Novel forsterite/polycaprolactone nanocomposite scaffold for tissue engineering applications

Novel highly porous nanocomposite scaffolds consisting of polycaprolactone (PCL) and forsterite nanopowder were prepared by a solvent-casting/particle-leaching method. In addition, the effects of forsterite nanopowder contents on the structure of the scaffolds were investigated to provide an appropriate composite for bone regenerative medicine. Results showed that the scaffolds exhibited high porosity (up to 92%) with open pores of 100-300 μm average diameters. This porosity increased with decreasing forsterite nanopowder content. In addition, the pore walls contained numerous micropores. Microstructure studies showed that the pores were well distributed throughout the structures. Furthermore, the bioactive forsterite nanoparticles were homogenously distributed within the PCL matrix of the scaffolds, which contained up to 30 wt.% forsterite nanopowder. This porous structure with micropores provides the properties required for bone tissue engineering applications.     

1,4-Dioxane enhances properties and biocompatibility of polyanionic collagen for tissue engineering applications

Polyanionic collagen obtained from bovine pericardial tissue submitted to alkaline hydrolysis is an acellular matrix with strong potential in tissue engineering. However, increasing the carboxyl content reduces fibril formation and thermal stability compared to the native tissues. In the present work, we propose a chemical protocol based on the association of alkaline hydrolysis with 1,4-dioxane treatment to either attenuate or revert the drastic structural modifications promoted by alkaline treatments. For the characterization of the polyanionic membranes treated with 1,4-dioxane, we found that (1) scanning electron microscopy (SEM) shows a stronger reorientation and aggregation of collagen microfibrils; (2) histological evaluation reveals recovering of the alignment of collagen fibers and reassociation with elastic fibers; (3) differential scanning calorimetry (DSC) shows an increase in thermal stability; and (4) in biocompatibility assays there is a normal attachment, morphology and proliferation associated with high survival of the mouse fibroblast cell line NIH3T3 in reconstituted membranes, which behave as native membranes. Our conclusions reinforce the ability of 1,4-dioxane to enhance the properties of negatively charged polyanionic collagen associated with its potential use as biomaterials for grafting, cationic drug- or cell-delivery systems and for the coating of cardiovascular devices.     

In vitro evaluation of biodegradable nHAP‐Chitosan‐Gelatin‐based scaffold for tissue engineering application

The present study focuses on fabrication and characterisation of porous composite scaffold containing hydroxyapatite (HAP), chitosan, and gelatin with an average pore size of 250–1010 nm for improving wound repair and regeneration by Electrospinning method. From the results of X ‐Ray Diffraction (XRD) study, the peaks correspond to crystallographic structure of HAP powder. The presence of functional group bonds of HAP powder, Chitosan and scaffold was studied using Fourier Transform Infrared Spectroscopy (FTIR). The surface morphology of the scaffold was observed using Scanning Electron Microscope (SEM). The Bioactivity of the Nano composite scaffolds was studied using simulated body fluid solution at 37 ± 1°C. The biodegradability test was studied using Tris‐Buffer solution for the prepared nanocomposites [nano Chitosan, nano Chitosan gelatin, Nano based Hydroxyapatite Chitosan gelatin]. The cell migration and potential biocompatibility of nHAP‐chitosan‐gelatin scaffold was assessed via wound scratch assay and were compared to povedeen as control. Cytocompatibility evaluation for Vero Cells using wound scratch assay showed that the fabricated porous nanocomposite scaffold possess higher cell proliferation and growth than that of povedeen. Thus, the study showed that the developed nanocomposite scaffolds are potential candidates for regenerating damaged cell tissue in wound healing process.Inspec keywords: nanofabrication, tissue engineering, electrospinning, wounds, cellular biophysics, scanning electron microscopy, surface morphology, X‐ray diffraction, biomedical materials, nanomedicine, porosity, biodegradable materials, nanoporous materials, calcium compounds, gelatin, nanocomposites, Fourier transform infrared spectra, nanoparticles, precipitation (physical chemistry)Other keywords: average pore size, wound repair, crystallographic structure, HAP powder, functional group bonds, simulated body fluid solution, biodegradability test, Tris‐Buffer solution, cell migration, wound scratch assay, tissue engineering, electrospinning method, X‐ray diffraction, Fourier transform infrared spectroscopy, scanning electron microscopy, biocompatibility, cytocompatibility, porous nanocomposite scaffold, cell tissue, nHAP‐chitosan‐gelatin scaffold composites, wet chemical precipitation method, surface morphology, nanohydroxyapatite‐nanochitosan‐gelatin scaffold composites, cell proliferation, wound healing, (Ca₁₀ (PO₄)₆ (OH)₂)     

