Biomaterials (v.29, #27)

Porous titanium scaffolds fabricated using a rapid prototyping and powder metallurgy technique by Garrett E. Ryan; Abhay S. Pandit; Dimitrios P. Apatsidis (3625-3635).
One of the main issues in orthopaedic implant design is the fabrication of scaffolds that closely mimic the biomechanical properties of the surrounding bone. This research reports on a multi-stage rapid prototyping technique that was successfully developed to produce porous titanium scaffolds with fully interconnected pore networks and reproducible porosity and pore size. The scaffolds' porous characteristics were governed by a sacrificial wax template, fabricated using a commercial 3D-printer. Powder metallurgy processes were employed to generate the titanium scaffolds by filling around the wax template with titanium slurry. In the attempt to optimise the powder metallurgy technique, variations in slurry concentration, compaction pressure and sintering temperature were investigated. By altering the wax design template, pore sizes ranging from 200 to 400 μm were achieved. Scaffolds with porosities of 66.8 ± 3.6% revealed compression strengths of 104.4 ± 22.5 MPa in the axial direction and 23.5 ± 9.6 MPa in the transverse direction demonstrating their anisotropic nature. Scaffold topography was characterised using scanning electron microscopy and microcomputed tomography. Three-dimensional reconstruction enabled the main architectural parameters such as pore size, interconnecting porosity, level of anisotropy and level of structural disorder to be determined. The titanium scaffolds were compared to their intended designs, as governed by their sacrificial wax templates. Although discrepancies in architectural parameters existed between the intended and the actual scaffolds, overall the results indicate that the porous titanium scaffolds have the properties to be potentially employed in orthopaedic applications.
Keywords: Titanium; Scaffold; Rapid prototyping; Three-dimensional printing; Microstructure;

Fracture toughness, strength and slow crack growth in a ceria stabilized zirconia–alumina nanocomposite for medical applications by Rajaa Benzaid; Jerome Chevalier; Malika Saâdaoui; Gilbert Fantozzi; Masahiro Nawa; Luis Antonio Diaz; Ramon Torrecillas (3636-3641).
Mechanical properties and slow crack growth (SCG) behavior of a 10Ce-TZP/Al2O3 nanocomposite currently developed as a biomaterial are considered. Fracture toughness is determined for sharp, long (double torsion) and short (indentation) cracks and a good agreement is found between the two types of cracks. The main toughening mechanism in the nanocomposite is the tetragonal to monoclinic phase transformation of the ceria-stabilized zirconia (Ce-TZP) phase. Transformation at the surface of ground specimens leads to surface compressive induced stresses and an increase in strength. Crack velocity curves (VK I curves) are obtained under static and cyclic fatigue using the double torsion method. The static VK I curve in air reveals the three stages characteristic of stress corrosion with a threshold K I0  ∼ 4.5 MPa m1/2 and a fracture toughness of 8.8 MPa m1/2 significantly higher than those of currently used inert bioceramics (i.e. alumina and Y-TZP). A crack growth accelerating effect is shown under cyclic loading, correlated with a decrease in the threshold. However, the cyclic fatigue threshold (4 MPa m1/2) still stands above that of current biomedical grade alumina and zirconia.
Keywords: Mechanical properties; Crack; Alumina; Zirconia; Nanocomposite; Fatigue;

Synthesis, characterization and in vitro biological properties of O-methyl free N,N,N-trimethylated chitosan by Rolf J. Verheul; Maryam Amidi; Steffen van der Wal; Elly van Riet; Wim Jiskoot; Wim E. Hennink (3642-3649).
N,N,N-Trimethylated chitosan (TMC) with varying degree of quaternization (DQ) is currently being investigated in mucosal drug, vaccine and in gene delivery. However, besides N-methylation, O-methylation and chain scission occur during the synthesis of this polymer. Since both side reactions may affect the polymer characteristics, there is a need for TMCs without O-methylation and disparities in chain lengths while varying the DQ. In this study, O-methyl free TMC with varying DQs was successfully synthesized by using a two-step method. First, chitosan was quantitatively dimethylated using formic acid and formaldehyde. Then, in the presence of an excess amount of iodomethane, TMC was obtained with different DQs by varying reaction time. TMC obtained by this two-step method showed no detectable O-methylation (1H NMR) and a slight increase in molecular weight with increasing DQ (GPC), implying that no chain scission occurred during synthesis. The solubility in aqueous solutions at pH 7 of O-methyl free TMC with DQ < 24% was less as compared to O-methylated TMC with the same DQ. On the other hand, O-methyl free TMC with DQ > 33% had a good aqueous solubility. On Caco-2 cells, O-methyl free TMCs demonstrated a larger decrease in transepithelial electrical resistance (TEER) than O-methylated TMCs. Also, with increasing DQ, an increase in cytotoxicity (MTT) and membrane permeability (LDH) was observed.
Keywords: Trimethylated chitosan; Chitosan; In vitro cytotoxicity; MTT assay; LDH assay; TEER;

