Substrates For Tissue Engineering Research Articles

Physicochemical and cell adhesion properties of chitosan films prepared from sugar and phosphate-containing solutions

This work was aimed at investigating a series of chitosan films obtained from chitosan, chitosan-phosphate, chitosan-phosphate- d-(+)raffinose or chitosan-phosphate- d-(+)sucrose solutions to preliminarily select a suitable biomaterial for developing a cell substrate for tissue engineering. The prepared films were characterized in terms of physicochemical properties (FT-IR, XRD, optical microscopy, wettability, water absorption, and tensile stress) and effects on proliferation of different types of human cells (endothelial, HUVEC; fibroblast, WI-38). The obtained results indicated that the presence of sucrose or raffinose at high concentration along with phosphate salts in the chitosan film-forming solution affords smooth, amorphous and highly hydrophilic materials in the form of soft and elastic film with optimal cytocompatibility. Owing to improved physicochemical and mechanical properties as well as affinity for differentiated human cells, these novel chitosan films appear as promising candidate biomaterials for tissue regeneration and repair. The major finding is the possibility to improve the biocompatibility of chitosan films by simply modifying their solid state characteristics.

Increased response of Vero cells to PHBV matrices treated by plasma

The copolymers poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) (PHBV) are being intensely studied as a tissue engineering substrate. It is known that poly 3-hydroxybutyric acids (PHBs) and their copolymers are quite hydrophobic polyesters. Plasma-surface modification is an effective and economical surface treatment technique for many materials and of growing interest in biomedical engineering. In this study we investigate the advantages of oxygen and nitrogen plasma treatment to modify the PHBV surface to enable the acceleration of Vero cell adhesion and proliferation. PHBV was dissolved in methylene chloride at room temperature. The PHBV membranes were modified by oxygen or nitrogen-plasma treatments using a plasma generator. The membranes were sterilized by UV irradiation for 30 min and placed in 96-well plates. Vero cells were seeded onto the membranes and their proliferation onto the matrices was also determined by cytotoxicity and cell adhesion assay. After 2, 24, 48 and 120 h of incubation, growth of fibroblasts on matrices was observed by scanning electron microscopy (SEM). The analyses of the membranes indicated that the plasma treatment decreased the contact angle and increased the surface roughness; it also changed surface morphology, and consequently, enhanced the hydrophilic behavior of PHBV polymers. SEM analysis of Vero cells adhered to PHBV treated by plasma showed that the modified surface had allowed better cell attachment, spreading and growth than the untreated membrane. This combination of surface treatment and polymer chemistry is a valuable guide to prepare an appropriate surface for tissue engineering application.

Effect of human corneal keratocytes and retinal pigment epithelial cells on the mechanical properties of micropatterned collagen films

Collagen-based micropatterned films were seeded with human corneal keratocyte and epithelial cells to study their mechanical properties as tissue engineering substrates. The patterns were in the form of parallel channels with slanted walls. Influence of cell presence, type and growth on the mechanical properties of the films was investigated. Unseeded films showed gradual strength reduction from an initial value of 0.046 N/mm, possibly due to degradation, down to 0.032±0.012 N/mm in 2 weeks. Keratocyte growth was found to significantly improve the mechanical behavior of the films upon 1 week of incubation (0.067±0.017 N/mm) and the improvement continued gradually over the next 2 weeks. Films seeded with D407 retinal pigment epithelial cells, on the other hand, experienced a decrease (0.023±0.011 N/mm), followed by a slight increase in mechanical properties in the 21-day period. A steady increase in the number of keratocytes along the channels, cytoskeleton alignment and extracellular matrix (ECM) secretion restricted to the channels was observed. Increase in strength observed with keratocytes and, to a lesser extent, with the epithelial cells can be attributed to directional ECM synthesis and the orientation of the cells and their cytoskeleton which contribute to the strength in the direction of the channels. This study showed that cell, especially keratocyte, presence compensates for the degradation of collagen films and improve the overall mechanical properties of the engineered tissue.

