Polyester scaffolds with bimodal pore size distribution for tissue engineering

This paper presents a method for the preparation of porous poly(L-lactide)/poly[(L-lactide)-co-glycolide] scaffolds for tissue engineering. Scaffolds were prepared by a mold pressing-salt leaching technique from structured microparticles. The total porosity was in the range 70-85%. The pore size distribution was bimodal. Large pores, susceptible for osteoblasts growth and proliferation had the dimensions 50-400 microm. Small pores, dedicated to the diffusion of nutrients or/and metabolites of bone forming cells, as well as the products of hydrolysis of polyesters from the walls of the scaffold, had sizes in the range 2 nm-5 microm. The scaffolds had good mechanical strength (compressive modulus equal to 41 MPa and a strength of 1.64 MPa for 74% porosity). Scaffolds were tested in vitro with human osteoblast-like cells (MG-63). It was found that the viability of cells seeded within the scaffolds obtained using the mold pressing-salt leaching technique from structured microparticles was better when compared to cells cultured in scaffolds obtained by traditional methods. After 34 d of culture, cells within the tested scaffolds were organized in a tissue-like structure. Photos of section of macro- and mesoporous PLLA/PLGA scaffold containing 50 wt.-% of PLGA microspheres after 34 d of culture. Dark spots mark MG-63 cells, white areas belong to the scaffold. The specimen was stained with haematoxylin/eosin. Bar = 100 microm.     

Nano-fibrous scaffolds for tissue engineering

With the ability to form nano-fibrous structures, a drive to mimic the extracellular matrix (ECM) and form scaffolds that are an artificial extracellular matrix suitable for tissue formation has begun. These nano-fibrous scaffolds attempt to mimic collagen, a natural extracellular matrix component, and could potentially provide a better environment for tissue formation in tissue engineering systems. Three different approaches toward the formation of nano-fibrous materials have emerged: self-assembly, electrospinning and phase separation. Each of these approaches is very different and has a unique set of characteristics, which lends to its development as a scaffolding system. For instance, self-assembly can generate small diameter nano-fibers in the lowest end of the range of natural extracellular matrix collagen, while electrospinning has only generated large diameter nano-fibers on the upper end of the range of natural extracellular matrix collagen. Phase separation, on the other hand, has generated nano-fibers in the same range as natural extracellular matrix collagen and allows for the design of macropore structures. These attempts at an artificial extracellular matrix have the potential to accommodate cells and guide their growth and subsequent tissue regeneration.     

Monolithic biocompatible and biodegradable scaffolds for tissue engineering

The state of the art in polymeric materials for tissue engineering as well as the needs and concerns for future medical applications are outlined and discussed and brought into relation to recent developments in polymer chemistry. Particularly, the recent developments in micro‐ and nano‐structured polymeric monoliths designed for these purposes will be discussed. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 2219–2227, 2009     

Nondestructive technique for the characterization of the pore size distribution of soft porous constructs for tissue engineering

Polymer scaffolds tailored for tissue engineering applications possessing the desired pore structure require reproducible fabrication techniques. Nondestructive, quantitative methods for pore characterization are required to determine the pore size and its distribution. In this study, a promising alternative to traditional pore size characterization techniques is presented. We introduce a quantitative, nondestructive and inexpensive method to determine the pore size distribution of large soft porous solids based on the on the displacement of a liquid, that spreads without limits though a porous medium, by nitrogen. The capillary pressure is measured and related to the pore sizes as well as the pore size distribution of the narrowest bottlenecks of the largest interconnected pores in a porous medium. The measured pore diameters correspond to the narrowest bottleneck of the largest pores connecting the bottom with the top surface of a given porous solid. The applicability and reproducibility of the breakthrough technique is demonstrated on two polyurethane foams, manufactured using the thermally induced phase separation (TIPS) process, with almost identical overall porosity (60-70%) but very different pore morphology. By selecting different quenching temperatures to induce polymer phase separation, the pore structure could be regulated while maintaining the overall porosity. Depending on the quenching temperature, the foams exhibited either longitudinally oriented tubular macropores interconnected with micropores or independent macropores connected to adjacent pores via openings in the pore walls. The pore size and its distribution obtained by the breakthrough test were in excellent agreement to conventional characterization techniques, such as scanning electron microscopy combined with image analysis, BET technique, and mercury intrusion porosimetry. This technique is suitable for the characterization of the micro- and macropore structure of soft porous solids intended for tissue engineering applications. The method is sensitive for the smallest bottlenecks of the largest continuous pores throughout the scaffold that contributes to fluid flow.     

