Preparation and modification of collagen-based porous scaffold for tissue engineering

In the effort to generate cartilage tissues using mesenchymal stem cells, porous scaffolds with prescribed biomechanical properties were prepared. Scaffolds with interconnected pores were prepared via lyophilisation of frozen hydrogels made from collagen modified with chitosan nanofibres, hyaluronic acid, copolymers based on poly(ethylene glycol) (PEG), poly(lactic-co-glycolic acid) (PLGA), and itaconic acid (ITA), and hydroxyapatite nanoparticles. The modified collagen compositions were cross-linked using N-(3-dimethylamino propyl)-N′-ethylcarbodiimide hydrochloride (EDC) combined with N-hydroxysuccinimide (NHS) in water solution. Basic physicochemical and mechanical properties were measured and an attempt to relate these properties to the molecular and supermolecular structure of the modified collagen compositions was carried out. Scaffolds containing hydrophilic chitosan nanofibres showed the highest swelling ratio (SR = 20–25) of all the materials investigated, while collagen modified with an amphiphilic PLGA-PEG-PLGA copolymer or functionalised with ITA exhibited the lowest swelling ratio (SR = 5–8). The best resistance to hydrolytic degradation was obtained for hydroxyapatite containing scaffolds. On the other hand, the fastest degradation rate was observed for synthetic copolymer-containing scaffolds. The results showed that the addition of hydroxyapatite or hyaluronic acid to the collagen matrix increases the rigidity in comparison to the collagen-chitosan scaffold. Collagen scaffold modified with hyaluronic acid presented reduced deformation at break while the presence of hydroxypatatite enhanced the scaffold deformation under tensile loading. The tensile elastic modulus of chitosan nanofibre collagen scaffold was the lowest but closest to the articular cartilage; however, the strength and deformation to failure increased up to 200 %. Presented at the 1st Bratislava Young Polymer Scientists Workshop, Bratislava, 20–23 August 2007.     

Fabrication of biodegradable poly(trimethylene carbonate) networks for potential tissue engineering scaffold applications

Biodegradable poly(trimethylene carbonate) (PTMC) networks were prepared by photopolymerization of linear (L)‐ and star (S)‐shaped PTMC macromonomers for potential tissue engineering scaffold applications. The L‐ (M_n, 6400) and S‐shaped (M_n, 5880) PTMC macromonomers were synthesized using 1,4‐butane diol and 2‐ethyl‐ 2‐hydroxyl‐propane‐1,3‐diol co‐initiated ring‐opening polymerization of trimethylene carbonate (TMC) in the presence of stannous octoate and subsequent acrylation with acryloyl chloride. Chemical structures of the PTMC macromonomers and their corresponding networks were characterized by ¹H NMR and ¹³C NMR spectroscopy. The human endothelial cell line, EA.hy926 was used to test the biocompatibility, cell adhesion, and proliferation behavior of both PTMC networks. The PTMC networks made from the S‐shaped macromonomers exhibited superior cell adhesion and proliferation behavior than those made of the linear macromonomers. Copyright © 2008 John Wiley & Sons, Ltd.     

Immobilization of gelatin onto natural nanofibers for tissue engineering scaffold applications without utilization of any crosslinking agent

Bacterial cellulose was oxidized by periodate oxidation to give rise to 2,3-dialdehyde bacterial cellulose (DABC) with 60.3 ± 0.5 % aldehyde content, which was further reacted with gelatin (Gel) for the immobilization of Gel to form DABC/Gel nanocomposites. The scanning electron microscopy and transmission electron microscopy revealed that DABC/Gel exhibited the refined 3D nano-network structures and the average thickness of Gel coatings in the composites was about 75 nm. FTIR and XPS were utilized to analyze the functional groups and chemical states of DABC/Gel nanocomposites. The results inferred that Gel was fixed on DABC nanofibers via the Schiff base reaction between –NH₂ in Gel and –CHO in DABC backbone. NIH3T3 mice fibroblast cells were used for determining the cytocompatibility of the scaffolds. The morphology of the cells was observed through optical inverted microscopy. The results show that DABC/Gel can be used as scaffold material in tissue engineering.     

