Biomaterials (v.26, #13)

Calendar (I).

Tensile testing of a single ultrafine polymeric fiber by E.P.S. Tan; S.Y. Ng; C.T. Lim (1453-1456).
Due to the difficulty in handling micro and nanoscale fibers and measuring the small load required for deformation, mechanical properties of these fibers have not been widely characterized. In this study, tensile test of a single-strand polycaprolactone electrospun ultrafine fiber was performed using a nano tensile tester. The tested fiber exhibited the characteristic low strength and low modulus but high extensibility at room temperature. The mechanical properties were also found to be dependent on fiber diameter. Fibers with smaller diameter had higher strength but lower ductility due to the higher ‘draw ratio’ that was applied during the electrospinning process.
Keywords: Electrospinning; Nano and microscale fiber; Polymer; PCL; Tensile test; Mechanical properties;

In an effort to improve the long-term patency of vascular prostheses several groups now advocate seeding autologous endothelial cells (ECs) onto the lumen of the vessel prior to implantation, a procedure that involves pre-treating the prosthesis material with fibrin, collagen and/or other matrix molecules to promote cell attachment and retention. In this study, we examined the degree to which human umbilical venous endothelial cells (HUVECs) adhered to three materials commonly used polymeric vascular prosthesis that had been coated with the same commercial extra cellular matrix proteins, and after exposure to fluid shear stresses representative of femoro-distal bypass in a cone-and-plate shearing device. We quantified cell number, area of coverage and degree of cell spreading using image analysis techniques.The response of cells that adhered to the surface of each material, and following exposure to fluid shear stress, depended on surface treatment, topology and cell type. Whereas collagen coating improved primary cellular adhesion and coverage significantly, the degree of spreading depended on the underlying surface structure and on the application of the shear stress. In some cases, fewer than 30% of cells remained on the surface after only 1-h exposure to physiological levels of shear stress. The proportion of the surface that was covered by cells also decreased, despite an increase in the degree to which individual cells spread on exposure to shear stress. Moreover, the behaviour of HUVECs was distinct from that of fibroblasts, in that the human ECs were able to adapt to their environment by spreading to a much greater extent in response to shear. The quality of HUVEC attachment, as measured by extent of cell coverage and resistance to fluid shear stress, was greatest on expanded polytetrafluoroethylene samples that had been impregnated with Type I/III collagen.
Keywords: Cell adhesion; Cell spreading; Cell culture; Endothelialisation; Tissue engineering;

The successful development of biomaterials must take into consideration how those surfaces will interact with in vivo processes such as adsorption of endogenous proteins. In this study, we examined whether modifying highly adsorbent materials like hydroxyapatite (HA) with RGD peptides would improve mesenchymal stem cell (MSC) adhesion. We found that RGD, alone, was not sufficient to promote full cell spreading. However, given that RGD-modified HA will likely adsorb osteogenic serum proteins in vivo, we evaluated MSC behavior on HA pre-coated with RGD, then over-coated with serum (RGD/FBS). Interestingly, RGD/FBS coatings additively stimulated MSC attachment and spreading compared to either coating alone, but only at low RGD coating concentrations. High RGD concentrations inhibited cell attachment, and completely eliminated cell spreading on RGD/FBS surfaces. To better understand the mechanism by which RGD and adsorbed serum proteins interactively regulate cell behavior, we monitored the deposition of fibronectin (FN) from serum onto HA pre-coated with increasing RGD concentrations. These studies showed that high RGD concentrations did not inhibit FN adsorption, therefore cell spreading is attenuated by mechanisms other than lack of FN availability. Collectively, our results suggest a potential therapeutic benefit for functionalizing HA with RGD, however such a benefit will likely depend upon the RGD density.
Keywords: Cell adhesion; Hydroxyapatite; Mesenchymal stem cell; Protein adsorption; RGD peptide; Fibronectin; Integrin; Bone;