Cellulose-based materials as scaffolds for tissue engineering

Two types of cellulose-based materials, 6-carboxycellulose with 2.1 or 6.6 wt% of –COOH groups, were prepared and tested for potential use in tissue engineering. The materials were functionalized with arginine, i.e. an amino acid with a basic side chain, or with chitosan, in order to balance the relatively acid character of oxidized cellulose molecules, and were seeded with vascular smooth muscle cells (VSMC). The cell adhesion and growth were then evaluated directly on the materials, and also on the underlying polystyrene culture dishes. Of these two types of studied materials, 6-carboxycellulose with 2.1 wt% of –COOH groups was more appropriate for cell colonization. The cells on this material achieved an elongated shape, while they were spherical in shape on the other materials. The number of cells and the concentration (per mg of protein) of contractile proteins alpha-actin and SM1 and SM2 myosins, i.e. markers of the phenotypic maturation of VSMC, were also significantly higher on this material. Functionalization of the material with arginine and chitosan further improved the phenotypic maturation of VSMC. Chitosan also improved the adhesion and growth of these cells. In comparison with the control polystyrene dishes, the proliferation of cells on our cellulose-based materials was relatively low. This suggests that these materials can be used in applications where high proliferation activity of cells is not desirable, e.g. proliferation of VSMC on vascular prostheses. Alternatively, the cell proliferation might be enhanced by another more efficient modification, which would require further research.     

Thermally produced biodegradable scaffolds for cartilage tissue engineering

A novel process was developed to fabricate biodegradable polymer scaffolds for tissue engineering applications, without using organic solvents. Solvent residues in scaffolds fabricated by processes involving organic solvents may damage cells transplanted onto the scaffolds or tissue near the transplantation site. Poly(L-lactic acid) (PLLA) powder and NaCl particles in a mold were compressed and subsequently heated at 180 degrees C (near the PLLA melting temperature) for 3 min. The heat treatment caused the polymer particles to fuse and form a continuous matrix containing entrapped NaCl particles. After dissolving the NaCl salts, which served as a porogen, porous biodegradable PLLA scaffolds were formed. The scaffold porosity and pore size were controlled by adjusting the NaCl/PLLA weight ratio and the NaCl particle size. The characteristics of the scaffolds were compared to those of scaffolds fabricated using a conventional solvent casting/particulate leaching (SC/PL) process, in terms of pore structure, pore-size distribution, and mechanical properties. A scanning electron microscopic examination showed highly interconnected and open pore structures in the scaffolds fabricated using the thermal process, whereas the SC/PL process yielded scaffolds with less interconnected and closed pore structures. Mercury intrusion porosimetry revealed that the thermally produced scaffolds had a much more uniform distribution of pore sizes than the SC/PL process. The utility of the thermally produced scaffolds was demonstrated by engineering cartilaginous tissues in vivo. In summary, the thermal process developed in this study yields tissue-engineering scaffolds with more favorable characteristics, with respect to, freedom from organic solvents, pore structure, and size distribution than the SC/PL process. Moreover, the thermal process could also be used to fabricate scaffolds from polymers that are insoluble in organic solvents, such as poly(glycolic acid). Cartilage tissue regenerated from thermally produced PLLA scaffold.     