The effect of extensible PEG tethers on shielding between grafted thermo-responsive polymer chains and integrin–RGD binding by Mitsuhiro Ebara; Masayuki Yamato; Takao Aoyagi; Akihiko Kikuchi; Kiyotaka Sakai; Teruo Okano (3650-3655).
The affinity control of integrin–RGD (Arg-Gly-Asp) binding by a thermal “on–off” switch has been achieved using newly designed surfaces presenting grafted temperature-responsive poly(N-isopropylacrylamide-co-2-carboxyisopropylacrylamide) copolymers functionalized with synthetic peptides. The prepared surface was designed to expose the tethered peptides available for cell binding at active “on” state above the lower critical solution temperature (LCST). The fully extended chains, on the other hand, masked the peptides completely and the cells started to detach from the surfaces at inactive “off” sate below the LCST. This paper elucidates the shielding effect of the grafted polymer chains on the dissociation of integrin–RGD binding below the LCST. To assess the ability of the polymer-shielding, extensible poly(ethylene glycol) (PEG) tethers were introduced between peptides and the grafted polymers. PEG chains allow peptides to be tethered to surfaces via functional PEG end-groups, leading to active “on” state even below the LCST. The time required to release cells from the surface was found to be longer when peptides were coupled to an extensible tether ends, suggesting that the surfaces can engender cell attachment through adhesive moieties covalently bound to the free ends of PEG chains. These results indicate that architectural changes on the nanometer length scale are crucial for controlling integrin–RGD binding and one of the main factors causing cell detachment is the shielding effect of the grafted polymer chains.
Keywords: Poly(N-isopropylacrylamide); 2-Carboxyisopropylacrylamide; Temperature-responsive surface; RGD peptides; Affinity control;

Selective endothelial cell attachment to peptide-modified terpolymers by Anka N. Veleva; Daniel E. Heath; Stuart L. Cooper; Cam Patterson (3656-3661).
In a previous report we screened a combinatorial peptide library to identify novel ligands that bind with high affinity and specificity to human blood outgrowth endothelial cells (HBOEC). In this study we demonstrate the use of the phage display-selected-HBOEC-specific peptides as a tool to direct and modulate endothelial cell (EC) behavior with a focus on designing functional biomaterials intended for use in cardiovascular applications. First, we ensured that our peptide ligands did not interfere with EC function as tested by proliferation, migration, tube formation, and response to vascular endothelial growth factor. Second, peptides that supported EC function were incorporated into methacrylic terpolymers via chain transfer free radical polymerization. The HBOEC-specific peptide, TPSLEQRTVYAK, when covalently coupled to a terpolymer matrix, retained binding affinity towards HBOEC in a serum-free medium. Under the same binding conditions, the attachment of human umbilical vein endothelial cells (HUVEC) was limited, thus establishing HBOEC specificity. To our knowledge, this is the first report demonstrating specificity in binding to peptide-modified biomaterials of mature EC, i.e., HUVEC, and EC of progenitor origin such as HBOEC. The findings from this work could facilitate the development of autologous cell therapies with which to treat cardiovascular disease.
Keywords: Phage display-selected-peptide ligands; Cell-specific adhesion; HBOEC; Bioactive terpolymers; Autologous cell therapies;