Novel Nanostructured Hydroxyl-Paracyclophane Thin Films

We have recently developed nanostructured polymer thin films (STFs) that are hierarchical and spatially organized. STFs were prepared from hydroxyl-paracyclophane precursors for depositing ordered nanoporous materials. STFs offer the possibility of fabricating polymer films exhibiting tunable porosity by systematically varying and controlling both the surface chemistry and topology. The strength of our technology is the ability to control surface topology, chemistry and film morphology at the same time. Functionalized STF materials provide many technological advantages such as the controlled delivery of drugs, encapsulation of enzymes, and tissue engineering substrates of specific cells.

Surface modification of PLGA/β-TCP scaffold for bone tissue engineering: Hybridization with collagen and apatite

The objective of this experiment is to produce a novel scaffold composed of poly (lactic-co-glycolic acid) (PLGA)/β-tricalciumphosphate (β-TCP) skeleton covered with apatite/collagen sponge. BMSCs were used to evaluate the cell behavior on the hybrid scaffold. Cell morphology on the surfaces of PLGA/β-TCP skeleton and PLGA/β-TCP skeleton with apatite/collagen sponge composite coating was studied by scanning electron microscopy (SEM). Cell proliferation was assessed by cell number counting. Differentiated BMSCs function was monitored by measuring alkaline phosphates activity of the cells. These results demonstrated that the apatite/collagen sponge composite coating could improve cell proliferation and ALP activity of the hybrid scaffolds and increase its hydrophilicity after hybridization. The experiment suggested that the PLGA/β-TCP skeleton with apatite/collagen sponge composite coating is promising as a candidate 3D substrate for bone tissue engineering.

High aspect ratio PDMS replication through proton beam fabricated Ni masters

In application areas where multiple samples are required (for example tissue engineering substrates), proton beam writing (PBW) is a suitable technique to fabricate high quality metal masters. These masters can then be used to replicate multiple copies in polymers, either through nanoimprinting or softlithography. Since poly(dimethyl siloxane) (PDMS) is a compatible material in tissue engineering we explore PDMS casting on Ni masters as an alternative way to replicate high aspect ratio micro structures. Ni masters with grooves spaced 2.5 μm apart, and 13 μm deep were successfully replicated in PDMS: These PDMS structures, which have aspect ratio of more than 5, are comparable to the best high aspect ratios reported in PDMS replication.

3D culture of bone-derived cells immobilised in alginate following light-triggered gelation

Photoreactive liposomes have been exploited as a means of developing 3D tissue constructs. Liposomes formulated using the photosensitive lipid 1,2-bis(4-( n-butyl)phenylazo-4′-phenylbutyroyl)phosphatidylcholine (Bis Azo PC), which undergoes conformational change on stimulation with long wavelength ultraviolet light, were prepared with entrapped CaCl 2 before being incorporated into a 4% alginate solution. It was shown that stimulation of the photosensitive lipid using a light emitting diode (LED) (peak emission at 385 nm, dose equivalent to 9 mJ/cm 2) caused the release of liposome-entrapped CaCl 2, resulting in cross-linking of the alginate solution and immobilisation of bone-derived cells over a range of seeding densities, approximately 97% of which remained viable for periods of up to 14 days in culture. Entrapment volumes of a variety of liposome types were evaluated and interdigitating fusion vesicles were identified as having the highest payload (24%), however the inclusion of cholesterol as a means of shifting Bis Azo PC sensitivity into the visible light wavelengths resulted in an approximately 10-fold reduction in calcium entrapment. This application of light-sensitised liposomes offers the potential to create complex tissue engineering substrates containing cells immobilised in precise locations, in contrast with substrates onto which cells are seeded post-production.

Electrospinning of keratin/poly(ethylene oxide)blend nanofibers

AbstractResearch on the electrospinning of nanofibers has increased in recent years because of the number of potential applications in different areas, ranging from technical textiles (e.g., filters, composite reinforcements, and protective fabrics) to biomedical commodities and devices such as bandages, membranes, bioactive surfaces, and porous substrates for tissue engineering, for which biocompatible polymers play an essential role. In this work, wool keratin/poly(ethylene oxide) nanofibers were electrospun from aqueous solutions of polymer blends under different operating conditions. The filaments were characterized with scanning electron microscopy, Fourier transform infrared, and differential scanning calorimetry analyses and compared with films of the same materials produced via casting with the aim of investigating structural changes due to the electrospinning process. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 104: 863–870, 2007

Soft biological materials and their impact on cell function.