Magnesium-zinc-graphene oxide nanocomposite scaffolds for bone tissue engineering

The aim of this study was to fabricate and evaluate magnesium-zinc-graphene oxide nanocomposite scaffolds for bone tissue engineering. For this reason, Mg-6Zn, Mg-6Zn-1GO, and Mg-6Zn-2GO scaffolds were fabricated by the powder metallurgy method. The porosity level and also the pore size of the scaffolds were evaluated by SEM which varied from 40 to 46% and 200 to 500 μm, respectively. The chemical composition and microstructure of the scaffolds were characterized by XRD and SEM equipped with EDS; the presence of Mg, Zn, C, and O elements in the structure of the scaffolds was shown. Also, the elemental map confirmed the existence of magnesium, zinc, carbon, and oxygen in the structure of the scaffold. The mechanical properties of the scaffolds were investigated by the compression test; the results showed that by the addition of graphene oxide to the structure, the compressive strength of the samples increased from 5 to 8 MPa. Electrochemical corrosion polarization tests were conducted to evaluate the corrosion resistance of the samples immersed in simulated body fluid (SBF). Furthermore, the biodegradability of the scaffolds was determined by immersion of the samples in phosphate-buffered saline (PBS). The results demonstrated that the polarization resistance value and the corrosion rate for different formulations including Mg-6Zn, Mg-6Zn-1GO, and Mg-6Zn-2GO were 41.58, 35.48, and 55.40 Ω.cm² followed by 10.60, 14.83, and 9.06 mm.year^?1, respectively. Based on the results, the Mg-6Zn-2GO formulation presented the best corrosion resistance among the samples were investigated, which confirmed the results of the immersion test. Moreover, the MTT assay proved that the extract of Mg-6Zn-2GO scaffolds was not cytotoxic in contact with L-929 cells which validated the studied scaffolds for bone tissue applications.     

Original method of imprinting pores in scaffolds for tissue engineering

Results of the preparation of biodegradable porous scaffolds using an original modification of a wet phase inversion method were presented. Influence of gelatin non‐woven as a non‐classic pore precursor and polyvinylpyrrolidone, Pluronic as classic pore precursors on the structure of obtained scaffolds was analyzed. It was shown that the addition of gelatin non‐wovens enables the preparation of scaffolds, which allow for the growth of cells (size, distribution, and shape of pores). Mechanical properties of the obtained cell scaffolds were determined. The influence of pore precursors on mass absorption of scaffolds against isopropanol and plasma was investigated. Interaction of scaffolds with a T‐lymphocyte line (Jurkat) and with fibroblasts (L929) was investigated. Obtained scaffolds are not cytotoxic and can be used as implants, for example, the regeneration of cartilage tissue.     

Control of pore size and pore uniformity in films based on self‐assembling block copolymers

Blends of self‐assembling polystyrene‐block‐poly(4‐vinyl pyridine) (PS‐b‐P4VP) diblock‐copolymers and poly(4‐vinyl pyridine) (P4VP) homopolymers were used to fabricate isoporous and nanoporous films. Block copolymers (BCP) self‐assembled into a structure where the minority component forms very uniform cylinders, while homopolymers, resided in the core of the cylinders. Selective removal of the homopolymers by ethanol immersion led to the formation of well‐ordered pores. In films without added homopolymer, just immersion in ethanol and subsequent swelling of the P4VP blocks was found to be sufficient to create pores. Pore sizes were tuned between 10 and 50 nm by simply varying the homopolymer content and the molecular weight of the block‐copolymer. Uniformity was lost when the average pore size exceeded 30 nm because of macrophase separation. However, preparation of films from low M_W diblock copolymers showed that it is possible to have excellent pore size control and a high porosity, while retaining a low pore size distribution. © 2014 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2014 , 52, 1568–1579     

Nanocellulose based hydrogel or aerogel scaffolds for tissue engineering

Cellulose - Some structures and properties of hydrogel or aerogel tissue engineering scaffolds can be regulated by adding nanocellulose due to its biocompatibility, biodegradability, hydrophilic...     

