Biomaterials (v.26, #36)
Get a grip: integrins in cellâ€“biomaterial interactions
by A.J. Garcia Andrés J. García (pp. 7525-7529).
Integrin adhesion receptors have emerged as central regulators of cellâ€“biomaterial interactions. This opinion paper discusses how integrins control cellular and host responses to biomaterials and new strategies to manipulate these adhesive interactions in order to elicit specific cellular responses.
Keywords: Integrins; Cell adhesion; Extracellular matrix; Signaling; Inflammation; Biomimetic materials; RGD
Ligament tissue engineering: An evolutionary materials science approach
by Cato T. Laurencin; Joseph W. Freeman (pp. 7530-7536).
The anterior cruciate ligament (ACL) is important for knee stabilization. Unfortunately, it is also the most commonly injured intra-articular ligament. Due to poor vascularization, the ACL has inferior healing capability and is usually replaced after significant damage has occurred. Currently available replacements have a host of limitations, this has prompted the search for tissue-engineered solutions for ACL repair. Presently investigated scaffolds range from twisted fiber architectures composed of silk fibers to complex three-dimensional braided structures composed of poly (l-lactic acid) fibers. The purpose of these tissue-engineered constructs is to apply approaches such as the use of porous scaffolds, use of cells, and the application of growth factors to promote ligament tissue regeneration while providing mechanical properties similar to natural ligament.
Keywords: Anterior cruciate ligament; Tissue engineering; Scaffold
Towards a methodology for the effective surface modification of porous polymer scaffolds
by Laleh Safinia; Nathalie Datan; Hohse Marek Höhse; Athanassios Mantalaris; Alexander Bismarck (pp. 7537-7547).
A novel low-pressure radio-frequency plasma treatment protocol was developed to achieve the effective through-thickness surface modification of large porous poly (d,l-lactide) (PDLLA) polymer scaffolds using air or water: ammonia plasma treatments. Polymer films were modified as controls. Scanning electron micrographs and maximum bubble point measurements demonstrated that the PDLLA foams have the high porosity, void fraction and interconnected pores required for use as tissue engineering scaffolds. The polymer surface of the virgin polymer does contain acidic functional groups but is hydrophobic.Following exposure to air or water: ammonia plasma, an increased number of polar functional groups and improved wetting behaviour, i.e. hydrophilicity, of wet surfaces was detected. The number of polar surface functional groups increased (hence the decrease in water contact angles) with increasing exposure time to plasma. The change in surface composition and wettablility of wet polymer constructs was characterised by zeta potential and contact angle measurements. The hydrophobic recovery of the treated PDLLA polymer surfaces was also studied. Storage of the treated polymer constructs in ambient air caused an appreciable hydrophobic recovery, whereas in water only partial hydrophobic recovery occurred. However, in both cases the initial surface characteristics decay as function of time.
Keywords: Scaffold; Polylactic acid; Plasma; Surface modification; Contact angle; Wettability hydrophilicity
Local structure of channel ions in carbonate apatite
by Michael E. Fleet; Xiaoyang Liu (pp. 7548-7554).
Refinement of the single-crystal X-ray diffraction structure of a type A carbonate apatite [CAp; Ca10(PO4)6â€“ y(CO3) x+(3/2) y(OH)2âˆ’2 x,x=0.75,y=0.0; space groupP3Â¯] has been continued with independent positional and isotropic displacement parameters for the carbonate oxygen atoms, reducing the residual indices significantly (R=0.024,Rw=0.020) and confirming the earlier structure assignment. The carbonate ion is located in the apatite channel at zâ‰ˆ0.5, and oriented with two oxygen atoms close to the c-axis. Rigid body refinement, giving a preferred structure, used a novel procedure for defining the ideal equilateral triangular geometry of the channel carbonate ion. Resolution of the channel carbonate ions in type A-B CAp (x=0.69,y=0.57; P63/m) is also improved. Channel carbonate ions in CAp are canted, rotated and displaced to optimize Ca2â€“O bond distances. The rotation of the A1 carbonate in type A-B CAp is opposite to that of the channel carbonate in type A CAp, due mainly to the accommodation of a second channel carbonate ion (A2). These structures simulate the local structure of type A carbonate in hydroxyapatite of bone and dental enamel.
Keywords: Apatite structure; Carbonate apatite; Hydroxyapatite; Bone; Crystallography
Synthesis of degradable poly(l-lactide- co-ethylene glycol) porous tubes by liquidâ€“liquid centrifugal casting for use as nerve guidance channels
by Alex Goraltchouk; Thomas Freier; Molly S. Shoichet (pp. 7555-7563).