Biomimetic strengthening polylactide scaffold materials for bone tissue engineering

In this paper, a new polylactide (PLA)-based scaffold composite by biomimetic synthesis was designed. The novel composite mainly consists of nano-hydroxyapatite (n-HA), which is the main inorganic content in natural bone tissue for the PLA. The crystal degree of the n-HA in the composite is low and the crystal size is very small, which is similar to that of natural bone. The compressive strength of the composite is higher than that of the PLA scaffold. Using the osteoblast culture technique, we detected cell behaviors on the biomaterial in vitro by SEM, and the cell affinity of the composite was found to be higher than that of the PLA scaffold. The biomimetic three-dimensional porous composite can serve as a kind of excellent scaffold material for bone tissue engineering because of its microstructure and properties. Translated from Journal of Hunan University (Natural Sciences), 2006, 33(2) (in Chinese)     

Preparation of 3D fibroin/chitosan blend porous scaffold for tissue engineering via a simplified method

In this work, we developed a simple and flexible method to manufacture a 3D porous scaffold based on the blend of regenerated silk fibroin (RSF) and chitosan (CS). No crosslinker or other toxic reagents were used in this method. The pores of resulted 3D scaffolds were connected with each other, and their sizes could be easily controlled by the concentration of the mixed solution. Compared with pure RSF scaffolds, the water absorptivities of these RSF/CS blend scaffolds with significantly enhanced mechanical properties were greatly increased. The results of MTT and RT-PCR tests indicated that the chondrocytes grew very well in these blend RSF/CS porous scaffolds. This suggested that the RSF/CS blend scaffold prepared by this new method could be a promising candidate for applications in tissue engineering.     

Preparation of chitosan scaffolds loaded with dexamethasone for tissue engineering applications using supercritical fluid technology

Supercritical fluid impregnation was tested to prepare a new scaffold loaded with a bioactive compound. Dexamethasone is used in osteogenic media to direct the differentiation of stem cells towards the osteogenic lineage. Dexamethasone was impregnated in chitosan scaffolds at different operating conditions, in order to optimize the impregnation process. Pressure and temperature affect the carbon dioxide density and influence the swelling of the polymer and the drug solubility in the fluid phase, therefore these are two important parameters that were studied in this work. Chitosan sponges prepared by freeze drying were impregnated with the active compound at pressures from 8.0 up to 14.0 MPa and temperatures from 35 up to 55 °C. The effect of the impregnation contact time (3 h and 6 h) was also evaluated. From the experiments performed we can conclude that the yield of impregnation is lower when increasing pressure and temperature. The contact time will mainly influence the amount of drug impregnated in the scaffold and for higher contact times the impregnation yield is also higher. Scanning electron microscopy shows particles of dexamethasone in the bulk of the scaffold, which confirms the feasibility of the supercritical fluid impregnation technology for the preparation of delivery devices. The loading capacity of the scaffolds was determined by spectroscopic analysis and the highest loading was achieved for the experiments performed at 8.0 MPa and 35 °C. Furthermore, in vitro drug release studies were carried out and the results show that dexamethasone was sustainably released. Supercritical fluid impregnation proved to be feasible for the preparation of a drug delivery system for bone tissue engineering purposes.     

