Biomaterials (v.27, #6)

Calendar (I).

The induction of bone formation starts by erecting scaffolds of smart biomimetic matrices acting as insoluble signals affecting the release of soluble osteogenic molecular signals. The cascade of bone differentiation by induction develops as a mosaic structure singly initiated by the osteogenic proteins of the transforming growth factor-β (TGF-β) supergene family. The osteogenic signals when combined with an insoluble signal or substratum initiate de novo bone formation by induction and are deployed singly, synergistically and synchronously to sculpt the architecture of the mineralized bone/bone marrow organ. The osteogenic proteins of the TGF-β superfamily are the common molecular initiators deployed for embryonic development and the induction of bone in postnatal osteogenesis, whereby molecules exploited in embryonic development are re-deployed in postnatal tissue morphogenesis as a recapitulation of embryonic development. The pleiotropy of the osteogenic proteins of the TGF-β superfamily is highlighted by the apparent redundancy of molecular signals initiating bone formation by induction including the TGF-β isoforms per se, powerful inducers of endochondral bone but in the primate only. Bone induction by the TGF-β isoforms in the primate is site and tissue specific with substantial endochondral bone induction in heterotopic sites but with absent osteoinductivity in orthotopic calvarial sites on day 30 and only limited osteogenesis pericranially on day 90. Ebaf/Lefty-A, a novel member of the TGF-β superfamily, induces chondrogenesis in calvarial defects of Papio ursinus and bone regeneration across the defect on day 30 and 90, respectively. The strikingly pleiotropic effects of the bone morphogenetic and osteogenic proteins (BMPs/OPs) spring from amino acid sequence variations in the carboxy-terminal domain and in the transduction of distinct signalling pathways by individual Smad proteins after transmembrane serine/threonine kinase complexes of type I and II receptors. Predictable bone regeneration in clinical contexts requires information concerning the expression and cross regulation of gene products of the TGF-β superfamily. OP-1, BMP-3, TGF-β1 and type IV collagen mRNAs expression correlates to the morphological induction and maintenance of engineered ossicles by the hOP-1 osteogenic devices in the non-human primate P. ursinus. Amino-acid sequence variations amongst BMPs/OPs in the carboxy terminal domain confer the structure/activity profile responsible for the pleiotropic activity that controls tissue induction and morphogenesis of a variety of tissues and organs by different BMPs/OPs which are helping to engineer skeletal tissue regeneration in molecular terms.
Keywords: Osteogenic proteins; TGF-β superfamily; Redundancy; Structure/activity profile; Species and site tissue specificity;

Hydroxyapatite and ‘duplex’ hydroxyapatite + titania bond coat layers were deposited onto Ti6Al4 V substrates by atmospheric plasma spraying (APS) at moderate plasma enthalpies. From as-sprayed coatings and coatings incubated in simulated body fluid (r-SBF) electron-transparent samples were generated by focused ion beam (FIB) excavation and investigated by STEM/TEM in conjuction with energy-dispersive X-ray analysis (EDX), electron diffraction (ED), and electron energy loss spectroscopy (EELS).Adjacent to the metal surface a thin layer of amorphous calcium phosphate (ACP) was deposited whose Ca/P ratio is determined by the presence or absence of the bond coat. No clear indication of a Ca–Ti oxide reaction layer was found at the interface titania bond coat/calcium phosphate. After in vitro incubation of duplex coatings for 24 weeks Ca-deficient defect apatite needles precipitated from ACP. During incubation of hydroxyapatite without a bond coat for 1 week diffusion bands were formed within the ACP of 1–2 μm width parallel to the interface metal/coating, presumably by a dissolution–precipitation sequence.
Keywords: Hydroxyapatite coatings; Calcium phosphate; In vitro test; Electron microscopy; Focused ion beam excavation;