Biodegradable nerve guidance channels are advantageous, obviating the need for their removal after regeneration; however, most channels lack the appropriate mechanical properties for soft tissue implantation and/or degrade too quickly, resulting in reduced regeneration and necessitating the need for the design of polymers with tunable degradation profiles and mechanical properties. We designed a series of biodegradable polymeric hydrogel tubes consisting ofl-lactide (LLA) and polyethylene glycol (PEG) where both the ratio of LLA to PEG and PEG molar mass were varied. By adjusting the PEG:LLA ratio and the molecular weight of the PEG oligomer we were able to control degradation and mechanical properties of our polymers. By incorporating methacrylate (MA) groups on both termini of the linear oligomers, porous tubes were synthesized by a redox-initiated free radical mechanism during a liquidâ€“liquid centrifugal casting process. The tube wall had a bead-like morphology, as determined by SEM, which was reminiscent of previous porous hydrogel tubes synthesized by the same method. Tubes swelled with degradation to 160vol%, or 640wt%, and an increased radius calculated at 1.26 times. Those tubes with greater PEG content and PEG molar mass degraded faster than those with greater LLA content, as was expected. Interestingly, the wall morphology changed with degradation to a fiber-like structure and the mechanical properties decreased with degradation. By correlating the accelerated degradation study to a physiologic one, these porous hydrogel tubes were stable for an equivalent of 1.5 months, after which the mechanical properties began to deteriorate. This study demonstrates how porous hydrogel tubes can be designed to meet tissue regeneration criteria by tuning the formulation chemistry and specifically how the ratio of hydrophobic/crystalline LLA and hydrophilic/amorphous PEG impact tube properties.
Keywords: Poly(lactide); Poly(ethylene glycol); Centrifugal casting; Polymer tubes; Nerve guidance channels
A three-layered nano-carbonated hydroxyapatite/collagen/PLGA composite membrane for guided tissue regeneration
by Susan Liao; Wei Wang; Motohiro Uo; Shoji Ohkawa; Tsukasa Akasaka; Kazuchika Tamura; Fuzhai Cui; Fumio Watari (pp. 7564-7571).
Functional graded materials (FGM) provided us one new concept for guided tissue regeneration (GTR) membrane design with graded component and graded structure where one face of the membrane is porous thereby allowing cell growth thereon and the opposite face of the membrane is smooth, thereby inhibiting cell adhesion in periodontal therapy. The goal of the present study was to develop a three-layered graded membrane, with one face of 8% nano-carbonated hydroxyapatite/collagen/poly(lactic- co-glycolic acid) (nCHAC/PLGA) porous membrane, the opposite face of pure PLGA non-porous membrane, the middle layer of 4% nCHAC/PLGA as the transition through layer-by-layer casting method. Then the three layers were combined well with each other with flexibility and enough high mechanical strength as membrane because the three layers all contained PLGA polymer that can be easily used for practical medical application. This high biocompatibility and osteoconductivity of this biodegraded composite membrane was enhanced by the nCHAC addition, for the same component and nano-level crystal size with natural bone tissue. The osteoblastic MC3T3-E1 cells were cultured on the three-layered composite membrane, the primary result shows the positive response compared with pure PLGA membrane.
Keywords: Nano-carbonated hydroxyapatite/collagen composite; GTR; Biodegrade
Effects of phosphatidylserine coatings on titanium on inflammatory cells and cell-induced mineralisation in vitro
by Michela Bosetti; Andrew W. Lloyd; Matteo Santin; Steve P. Denyer; M. Cannas (pp. 7572-7578).
Ideally an active bone biomaterial should increase the mineralisation rate at the bone healing sites, keeping at the same time the inflammation process to levels required for tissue regeneration. Our studies suggest that in addition to improving the nucleation process for new bone formation, coating titanium with phospholipids may reduce the inflammatory response, which was shown to vary depending on the formulation employed. As phosphatidylserine reduced the inflammatory response to the greatest extent, in the second part of this study we examined its effect on osteoblast mineralisation. These studies demonstrated that phosphatidylserine improves the nucleation process for bone formation, by promoting the formation of bone-like tissue, so the high mineralisation potential of phosphatidylserine-coated titanium, together with the lower level of inflammatory response, supports the further development of this technology for coating osteointegrative devices.
Keywords: Mineralisation; Phosphatidylserine; Titanium; Inflammation
The effect of surface chemistry modification of titanium alloy on signalling pathways in human osteoblasts
by H. Zreiqat; Stella M. Valenzuela; Besim Ben Nissan; Richard Roest; Christine Knabe; Ralf J. Radlanski; Herbert Renz; Peter J. Evans (pp. 7579-7586).
