for blood vessel tissue engineering.

Biodegradable polymer nanofibres have been extensively studied as cell culture scaffolds in tissue engineering. However, long-term in vitro studies of cell-nanofibre interactions were rarely reported and successful organ regeneration using tissue engineering techniques may take months (e.g. blood vessel tissue engineering). Understanding the long-term interaction between cells and nanofibrous scaffolds (NFS) is crucial in material selection, design and processing of the tissue engineering scaffolds. In this study, poly(L-lactide-co-epsilon-caprolactone) [P(LLA-CL)] (70:30) copolymer NFS were produced by electrospinning. Porcine coronary artery smooth muscle cells (PCASMCs) were seeded and cultured on the scaffold to evaluate cell-nanofibre interactions for up to 105 days. A favourable interaction between this scaffold and PCASMCs was demonstrated by cell viability assay, scanning electron microscopy, histological staining and extracellular matrix (ECM) secretion. Degradation behaviours of the scaffolds with or without PCASMC culture were determined by mechanical testing and gel permeation chromatography (GPC). The results showed that the PCASMCs attached and proliferated well on the P(LLA-CL) NFS. Large amount of ECM protein secretion was observed after 50 days of culture. Multilayers of aligned oriented PCASMCs were formed on the scaffold after two months of in vitro culture. In the degradation study, the PCASMCs were not shown to significantly increase the degradation rate of the scaffolds for up to 105 days of culture. The in vitro degradation time of the scaffold could be as long as eight months by extrapolating the results from GPC. These observations further supported the potential use of the P(LLA-CL) nanofibre in blood vessel tissue engineering.     

Fabrication and characterization of gold nanoparticle-doped electrospun PCL/chitosan nanofibrous scaffolds for nerve tissue engineering

In the field of nerve tissue engineering, nanofibrous scaffolds could be a promising candidate when they are incorporated with electrical cues. Unique physico-chemical properties of gold nanoparticles (AuNPs) make them an appropriate component for increasing the conductivity of scaffolds to enhance the electrical signal transfer between neural cells. The aim of this study was fabrication of AuNPs-doped nanofibrous scaffolds for peripheral nerve tissue engineering. Polycaprolactone (PCL)/chitosan mixtures with different concentrations of chitosan (0.5, 1 and 1.5) were electrospun to obtain nanofibrous scaffolds. AuNPs were synthesized by the reduction of HAuCl₄ using chitosan as a reducing/stabilizing agent. A uniform distribution of AuNPs with spherical shape was achieved throughout the PCL/chitosan matrix. The UV–Vis spectrum revealed that the amount of gold ions absorbed by nanofibrous scaffolds is in direct relationship with their chitosan content. Evaluation of electrical property showed that inclusion of AuNPs significantly enhanced the conductivity of scaffolds. Finally, after 5 days of culture, biological response of Schwann cells on the AuNPs-doped scaffolds was superior to that on as-prepared scaffolds in terms of improved cell attachment and higher proliferation. It can be concluded that the prepared AuNPs-doped scaffolds can be used to promote peripheral nerve regeneration.

Open image in new window

     

The influence of fiber thickness, wall thickness and gap distance on the spiral nanofibrous scaffolds for bone tissue engineering

We have developed a 3D nanofibrous spiral scaffold for bone tissue engineering which has shown enhanced cell attachment, proliferation and differentiation compared to traditional cylindrical scaffolds due to the spiral structures and the nanofiber incorporation. Some important parameters of these spiral scaffolds including gap distance, wall thickness and especially fiber thickness are crucial to the performance of the spiral structured scaffolds. In this study, we investigated the fiber thickness, gap distance and wall thickness of the spiral structure on the behavior of osteoblast cells. The human osteoblast cells are seeded on spiral structured scaffolds with various fiber thickness, gap distance and wall thickness and cell attachment, proliferation, differentiation and mineralized matrix deposition on the scaffolds are evaluated. It was found that increasing the thickness of nanofiber layer not only limited the cell infiltration into the scaffolds, but also restrained the osteoblastic cell phenotype development. Moreover, the geometric effect studies indicated that scaffolds with the thinner wall and gap distance 0.2 mm show the best bioactivity for osteoblasts.     