Novel photopolymerizable biodegradable triblock polymers for tissue engineering scaffolds: synthesis and characterization

For use in micro-patterned scaffolds in tissue engineering, novel diacrylated triblock macromers (PLA-b-PCL-b-PLA, PGA-b-PCL-b-PGA and PCL-b-PEO-b-PCL) were synthesized and characterized by Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance spectroscopy (NMR) and gel permeation chromatography (GPC). All diacrylated polymers were designed as triblock copolymers and involved biodegradable blocks of relatively non-polar epsilon-caprolactone (CL) and polar monomers such as glycolide (GA), lactide (LA) or ethylene oxide (EO). All triblock polymers were prepared in molecular weights of a few kilo daltons via the anionic ring-opening polymerization (ROP) of the corresponding lactide, glycolide or caprolactone using stannous octoate [Sn(Oct)(2)] as catalyst. The polymers had low polydispersity indices, ranging from 1.23 to 1.56. Biodegradable polymeric networks were prepared with conversions of 72-84% via photopolymerization of the triblock diacrylated polymers with 2,2-dimethoxy-2-phenylacetophenone (DMPA) as photoinitiator. PLA-b-PCL-b-PLA copolymers crumbled easily and were not suitable for micro-patterning. PGA-b-PCL-b-PGA copolymers had higher water contact angles than PCL-b-PEO-b-PCL and were also cytocompatible with Fibroblasts 3T3.     

Electrospun nanofibrous polycaprolactone scaffolds for tissue engineering of annulus fibrosus

The annulus fibrosus comprises concentric lamellae that can be damaged due to intervertebral disc degeneration; to provide permanent repair of these acquired structural defects, one solution is to fabricate scaffolds that are designed to support the growth of annulus fibrosus cells. In this study, electrospun nanofibrous scaffolds of polycaprolactone are fabricated in random, aligned, and round-end configurations. Primary porcine annulus fibrosus cells are grown on the scaffolds and evaluated for attachment, proliferation, and production of extracellular matrix. The scaffold consisting of round-end nanofibers substantially outperforms the random and aligned scaffolds on cell adhesion; additionally, the scaffold with aligned nanofibers strongly affects the orientation of cells.     

Microfluidic chip-based fabrication of PLGA microfiber scaffolds for tissue engineering

In this paper, we have developed a method to produce poly(lactic- co-glycolic acid) (PLGA) microfibers within a microfluidic chip for the generation of 3D tissue engineering scaffolds. The synthesis of PLGA fibers was achieved by using a polydimethylsiloxane (PDMS)-based microfluidic spinning device in which linear streams of PLGA dissolved in dimethyl sulfoxide (DMSO) were precipitated in a glycerol-containing water solution. By changing the flow rate of PLGA solution from 1 to 50 microL/min with a sheath flow rate of 250 or 1000 microL/min, fibers were formed with diameters that ranged from 20 to 230 microm. The PLGA fibers were comprised of a dense outer surface and a highly porous interior. To evaluate the applicability of PLGA microfibers generated in this process as a cell culture scaffold, L929 fibroblasts were seeded on the PLGA fibers either as-fabricated or coated with fibronectin. L929 fibroblasts showed no significant difference in proliferation on both PLGA microfibers after 5 days of culture. As a test for application as nerve guide, neural progenitor cells were cultured and the neural axons elongated along the PLGA microfibers. Thus our experiments suggest that microfluidic chip-based PLGA microfiber fabrication may be useful for 3D cell culture tissue engineering applications.     

Tailoring the porosity and morphology of gelatin-methacrylate polyHIPE scaffolds for tissue engineering applications

Gelatin is a natural protein with many desirable properties for application as a biomaterial, including scaffolding for tissue engineering. In this work gelatin A with a molecular weight in the range 50-100 kg mol-1 was modified with methacrylic anhydride and processed into a concentrated oil-in-water emulsion. Polymerization of the continuous phase gave rise to a polyHIPE, a porous material possessing a highly interconnected, trabecular morphology. In the paper, we focused on the goal of obtaining matrixes characterized by suitable sizes of both voids and interconnects, to allow an in depth colonization from transplanted cells. In this respect, we investigated the role of the volume percentage of the dispersed phase and the effect of additives. It was established that high pore volumes (>or=90%) are to be preferred, because they allow the production of solid foams characterized by average void and interconnect diameters of approximately 20 and 10 microm, respectively. These values are still inadequate for the intended application of these scaffolds but represent a good starting point for further improvements. These were achieved through the use of additives, namely sodium chloride and dimethyl sulfoxide, which partially destabilized the precursor emulsion and allowed a solid foam to be obtained with void and interconnect diameters in the range of 30-150 microm and 10-50 microm, respectively.     