Electrospun polypyrrole-coated polycaprolactone nanoyarn nerve guidance conduits for nerve tissue engineering

Nerve guidance conduits (NGCs) can provide suitable microenvironment for nerve repair and promote the proliferation and migration of Schwann cells (SCs). Thus, we developed nerve guidance conduits (NGCs) with polypyrrole-coated polycaprolactone nanoyarns (PPy-PCL-NYs) as fillers in this study. PCL-NYs with the oriented structure were prepared with a double-needle electrospinning system and then PPy was coated on PCL-NYs via the in situ chemical polymerization. Subsequently, PCL nanofibers were collected around nanoyarns by the conventional electrospinning process as the outer layer to obtain PPy-PCL-NY nerve guidance conduits (PPy-PCLNY NGCs). PPy-PCL-NYs were analyzed by SEM, FTIR and XPS. Results showed that PPy was homogeneously and uniformly deposited on the surface of PCL-NY. Strain-stress curves and the Young’s modulus of PPy-PCL-NYs were investigated compared with those of non-coated PCL-NYs. Studies on biocompatibility with SCs indicated that PPy-PCL-NY NGCs were more conducive to the proliferation of SCs than PCL-NY NGCs. In summary, PPy-PCL-NY NGCs show the promising potential for nerve tissue engineering repair and regeneration.     

Characterisation of a collagen membrane for its potential use in cardiovascular tissue engineering applications

In this study, Biomend, a collagen membrane conventionally used in the regeneration of periodontal tissue, is investigated for its possible use in the field of cardiovascular tissue engineering. A key requirement of most potential tissue engineering scaffolds is that degradation occurs in tandem with tissue regeneration and extra cellular matrix remodelling. To this end, it is crucial to understand the degradation mechanics and mechanisms of the material and to investigate the practicability of using Biomend as a possible scaffold material. With this in mind, methodologies for the initial characterisation of the scaffold material were determined. The mechanical properties of Biomend samples, subjected to various degrees of hydration and enzymatic degradation, were examined primarily through tensile testing experiments. The effects of enzymatic degradation were monitored quantitatively, by observing weight loss, and visually, by studying micrographs. Cell adhesion and viability were of primary concern. Confocal laser scanning microscopy was employed to illustrate endothelialisation on the surface of this collagen membrane. Fluorescence microscopy was used to visualise cell viability on the membrane surface. These images, coupled with assays to measure cell activity, suggest that Biomend is not a suitable substrate to allow endothelialisation. In summary, this collagen membrane has suitable mechanical properties with the potential to control its degradation rate. However, since poor endothelial cell viability was observed on the membrane, it may not be suitable for use in cardiovascular tissue engineering applications.     

Electrospun bioactive nanocomposite scaffolds of polycaprolactone and nanohydroxyapatite for bone tissue engineering