Binding of C3 fragments on top of adsorbed plasma proteins during complement activation on a model biomaterial surface by Jonas Andersson; Kristina Nilsson Ekdahl; John D. Lambris; Bo Nilsson (1477-1485).
In the present study we investigate whether complement activation in blood in contact with a model biomaterial surface (polystyrene) occurs directly on the material surface or on top of an adsorbed plasma protein layer. Quartz crystal microbalance-dissipation analysis (QCM-D) complemented with enzyme immunoassays and Western blotting were used. QCM-D showed that the surface was immediately covered with a plasma protein film of approximately 8 nm. Complement activation that started concomitantly with the adsorption of the protein film was triggered by a self-limiting classical pathway activation. After adsorption of the protein film, alternative pathway activation provided the bulk of the C3b deposition that added 25% more mass to the surface. The build up of alternative pathway convertase complexes using purified C3 and factors B and D on different protein films as monitored by QCM-D showed that only adsorbed albumin, IgG, but not fibrinogen, allowed C3b binding, convertase assembly and amplification. Western blotting of eluted proteins from the material surface demonstrated that the C3 fragments were covalently bound to other proteins. This is consistent with a model in which the activation is triggered by initiating convertases formed by means of the initially adsorbed proteins and the main C3b binding is mediated by the alternative pathway on top of the adsorbed protein film.
Keywords: Complement; Complement activation; Biomaterials; Proteins adsorption; C3b binding;

A technique for preparing protein gradients on polymeric surfaces: by Bin Li; Yuexia Ma; Shu Wang; Peter M. Moran (1487-1495).
A technique for preparing micropatterns and gradients of proteins on polymeric substrates has been developed in this work. Peroxides were generated on the substrate surface by UV preirradiation and they initiated graft polymerization of acrylic acid (AA) onto the surface upon a second UV irradiation. Micropatterns and gradients of poly(acrylic acid) (PAA) were formed when the substrate was placed under or moved with respect to a photomask during UV preirradiation. Protein micropatterns and gradients were fabricated on the surface by covalently linking to the carboxyl groups on PAA chains. To test cell response to the protein gradient surfaces, PC12 pheochromocytoma cells were cultured on laminin-bound substrates in serum-free medium supplemented with nerve growth factor (NGF). It is found that both the attachment and neurite outgrowth behaviors of PC12 cells were dependent on the surface laminin density. However, the unreacted carboxyl groups on the polymer surface negatively affected PC12 cells. This weakened the positive influence from laminin.
Keywords: Acrylic acid; Graft polymerization; Micropatterning; Protein gradient; Laminin; PC12 pheochromocytoma cells;

Craniofacial muscle engineering using a 3-dimensional phosphate glass fibre construct by R. Shah; A.C.M. Sinanan; J.C. Knowles; N.P. Hunt; M.P. Lewis (1497-1505).
The current technique to replace missing craniofacial skeletal muscle is the surgical transfer of local or free flaps. This is associated with donor site morbidity, possible tissue rejection and limited supply. The alternative is to engineer autologous skeletal muscle in vitro, which can then be re-implanted into the patient. A variety of biomaterials have been used to engineer skeletal muscle with limited success. This study investigated the use of phosphate-based glass fibres as a potential scaffold material for the in vitro engineering of craniofacial skeletal muscle.Human masseter (one of the muscles of mastication)—derived cell cultures were used to seed the glass fibres, which were arranged into various configurations. Growth factors and matrix components were to used to manipulate the in vitro environment. Outcome was determined with the aid of microscopy, time-lapse footage, immunofluorescence imaging and CyQUANT proliferation, creatine kinase and protein assays. A 3-dimensional mesh arrangement of the glass fibres was the best at encouraging cell attachment and proliferation. In addition, increasing the density of the seeded cells and using Matrigel and insulin-like growth factor I enhanced the formation of prototypic muscle fibres. In conclusion, phosphate-based glass fibres can support the in vitro engineering of human craniofacial muscle.
Keywords: Craniofacial muscle; Phosphate glasses; Skeletal muscle; Tissue engineering;