Establishing and maintaining mature bone at the boneâ€“device interface is critical to the long-term success of prosthesis. Poor cell adhesion to orthopaedic and dental implants results in implant failure. Considerable effort has been devoted to alter the surface characteristics of these biomaterials in order to improve the initial interlocking of the device and skeleton. We investigated the effect of surface chemistry modification of titanium alloy (Tiâ€“6Alâ€“4V) with zinc, magnesium or alkoxide-derived hydroxy carbonate apatite (CHAP) on the regulation of key intracellular signalling proteins in human bone-derived cells (HBDC) cultured on these modified Tiâ€“6Alâ€“4V surfaces. Western blotting demonstrated that modifying Tiâ€“6Alâ€“4V with CHAP or Mg results in modulation of key intracellular signalling proteins. We showed an enhanced activation of Shc, a common point of integration between integrins and the Ras/Mapkinase pathway. Mapkinase pathway was also upregulated, suggesting its role in mediating osteoblastic cell interactions with biomaterials. The signalling pathway involving c-fos (member of the activated protein-1) was also shown to be upregulated in osteoblasts cultured on the Mg and CHAP modified Tiâ€“6Alâ€“4V. Thus surface modification with CHAP or Mg may contribute to successful osteoblast function and differentiation at the skeletal tissueâ€“device interface.
Keywords: Hydroxyapatite; Solâ€“gel coatings; Titanium alloy; Intracellular signalling proteins; Osteoblasts
Nano-C60 cytotoxicity is due to lipid peroxidation
by Christie M. Sayes; Andre M. Gobin; Kevin D. Ausman; Joe Mendez; Jennifer L. West; Vicki L. Colvin (pp. 7587-7595).
This study examines the biological effects of water-soluble fullerene aggregates in an effort to evaluate the fundamental mechanisms that contribute to the cytotoxicity of a classic engineered nanomaterial. For this work we used a water-soluble fullerene species, nano-C60, a fullerene aggregate that readily forms when pristine C60 is added to water. Nano-C60 was cytotoxic to human dermal fibroblasts, human liver carcinoma cells (HepG2), and neuronal human astrocytes at doses â©¾50ppb (LC50=2â€“50ppb, depending on cell type) after 48h exposure. This water-soluble nano-C60 colloidal suspension disrupts normal cellular function through lipid peroxidation; reactive oxygen species are responsible for the membrane damage. Cellular viability was determined through live/dead staining and LDH release. DNA concentration and mitochondrial activity were not affected by the nano-C60 inoculations to cells in culture. The integrity of cellular membrane was examined by monitoring the peroxy-radicals on the lipid bilayer. Subsequently, glutathione production was measured to assess the cell's reaction to membrane oxidation. The damage to cell membranes was observed both with chemical assays, and confirmed physically by visualizing membrane permeability with high molecular weight dyes. With the addition of an antioxidant,l-ascorbic acid, the oxidative damage and resultant toxicity of nano-C60 was completely prevented.
Keywords: Nanoparticle; Cytotoxicity; Nano-C; 60; Membrane oxidation
Proliferative and re-defferentiative effects of photo-immobilized micro-patterned hyaluronan surfaces on chondrocyte cells
by Rolando Barbucci; Paola Torricelli; Milena Fini; Daniela Pasqui; Pietro Favia; Eloisa Sardella; Riccardo dâ€™Agostino; Roberto Giardino (pp. 7596-7605).
A photo-immobilisation procedure was utilised to create two different micro-patterned surfaces (tracks 25 and 5Î¼m wide) of hyaluronan (Hyal) on polyethylene-terephthalate (PET) previously plasma activated. Aim of the study was to investigate the proliferation and re-differentiation capacity of articular chondrocytes cultured on micro-patterned Hyal, compared to homogeneous Hyal and plain plasma-treated (pt-)PET substrates. Cytotoxicity, cell proliferation, activation and differentiation of articular knee cartilage chondrocytes (Mongrel sheep) were evaluated after 14 days of culture. It was found that micro-patterned Hyal surfaces induced the adhesion, migration and alignment of chondrocytes, as shown by light and scanning electron microscopy. Furthermore, the same surfaces induced chondrocyte differentiation, with a significant increase of aggrecan and collagen type II production, while homogeneous Hyal and pt-PET surfaces did not.