Polylactic acid fibre-reinforced polycaprolactone scaffolds for bone tissue engineering by Vincenzo Guarino; Filippo Causa; Paola Taddei; Michele di Foggia; Gabriela Ciapetti; Desirèe Martini; Concezio Fagnano; Nicola Baldini; Luigi Ambrosio (3662-3670).
The employment of composite scaffolds with a well-organized architecture and multi-scale porosity certainly represents a valuable approach for achieving a tissue engineered construct to reproduce the middle and long-term behaviour of hierarchically complex tissues such as spongy bone. In this paper, fibre-reinforced composites scaffold for bone tissue engineering applications is described. These are composed of poly-l-lactide acid (PLLA) fibres embedded in a porous poly(ɛ-caprolactone) matrix, and were obtained by synergistic use of phase inversion/particulate leaching technique and filament winding technology. Porosity degree as high as 79.7% was achieved, the bimodal pore size distribution showing peaks at ca 10 and 200 μm diameter, respectively, accounting for 53.7% and 46.3% of the total porosity. In vitro degradation was carried out in PBS and SBF without significant degradation of the scaffold after 35 days, while in NaOH solution, a linear increase of weight lost was observed with preferential degradation of PLLA component. Subsequently, marrow stromal cells (MSC) and human osteoblasts (HOB) reached a plateau at 3 weeks, while at 5 weeks the number of cells was almost the same. Human marrow stromal cell and trabecular osteoblasts rapidly proliferate on the scaffold up to 3 weeks, promoting an oriented migration of bone cells along the fibre arrangement. Moreover, the role of seeded HOB and MSC on composite degradation mechanism was assessed by demonstrating a more relevant contribution to PLLA degradation of MSC when compared to HOB. The novel PCL/PLLA composite scaffolds thus showed promise whenever tuneable porosity, controlled degradability and guided cell–material interaction are simultaneously requested.
Keywords: Fibrous composite; Scaffolds; Degradation; Progenitor cells; Bone tissue engineering;

Injectable dendritic cell-carrying alginate gels for immunization and immunotherapy by Yuki Hori; Amy M. Winans; Catherine C. Huang; Elizabeth M. Horrigan; Darrell J. Irvine (3671-3682).
Dendritic cell vaccines, in which antigen-loaded dendritic cells (DCs) are injected directly into patients to trigger immune responses, are in development as a treatment for cancer and some infectious diseases. In this study, we tested the concept of delivering DCs in an injectable hydrogel matrix, with the aim of harboring dendritic cells for prolonged time periods at a defined site and trapping/concentrating factors secreted by DCs to establish an inflammatory milieu in situ. To achieve these goals, a self-gelling formulation of alginate was developed, obtained by mixing calcium-loaded alginate microspheres with soluble alginate solution and dendritic cells, a formulation that rapidly gelled in vivo. When injected subcutaneously in mice, these alginate ‘vaccination nodes’ containing activated DCs attracted both host dendritic cells and a large number of T cells to the injection sites over a week in vivo, while some of the inoculated DCs trafficked to the draining lymph nodes. Using an adoptive transfer model to track a defined population of T cells responding to immunization with antigen-loaded DCs, we show that DC/alginate immunization led to recruitment of activated antigen-specific T cells to the alginate matrix, in a manner dependent on the presence of the DCs. This gel/DC immunization system may thus be of interest for immunotherapy to direct the accumulation of immune cells at solid tumors or infection sites in the presence of supporting factors co-delivered by the hydrogel matrix.
Keywords: Dendritic cells; Alginate; Hydrogels; Inflammatory response; Injectable gel;

Ligament regeneration using a knitted silk scaffold combined with collagen matrix by Xiao Chen; Yi-Ying Qi; Lin-Lin Wang; Zi Yin; Guo-Li Yin; Xiao-Hui Zou; Hong-Wei Ouyang (3683-3692).
This study was aimed to develop a new practical ligament scaffold by synergistic incorporation of silk fibers, a knitted structure, and a collagen matrix. The efficacy for ligament tissue engineering was investigated in vitro and in animal models. Cells cultured on a collagen substrate expressed ligament matrix genes at higher levels than those on a silk substrate. The silk scaffold elicited little inflammatory reaction and degraded slowly after subcutaneous implantation in a mouse model. In the rabbit MCL defect model, MCLs treated with a silk + collagen scaffold deposited more collagen, had better mechanical properties, and showed more native microstructure with larger diameter collagen fibrils and stronger scaffold–ligament interface healing than untreated MCLs and those treated with silk scaffolds. These results demonstrated that the knitted silk + collagen sponge scaffold improves structural and functional ligament repair by regulating ligament matrix gene expression and collagen fibril assembly. The findings are the first to highlight the important roles of biomaterials in ligament regeneration biology. Also, the concept of an “internal-space-preservation” scaffold is proposed for the tissue repair under physical loading.
Keywords: Ligament; Tissue engineering; Knitted silk scaffold; Collagen matrix;