Nanocomposite scaffolds based on nanofibrous poly(epsilon-caprolactone) (PCL) and nanohydroxyapatite (nanoHA) with different compositions (wt%) were prepared by electrostatic co-spinning to mimic the nano-features of the natural extracellular matrix (ECM). NanoHA was found to be well dispersed in polymers up to the addition of 20 wt%, after ultrasonication. The composite scaffolds were characterized for structure and morphology using XRD, EDX, SEM, and DSC. The scaffolds have a porous nanofibrous morphology with fibers (majority) having diameters in the range of 450-650 nm, depending on composition, and interconnected pore structures. SEM, EDX, and XRD analyses have confirmed the presence of nanoHA in the fibers. As the nanoHA content in the fibers increases, the surface of fibers becomes rougher. The mechanical (tensile) property measurement of the electrospun composites reveals that as the nanoHA content increases, the ultimate strength increases from 1.68 MPa for pure PCL to 2.17, 2.65, 3.91, and 5.49 MPa for PCL/nanoHA composites with the addition of 5, 10, 15, and 20 wt% nanoHA, respectively. Similarly the tensile modulus also increases gradually from 6.12 MPa to 21.05 MPa with the increase of nanoHA content in the PCL/nanoHA fibers, revealing an increase in stiffness of the fibers due to the presence of HA. DSC analysis reveals that as nanoHA in the composite scaffolds increases, the melting point slightly increases due to the good dispersion and interface bonding between PCL and nanoHA.     

New hydrogels based on maleilated collagen with potential applications in tissue engineering

New hydrogels based on maleic anhydride (MA) modified collagen were prepared with the aim of overcoming the high degradation rate displayed by collagen that is not otherwise chemically crosslinked. Semi-interpenetrated matrices were obtained by free radical polymerization of maleilated collagen (CM) and 2-hydroxyethyl methacrylate (HEMA) in the presence of ammonium persulfate (APS) and N,N,N′,N′-tetramethylethylenediamine (TEMED) as initiating system. The resulting matrices (CMH) had a sharp decrease in degradation, when compared to pure collagen. FTIR and H¹ NMR spectroscopies were used to confirm the incorporation of MA on the collagen peptide chains. The final composition of CMH was found to be strongly dependent by the concentration of maleilated collagen. The morphology of the hydrogels was studied by Scanning electron microscopy (SEM) and the macro-gel structure was confirmed. Water uptake of the synthetised hydrogels is influenced by both composition and the porosity of the matrices.     

Pluronic F127 blended polycaprolactone scaffolds via e-jetting for esophageal tissue engineering

Several attempts have been made to fabricate esophageal tissue engineering scaffolds. However, most of these scaffolds possess randomly oriented fibres and uncontrollable pore sizes. In order to mimic the native esophageal tissue structure, electro-hydrodynamic jetting (e-jetting) was used in this study to fabricate scaffolds with aligned fibres and controlled pore size. A hydrophilic additive, Pluronic F127 (F127), was blended with polycaprolactone (PCL) to improve the wettability of the scaffolds and hence the cell adhesion. PCL/F127 composite scaffolds with different weight ratios (0–12%) were fabricated. The wettability, phase composition, and the mechanical properties of the fabricated scaffolds were investigated. The results show that the e-jetted scaffolds have controllable fibres orientated in two perpendicular directions, which are similar to the human esophagus structure and suitable pore size for cell infiltration. In addition, the scaffolds with 8% F127 exhibited better wettability (contact angle of 14°) and an ultimate tensile strength (1.2?MPa) that mimics the native esophageal tissue. Furthermore, primary human esophageal fibroblasts were seeded on the e-jetted scaffolds. PCL/F127 scaffolds showed enhanced cell proliferation and expression of the vascular endothelial growth factor (VEGF) compared to pristine PCL scaffolds (1.5- and 25.8- fold increase, respectively; P?<?0.001). An in vitro wound model made using the PCL/F127 scaffolds showed better cell migration than the PCL scaffolds. In summary, the PCL/F127 e-jetted scaffolds offer a promising strategy for the esophagus tissue repair.

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Characterization of calcium phosphate layers grown on polycaprolactone for tissue engineering purposes

Composites fabricated by biomimetic mineral precipitation on polymeric substrates are of interest for tissue engineering. As biological properties of such mineral layers vary with slight changes in composition, a good physical characterization is necessary in order to study their biological activity. In this work polycaprolactone sheets were subjected to air plasma treatment followed by nucleation of calcium phosphate seeds to activate the growth of an apatite-like coating when immersing in simulated body fluid. Two compositions of the SBF were prepared, one of them highly carbonated and the other with no carbonate or magnesium ions. Immersion of PCL in the high carbonate composition produced a low-crystallinity apatite-like layer while the absence of carbonate and magnesium ions yielded a high crystallinity apatite with low Ca/P ratio that is likely partially hydrolyzed octacalciumphosphate (OCP). The morphology, crystal structure and composition of both types of coatings were characterized; osteoblast-like cell adhesion behaviour on different surfaces was observed by fluorescence and electron microscopy.     