Most organs and biological tissues are soft viscoelastic materials with elastic moduli ranging from on the order of 100 Pa for the brain to 100 000 Pa for soft cartilage. Biocompatible synthetic materials already have many applications, but combining chemical compatibility with physiologically appropriate mechanical properties will increase their potential for use both as implants and as substrates for tissue engineering. Understanding and controlling mechanical properties, specifically softness, is important for appropriate physiological function in numerous contexts. The mechanical properties of the substrate on which, or within which, cells are placed can have as large an impact as chemical stimuli on cell morphology, differentiation, motility, and commitment to live or die.

Monocytic U937 Adhesion, Tumor Necrosis Factor-Alpha and Interleukin-1 Beta Expression in Response to Gelatin-Based Networks Grafted with Arginine-Glycine-Aspartic Acid and Proline-Histidine-Serine-Arginine-Asparagine Oligopeptides

In this study we synthesized gelatin-based, tissue-engineering, interpenetrating network (IPN) scaffolds immobilized with fibronectin (FN)-derived peptides to assess monocyte-biomaterial interaction. Human promonocytic U937 cells were seeded onto peptide-grafted IPN or tissue-culture polystyrene plate (TCPS) pre-adsorbed with FN or FN-derived peptides. The presence of RGD influenced U937 density on IPN. Interleukin-1 beta (IL-1beta) messenger ribonucleic acid (mRNA) expression in adherent U937 on treated TCPS was slightly upregulated at 4 h. Tumor necrosis factor alpha (TNF-alpha) and IL-1beta mRNA expression in adherent U937 on all IPNs was generally downregulated at 4 h. This downregulation of IL-1beta mRNA apparently varied in IPNs grafted with different ligand and was still present at 24 h. TNF-alpha and IL-1beta proteins released from U937 on treated TCPS were comparable with the control at 24 h, but TNF-alpha and IL-1beta protein expression in U937 on IPNs was lower at 24 h than on the TCPS control. The results indicate that the tissue-engineering substrate and the bioactive peptides modulate the initial U937 adhesion and the subsequent inflammatory cytokine gene and protein expression.

Optimisation of biochemical condition and substrates in vitro for tissue engineering of ligament.

In this work, we analysed the effect of growth factors on in vitro cell proliferation and collagens synthesis by fibroblasts cultured for 72 h on different substrates (silicon sheet with or without 1% gelatin, and glass as control surface) for ligament tissue engineering. A human fibroblast cell line (CRL-2703) was used. The synthesis of type I and type III collagens were evaluated qualitatively and quantitatively by RT-PCR and confocal microscopy, respectively. Cell proliferation was evaluated by two methods: (1) MTT assay (2) cell cycle analysis. It was found that PDGF-AB stimulate the proliferation of fibroblast cultured on gelatin coated silicon sheet in dose dependant manner with a maximum effect at 10 ng ml−1. The exogenous TGF-β1 induced the expression of type I and type III collagens in a dose and substrate-dependant manner. We deduce from this work that biochemical conditions and substrates have an important impact for optimisation of the tissue neo synthesis.

Cartilage-like Tissue Engineering Using Silk Scaffolds and Mesenchymal Stem Cells

Silk fibroin scaffolds were studied as a new biomaterial option for tissue-engineered cartilage-like tissue. Human bone marrow-derived mesenchymal stem cells (MSCs) were seeded on silk, collagen, and crosslinked collagen scaffolds and cultured for 21 days in serum-free chondrogenic medium. Cells proliferated more rapidly on the silk fibroin scaffolds than on the collagen matrices. The total content of glycosaminoglycan deposition was three times higher on silk as compared to collagen scaffolds. Glycosaminoglycan deposition coincided with overexpression of collagen type II and aggrecan genes. Cartilage-like tissue was homogeneously distributed throughout the entire silk scaffolds, while on the collagen and crosslinked collagen systems tissue formation was restricted to the outer rim, leaving a doughnut appearance. Round or angular-shaped cells resided in deep lacunae in the silk systems and stained positively for collagen type II. The aggregate modulus of the tissue-engineered cartilage constructs was more than 2-fold higher than that of the unseeded silk scaffold controls. These results suggest that silk fibroin scaffolds are suitable biomaterial substrates for autologous cartilage tissue engineering in serum-free medium and enable mechanical improvements along with compositional features suitable for durable implants to generate or regenerate cartilage.