A series of self-crosslinkable and biodegradable polymers, poly(caprolactone fumarate) (PCLF), poly(ethylene glycol fumarate) (PEGF), and their copolymer PEGF-co-PCLF, has been developed for tissue engineering applications using a novel synthesis method. The current method employs potassium carbonate (K2CO3), other than the previously reported triethylamine, as the proton scavenger. The new synthetic route is more convenient and less time-consuming to carry out, and the synthesized polymers have a much lighter color, which renders them more suitable for self-crosslinking via photo-initiation. This group of polymers are essentially copolymers of fumaryl chloride, which contains double bonds for in situ crosslinking, with poly(ε-caprolactone) (PCL) or/and poly(ethylene glycol) (PEG) that has a flexible chain to facilitate self-crosslinking. Both PCLF and PEGF, and their amphiphilic copolymer PEGF-co-PCLF could be self-crosslinked or photocrosslinked to produce scaffolds without the use of a crosslinking agent. Our results suggest that these polymers are potentially useful as injectable, self-crosslinkable, and photo-crosslinkable materials for diverse tissue engineering applications.
Keywords: Polymerization; Polycaprolactone; Polyethylene oxide; Cross-linking; Amphiphilic polymer network;

Interaction of B50 rat hippocampal cells with stain-etched porous silicon by Andrei V. Sapelkin; Susan C. Bayliss; Bayram Unal; Amanda Charalambou (842-846).
We present a scanning electron microscopy study of interaction of porous silicon prepared by stain etching with rat hippocampal neurons (B50, immortalised line). Porous Si has been prepared by stain etching both from crystalline and polycrystalline substrates. Two types of patterns have been examined: (i) 100 μm square pads separated by 100 μm of untreated space and (ii) 30 μm and 100 μm stripes separated by 300 μm of untreated surface. In all cases cells show clear preference in adhesion to porous Si over untreated surface. We observe a much closer attachment of the cell to the surface of porous Si than has been anticipated before with the gap between a cell and porous surface of no more than 20 nm. This study demonstrates the influence of surface topology on proliferation of a neuron network, thus suggesting that a degree of control over cell growth pattern can be achieved using porous material alone. We show that the network pattern on porous Si is influenced by a single neuron response to the surface nature and topology.
Keywords: SEM; Microstructure; Neural network; Neural cell; B50; Surface topography;

Biomimetic poly(2-methacryloyloxyethyl phosphorylcholine) (poly(MPC)) brushes with graft density 0.06–0.39 chains/nm2 and chain length 5–200 monomer units were prepared from silicon wafer surfaces by combining self-assembly of initiator and surface-initiated atom transfer radical polymerization (ATRP). Water contact angle, X-ray photoelectron spectroscopy, and atomic force microscopy were used to characterize the modified surfaces. These surfaces with well-controlled poly(MPC) brushes were tested for protein repelling performance. Fibrinogen adsorption from tris-buffered saline at pH 7.4 decreased significantly with increasing graft density and/or chain length of poly(MPC) and reached a level of <10 ng/cm2 at graft density ⩾0.29 chains/nm2 and chain length ⩾100 units, compared to ca. 570 ng/cm2 for the unmodified samples. While the fibrinogen adsorption was determined by both graft density and chain length, it showed a stronger dependence on graft density than on chain length.
Keywords: Surface modification; Non-biofouling; Protein adsorption; Biomimetic polymer; Atom transfer radical polymerization; Grafting;

Polysulfone-graft-poly(ethylene glycol) graft copolymers for surface modification of polysulfone membranes by Jane Y. Park; Metin H. Acar; Ariya Akthakul; William Kuhlman; Anne M. Mayes (856-865).
Amphiphilic graft copolymers having polysulfone (PSf) backbones and poly(ethylene glycol) (PEG) side chains were synthesized via reaction of an alkoxide formed from PEG and a base (sodium hydride) with chloromethylated polysulfone. The resulting polysulfone-graft-poly(ethylene glycol), PSf-g-PEG, materials were hydrophilic but water insoluble, rendering them potentially useful as biomaterial coatings. PSf-g-PEG films exhibited high resistance to protein adsorption and cell attachment. When used as an additive in PSf membranes prepared by immersion precipitation, the graft copolymer preferentially segregates to the membrane surface, delivering enhanced wettability, porosity and protein resistance compared to unmodified PSf membranes. The surface properties of PSf-g-PEG modified membranes render them desirable candidates for hemodialysis.
Keywords: Polysulfone; Polyethylene glycol; Surface modification; Protein resistance; Membrane; Dialysis;