Novel elastic material from collagen for tissue engineering

Elastic collagen gel (e-gel) was prepared from salmon atelocollagen fibrillar gel reinforced by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) mediated cross-linking (f-gel). The preparation consisted of a simple heat treatment of the f-gel at 80 °C, in which the f-gel drastically shrank and the collagen fibril structure was deformed. The e-gel obtained showed rubber-like elasticity; its stress–strain behavior little changed through repeated stretching. The elongation at the breaking point was approximately 230%. Furthermore, normal human osteoblasts showed good attachment and proliferation on the e-gel. These results suggest its potential to be utilized for the development of tissue engineering.     

Extruded collagen fibres for tissue engineering applications: effect of crosslinking method on mechanical and biological properties

Reconstituted collagen fibres are promising candidates for tendon and ligament tissue regeneration. The crosslinking procedure determines the fibres’ mechanical properties, degradation rate, and cell–fibre interactions. We aimed to compare mechanical and biological properties of collagen fibres resulting from two different types of crosslinking chemistry based on 1-ethyl-3-(3-dimethyllaminopropyl)carbodiimide (EDC). Fibres were crosslinked with either EDC or with EDC and ethylene-glycol-diglycidyl-ether (EDC/EGDE). Single fibres were mechanically tested to failure and bundles of fibres were seeded with tendon fibroblasts (TFs) and cell attachment and proliferation were determined over 14 days in culture. Collagen type I and tenascin-C production were assessed by immunohistochemistry and dot-blotting. EDC chemistry resulted in fibres with average mechanical properties but the highest cell proliferation rate and matrix protein production. EDC/EGDE chemistry resulted in fibres with improved mechanical properties but with a lower biocompatibility profile. Both chemistries may provide useful structures for scaffolding regeneration of tendon and ligament tissue and will be evaluated for in vivo tendon regeneration in future experiments.     

One‐step method for the preparation of poly(methyl methacrylate) modified titanium‐bioactive glass three‐dimensional scaffolds for bone tissue engineering

A novel three‐dimensional (3D) titanium (Ti)‐doping meso‐macroporous bioactive glasses (BGs)/poly(methyl methacrylate) (PMMA) composite was synthesised using PMMA and EO₂₀ PO₇₀ EO₂₀ (P₁₂₃) as the macroporous and mesoporous templates, respectively. Unlike the usual calcination method, the acid steam technique was used to improve the polycondensation of Ti‐BGs, and then PMMA was partially extracted via chloroform to induce the macroporous structure. Simultaneously, the residual PMMA which remained in the wall enhanced the compressive strength to 2.4 MPa (0.3 MPa for pure BGs). It is a simple and green method to prepare the macro‐mesoporous Ti‐BGs/PMMA. The materials showed the 3D interconnected hierarchical structure (250 and 3.4 nm), making the fast inducing‐hydroxyapatite growth and the controlled drug release. Besides mentioned above, the good antimicrobial property and biocompatible of the scaffold also ensure it is further of clinical use. Herein, the fabricated materials are expected to have potential application on bone tissue regeneration.Inspec keywords: titanium, bone, tissue engineering, glass, materials preparation, biomedical materials, polymers, porous materials, drug delivery systems, nanomedicineOther keywords: poly(methyl methacrylate), PMMA preparation, 3D titanium‐bioactive glass scaffold, bone tissue engineering, titanium‐doping mesomacroporous bioactive glass, bioactive glass‐PMMA composite, macroporous template, mesoporous template, calcination method, acid steam technique, titanium‐bioactive glass polycondensation, macroporous structure, green method, macromesoporous titanium‐bioactive glass‐PMMA, 3D interconnected hierarchical structure, fast inducing‐hydroxyapatite growth, controlled drug release, bone tissue regeneration, Ti     