Bioactive borosilicate glass scaffolds: improvement on the strength of glass-based scaffolds for tissue engineering

Three-dimensional macroporous scaffolds with the pore size of 200-500 mum were fabricated by replication method using bioactive borosilicate glass from Na(2)O-K(2)O-MgO-CaO-SiO(2)-P(2)O(5)-B(2)O(3) system. The effects of the strength of the strut in reticulated scaffold, as well as the geometrical parameter of the scaffold on the strength of reticulated scaffold were investigated. Scanning electron microscope (SEM) and X-ray diffraction (XRD) results show that the solidified glass struts in the reticulated scaffold could be obtained through a sufficient vicious flow of glass, during the fabrication. By increasing the solid content in slurries, from which the scaffold was made, the load-bearing units of the reticulated scaffold switch from struts to the walls between the pores, and the compressive strength dramatically climbs higher than the theoretical strength calculated by Gibson model. In particular, the compressive strength of the reticulated scaffold, as high as approximately 10 MPa with the porosity of approximately 70%, is close to the reported compressive values of human cancellous bone. This indicates the bioactive borosilicate glass-based scaffold is a promising candidate for bone tissue engineering.     

组织工程用高分子骨架研究的进展

组织工程是一个新兴的交叉学科。结合目前组织工程用高分子骨架研究和发展的最新状况，探讨了高分子骨架材料的本体结构、性能及表面形态与分子结构要求，并对常用的骨架制备方法进行了概述。     

Chitosan particles agglomerated scaffolds for cartilage and osteochondral tissue engineering approaches with adipose tissue derived stem cells

It is well accepted that natural tissue regeneration is unlikely to occur if the cells are not supplied with an extracellular matrix (ECM) substitute. With this goal, several different methodologies have been used to produce a variety of 3D scaffolds as artificial ECM substitutes suitable for bone and cartilage tissue engineering. Furthermore, osteochondral tissue engineering presents new challenges since the combination of scaffolding and co-culture requirements from both bone and cartilage applications is required in order to achieve a successful osteochondral construct. In this paper, an innovative processing route based on a chitosan particles aggregation methodology for the production of cartilage and osteochondral tissue engineering scaffolds is reported. An extensive characterization is presented including a morphological evaluation using Micro-Computed Tomography (μCT) and 3D virtual models built with an image processing software. Mechanical and water uptake characterizations were also carried out, evidencing the potential of the developed scaffolds for the proposed applications. Cytotoxicity tests show that the developed chitosan particles agglomerated scaffolds do not exert toxic effects on cells. Furthermore, osteochondral bilayered scaffolds could also be developed. Preliminary seeding of mesenchymal stem cells isolated from human adipose tissue was performed aiming at developing solutions for chondrogenic and osteogenic differentiation for osteochondral tissue engineering applications. An erratum to this article is available at .     

Chitosan particles agglomerated scaffolds for cartilage and osteochondral tissue engineering approaches with adipose tissue derived stem cells

In the XML file of the original article, H. Redl’s affiliation is incorrect. It is listed correctly in both the paper and PDF versions of the article, and can be found below: The online version of the original article can be found at     

Processing and characterization of innovative scaffolds for bone tissue engineering