Fabricating tubular scaffolds with a radial pore size gradient by a spinning technique by Brendan A. Harley; Abel Z. Hastings; Ioannis V. Yannas; Alessandro Sannino (866-874).
A novel fabrication process has been developed to produce collagen-based, porous tubular scaffolds to facilitate the study of myofibroblast migration during peripheral nerve regeneration; however, this fabrication technique offers broader appeal for the production of a variety of tubular structures without the use of a complicated mold system. A collagen-glycosaminoglycan (CG) suspension in acetic acid was spun in a cylindrical copper mold about its longitudinal axis at variable angular velocities and for different times, resulting in variable relative sedimentation of the CG content towards the mold outer edge; after the specified spinning time, the spinning mold was placed into a bath of liquid nitrogen where the CG suspension was rapidly frozen. Due to the rapid solidification, the CG content remained sedimented while an interconnected network of ice crystals formed throughout. Sublimation of the frozen mass removed the solvent (acetic acid) content, producing a porous, tubular structure defined by sedimentation and ice crystal nucleation processes. A porous, tubular scaffold with a sharply defined inner tube wall can be produced; further, increasing the spinning time and/or spinning velocity increases the sedimentation effect leading to the production of a hollow tube with a larger inner diameter. The tube walls display a radially aligned pore structure, even in cases where sedimentation was not sufficient to produce a hollow tube. A gradient of porosity along the tube radius was also observed in cases of extreme sedimentation: the pore structure of the external portion of the tube wall had a larger solid volume fraction and a smaller mean pore size compared to the internal portion of the tube. This tubular structure may allow preferential cell migration from the inner tube lumen towards the outer tube edge while blocking cell entrance into the tube through its outer surface due to increased scaffold relative density and decreased pore size.
Keywords: Collagen; Porosity; Gradient; Nerve regeneration; Myofibroblast; Migration;

Current research in the field of tissue engineering utilizes biomaterial scaffolds, cells, and growth factors for the creation of a functional, biologically active tissue. This study examined the effect of two commercially available, three-dimensional scaffolds, ultraporous β-tricalcium phosphate ceramics (β-TCP, Vitoss) and open-celled poly(lactic acid) foams (OPLA, Drilac), on the osteogenic differentiation potential of human dermal fibroblasts. Serum-free, chemically-defined medium containing the metabolic factor 1α,25-dihydroxyvitamin D3 was used to promote an osteogenic phenotype in these cells. Osteoblast differentiation was assessed using PCR and immunohistochemical methods to detect gene and protein expression for the osteoblast markers alkaline phosphatase, osteopontin, and osteocalcin. Dermal fibroblasts cultured on β-TCP scaffolds in chemically-defined medium with vitamin D3 exhibited up-regulated gene and protein expression compared to cells cultured on OPLA scaffolds. These results suggest that Vitoss (β-TCP) scaffolds seeded with dermal fibroblasts and maintained in chemically-defined medium with vitamin D3 are better suited for bone tissue engineering applications than Drilac (OPLA) foams.
Keywords: Bone tissue engineering; Calcium phosphate; Fibroblast; Osteoblast; Polylactic acid;

Tissue engineering of bone on micropatterned biodegradable polyester films by Halime Kenar; Gamze Torun Köse; Vasif Hasirci (885-895).
In this study, the effect of cell alignment on proliferation and phenotype expression of rat bone marrow derived osteoblasts on micropatterned (MP) PHBV and P(l/d,l)LA films with 27 μm wide parallel microgrooves was investigated. Immobilization of fibrinogen (Fb) on film surface by adsorption increased hydrophilicity, while covalent immobilization decreased it. Amount of Fb immobilized was significantly higher upon covalent bonding (153.1±42.4 μg Fb/cm2) than when adsorbed (10.0±3.3 μg Fb/cm2). It was observed that the presence of MP did not influence cell proliferation in the long run. Osteoblasts on MP films with adsorbed (MP Fba) and covalently immobilized Fb (MP Fbi) aligned parallel to the groove axis with mean deviation angles of 10.59±23.47 and 29.02±33.03°, respectively, while on tissue culture polystyrene (TCP), on unpatterned films (UNP) and on UNP with adsorbed Fb (UNP Fba) alignment with an arbitrary axis was much higher: 46.66±24.98, 48.72±31.19, 47.74±27.29 degrees, respectively. Fb-free MP films were not effective in cell alignment, and clumps were formed. Cell alignment achieved on MP Fba films did not influence cell proliferation, but increased differentiation, as shown by ALP activity per cell and the evenness and the amount of calcium phosphate deposition. It was concluded that orientation of cells was influential on their differentiation and also, MP cell carriers with chemical cues on their surfaces are important in improving tissue repair.
Keywords: Tissue engineering; Micropatterned surfaces; Biodegradable films; Cell guidance; Osteoblastic function;