A hydrogel scaffold of well-defined geometry was created and modified with laminin-derived peptides in an aqueous solution, thereby maintaining the geometry of the scaffold while introducing bioactive peptides that enhance cell adhesion and neurite outgrowth. By combining a fiber templating technique to create longitudinal channels with peptide modification, we were able to synthesize a scaffold that guided cell adhesion and neurite outgrowth of primary neurons. Scaffolds were designed to have numerous longitudinally oriented channels with an average channel diameter of 196±6 μm to ultimately promote fasciculation of regenerating cables and a compressive modulus of 192±8 kPa to match the modulus of the soft nerve tissue. Copolymerization of 2-hydroxylethyl methacrylate (HEMA) with 2-aminoethyl methacrylate (AEMA) scaffolds, provided primary amine groups to which two sulfhydryl terminated, laminin-derived oligopeptides, CDPGYIGSR and CQAASIKVAV, were covalently bound using the sulfo-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) crosslinking agent. The concentration of peptides on the scaffolds was measured at 106±4 μmol/cm2 using the ninhydrin method and 92±9 μmol/cm2 using the BCA protein assay. The peptide modified P(HEMA-co-AEMA) scaffolds were easily fabricated in aqueous conditions, highly reproducible, well-defined, and enhanced neural cell adhesion and guided neurite outgrowth of primary chick dorsal root ganglia neurons relative to non-peptide-modified controls. The copolymerization of AEMA with HEMA can be extended to other radically polymerized monomers and is advantageous as it facilitates scaffold modification in aqueous solutions thereby obviating the use of organic solvents which can be cytotoxic and often disrupt scaffold geometry. The combination of well-defined chemical and physical stimuli described herein provides a means for guided regeneration both in vitro and in vivo.
Keywords: Scaffold; PolyHEMA; Surface modification; Peptide; Cell adhesion; Nerve guidance; Tissue engineering;

Microencapsulation of living cells in semi-permeable membranes with covalently cross-linked layers by Julie Dusseault; Francois A. Leblond; Robert Robitaille; Guillaume Jourdan; Josée Tessier; Martin Ménard; Nathalie Henley; Jean-Pierre Hallé (1515-1522).
Microencapsulation in semi-permeable membranes protects transplanted cells against immune destruction. Microcapsule strength is critical. We describe a method to microencapsulate living cells in alginate-poly-l-lysine (PLL)-alginate membranes with covalent links between adjacent layers of microcapsule membranes, while preserving the desired membrane molecular weight cut-off (MWCO) and microencapsulated cell viability. A heterobifunctional photoactivatable cross-linker, N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOS) was used. The N-hydroxysuccinimide ester group of ANB-NOS was covalently linked to PLL. Islets of Langerhans were immobilized in alginate beads, incubated in PLL-ANB-NOS and again in alginate. Upon illumination with UVA, covalent links were created between the phenyl azide residue of ANB-NOS and alginate from both the core bead and the outer coating. Covalently linked microcapsules remained intact after 3 years in a strong alkaline buffer (pH 12), whereas standard microcapsules disappeared within 45 s in the same solution. A standardized mechanical stress broke 22-fold more standard than covalently linked microcapsules. The MWCO and microencapsulated cell viability were similar with standard and covalently linked microcapsules. These microcapsules, extremely resistant to chemical and mechanical stresses, will be useful in numerous applications.
Keywords: Islet; Microencapsulation; Alginate; Mechanical properties; Transplantation; Diabetes;

Fiber-based tissue-engineered scaffold for ligament replacement: design considerations and in vitro evaluation by James A. Cooper; Helen H. Lu; Frank K. Ko; Joseph W. Freeman; Cato T. Laurencin (1523-1532).
The anterior cruciate ligament (ACL) is the major intraarticular ligamentous structure of the knee, which functions as a joint stabilizer. It is the most commonly injured ligament of the knee, with over 150,000 ACL surgeries performed annually in the United States. Due to limitations associated with current grafts for ACL reconstruction, there is a significant demand for alternative graft systems. We report here the development of a biodegradable, tissue-engineered ACL graft. Several design parameters including construct architecture, porosity, degradability, and cell source were examined. This graft system is based on polymeric fibers of polylactide-co-glycolide 10:90, and it was fabricated using a novel, three-dimensional braiding technology. The resultant micro-porous scaffold exhibited optimal pore diameters (175–233 μm) for ligament tissue ingrowth, and initial mechanical properties of the construct approximate those of the native ligament.
Keywords: Porosity; Degradable; Polymer; Anterior cruciate ligament; Ligament repair; Tissue engineering; Ligament and ligament tissue engineering;