Dendrimer hydrazides as multivalent transient inter-cellular linkers by Deqiang Zhao; Siew-Min Ong; Zhilian Yue; Zhiyong Jiang; Yi-Chin Toh; Majad Khan; Jiahua Shi; Choon-Hong Tan; J. Paul Chen; Hanry Yu (3693-3702).
Three-dimensional (3D) multi-cellular aggregates (MCAs), as a model scaffold-free tissue construct, are useful for engineering cell-dense and matrix-poor tissues for repair and regeneration applications. To facilitate rapid MCA formation with high degrees of linker consistency and performance, we synthesized a class of dendrimer hydrazides with 8, 16 and 32 arms that can react with the aldehyde on the modified cell surfaces to form MCAs. DAB-AM-16 hydrazide with 32 arms demonstrated the best cell aggregation ability as compared to the dendrimer hydrazides with fewer arms, facilitating MCA formation at lower linker concentrations, minimizing cytotoxicity. Characterization of the MCAs formed with 2 μm of DAB-AM-16 hydrazide indicated that the cells proliferated well, maintained 3D cell–cell interaction and 3D cell morphology even as the inter-cellular linker gradually disappeared from the cell surfaces. Cells cultured as MCAs also demonstrated improved cell functions than the cells cultured in 2D monolayer. The dendrimer hydrazides would be a class of consistent, economical, and high performance multivalent transient inter-cellular linkers useful in forming scaffold-free 3D tissue constructs for soft-tissue engineering and regenerative medicine.
Keywords: Tissue construct; Cell glue; Dendrimer; Multivalent linker; Organ printing;

The discovery of the endothelial progenitor cell (EPC) has led to an intensive research effort into progenitor cell-based tissue engineering of (small-diameter) blood vessels. Herein, EPC are differentiated to vascular endothelial cells and serve as the inner lining of bioartificial vessels. As yet, a reliable source of vascular smooth muscle progenitor cells has not been identified. Currently, smooth muscle cells (SMC) are obtained from vascular tissue biopsies and introduce new vascular pathologies to the patient. However, since SMC are mesenchymal cells, endothelial-to-mesenchymal transdifferentiation (EnMT) may be a novel source of SMC. Here we describe the differentiation of smooth muscle-like cells through EnMT. Human umbilical cord endothelial cells (HUVEC) were cultured either under conditions favoring endothelial cell growth or under conditions favoring mesenchymal differentiation (TGF-β and PDGF-BB). Expression of smooth muscle protein 22α and α-smooth muscle actin was induced in HUVEC cultured in mesenchymal differentiation media, whereas hardly any expression of these markers was found on genuine HUVEC. Transdifferentiated endothelial cells lost the ability to prevent thrombin formation in an in vitro coagulation assay, had increased migratory capacity towards PDGF-BB and gained contractile behavior similar to genuine vascular smooth muscle cells. Furthermore, we showed that EnMT could be induced in three-dimensional (3D) collagen sponges. In conclusion, we show that HUVEC can efficiently transdifferentiate into smooth muscle-like cells through endothelial-to-mesenchymal transdifferentiation. Therefore, EnMT might be used in future progenitor cell-based vascular tissue engineering approaches to obtain vascular smooth muscle cells, and circumvent a number of limitations encountered in current vascular tissue engineering strategies.
Keywords: HUVEC; TGF-β; PDGF-BB; Endothelial-to-mesenchymal transdifferentiation (EnMT); Vascular tissue engineering; Smooth muscle cell;