Bone tissue engineering evaluation based on rat calvaria stromal cells cultured on modified PLGA scaffolds by Yu-Chun Wu; Shyh-Yu Shaw; Hong-Ru Lin; Tzer-Min Lee; Chyun-Yu Yang (896-904).
Using natural materials to coat the scaffolds used for tissue-engineered bone-repair techniques is expected to increase osteoblast adhesion to the scaffold and to express normal physiological function. To test this hypothesis, we therefore modified poly(dl-lactic-co-glycolic acid) (PLGA) substrate by coating it with natural biomaterial solutions of collagen, chitosan, or N-succinyl-chitosan, and then used these three combinations as scaffolds to evaluate their effects on osteoblast attachment, proliferation, and differentiation. The results demonstrated that the pore size of scaffolds ranging from 125–500 μm did not affect the osteoblast phenotype; however, the surface modification of the scaffolds coated with these natural biomaterials did. Collagen increased cell attachment and proliferation, but chitosan and N-succinyl-chitosan decreased them. Chitosan and N-succinyl-chitosan increased differentiation, but collagen decreased it. These results provide us a new strategy for modifying microenvironments to increase osteoblast adhesion, proliferation, and differentiation on PLGA scaffolds, a strategy that might be useful for tissue regeneration.
Keywords: Bone tissue engineering; Poly(lactic-co-glycolic acid); Chitosan; N-succinyl-chitosan; Collagen; Scaffold;

Architecture and properties of anisotropic polymer composite scaffolds for bone tissue engineering by Laurence Marcelle Mathieu; Thomas L. Mueller; Pierre-Etienne Bourban; Dominique P. Pioletti; Ralph Müller; Jan-Anders E. Månson (905-916).
Bone is a complex porous composite structure with specific characteristics such as viscoelasticity and anisotropy, both in morphology and mechanical properties. Bone defects are regularly filled with artificial tissue grafts, which should ideally have properties similar to those of natural bone. Open cell composite foams made of bioresorbable poly(l-lactic acid) (PLA) and ceramic fillers, hydroxyapatite (HA) or β-tricalcium phosphate (β-TCP), were processed by supercritical CO2 foaming. Their internal 3D-structure was then analysed by micro-computed tomography (μCT), which evidenced anisotropy in morphology with pores oriented in the foaming direction. Furthermore compressive tests demonstrated anisotropy in mechanical behaviour, with an axial modulus up to 1.5 times greater than the transverse modulus. Composite scaffolds also showed viscoelastic behaviour with increased modulus for higher strain rates. Such scaffolds prepared by gas foaming of polymer composite materials therefore possess suitable architecture and properties for bone tissue engineering applications.
Keywords: Composite; Scaffold; Compression; MicroCT; Anisotropy;

Adhesive/abrasive wear in ultra-high molecular weight polyethylene (UHMWPE) has been minimized by radiation cross-linking. Irradiation is typically followed by melting to eliminate residual free radicals that cause oxidative embrittlement. Irradiation and subsequent melting reduce the strength and fatigue resistance of the polymer. We determined the radiation dose dependence and decoupled the effects of post-irradiation melting on the crystallinity, mechanical properties and fatigue crack propagation resistance of room temperature irradiated UHMWPE from those of irradiation alone. Stiffness and yield strength, were largely not affected by increasing radiation dose but were affected by changes in crystallinity, whereas plastic properties, ultimate tensile strength and elongation at break, were dominated at different radiation dose ranges by changes in radiation dose or crystallinity. Fatigue crack propagation resistance was shown to decrease with increase in radiation dose and with decrease in crystalline content. Morphology of fracture surfaces revealed loss of ductility with increase in radiation dose and more detrimental effects on ductility at lower radiation doses after post-irradiation melting.
Keywords: Polyethylene; Arthroplasty; Plasticity; Electron beam; Fatigue; Cross-linking;