On-line fluorescent monitoring of the degradation of polymeric scaffolds for tissue engineering

Tissue engineering involves culturing, growing and assembling cells and newly generated matrix in polymeric scaffolds. To achieve a functional tissue in vitro, the cell-scaffold constructs are subjected to various stimulations during an incubation phase, which mimics the in vivo environment. In order to monitor the progression of tissue formation, there is a need for on-line and non-destructive methods of monitoring at the cellular and biomolecular level, for example, the assessment of scaffold degradation alongside the measure of matrix production. This study presents a proof of concept for monitoring scaffold degradation on-line within a culture environment. Using a mesoporous silica based approach, a pH sensitive fluorescent probe, fluorescein isothiocyanate (FITC), was incorporated into degradable polymeric scaffolds made from poly(L-lactic acid) which has a slow degradation rate, and poly(lactide-co-glycolide) which has a rapid degradation rate. The fluorescent probe was incorporated into thin films and three dimensional porous scaffolds demonstrating the capabilities of monitoring on-line. Following incubation, the intensity of fluorescence in the rapidly degrading scaffolds reduced with culture time in comparison to slow degrading polymeric scaffolds when observed qualitatively using fluorescent microscopy. The relationship between pH and fluorescent intensity was assessed, and the use of this technique for monitoring by-products via the solid scaffold by microscopy or through culture medium by a luminescence spectrometer is discussed. This study demonstrates that endowing scaffolds with a sensing element could provide an on-line and non-destructive monitoring method for tissue engineering.     

A flexible approach to the preparation of polymer scaffolds for tissue engineering

Porous polyester thermoset xerogels have been produced via sol-gel chemistry as a first step in the development of sol-gel derived tissue engineering scaffolds templated by replica molding and/or salt leaching. The pore structure of these untemplated thermosets is tunable and can be altered independent of or in tandem with alterations in composition. Cytocompatibility studies on these xerogels imply the effects of both pore size and materials chemistry, with fully aliphatic polyesters with large pore structures allowing the growth of mammalian cells. To the best of our knowledge, this represents the first report examining the preparation and potential of sol-gel derived porous polymer xerogels as tissue engineering scaffolds.     

Biomimetic polymer/apatite composite scaffolds for mineralized tissue engineering

The material surface must be considered in the design of scaffolds for bone tissue engineering so that it supports bone cells adhesion, proliferation and differentiation. A biomimetic approach has been developed as a 3D surface modification technique to grow partially carbonated hydroxyapatite (the bonelike mineral) in prefabricated, porous, polymer scaffolds using a simulated body fluid in our lab. For the rational design of scaffolding materials and optimization of the biomimetic process, this work focused on various materials and processing parameters in relation to apatite formation on 3D polymer scaffolds. The apatite nucleation and growth in the internal pores of poly(L-lactide) and poly(D,L-lactide) scaffolds were significantly faster than in those of poly(lactide-co-glycolide) scaffolds in simulated body fluids. The apatite distribution was significantly more uniform in the poly(L-lactide) scaffolds than in the poly(lactide-co-glycolide) scaffolds. After incubation in a simulated body fluid for 30 d, the mass of poly(L-lactide) scaffolds increased approximately 40%, whereas the mass of the poly(lactide-co-glycolide) scaffolds increased by about 15% (see Figure). A higher ionic concentration and higher pH value of the simulated body fluid enhanced apatite formation. The effects of surface functional groups on apatite nucleation and growth were found to be more complex in 3D scaffolds than on 2D films. Surprisingly enough, it was found that carboxyl groups significantly reduced the apatite formation, especially on the internal pore surfaces of 3D scaffolds. These findings are critically important in the rational selection of materials and surface design of 3D scaffolds for mineralized tissue engineering and may contribute to the understanding of biomineralization as well.SEM micrograph of a poly(L-lactide) scaffold.     