The role of adipose protein derived hydrogels in adipogenesis by Shiri Uriel; Jung-Ju Huang; Monica L. Moya; Megan E. Francis; Rui Wang; Shu-ying Chang; Ming-Huei Cheng; Eric M. Brey (3712-3719).
Biomaterials that induce adipogenesis may ultimately serve as alternatives to traditional tissue reconstruction and regeneration techniques. In addition, these materials can provide environments for studying factors that regulate adipogenesis. The present study investigates the potential of adipose-derived matrices to induce adipogenesis in vitro and in vivo. Solutions containing basement membrane proteins and growth factors were extracted from subcutaneous adipose tissue. These extracts could be induced to form gels by either incubating the solutions at 37 °C or adjusting the pH to 4.0. The adipose extracts promoted rapid preadipocyte aggregation and formation of lipid-loaded colonies in vitro. Differentiation on adipose-derived gels was greater than tissue culture dishes and the tumor-derived product Matrigel™ (p  < 0.05). Significant adipose formation was observed when adipose-derived gels were implanted around a rat epigastric pedicle bundle. Adipose levels in these gels were significantly greater than Matrigel™ (p  < 0.05). The duration of adipose formation depended on the mechanism for gelling the solutions, with acid gelled matrices having greater adipose levels at 6 weeks than temperature gelled matrices. These adipose-derived hydrogels promote rapid adipogenesis in vitro and in vivo. They may lead to new materials for adipose tissue engineering, and provide an environment for studying cell–matrix interactions in adipogenesis.
Keywords: Preadipocytes; Extracellular matrix; Basement membrane; Adipogenesis; Angiogenesis; Differentiation;

Nanofibrous polyhydroxyalkanoate matrices as cell growth supporting materials by Xiao-Tao Li; Yan Zhang; Guo-Qiang Chen (3720-3728).
Polyhydroxyalkanoates (PHAs) have been demonstrated to be a family of biopolymers with good biodegradability and biocompatibility. To mimic the real microenvironment of extracellular matrix (ECM) for cell growth, novel nanofiber matrices based on PHA polymers were prepared via a phase separation process. Three-dimensional interconnected fibrous networks were observed in these matrices with average fiber diameters of 50–500 nm, which are very similar to the major ECM component collagen. Compared with nanofiber matrix made of poly(l-lactide), the mechanical properties of PHA nanofiber matrices were significantly improved, especially those matrices of PHA blends PHB/PHBHHx containing polyhydroxybutyrate (PHB) and copolyesters PHBHHx consisting of 3-hydroxybutyrate and 3-hydroxyhexanoate, and PHB/P3HB4HB that are PHB blended with copolyesters of 3-hydroxybutyrate and 4-hydroxybutyrate, respectively. More importantly, cell attachment and growth of human keratinocyte cell line HaCat on the nanofiber PHA matrices showed a notable improvement over those on PHA matrices prepared via an ordinary solution casting method. It was therefore proposed that PHA nanofiber matrices combined the advantages of biodegradation, improved mechanical strengths and the nanostructure of a natural extracellular matrix, leading to a better cell compatibility, thus they can be used for future implant biomaterial development.
Keywords: PHB; Polyhydroxybutyrate; Nanofiber; Matrix; HaCat cells; Biocompatibility; Tissue engineering;

The use of trehalose-treated freeze-dried amniotic membrane for ocular surface reconstruction by Takahiro Nakamura; Eiichi Sekiyama; Maho Takaoka; Adam J. Bentley; Norihiko Yokoi; Nigel J. Fullwood; Shigeru Kinoshita (3729-3737).
The aim of this study was to evaluate the efficacy and safety of trehalose-treated freeze-dried amniotic membrane (TT-FDAM) for ocular surface reconstruction. Human AM deprived of amniotic epithelial cells was first incubated with 10% trehalose solution, and then freeze-dried, vacuum-packed, and sterilized with gamma-irradiation. The resultant newly developed TT-FDAM was characterized for its physical, biological, and morphological properties by comprehensive physical assays, immunohistochemistry, electron microscopy, cell adhesion assay, 3D cell culture, and an in vivo biocompatibility test. The adaptability of TT-FDAM was markedly improved as compared to FDAM. Immunohistochemistry for several extracellular matrix molecules revealed that the process of freeze-drying and irradiation apparently did not affect its biological properties, however, electron microscopy revealed that the detailed morphological appearance of TT-FDAM is more similar to that of native AM than to FDAM. Intracorneal and scleral-surface transplantation of TT-FDAM showed excellent biocompatibility with ocular surface tissues. Thus, TT-FDAM retained most of the physical, biological, and morphological characteristics of native AM, consequently it is a useful biomaterial for ocular surface reconstruction.
Keywords: Trehalose; Amniotic membrane; Freeze-dry; Ocular surface reconstruction; Biocompatibility;