Fibroblast populated dense collagen matrices: cell migration, cell density and metalloproteinases expression by Christophe Helary; Alexandrine Foucault-Bertaud; Gaston Godeau; Bernard Coulomb; Marie Madeleine Giraud Guille (1533-1543).
Dense collagen matrices obtained by using the property of type I collagen to form liquid crystals at high concentrations, were shown to be colonized by human dermal fibroblasts (Biomaterials 23 (2002) 27). In order to evaluate them as possible tissue substitutes, we investigated in this study the mechanism of cell colonization.Fibroblasts were seeded at the surface of collagen matrices at concentrations of 5 and 40 mg/ml. Cell density and migration were estimated from histological sections over 28 days within 500 μm thick matrices. At day 14, migration started in the 40 mg/ml matrices, attaining 320 μm in distance and 5500 cell/mm3 in density at day 28. As zymography and western blot techniques demonstrated production of collagenase 1 (MMP1) and gelatinase A (MMP2) in culture medium, collagen hydrolysis was required for cells to penetrate the collagen network.Furthermore, the presence of MMP1 and MMP2 and their tissue inhibitors TIMP1 and TIMP2 was revealed by immunohistochemistry.We presently show that 40 mg/ml collagen matrices are colonized by human dermal fibroblasts and reach, at day 28, a density close to that measured in human dermis.
Keywords: Collagen; 3D Matrix; Human dermal fibroblasts; Matrix metalloproteinases;

The effect of the topical administration of bioactive glass on inflammatory markers of human experimental gingivitis by Jörg Eberhard; Nikolaus Reimers; Hendrik Dommisch; Johanna Hacker; Sandra Freitag; Yahya Acil; Hans-Karl Albers; Sören Jepsen (1545-1551).
Recent studies demonstrated that bioactive glass attenuated inflammatory reactions and bacterial growth in vitro. The aim of the present clinical study was to evaluate the effects of local bioactive glass-administration in vivo in subjects with experimental gingivitis. In each individual, contralateral teeth served as test and control over a 21-day non-hygiene (preventive phase) and a 7-day therapeutic phase. A 45S5 bioactive glass (10% solution) was applied daily (2×1 h) on the test teeth during the preventive and therapeutic phase of the study. Inflammation assessment was based on the plaque index record (PI), on the bleeding frequency (BOP) and the gingival crevicular fluid volume (GCF). Interleukin-1β levels (IL-1β) in the GCF were measured by ELISA. Bacterial deposits on teeth increased during the 21-days non-brushing period, with no difference in plaque accumulation between test and control sides. BOP levels were significantly reduced during the therapeutic phase by the application of bioactive glass. GCF-values showed a significant reduction on the test compared to control side during the preventive and therapeutic phase. IL-1β counts decreased during the last 7 days of the study on the test side, but no significance was given. The topical application of 45S5 bioactive glass in humans with experimental gingivitis attenuated the clinical signs of inflammation, although the bacterial accumulation was not inhibited in this clinical trial.
Keywords: Bioactive glass; Experimental gingivitis; Inflammatory markers; Clinical study; Bacterial plaques;

Injectability of calcium phosphate pastes by Marc Bohner; Gamal Baroud (1553-1563).
A theoretical model was developed to assess ways to improve the injectability of calcium phosphate pastes. The theoretical results were then compared to experimental data obtained on calcium phosphate slips. The theoretical approach predicted that the injectability of a cement paste could be improved by an increase of the liquid-to-powder ratio, and a decrease of the particle size and the plastic limit (PL) of the powder. The theoretical results were confirmed by experimental data. Interestingly, an increase of the viscosity of the mixing liquid with small additions of xanthan had a positive effect on the paste injectability. This effect could be due to a change of the PL of the powder or to the lubricating effect of the polymer.
Keywords: Model; Injectability; Calcium phosphate; Cement; Plastic limit;

The aim of this study was to modify the surfaces of superparamagnetic iron oxide nanoparticles (SPION) with pullulan in order to reduce the cytotoxicity and enhance the cellular uptake of the nanoparticles. In this study, we have prepared and characterised the pullulan coated superparamagnetic iron oxide nanoparticles (Pn-SPION) of size around 40–45 nm with magnetite inner core and hydrophilic outer shell of pullulan. We have investigated the effect of cellular uptake of uncoated and Pn-SPION on cell adhesion/viability, cytotoxicity, morphology and cytoskeleton organisation of human fibroblasts. Cell cytotoxicity/adhesion studies of SPIONs on human dermal fibroblasts showed that the particles are toxic and their internalisation resulted in disruption of cytoskeleton organisation of cells. On the other hand, Pn-SPIONs were found to be non-toxic and induced changes in cytoskeleton organisation different from that observed with SPION. Transmission electron microscopy results indicated that the SPION and Pn-SPION were internalised into cells via different mechanisms, thereby suggesting that the particle endocytosis behaviour is dependent on the surface characteristics of the nanoparticles.
Keywords: Drug delivery; Magnetic nanoparticle; Surface modification; Cell adhesion; TEM; Cytotoxicity;