Membrane emulsification using membranes of regular pore spacing: Droplet size and uniformity in the presence of surface shear

During membrane emulsification it is shown that the size of the drops formed at the membrane surface may increase with increasing dispersed phase injection rate through the membrane, or it may decrease, depending on the prevailing conditions. This is illustrated using a paddle stirrer positioned above flat disc membranes with regular arrays of pores of 20 μm diameter and spacing between the pores of 80 and 200 μm. In the former case an additional mechanism for drop detachment is the push-off force, which is determined by the geometry of the drops as they deform at the membrane surface. When dispersing sunflower oil in to aqueous solutions containing Tween 20, drop sizes between 60 and 200 μm were produced, and in the case of the membrane when the push-off force was working the Coefficient of Variation of the drops formed was below 10%. The push-off force may be added to the shear-drag force to predict drop detachment. For the 200 μm pore spaced membrane this force is much less prominent than the 80 μm spaced membrane. The capillary-shear model has been modified to include this push-off force. The experimental study required the use of very low dispersed phase injection rates as well as very high rates. Hence, two different types of pumps were used to provide these: a peristaltic and syringe pumps. A small study comparing the drop size, and size distributions, showed that the pump type did not influence the drops produced by the membrane emulsification process.     

Heparin-conjugated scaffolds with pore structure of inverted colloidal crystals for cartilage regeneration

A uniform de novo production of neocartilage is a critical issue in the fabrication of tissue-engineered diarthrodial substitutes. The aim of this work is to develop homogeneous chondrogenesis in heparinized scaffolds with pores of inverted colloidal crystal (ICC) geometry. Monodispersed polystyrene microspheres were self-assembled by floating in the medium containing ethylene glycol, dried, annealed and infiltrated with heparin/chitin/chitosan gels. The results indicated that the colloidal template was in a structure of hexagonal arrays. In addition, the regularity of the organized pores in the scaffolds reduced when the concentration of ethylene glycol decreased. An increase in the weight percentage of heparin enhanced the viability of bovine knee chondrocytes (BKCs) in ICC matrices. Over 4 weeks of cultivation, the amount of cartilaginous components including BKCs, glycosaminoglycans (GAGs) and collagen enhanced with time. Moreover, an increase in the weight percentage of heparin promoted the production of BKCs, GAGs and collagen in ICC constructs. Histological and immunochemical staining of the cultured ICC constructs revealed minor differences in BKCs, GAGs and type II collagen between the peripheral and core regions. Therefore, the ordered pores in the heparinized ICC constructs could favor the chondrocyte culture to regenerate a uniform distribution of cartilage.     

Porous-conductive chitosan scaffolds for tissue engineering, 1. Preparation and characterization

Novel porous-conductive chitosan scaffolds were fabricated by incorporating conductive polypyrrole (PPy) particles into a chitosan matrix and employing a phase separation technique to build pores inside the scaffolds. Conductive polypyrrole particles were prepared with a microemulsion method using FeCl3 as a dopant. The preparation conditions were optimized to obtain scaffolds with controlled pore size and porosity. The conductivity of the scaffolds was investigated using a standard four-point probe technique. It was found that several kinds of scaffolds showed a conductivity close to 10(-3) S.cm(-1) with a low polypyrrole loading of around 2 wt.-%. The main mechanical properties, such as tensile strength, breaking elongation and Young's modulus of the scaffolds, were examined both in the dry and in the hydrated states. The results indicated that a few different kinds of scaffolds exhibited the desired mechanical strength for some tissue engineering applications. The miscibility of polypyrrole and chitosan was also evaluated using a dynamic mechanical method. The presence of significant phase separation was detected in non-porous PPy/chitosan scaffolds but enhanced miscibility in porous PPy/chitosan scaffolds was observed.