A new protocol, based on a modified replication method, is proposed to obtain bioactive glass scaffolds. The main feature of these samples, named "shell scaffolds", is their external surface that, like a compact and porous shell, provides both high permeability to fluids and mechanical support. In this work, two different scaffolds were prepared using the following slurry components: 59 % water, 29 % 45S5 Bioglass(?) and 12 % polyvinylic binder and 51 % water, 34 % 45S5 Bioglass(?), 10 % polyvinylic binder and 5 % polyethylene. All the proposed samples were characterized by a widespread microporosity and an interconnected macroporosity, with a total porosity of 80 % vol. After immersion in a simulated body fluid (SBF), the scaffolds showed strong ability to develop hydroxyapatite, enhanced by the high specific surface of the porous systems. Moreover preliminary biological evaluations suggested a promising role of the shell scaffolds for applications in bone tissue regeneration. As regards the mechanical behaviour, the shell scaffolds could be easily handled without damages, due to their resistant external surface. More specifically, they possessed suitable mechanical properties for bone regeneration, as proved by compression tests performed before and after immersion in SBF.     

Evaluation of synovium-derived mesenchymal stem cells and 3D printed nanocomposite scaffolds for tissue engineering

Abstract

Stem cells and scaffolds play a very important role in tissue engineering. Here, we isolated synovium-derived mesenchymal stem cells (SMSCs) from synovial membrane tissue and characterized stem-cell properties. Gelatin nanoparticles (NP) were prepared using a two-step desolvation method and then pre-mixed into different host matrix (silk fibroin (SF), gelatin (Gel), or SF–Gel mixture) to generate various 3D printed nanocomposite scaffolds (NP/SF, NP/SF–Gel, NP/Gel-1, and NP/Gel-2). The microstructure was examined by scanning electron microscopy. Biocompatibility assessment was performed through CCK-8 assay by coculturing with SMSCs at 1, 3, 7 and 14 days. According to the results, SMSCs are similar to other MSCs in their surface epitope expression, which are negative for CD45 and positive for CD44, CD90, and CD105. After incubation in lineage-specific medium, SMSCs could differentiate into chondrocytes, osteocytes and adipocytes. 3D printed nanocomposite scaffolds exhibited a good biocompatibility in the process of coculturing with SMSCs and had no negative effect on cell behavior. The study provides a strategy to obtain SMSCs and fabricate 3D printed nanocomposite scaffolds, the combination of which could be used for practical applications in tissue engineering.     

Evaluation of synovium-derived mesenchymal stem cells and 3D printed nanocomposite scaffolds for tissue engineering

Stem cells and scaffolds play a very important role in tissue engineering. Here, we isolated synovium-derived mesenchymal stem cells (SMSCs) from synovial membrane tissue and characterized stem-cell properties. Gelatin nanoparticles (NP) were prepared using a two-step desolvation method and then pre-mixed into different host matrix (silk fibroin (SF), gelatin (Gel), or SF–Gel mixture) to generate various 3D printed nanocomposite scaffolds (NP/SF, NP/SF–Gel, NP/Gel-1, and NP/Gel-2). The microstructure was examined by scanning electron microscopy. Biocompatibility assessment was performed through CCK-8 assay by coculturing with SMSCs at 1, 3, 7 and 14 days. According to the results, SMSCs are similar to other MSCs in their surface epitope expression, which are negative for CD45 and positive for CD44, CD90, and CD105. After incubation in lineage-specific medium, SMSCs could differentiate into chondrocytes, osteocytes and adipocytes. 3D printed nanocomposite scaffolds exhibited a good biocompatibility in the process of coculturing with SMSCs and had no negative effect on cell behavior. The study provides a strategy to obtain SMSCs and fabricate 3D printed nanocomposite scaffolds, the combination of which could be used for practical applications in tissue engineering.     