DNA delivery from hyaluronic acid-collagen hydrogels via a substrate-mediated approach by Tatiana Segura; Peter H. Chung; Lonnie D. Shea (1575-1584).
Efficient and controlled gene delivery from biodegradable materials can be employed to stimulate cellular processes that lead to tissue regeneration. In this report, a substrate-mediated approach was developed to deliver DNA from hyaluronic acid-collagen hydrogels. The hydrogels were formed by crosslinking HA with poly(ethylene glycol) diglycidyl ether. Poly(ethylene imine)(PEI)/DNA complexes were immobilized to the substrate using either biotin/neutravidin or non-specific adsorption. Complexes were formed in the presence or absence of salt to regulate complex size, and resulted in complexes with z-average diameters of 1221.7±152.3 and 139.4±1.3 nm, respectively. During 48-h incubation in PBS or hyaluronidase, DNA was released slowly from the hydrogel substrate (<30% of immobilized DNA), which was enhanced by incubation with conditioned media (≈50% of immobilized DNA). Transgene expression mediated by immobilized, large diameter complexes was 3 to 7-fold greater than for small diameter complexes. However, the percentage of cells expressing the transgene was greater for small diameter complexes (48.7%) than for large diameter complexes (22.3%). Spatially controlled gene transfer was achieved by topographically patterning the hydrogel to pattern cell adhesion. Biomaterial-based gene delivery can be applicable to numerous tissue engineering applications, or as a tool to examine tissue formation.
Keywords: Gene delivery; Polyethyleneimine; Neutravidin; Biotin; Reverse transfection; Solid plase;

Delivery of a vector encoding mouse hyaluronan synthase 2 via a crosslinked hyaluronan film by Angela P. Kim; Paige Yellen; Yang H. Yun; Evren Azeloglu; Weiliam Chen (1585-1593).
We have developed a crosslinked hyaluronic acid (HA) film with DNA incorporated within its structure and have characterized this system for its efficacy in sustained transferring of a vector encoding mouse hyaluronan synthase 2 (Has2). Analysis of the DNA release kinetics indicated that the HA films degraded when treated with hyaluronidase and that they released DNA over a prolonged period of time. Gel electrophoresis revealed that this DNA was intact and immunohistochemical analysis verified the transfection capabilities of DNA release samples. The ability of released DNA encoding Has2 to promote HA synthesis was confirmed by quantifying the amount of HA produced by COS-1 cells that were transfected with release samples. The intended future application of the HA films is in prevention of post-operative peritoneal adhesions. In addition to serving as a physical barrier, the film would function as a vehicle for sustained delivery of DNA encoding Has2, which would promote the synthesis of HA in transfected tissues.
Keywords: Hyaluronic acid; HA synthase; Post-operative adhesions; Gene delivery;

To study how crystal growth in dental enamel is controlled by the components of the extracellular matrix, we investigated the functional roles of amelogenins and fluoride ions in apatite formation occurring through an octacalcium phosphate (OCP)-precursor pathway. Using a cation selective membrane system as a model of tooth enamel formation, we evaluated the resulting mineral habit grown in native porcine amelogenins and fluoride ions. In the absence of amelogenin and in the presence of 1 or 2 ppm F, we obtained OCP+apatite and apatite, respectively. Without amelogenins, the crystals were hexagonal prisms and cones with diameters of ∼100–200 nm. In the presence of 10% amelogenins and in the absence of fluoride, rod-like OCP with a diameter of 35 nm were obtained. Remarkably, a combination of amelogenin and fluoride created the formation of rod-like apatite crystals with dimensions similar to the former crystals. These observations indicate a cooperative role of amelogenin and fluoride in the regulation of habit, size orientation and phase of the calcium-phosphate crystals, resulting in the formation of fine rod-like apatite whose habit and orientation were similar to that of authentic tooth enamel crystals. The significant modulating effect of the amelogenin matrix combined with fluoride ions suggests the potential for this artificial system to contribute to the engineering of novel enamel-like biomaterials in vitro.
Keywords: Tooth enamel; Apatite; Amelogenins; Fluoride; Biomimetic material;