Mesoscale Pincushions, Microrings, and Microdots Prepared by Heating and Peeling of Self-Organized Honeycomb-Patterned Films Deposited on a Solid Substrate

We describe here a preparation of pincushion structures with holes, hexagonally arranged microrings, and microdots by simple heating and peeling of self-organized honeycomb-patterned films. We have reported that the honeycomb-patterned films can be prepared by casting the solution of an amphiphilic polymer and a hydrophobic polymer under humid conditions. When annealing the honeycomb-patterned films prepared from an amphiphilic copolymer and poly(bisphenol A carbonate), we obtained a variety of mesoscale structures, depending on the heating temperatures. We revealed that these microstructures were formed by using the phase-separation structures in the self-organized honeycomb-patterned films. These micropatterns can be utilized for the template for microelectrodes, superhydrophobic surfaces, photonic crystals, and as a substrate for tissue engineering.

In Vitro Evaluation of Poly[Bis(Ethyl Alanato)Phosphazene] as a Scaffold for Bone Tissue Engineering

Polyphosphazenes with amino acid ester as side groups are biocompatible polymers that could provide valid scaffolds for cell growth. In the present study we investigate the adhesion and growth of osteoblasts obtained from rat bone marrow on matrices composed of thin fibers of poly[bis(ethyl alanato)phosphazene] (PAlaP), poly(d,l-lactic acid) (PDLLA), or PAlaP/PDLLA blend. Our data show that scaffolds of PAlaP or PAlaP/PDLLA blend enhanced the cell adhesion and growth in comparison with that observed in cultures seeded on polystyrene tissue culture plates. Although collagenase-digestible protein synthesis remained unchanged, all scaffolds induced a decrease in alkaline phosphatase activity, suggesting that osteoblasts are in the proliferation phase. Both PAlaP and PAlaP blended with PDLLA may represent a new and interesting substrate for bone tissue engineering.

The thermal analysis of films in the 21st century: Relevance to cell culture, biochips and roll-to-roll circuits

Films may be considered as wide fibers, or as unique material geometry possessing two dimensional symmetry (X, Y≫Z). Potentially different uniformity issues along the machine (long) and transverse directions of the film, characterization of Z-axis performance—especially as films become “thin” and the characterization/identification of surface modification, introduce the need for careful sampling strategies if the resulting thermal analysis data is to be reflective of either process history or end-use performance or both. Two dimensional imaging by a combination of techniques—i.e., DSC–WAXS–FTIR–AFM exploit the convenience of the sample geometry, while aiding in the definition of structural complexity. Molecular spectroscopy techniques (DMA, TSC) provide a sensitive and instructive tool for examining novel surface or interface chemistry. Characterization under biorelevant conditions (37°C, aqueous, standard saline, presence of adhesive and other proteins, presence of cells) is critical for the generation of meaningful data on films to be utilized biologically (cell culture, tissue engineering substrates, biochips). Of special interest are strategies for accelerated aging that allow prediction of biological or biorelevant performance.

PLLA scaffolds with biomimetic apatite coating and biomimetic apatite/collagen composite coating to enhance osteoblast-like cells attachment and activity

Novel scaffolds composed of poly( l-lactic acid) (PLLA) skeleton covered with bonelike apatite or apatite/collagen composite were produced via a combined phase-separation technique and an accelerated biomimetic coating process. Saos-2 osteoblast-like cells were used to evaluate the cellular behaviors on these biomimetic coatings. Cell morphologies on the surfaces of PLLA scaffolds, PLLA scaffolds with apatite coating, and PLLA scaffolds with apatite/collagen composite coating were studied by scanning electron microscopy (SEM). Cell viability was assessed by the MTT assay. Differentiated osteoblastic function was monitored by measuring alkaline phosphatase activity. These results suggested that the apatite coating and apatite/collagen composite coating generated through the accelerated biomimetic processes could improve the interactions between osteoblasts and the polymeric scaffolds. The apatite/collagen composite coating was more effective than apatite coating in improving such interactions. The PLLA scaffold with apatite/collagen composite coating is promising as a candidate 3D substrate for bone tissue engineering.