Analysis of OPLA scaffolds for bone engineering constructs using human jaw periosteal cells

For bone regeneration constructs using human jaw periosteal cells (JPC) the extent of osteoinductive ability of different three-dimensional scaffolds is not yet established. We analyzed open-cell polylactic acid (OPLA) scaffolds for their suitability as bone engineering constructs using human JPC. Cell adhesion and spreading was visualized on the surface of scaffolds by scanning electron microscopy. JPC proliferation within OPLA scaffolds was compared with proliferation within collagen and calcium phosphate scaffolds. We found a significant increase of proliferation rates in OPLA scaffolds versus Coll/CaP scaffolds at three time points. Live-measurements of oxygen consumption within the cell-seeded scaffolds indicate that the in vitro culturing time should not exceed 12–15 days. OPLA scaffolds, which were turned out to be the most beneficial for JPC growth, were chosen for osteogenic differentiation experiments with or without BMP-2. Gene expression analyses demonstrated induction of several osteogenic genes (alkaline phosphatase, osterix, Runx-2 and insulin-like growth factor) within the 3D-scaffolds after 12 days of in vitro culturing. Element analysis by EDX spectrometry of arising nodules during osteogenesis demonstrated that JPC growing within OPLA scaffolds are able to form CaP particles. We conclude that OPLA scaffolds provide a promising environment for bone substitutes using human JPC.     

骨组织工程多孔支架材料性质及制备技术

多孔性生物可降解支架的选择和制备是组织工程技术成功运用的关键。从骨架的材料要求、常用的骨架材料、骨架的制备技术等几个方面对组织工程和生物降解支架的研究进行了综述 ,并对该研究的前景进行了展望     

Nano- and micro-fiber combined scaffolds: A new architecture for bone tissue engineering

One possible interesting way of designing a scaffold for bone tissue engineering is to base it on trying to mimic the biophysical structure of natural extracellular matrix (ECM). This work was developed in order to produce scaffolds for supporting bone cells. Nano and micro fiber combined scaffolds were originally produced from starch based biomaterials by means of a fiber bonding and a electrospinning, two step methodology. The cell culture studies with SaOs-2 human osteoblast-like cell line and rat bone marrow stromal cells demonstrated that presence of nanofibers influenced cell shape and cytoskeletal organization of the cells on the nano/micro combined scaffolds. Moreover, cell viability and Alkaline Phosphatase (ALP) activity for both cell types was found to be higher in nano/micro combined scaffolds than in control scaffolds based on fiber meshes without nanofibers. Consequently, the developed structures are believed have a great potential on the 3D organization and guidance of cells that is provided for engineering of 3-dimensional bone tissues.     

Electrospinning of aligned biodegradable polymer fibers and composite fibers for tissue engineering applications

Fibrous membranes of aligned poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) fibers have been made through electrospinning. A high-speed rotating drum was used as the fiber collector while the electric field was manipulated by using five knife-edged auxiliary electrodes. It was found that a high drum rotating speed of 3000 rpm could lead to a nearly perfect alignment of PHBV fibers during electrospinning. Multilayered fibrous structures with each layer having a different direction of fiber alignment could also be constructed through electrospinning. The electrospun PHBV fibers were further modified by incorporating carbonated hydroxyapatite (HA) nanospheres (up to 20% of HA) in the fibers. The fibrous membranes made of aligned PHBV fibers and made of HA/PHBV composite fibers should be very useful for the tissue engineering of different human body tissues.     

组织工程海藻酸钙多孔支架的制备和研究

采用浇铸/冷冻干燥的方法制备出一种可组装于神经导管内的组织工程海藻酸钙多孔支架.通过IR、SEM、不同浓度交联剂对凝胶化过程的平衡溶胀比、抗压强度以及多孔支架在SBF液中的含水率和体积膨胀度的分析,对7海藻酸钙多孔支架的性能进行了讨论.结果表明,海藻酸钠多孔支架在氯化钙溶液浸渍中能快速反应生成海藻酸钙,并在凝胶化反应过程中可保持原有呈一定方向排列、孔隙连通和层状的多孔结构;多孔支架随交联剂中Ca2+浓度的增加,形成的海藻酸钙多孔支架的抗压强度提高,使支架的稳定性增加;海藻酸钙多孔支架在SBF中的含水率>90%,体积的膨胀度与交联剂中Ca2+浓度成反比.     