Keywords: Chondrocyte; Micropatterning; Polyethylene terephthalate; Hyaluronic acid/hyaluronan; Bioactivity
Fabrication of collagen-coated biodegradable polymer nanofiber mesh and its potential for endothelial cells growth
by Wei He; ZuWei Ma; Thomas Yong; Wee Eong Teo; Seeram Ramakrishna (pp. 7606-7615).
Endothelialization of biomaterials is a promising way to prevent intimal hyperplasia of small-diameter vascular grafts. The aim of this study was to design a nanofiber mesh (NFM) that facilitates viability, attachment and phenotypic maintenance of human coronary artery endothelial cells (HCAECs). Collagen-coated poly(l-lactic acid)- co-poly( Îµ-caprolactone) P(LLA-CL 70:30) NFM with a porosity of 64â€“67% and a fiber diameter of 470Â±130nm was fabricated using electrospinning followed by plasma treatment and collagen coating. The structure of the NFM was observed by SEM and TEM, and mechanical property was studied by tensile test. The presence of collagen on the P(LLA-CL) NFM surface was verified by X-ray photoelectron spectroscopy (XPS) and quantified by colorimetric method. Spatial distribution of the collagen in the NFM was visualized by labelling with fluorescent probe. The collagen-coated P(LLA-CL) NFM enhanced the spreading, viability and attachment of HCAECs, and moreover, preserve HCAEC's phenotype. The P(LLA-CL) NFM is a potential material for tissue engineered vascular graft.
Keywords: Nanofiber; PLLA; PCL; Collagen; Coating; Endothelial cells; Tissue engineering; Vascular grafts
In vitro characterization of chitosanâ€“gelatin scaffolds for tissue engineering
by Yan Huang; Stella Onyeri; Mbonda Siewe; Aliakbar Moshfeghian; Sundararajan V. Madihally (pp. 7616-7627).
Recently, chitosanâ€“gelatin scaffolds have gained much attention in various tissue engineering applications. However, the underlying cellâ€“matrix interactions remain unclear in addition to the scaffold degradation and mechanical characteristics. In this study, we evaluated (i) the degradation kinetics of chitosan and chitosanâ€“gelatin scaffolds in the presence of 10mg/L of lysozyme for dimensional stability, weight loss, and pH changes for a period of 2 months, (ii) tensile and compressive properties of films and scaffolds in wet state at 37Â°C, (iii) viability of fibroblasts and human umbilical vein endothelial cells (HUVECs) on scaffolds, and (iv) the alteration in cell spreading characteristics, cytoskeletal actin distribution, focal adhesion kinase (FAK) distribution and PECAM-1 expression of HUVECs under static and 4.5, 8.5, 13 and 18dyn/cm2 shear stress conditions. Degradation results showed that gelatin-containing chitosan scaffolds had faster degradation rate and significant loss of material than chitosan. Mechanical properties of chitosan are affected by the addition of gelatin although there was no clear trend. Three-dimensional chitosan and chitosanâ€“gelatin scaffolds supported fibroblast viability equally. However, chitosan membranes decreased cell-spreading area, disrupted F-actin and localized FAK in the nucleus of HUVECs. Importantly, the lowest shear stress tested (4.5dyn/cm2) for 3h washed away cells on chitosan suggesting weak cell adhesion. In the blends, effect of gelatin was dominant; actin and FAK distribution were comparable to gelatin in static culture. However, at higher shear stresses, presence of chitosan inhibited shear-induced increase in cell spreading and weakened cell adhesive strength. No significant differences were observed in PECAM-1 expression. In summary, these results showed significant influence of blending gelatin with chitosan on scaffold properties and cellular behavior.
Keywords: Chitosan; Fibroblasts; Pore orientation; Cytoskeleton; Controlled rate freezing; Gelatin; Scaffolds; Cell adhesion; Shear stress; Degradation; Lysozyme; PECAM-1; Actin; FAK; Parallel flow reactor; Parallel flow chamber
Fabrication of tubular tissue constructs by centrifugal casting of cells suspended in an in situ crosslinkable hyaluronan-gelatin hydrogel
by Vladimir Mironov; Vladimir Kasyanov; Xiao Zheng Shu; Carol Eisenberg; Leonard Eisenberg; Steve Gonda; Thomas Trusk; Roger R. Markwald; Glenn D. Prestwich (pp. 7628-7635).