Fluoride modification effects on osteoblast behavior and bone formation at TiO2 grit-blasted c.p. titanium endosseous implants by Lyndon F. Cooper; Yongsheng Zhou; Jun Takebe; Juanli Guo; Armin Abron; Anders Holmén; Jan Eirik Ellingsen (926-936).
Increasing bone formation at endosseous titanium implants may be achieved by modification of topographically enhanced surfaces. The aim of this study was to determine the effect of fluoride ion modification of TiO2 grit -blasted, c.p. titanium implants on osteoblastic differentiation and interfacial bone formation by parallel in vitro and in vivo investigations. Human mesenchymal stem cells (Osiris Therapeutics, Inc.) were cultured on TiO2 grit -blasted c.p.titanium disks with and without fluoride ion modification. Cell adhesion, proliferation, and osteoblastic gene expression was measured by scanning electron microscopy, tritiated-thymidine uptake into insoluble DNA, and reverse transcription polymerase chain reaction detection of mRNAs encoding collagen 1, osteopontin, bone sialoprotein, osteocalcin and BMP-2. After 24 h, there were no differences in cell adhesion among the surfaces tested. Fluoride-treated surfaces supported greater proliferation and increased bone sialoprotein and BMP-2 expression. Additionally, 12 TiO2 grit-blasted and 12 fluoride ion modified implants were placed randomly into medial and distal osteotomies prepared in the tibia of 300 g Sprague Dawley rats. After 21 days, the tibiae were harvested and 100 μm ground sections were examined by backscatter scanning electron microscopy. The bone-to-implant contact formed at TiO2 grit-blasted and fluoride-treated versus TiO2 grit -blasted surfaces was 55.45% versus 34.21% ( p < 0.027 ), respectively. Fluoride ion modification of the TiO2 grit -blasted surface enhanced osteoblastic differentiation in vitro and interfacial bone formation in vivo. This parallel in vitro and in vivo investigation demonstrates that fluoride ion modification enhanced osteoblastic differentiation and interfacial bone formation. The mechanism(s) by which fluoride ion modification of c.p.titanium enhanced osteoblastic differentiation and osseointegration merit careful investigation.
Keywords: Osseointegration; Surface modification; Cell culture; Titanium; Endosseous implant; Stem cell;

A delivery strategy for rotenone microspheres in an animal model of Parkinson's disease by Jun Huang; Huaqing Liu; Wangwen Gu; Zhou Yan; Zhenghong Xu; Yongxin Yang; Xingzu Zhu; Yaping Li (937-946).
In order to study the pathogenesis of Parkinson's disease (PD), and explore therapeutic drug or approaches, the accurate animal model of PD with inexpensive, biocompatible and convenient administration was necessary. The aim of the present work was to investigate a delivery strategy for rotenone microspheres in an animal model of PD. The rotenone microspheres were prepared by solvent evaporation technique. The rotenone microspheres showed high entrapment efficiency (97.4±2.2%) with particle size about 100 μm. In vitro release of rotenone microspheres demonstrated different profiles from medium with different pH or concentration of isopropyl alcohol. The most consistent medium with in vivo rotenone levels in rat plasma was PBS (pH 5.8) with 20% isopropyl alcohol, and the cumulated release amount of rotenone over 30 days was 95.4% in it. The rotenone microspheres (90 mg/kg) produced typical PD symptoms in rats, for example, the cataleptic behavior test demonstrated a obviously prolonged descent latency compared with control animals after administration, and the tyrosine hydroxylase (TH) immunohistochemistry tests showed typical histological evidence of selective degeneration of the nigrostriatal dopaminergic system (striatum and substantia nigra) in rotenone microspheres-treated rats. In addition, this delivery system for rotenone model showed many noticeable advantages such as inexpensive, biocompatible and expedient administration by direct subcutaneous injection. This information suggested that rotenone microspheres as a delivery strategy for setting up an ideal animal model of PD was feasible.
Keywords: Parkinson's disease; Rotenone; Microspheres; PLGA; Drug delivery;