Heparinized hydroxyapatite/collagen three-dimensional scaffolds for tissue engineering

Currently, in bone tissue engineering research, the development of appropriate biomaterials for the regeneration of bony tissues is a major concern. Bone tissue is composed of a structural protein, collagen type I, on which calcium phosphate crystals are enclosed. For tissue engineering, one of the most applied strategies consists on the development and application of three dimensional porous scaffolds with similar composition to the bone. In this way, they can provide a physical support for cell attachment, proliferation, nutrient transport and new bone tissue infiltration. Hydroxyapatite is a calcium phosphate with a similar composition of bone and widely applied in several medical/dentistry fields. Therefore, in this study, hydroxyapatite three dimensional porous scaffolds were produced using the polymer replication method. Next, the porous scaffolds were homogeneously coated with a film of collagen type I by applying vacuum force. Yet, due to collagen degradability properties, it was necessary to perform an adequate crosslinking method. As a result, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) was employed as an efficient and non-toxic crosslinking method in this research. The composites were characterized by means of SEM, DSC and TNBS. Furthermore, heparin was incorporated in order to accomplish sustained delivery of a growth factor of interest namely, bone morphogenetic proteins (BMP-2). BMP-2 binding and release of non-heparinized and heparinized scaffolds was evaluated at specific time points. The incorporation of heparin leads to a reduced initial burst phase when compared to the non heparinized materials. The results show a beneficial effect with the incorporation of heparin and its potential as a localized drug delivery system for the sustained release of growth factors.     

壳聚糖支架在组织工程中的应用

综述了壳聚糖作为细胞生长载体在软骨组织工程，骨组织工程和皮肤组织工程等方面的应用进展，表明壳聚糖有望成为优异的组织工程支架材料。     

Porous scaffolds of polycaprolactone reinforced with in situ generated hydroxyapatite for bone tissue engineering

Polycaprolactone/hydroxyapatite (PCL/HA) composites were prepared by in situ generation of HA in the polymer solution starting from the precursors calcium nitrate tetrahydrate and ammonium dihydrogen phosphate via sol–gel process. Highly interconnected porosity was achieved by means of the salt-leaching technique using a mixture of sodium chloride and sodium bicarbonate as porogens. Structure and morphology of the PCL/HA composites were investigated by scanning electron microscopy, and mechanical properties were determined by means of tensile and compression tests. The possibility to employ the developed composites as scaffolds for bone tissue regeneration was assessed by cytotoxicity test of the PCL/HA composites extracts and cell adhesion and proliferation in vitro studies.     

Formulation and optimisation of lamivudine‐loaded Eudragit® S 100 polymer‐coated pectin microspheres for colon‐specific delivery

This investigation is to find a prolonged or delayed drug release system, exclusively for the treatment of hepatitis‐B to reduce the side effects, which arise when conventional solid dose forms are administered. To pursue this goal, lamivudine‐loaded Eudragit‐coated pectin microspheres have been formulated employing water/oil (W/O) emulsion evaporation strategy. The formulation was optimised using a 3⁴ factorial design. A drug to polymer ratio of 1:2, the surfactant of 1 ml, the volume of 50 ml of processing medium with a stirring speed of 2500 rpm were found to be the optimal parameters to obtain the lamivudine‐loaded Eudragit‐coated pectin microspheres formulation with a high drug entrapment efficiency of 89.44% ± 1.44%. The in vitro release kinetics of lamivudine was a suitable fit to the Higuchi model, indicating a diffusion‐controlled release with anomalous transport. The obtained microspheres were then subjected to different characterisation studies, including scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), and X‐ray diffraction (XRD). The results of this study clearly indicate that Eudragit‐coated pectin microspheres could be the promising controlled release carriers for colon‐specific delivery of lamivudine in the presence of rat cecal content.     