Achieving the optimal cell density and desired cell distribution in scaffolds is a major goal of cell seeding technologies in tissue engineering. In order to reach this goal, a novel centrifugal casting technology was developed using in situ crosslinkable hyaluronan-based (HA) synthetic extracellular matrix (sECM). Living cells were suspended in a viscous solution of thiol-modified HA and thiol-modified gelatin, a polyethyleneglycol diacrylate crosslinker was added, and a hydrogel was formed during rotation. The tubular tissue constructs consisting of a densely packed cell layer were fabricated with the rotation device operating at 2000rpm for 10min. The majority of cells suspended in the HA mixture before rotation were located inside the layer after centrifugal casting. Cells survived the effect of the centrifugal forces experienced under the rotational regime employed. The volume cell density (65.6%) approached the maximal possible volume density based on theoretical sphere packing models. Thus, centrifugal casting allows the fabrication of tubular constructs with the desired redistribution, composition and thickness of cell layers that makes the maximum efficient use of available cells. Centrifugal casting in this sECM would enable rapid fabrication of tissue-engineered vascular grafts, as well as other tubular and planar tissue-engineered constructs.
Keywords: Centrifugal casting; Thiol-modified gelatin; Glycosaminoglycan; Tubular construct; Synthetic extracellular matrix
Direct patterning of mammalian cells onto porous tissue engineering substrates using agarose stamps
by Molly M. Stevens; Michael Mayer; Daniel G. Anderson; Douglas B. Weibel; George M. Whitesides; Robert Langer (pp. 7636-7641).
This paper describes simple, inexpensive, and potentially generic methodology for generating patterns of mammalian cells on porous scaffolds for tissue engineering using replica printing. Circular patterns (diameter: 200, 700, and 1000Î¼m) of human osteoblasts were transferred directly from topographically patterned agarose stamps onto porous hydroxyapatite scaffolds or onto fibronectin-coated glass slides. The use of hydrogel stamps provided a â€œwetâ€?, biocompatible surface and maintained the viability of cells adsorbed on stamps during the patterning process. Stamps inked once with suspensions of cells allowed the repeated patterning of substrates. Direct stamping of human osteoblasts (and, potentially other mammalian cells) can be used to control the size, spacing, and geometry of patterns of cells printed on porous tissue engineering substrates. This approach may find use in controlling the spatial invasion of scaffolds, promoting the hierarchical organization of cells, and in controlling cellâ€“cell interactions as a step in preservation of phenotypes of cells.
Keywords: Hydroxyapatite; Cell printing; Osteoblast
Direct micro-patterning of biodegradable polymers using ultraviolet and femtosecond lasers
by Carlos A. Aguilar; Yi Lu; Samuel Mao; Shaochen Chen (pp. 7642-7649).
Thin films of biodegradable polymeric materials, poly(Îµ-caprolactone) (PCL) and poly(glycolic acid) (PGA) were micro-patterned using a Ti-sapphire femtosecond pulsed laser and ArF excimer UV laser in ambient conditions. The laser-patterned polymers were characterized using a scanning electron microscope (SEM), Fourier transform infrared spectroscopy in attenuated total reflectance mode (FTIR-ATR) and X-ray photoelectron spectroscopy (XPS). In-vitro degradation tests were performed and the laser-patterned samples showed to be within one standard deviation of the control samples. Our results demonstrate that both lasers are excellent tools for micro-patterning biodegradable polymers since the bulk properties of the material can remain intact and because the direct-write method is rapid, flexible, and a chemical-free process.
Keywords: Biodegradable polymer; Laser micromachining; Femtosecond laser; Excimer laser; Photothermal; Photochemical; Hydrolytic degradation
A transmission electron microscopy study of mineralization in age-induced transparent dentin
by Alexandra E. Porter; Ravi K. Nalla; Andrew Minor; Joerg R. Jinschek; Christian Kisielowski; Velimir Radmilovic; John H. Kinney; Antoni P. Tomsia; R.O. Ritchie (pp. 7650-7660).
It is known that fractures are more likely to occur in altered teeth, particularly following restoration or endodontic repair; consequently, it is important to understand the structure of altered forms of dentin, the most abundant tissue in the human tooth, in order to better define the increased propensity for such fractures. Transparent (or sclerotic) dentin, wherein the dentinal tubules become occluded with mineral as a natural progressive consequence of aging, is one such altered form. In the present study, high-resolution transmission electron microscopy is used to investigate the effect of aging on the mineral phase of dentin. Such studies revealed that the intertubular mineral crystallites were smaller in transparent dentin, and that the intratubular mineral (larger crystals deposited within the tubules) was chemically similar to the surrounding intertubular mineral. Exit-wave reconstructed lattice-plane images suggested that the intratubular mineral had nanometer-size grains. These observations support a â€œdissolution and reprecipitationâ€? mechanism for the formation of transparent dentin.
Keywords: Dentin; Transmission electron microscopy; Mineralization; Sclerotic; Transparent; Aging