Long term degradation of poly( ɛ-caprolactone) films in biologically related fluids

The aim of this work was to study the long term degradation behaviour of poly( ɛ-caprolactone) (PCL) films, potentially useful as substrates for tissue engineering, obtained by two different methods (compression moulding or casting in chloroform) in two biologically related media: phosphate buffered solution (PBS) and Dulbecco's modified Eagle's medium (DMEM). The films were characterized at different degradation times by differential scanning calorimetry (DSC), Scanning Electron Microscopy (SEM) and Fourier Transform Infrared Spectroscopy (FTIR). The molecular weight was determined by Gel Permeation Chromatography (GPC). Chemiluminescence (CL) was used to assess physical or chemical changes from the early stage of the degradation. A different behaviour is observed in samples immersed in PBS when compared with those treated in DMEM. In this medium, the degradation after one year and a half (18 months) shows that although the chemical structure has been modified, the layers become more fragile but maintain their consistency. A higher degradation rate is obtained for membranes obtained by casting with respect to those obtained by compression moulding.

A novel star PEG–derived surface coating for specific cell adhesion

In this study a novel approach for the coating and functionalization of substrates for cell culture and tissue engineering is presented. Glass, silicon, and titanium panes were coated with an ultrathin film (30 +/- 5 nm) of reactive star-shaped poly(ethylene glycol) prepolymers (Star PEG). Homogeneity of the films was checked by optical microscopy and scanning force microscopy. These coatings prevent unspecific protein adsorption as monitored by fluorescence microscopy and ellipsometry. In order to allow specific cell adhesion the films were modified with linear RGD peptides (gRGDsc) in different concentrations. After sterilization, fibroblast, SaOS, and human mesenchymal stem cells (hMSC) were seeded on these substrates. Cell adhesion, spreading, and survival was observed for up to 30 days on linear RGD peptide (gRGDsc)-modified coatings, whereas no cell adhesion could be detected on unmodified Star PEG layers. By variation of the RGD concentration within the film the amount of cells that became adhesive could be controlled. When differentiation conditions are used for cultivation of hMSCs the cells show the expression of osteogenic marker genes after 14 days which is comparable to cultivation on cell culture plastic. Thus, the Star PEG/RGD film did not negatively influence the differentiation process. The high flexibility of the system considering the incorporation of biologically active compounds opens a broad field of future experiments.

Stromal architecture and immune tolerance in additive corneal xenografts in rodents

To examine immune tolerance and corneal ultrastructure following additive corneal xenografts in rodents. We carried out surgical implantation of excised BALB/c mouse corneal tissue, either freshly isolated (n=6) or after storage at--20 degrees C for 1 week (n=7), into the corneas of Wistar rats at approximately mid-stromal depth. Corneal opacity and neovascularization were evaluated postoperatively, and stromal ultrastructure was observed by transmission electron microscopy. Corneal opacification and neovascularization in the weeks after surgery were less prevalent in grafts of frozen-then-thawed tissue than in grafts of fresh tissue. In a well tolerated frozen-then-thawed xenograft, the matrix architecture was normal throughout most of the recipient and donor tissue, but pronounced fibrillar disorganization was evident adjacent to Descemet's membrane. We attribute the improved tolerance of frozen-then-thawed xenografts over fresh xenografts to a reduced cellular immune response.

Evaluation of in Vitro Drug Release, pH Change, and Molecular Weight Degradation of Poly(L-lactic acid) and Poly(D,L-lactide-co-glycolide) Fibers

Biodegradable fibers of poly(L-lactic acid) (PLLA) and poly(D,L-lactide-co-glycolide) (PLGA) that encapsulated a water-soluble drug were created by a patented technique consisting of wet-spinning a water-in-oil emulsion. These fibers are 2.4% by mass drug, which is slowly released, making these fibers potential candidates for implantation as drug delivery devices and/or tissue-engineering substrates. Drug release kinetics and changes in molecular weight were investigated over time. This study demonstrated that drug release rates and molecular weight degradation are a function of the amount of aqueous phase added as an emulsion during fabrication. The type of polymer used (PLLA or PLGA) determines the molecular weight degradation rates, but has little effect on drug release kinetics.