Plasma-modified and polyethylene glycol-grafted polymers for potential tissue engineering applications

Modified and grafted polymers may serve as building blocks for creating artificial bioinspired nanostructured surfaces for tissue engineering. Polyethylene (PE) and polystyrene (PS) were modified by Ar plasma and the surface of the plasma activated polymers was grafted with polyethylene glycol (PEG). The changes in the surface wettability (contact angle) of the modified polymers were examined by goniometry. Atomic Force Microscopy (AFM) was used to determine the surface roughness and morphology and electrokinetical analysis (Zeta potential) characterized surface chemistry of the modified polymers. Plasma treatment and subsequent PEG grafting lead to dramatic changes in the polymer surface morphology, roughness and wettability. The plasma treated and PEG grafted polymers were seeded with rat vascular smooth muscle cells (VSMCs) and their adhesion and proliferation were studied. Biological tests, performed in vitro, show increased adhesion and proliferation of cells on modified polymers. Grafting with PEG increases cell proliferation, especially on PS. The cell proliferation was shown to be an increasing function of PEG molecular weight.     

Preparation and characterization of polycaprolactone-chitosan composites for tissue engineering applications

Highly porous scaffold plays an important role in bone tissue engineering, which becomes a promising alternative approach for bone repair since its emergence. The objective of this work was to blend poly (є-caprolactone) (PCL) with chitosan (CS) for the purpose of preparation of porous scaffold. A simple unique method was employed under room-temperature condition to blend the two components together without separation of two phases. The reaction leads to formation of sponge-like porous 5, 10, 15 and 20 wt% CS composites. XRD, IR and SEM were used to determine components and morphology of the composites. DSC studies indicated that the miscibility of the two components. And pore volume fractures of composites were determined by a simple method in which a pycnometer was used. The results show that CS is successfully commingled into PCL matrix, and adding CS into PCL will not damage the crystalline structure of PCL. The composite shows no signs of phase separation and presents a unique porous structure under SEM observation. The porosity of composite increased with the increase of the content of CS in the composite. The highest porosity reached to 92% when CS content increased to 20 wt%. The mechanism of formation of this unique porous structure is also discussed.     

Lamivudine‐conjugated and efavirenz‐loaded G2 dendrimers: Novel anti‐retroviral nano drug delivery systems

Infection with human immunodeficiency virus (HIV)‐1 causes immunological disorders and death worldwide which needs to be further assisted by novel anti‐retroviral drug delivery systems. Consequently, finding newer anti‐retroviral pharmaceuticals by using biocompatible, biodegradable nanomaterials comprising a nanoparticle as core and a therapeutic agent is of high global interest. In this experiment, a second generation of a negatively charged nano‐biopolymer linear globular G2 dendrimer was carefully conjugated and loaded with well‐known anti‐HIV drugs lamivudine and efavirenz, respectively. They were characterised by a variety of analytical methods such as Zetasizer, Fourier‐transform infrared spectroscopy, elemental analysis and liquid chromatography‐mass spectroscopy. Additionally, conjugated lamivudine and loaded efazirenz with globular PEGylated G2 dendrimer were tested on an HEK293 T cell infected by single‐cycle replicable HIV‐1 virion and evaluated using XTT test and HIV‐1 P24 protein load. The results showed that lamivudine‐conjugated G2 significantly decreased retroviral activity without any cell toxicity. This effect was more or less observed by efavirenz‐loaded G2. These nano‐constructs are strongly suggested for further in vivo anti‐HIV assays.     

Thermal-crosslinked porous chitosan scaffolds for soft tissue engineering applications

The aim of this study was to demonstrate the feasibility of using a steam autoclave process for sterilization and simultaneously thermal-crosslinking of lyophilized chitosan scaffolds. This process is of great interest in biomaterial development due to its simplicity and low toxicity. The steam autoclave process had no significant effect on the average pore diameter (~ 70 μm) and overall porosity (> 80%) of the resultant chitosan scaffolds, while the sterilized scaffolds possessed more homogenous pore size distribution. The sterilized chitosan scaffolds exhibited an enhanced compressive modulus (109.8 kPa) and comparable equilibrium swelling ratio (23.3). The resultant chitosan scaffolds could be used directly for in vitro cell culture without extra sterilization. The data of in vitro studies demonstrated that the scaffolds facilitated cell attachment and proliferation, indicating great potential for soft tissue engineering applications.