Dual-source dual-power electrospinning and characteristics of multifunctional scaffolds for bone tissue engineering

Electrospun tissue engineering scaffolds are attractive due to their distinctive advantages over other types of scaffolds. As both osteoinductivity and osteoconductivity play crucial roles in bone tissue engineering, scaffolds possessing both properties are desirable. In this investigation, novel bicomponent scaffolds were constructed via dual-source dual-power electrospinning (DSDPES). One scaffold component was emulsion electrospun poly(d,l-lactic acid) (PDLLA) nanofibers containing recombinant human bone morphogenetic protein (rhBMP-2), and the other scaffold component was electrospun calcium phosphate (Ca–P) particle/poly(lactic-co-glycolic acid) (PLGA) nanocomposite fibers. The mass ratio of rhBMP-2/PDLLA fibers to Ca–P/PLGA fibers in bicomponent scaffolds could be controlled in the DSDPES process by adjusting the number of syringes used to supply solutions for electrospinning. Through process optimization, both types of fibers could be evenly distributed in bicomponent scaffolds. The structure and properties of each type of fibers in the scaffolds were studied. The morphological and structural properties and wettability of scaffolds were assessed. The effects of emulsion composition for rhBMP-2/PDLLA fibers and mass ratio of fibrous components in bicomponent scaffolds on in vitro release of rhBMP-2 from scaffolds were investigated. In vitro degradation of scaffolds was also studied by monitoring their morphological changes, weight losses and decreases in average molecular weight of fiber matrix polymers.     

Fabrication and characterization of electrospun osteon mimicking scaffolds for bone tissue engineering

Skeletal loss and bone deficiencies are a major worldwide problem with over 600,000 procedures performed in the US alone annually, making bone one of the most transplanted tissues, second to blood only. Bone is a composite tissue composed of organic matrix, inorganic bone mineral, and water. Structurally bone is organized into two distinct types: trabecular (or cancellous) and cortical (or compact) bones. Trabecular bone is characterized by an extensive interconnected network of pores. Cortical bone is composed of tightly packed units, called osteons, oriented parallel along to the axis of the bone. While the majority of scaffolds attempt to replicate the structure of the trabecular bone, fewer attempts have been made to create scaffolds to mimic the structure of cortical bone. The aim of this study was to develop a technique to fabricate scaffolds that mimic the organization of an osteon, the structural unit of cortical bone. We successfully built a rotating stage for PGA fibers and utilized it for collecting electrospun nanofibers and creating scaffolds. Resulting scaffolds consisted of concentric layers of electrospun PLLA or gelatin/PLLA nanofibers wrapped around PGA microfiber core with diameters that ranged from 200 to 600 μm. Scaffolds were mineralized by incubation in 10× simulated body fluid, and scaffolds composed of 10%gelatin/PLLA had significantly higher amounts of calcium phosphate. The electrospun scaffolds also supported cellular attachment and proliferation of MC3T3 cells over the period of 28 days.     

Comparative study of PCL-HAp and PCL-bioglass composite scaffolds for bone tissue engineering

The aim of this work is to compare the effect of hydroxyapatite (HAp) or bioglass (BG) nanoparticles in a polycaprolactone composite scaffold aimed to bone regeneration. To allow a comparison of the influence of both types of fillers, scaffolds made of PCL or composites containing up to 20 % by weight HAp or BG were obtained. Scaffolds showed acceptable mechanical properties for its use and high interconnected porosity apt for cellular colonization. To study the effect of the different materials on pre-osteoblast cells differentiation, samples with 5 % mineral reinforcement, were cultured for up to 28 days in osteogenic medium. Cells proliferated in all scaffolds. Nevertheless, differentiation levels for the selected markers were higher in pure PCL scaffolds than in the composites; inclusion of bioactive particles showed no positive effects on cell differentiation. In osteogenic culture conditions, the presence of bioactive particles is thus not necessary in order to observe good differentiation.