An in vivo murine model of continuous intramedullary infusion of polyethylene particles by Ting Ma; Zhinong Huang; Pei-Gen Ren; Ryan McCally; Derek Lindsey; R.L. Smith; Stuart B. Goodman (3738-3742).
Wear debris affects both initial osseointegration and subsequent bone remodeling of total joint replacements (TJRs). To study the complex cascade associated with the continuous generation of particles, a robust animal model is essential. To date, an animal model that incorporates continuously delivered particles to an intramedullary orthopaedic implant has not been available. In this study, we successfully infused clinically relevant ultra high molecular weight polyethylene particles, previously isolated from joint simulator tests, to the intramedullary space of the mouse femur for 4 weeks using a subcutaneous osmotic pump. Reduction of bone volume following the 4-week infusion of UHMWPE was detected by microCT. UHMWPE particles also changed the level of Alkaline Phosphatase expression in the infused femurs. Continuous infusion of particles to the murine bone–implant interface simulated the clinical scenario of local polymer wear particle generation and delivery in humans and can be used to further study the biological processes associated with wear debris particles.
Keywords: Murine model; Continuous infusion; UHMWPE particles; Osteolysis;

The control of cell adhesion and viability by zinc oxide nanorods by Jiyeon Lee; B.S. Kang; Barrett Hicks; Thomas F. Chancellor; Byung Hwan Chu; Hung-Ta Wang; Benjamin G. Keselowsky; F. Ren; Tanmay P. Lele (3743-3749).
The ability to control the behavior of cells that interact with implanted biomaterials is desirable for the success of implanted devices such as biosensors or drug delivery devices. There is a need to develop materials that can limit the adhesion and viability of cells on implanted biomaterials. In this study, we investigated the use of zinc oxide (ZnO) nanorods for modulating the adhesion and viability of NIH 3T3 fibroblasts, umbilical vein endothelial cells, and capillary endothelial cells. Cells adhered far less to ZnO nanorods than the corresponding ZnO flat substrate. The few cells that adhered to ZnO nanorods were rounded and not viable compared to the flat ZnO substrate. Cells were unable to assemble focal adhesions and stress fibers on nanorods. Scanning electron microscopy indicated that cells were not able to assemble lamellipodia on nanorods. Time-lapse imaging revealed that cells that initially adhered to nanorods were not able to spread. This suggests that it is the lack of initial spreading, rather than long-term exposure to ZnO that causes cell death. We conclude that ZnO nanorods are potentially useful as an adhesion-resistant biomaterial capable of reducing viability in anchorage-dependent cells.
Keywords: Nanorods; Cell adhesion; Cell viability; Anti-adhesion;

Kinetics of the breakdown of cross-linked soy protein films for drug delivery by Lingyun Chen; Gabriel Remondetto; Mahmoud Rouabhia; Muriel Subirade (3750-3756).
The aim of the present work was to investigate the potential of soy protein isolate (SPI) films as controlled release systems for active compounds. Mechanical properties, dissolution and compound release kinetics of SPI films prepared with different concentrations of formaldehyde were measured over time in the absence or presence of digestive enzymes at gastric or intestinal pH. The effect of formaldehyde on tensile strength, elastic modulus, % elongation and swelling suggested that increasing its concentration increased film cross-linking density. Film bulk erosion in the presence of digestive enzymes followed first-order kinetics. Methylene blue or rifampicin release followed variable kinetics depending on compound solubility during a 1–2 h initial phase, followed by zero-order release. Cross-linking density appears to provide effective means of regulating the erosion and release rate of SPI films. SPI film networks displayed excellent compound binding capacity, especially for hydrophobic molecules, and hence potential for use in controlled release systems based on matrix erosion.
Keywords: Films; Soy protein; Cross-linking; Degradation; Drug delivery;