Preparation and characterization of coaxial electrospun thermoplastic polyurethane/collagen compound nanofibers for tissue engineering applications

Collagen functionalized thermoplastic polyurethane nanofibers (TPU/collagen) were successfully produced by coaxial electrospinning technique with a goal to develop biomedical scaffold. A series of tests were conducted to characterize the compound nanofiber and its membrane in this study. Surface morphology and interior structure of the ultrafine fibers were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and atomic force microscopy (AFM), whereas the fiber diameter distribution was also measured. The crosslinked membranes were also characterized by SEM. Porosities of different kinds of electrospun mats were determined. The surface chemistry and chemical composition of collagen/TPU coaxial nanofibrous membranes were verified by X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectrometry (FTIR). Mechanical measurements were carried out by applying tensile test loads to samples which were prepared from electrospun ultra fine non-woven fiber mats. The coaxial electrospun nanofibers were further investigated as a promising scaffold for PIECs culture. The results demonstrated that coaxial electrospun composite nanofibers had the characters of native extracellular matrix and may be used effectively as an alternative material for tissue engineering and functional biomaterials.     

Porous membranes of PLLA-PCL blend for tissue engineering applications

Porous membranes of polycaprolactone-poly(l-lactic acid) blends were prepared by a freeze-extraction process. This procedure was able to disperse homogeneously both components despite their amorphous phases being immiscible (as proven by the fact that the glass transition temperature of PCL in the blend is independent of blend composition) and both polymers crystallize. Thus, the porous membrane consists of amorphous and crystalline phases of both components. DSC and AFM were used to characterize the microstructure of the blends, whereas SEM and gravimetric methods enabled the porosity (around 70%) and pore architecture to be determined. Compression stress-strain experiments show the characteristic behaviour of porous materials with a yield stress that rapidly drops when the PCL content increases - whereas the deformation plateau zone enlarges.     

In vitro characterization of an electroactive carbon-nanotube-based nanofiber scaffold for tissue engineering

In an effort to reduce organ replacement and enhance tissue repair, there has been a tremendous effort to create biomechanically optimized scaffolds for tissue engineering applications. In contrast, the development and characterization of electroactive scaffolds has attracted little attention. Consequently, the creation and characterization of a carbon nanotube based poly(lactic acid) nanofiber scaffold is described herein. After 28 d in physiological solution at 37 °C, a change in the mass, chemical properties and polymer morphology is seen, while the mechanical properties and physical integrity are unaltered. No adverse cytotoxic affects are seen when mesenchymal stem cells are cultured in the presence of the scaffold. Taken together, these data auger well for electroactive tissue engineering.

     

Preparation and characterization of a scaffold for vascular tissue engineering by direct-assembling of collagen and cells in a cylindrical geometry

In the past 25 years, several efforts have been focused on developing vascular substitutes showing long-term patency when implanted in humans. In this study, we present our last findings in an ongoing project aiming to develop a tissue-regenerated blood vessel from collagen-based scaffolds. A collagen-based scaffold with adequate biological properties was developed by directly assembling collagen and cells in a cylindrical geometry. The technique mainly involves the use of a ventilated rotating tube allowing smooth muscle cells to grow in a cylindrical collagen matrix. Scanning electron microscopy (SEM), histology, and cell cycle analyses were carried out in order to assess the biological potential of the cell-based scaffold for further maturation. Compliance results showed that this technique allows cells to assemble in the collagen matrix, thus providing enough rigidity to the structure to be handled and mounted in a perfusion bioreactor for further growth and maturation.     

Evaluation of PEGylated fibrin as a three-dimensional biodegradable scaffold for ovarian tissue engineering

The most challenging task of creating a bioengineered ovary to restore fertility in cancer patients is choosing an appropriate biomaterial to encapsulate isolated preantral follicles and ovarian cells. In this study, as a biocompatible and biodegradable biomaterial containing fibrin-like bioactivity and manageable physical properties, PEGylated fibrin aims to encapsulate isolated ovarian stromal cells as a first step of creating an engineered ovarian tissue. For this purpose, human ovarian stromal cells were isolated from frozen-thawed ovarian tissue and cultured in the PEGylated fibrin hydrogels (PEG:Fib), which were fabricated by combining two different molar ratios of PEG:Fib (10:1 and 5:1) and two thrombin concentrations. The samples were analyzed at days 0 and 5 of in vitro for cell density, proliferation (Ki67), and apoptosis (caspase-3). Moreover, LIVE/DEAD and PrestoBlue assays assessed cell viability and proliferation on days 1, 3, and 5. The effect of PEGylation on the biodegradation behavior of fibrin was evaluated by measuring the remaining mass ratio of non-modified fibrin, PEG:Fib 10:1, and PEG:Fib 5:1 hydrogels after 1, 2, 3, 5, 8, 11, and 15 days. The results showed that PEGylated fibrin hydrogels enhanced scaffold stability and supported cell viability and proliferation. In addition, PEG:Fib 5:1 T50 indicated a significantly higher cell density dynamic and non-significantly lower expression of caspase-3 on day 5. Besides, uniformity of cell distribution inside the hydrogel and a tendency to a high rate of Ki67-positive cells was observed in PEG:Fib 10:1 T50 hydrogels. In conclusion, this study reveals the positive effects of PEGylated fibrin hydrogels on isolated human ovarian stromal cells. Based on such promising findings, we believe that this matrix should be tested to encapsulate isolated human ovarian follicles.     

Fabrication of poly(lactide‐co‐glycolide) scaffold embedded spatially with hydroxyapatite particles on pore walls for bone tissue engineering

Poly(lactide‐co‐glycolide) (PLGA) scaffolds embedded spatially with hydroxyapatite (HA) particles on the pore walls (PLGA/HA‐S) were fabricated by using HA‐coated paraffin spheres as porogens, which were prepared by Pickering emulsion. For comparisons, PLGA scaffolds loaded with same amount of HA particles (2%) in the matrix (PLGA/HA‐M) and pure PLGA scaffolds were prepared by using pure paraffin spheres as porogens. Although the three types of scaffolds had same pore size (450–600 µm) and similar porosity (90%–93%), the PLGA/HA‐S showed the highest compression modulus. The embedment of the HA particles on the pore walls endow the PLGA/HA‐S scaffold with a stronger ability of protein adsorption and mineralization as well as a larger mechanical strength against compression. In vitro culture of rat bone marrow stem cells revealed that cell morphology and proliferation ability were similar on all the scaffolds. However, the alkaline phosphatase activity was significantly improved for the cells cultured on the PLGA/HA‐S scaffolds. Therefore, the method for fabricating scaffolds with spatially embedded nanoparticles provides a new way to obtain the bioactive scaffolds for tissue engineering. Copyright © 2011 John Wiley & Sons, Ltd.     

Preparation of chitosan/hydroxyapatite guided membrane used for periodontal tissue regeneration

<正>In an effort to develop biomaterials to meet guided tissue regeneration(GTR) standards for periodontal tissue recovery,a homogeneous and transparent chitosan(CS)/hydroxyapatite(HA) membrane with potential applications as GTR barrier in periodontal therapy has been prepared via in situ compositing.The membrane has been designed to have a smoothrough asymmetric structure that meets the demand for GTR.Component and morphology of the membrane are characterized by XRD and SEM.It can be indicated that HA was in situ synthesized uniformly in the CS membrane.Mechanical experiments of the membranes with various HA contents show that their tensile strengths are adequate for periodontal therapy.Biological properties of the membrane have been performed by cell toxicity assays,hemolysis tests and animal experiments.Results indicate that the membrane has good biocompatibility and inductive effect for cell growth.Therefore this membrane can be potentially applied as GTR barrier membrane for periodontal tissue regeneration.     

Graphene oxide-thionine-Au nanostructure composites: Preparation and applications in non-enzymatic glucose sensing

Herein, we demonstrated a facile strategy for preparing high-loading Au nanoparticles using thionine functionalized graphene oxide as the supporting material. Glassy carbon electrodes modified with the as-obtained nanocomposites showed remarkably electrocatalytic activity towards the oxidation of glucose, leading to an enzymeless glucose sensor with a wide linear range and a lower detection limit of 0.05 μmol/L.     

Atmospheric plasma treatment of porous polymer constructs for tissue engineering applications

Porous 3D polymer scaffolds prepared by TIPS from PLGA (53:47) and PS are intrinsically hydrophobic which prohibits the wetting of such porous media by water. This limits the application of these materials for the fabrication of scaffolds as supports for cell adhesion/spreading. Here we demonstrate that the interior surfaces of polymer scaffolds can be effectively modified using atmospheric air plasma (AP). Polymer films (2D) were also modified as control. The surface properties of wet 2D and 3D scaffolds were characterised using zeta-potential and wettability measurements. These techniques were used as the primary screening methods to assess surface chemistry and the wettability of wet polymer constructs prior and after the surface treatment. The surfaces of the original polymers are rather hydrophobic as highlighted but contain acidic functional groups. Increased exposure to AP improved the water wetting of the treated surfaces because of the formation of a variety of oxygen and nitrogen containing functions. The morphology and pore structure was assessed using SEM and a liquid displacement test. The PLGA and PS foam samples have central regions which are open porous interconnected networks with maximum pore diameters of 49 microm for PLGA and 73 microm for PS foams.     

Magnetic mesoporous carbonated hydroxyapatite microspheres with hierarchical nanostructure for drug delivery systems

Magnetic mesoporous carbonated hydroxyapatite microspheres have been fabricated hydrothermally by using CaCO(3)/Fe(3)O(4) microspheres as sacrificial templates. The high drug-loading capacity and sustained drug release property suggest that the multifunctional microspheres have great potentials for bone-implantable drug-delivery applications.     

In situ mineralization of hydroxyapatite on poly(vinyl alcohol) monolithic scaffolds for tissue engineering

A poly(vinyl alcohol) (PVA)/hydroxyapatite (HAp) composite monolithic scaffold is prepared via thermally impacted non-solvent induced phase separation method, successively followed by an alternate soaking process. The morphology of the resulting composite monolith is observed by scanning electron microscopy (SEM). The formation of hydroxyapatite is confirmed by X-ray diffraction, SEM in combination with energy dispersive X-ray analysis, and Fourier transform infrared spectroscopy. The effects of soaking cycle and soaking time upon the formation of hydroxyapatite on the monolith surface are systematically investigated. With the increase of soaking cycle and soaking time, the amount of the formed hydroxyapatite increases. As the soaking cycle increases, the water uptake of the composite monolith decreases. The PVA/HAp composite monolith greatly has a promising application as scaffold of bone tissue engineering.     

Dual-syringe reactive electrospinning of cross-linked hyaluronic acid hydrogel nanofibers for tissue engineering applications

A facile fabrication of a cross-linked hyaluronic acid (HA) hydrogel nanofibers by a reactive electrospinning method is described. A thiolated HA derivative, 3,3'-dithiobis(propanoic dihydrazide)-modified HA (HA-DTPH), and poly(ethylene glycol) diacrylate (PEGDA) are selected as the cross-linking system. The cross-linking reaction occurs simultaneously during the electrospinning process using a dual-syringe mixing technique. Poly(ethylene oxide) (PEO) is added into the spinning solution as a viscosity modifier to facilitate the fiber formation and is selectively removed with water after the electrospinning process. The nanofibrous structure of the electrospun HA scaffold is well preserved after hydration with an average fiber diameter of 110 nm. A cell morphology study on fibronectin (FN)-adsorbed HA nanofibrous scaffolds shows that the NIH 3T3 fibroblasts migrate into the scaffold through the nanofibrous network, and demonstrate an elaborate three-dimensional dendritic morphology within the scaffold, which reflects the dimensions of the electrospun HA nanofibers. These results suggest the application of electrospun HA nanofibrous scaffolds as a potential material for wound healing and tissue regeneration. [image: see text] Laser scanning confocal microscopy demonstrates that the NIH3T3 fibroblast develops an extended 3D dendritic morphology within the fibronectin-adsorbed electrospun HA nanofibrous scaffold.