Biomaterials (v.31, #36)

Craniofacial reconstruction would benefit from a degradable adhesive capable of holding bone fragments in three-dimensional alignment and gradually being replaced by new bone without loss of alignment or volume changes. Modeled after a natural adhesive secreted by the sandcastle worm, we studied the biocompatibility of adhesive complex coacervates in vitro and in vivo with two different rat calvarial models. We found that the adhesive was non-cytotoxic and supported the attachment, spreading, and migration of a commonly used osteoblastic cell line over the course of several days. In animal studies we found that the adhesive was capable of maintaining three-dimensional bone alignment in freely moving rats over a 12 week indwelling period. Histological evidence indicated that the adhesive was gradually resorbed and replaced by new bone that became lamellar across the defect without loss of alignment, changes in volume, or changes in the adjacent uninjured bone. The presence of inflammatory cells was consistent with what has been reported with other craniofacial fixation methods including metal plates, screws, tacks, calcium phosphate cements and cyanoacrylate adhesives. Collectively, the results suggest that the new bioadhesive formulation is degradable, osteoconductive and appears suitable for use in the reconstruction of craniofacial fractures.
Keywords: Bone repair; Osseointegration; Osteoconduction; Biocompatibility; Foreign body response;

Cell–cell signaling in co-cultures of macrophages and fibroblasts by Dolly J. Holt; Lisa M. Chamberlain; David W. Grainger (9382-9394).
The foreign body response (FBR) comprises a general, ubiquitous host tissue-based reaction to implanted materials. In vitro cell-based models are frequently employed to study FBR mechanisms involving cell signaling responses to materials. However, these models often study only one cell type, identify only limited signals, and cannot accurately represent the complexity of in vivo inflammatory signaling. To address this issue, a cell co-culture system involving two primary effector cells of the FBR, macrophages and fibroblasts, was employed. Cell–cell signaling systems were monitored between these cell types, including long-term 1) culture of one cell type in conditioned media from the other cell type, 2) non-contacting cell co-cultures (paracrine signaling), and 3) contact co-cultures (juxtacrine signaling) of primary- and secondary-derived cells. Cell culture media and cell images were collected on Days 1, 2, 3, 7, 14, and 21 and changes in soluble protein secretion, cellular behavior, and morphology were assessed. Primary- and secondary-derived cells responded uniquely during each signaling scenario and to one another. In general higher in vitro fidelity to FBR-like responses was found in primary cell co-cultures compared to their mono-cultures and all secondary cell cultures.
Keywords: Foreign body response; Co-culture; In vitro signaling; Cytokines; Fibroblast; Macrophage;

Recombinant self-assembling peptides as biomaterials for tissue engineering by Stuart Kyle; Amalia Aggeli; Eileen Ingham; Michael J. McPherson (9395-9405).
Synthetic nanostructures based on self-assembling systems that aim to mimic natural extracellular matrix are now being used as substrates in tissue engineering applications. Peptides are excellent starting materials for the self-assembly process as they can be readily synthesised both chemically and biologically. P11-4 is an 11 amino acid peptide that undergoes triggered self-assembly to form a self-supporting hydrogel. It exists as unimers of random coil conformations in water above pH 7.5 but at low pH adopts an antiparallel β-sheet conformation. It also self-assembles under physiological conditions in a concentration-dependent manner. Here we describe an unimer P11-4 production system and the use of a simple site-directed mutagenesis approach to generate a series of other P11-family peptide expression vectors. We have developed an efficient purification strategy for these peptide biomaterials using a simple procedure involving chemical cleavage with cyanogen bromide then repeated filtration, lyophilisation and wash steps. We report peptide-fusion protein yields of ca. 4.64 g/L and we believe the highest reported recovery of a recombinant self-assembling peptide at 203 mg/L of pure recombinant P11-4. This peptide forms a self-supporting hydrogel under physiological conditions with essentially identical physico-chemical properties to the chemically synthesised peptide. Critically it also displays excellent cytocompatibility when tested with primary human dermal fibroblasts. This study demonstrates that high levels of a series of recombinant self-assembling peptides can be purified using a simple process for applications as scaffolds in tissue engineering.
Keywords: Recombinant expression; Self-assembly; Peptide; Hydrogel; Cytocompatibility;

In vivo ectopic chondrogenesis of BMSCs directed by mature chondrocytes by Xia Liu; Hengyun Sun; Dan Yan; Lu Zhang; Xiaojie Lv; Tianyi Liu; Wenjie Zhang; Wei Liu; Yilin Cao; Guangdong Zhou (9406-9414).
In vivo niche plays an important role in determining the fate of exogenously implanted stem cells. Due to the lack of a proper chondrogenic niche, stable ectopic chondrogenesis of mesenchymal stem cells (MSCs) in subcutaneous environments remains a great challenge. The clinical application of MSC-regenerated cartilage in repairing defects in subcutaneous cartilage such as nasal or auricular cartilage is thus severely limited. The creation of a chondrogenic niche in subcutaneous environments is the key to solving this problem. The current study demonstrates that bone marrow stromal cells (BMSCs) could form cartilage-like tissue in a subcutaneous environment when co-transplanted with articular chondrocytes, indicating that chondrocytes could create a chondrogenic niche to direct chondrogenesis of BMSCs. Then, a series of in vitro co-culture models revealed that it was the secretion of soluble factors by chondrocytes but not cell–cell contact that provided the chondrogenic signals. The subsequent studies further demonstrated that multiple factors currently used for chondroinduction (including TGF-β1, IGF-1 and BMP-2) were present in the supernatant of chondrocyte-engineered constructs. Furthermore, all of these factors were required for initiating chondrogenic differentiation and fulfilled their roles in a coordinated way. These results suggest that paracrine signaling of soluble chondrogenic factors provided by chondrocytes was an important mechanism in directing the in vivo ectopic chondrogenesis of BMSCs. The multiple co-culture systems established in this study provide new methods for directing committed differentiation of stem cells as well as new in vitro models for studying differentiation mechanism of stem cells determined by a tissue-specific niche.
Keywords: Bone marrow stromal cells; Chondrogenesis; Chondrocytes; Co-culture; Cytokines;

The discrimination of type I and type II collagen and the label-free imaging of engineered cartilage tissue by Ping-Jung Su; Wei-Liang Chen; Tsung-Hsien Li; Chen-Kuan Chou; Te-Hsuen Chen; Yi-Yun Ho; Chi-Hsiu Huang; Shwu-Jen Chang; Yi-You Huang; Hsuan-Shu Lee; Chen-Yuan Dong (9415-9421).
Using excitation polarization-resolved second harmonic generation (SHG) microscopy, we measured SHG intensity as a function of the excitation polarization angle for type I and type II collagens. We determined the second order susceptibility (χ(2)) tensor ratios of type I and II collagens at each pixel, and displayed the results as images. We found that the χ(2) tensor ratios can be used to distinguish the two types of collagen. In particular, we obtained χ zzz/χ zxx = 1.40 ± 0.04 and χ xzx/χ zxx = 0.53 ± 0.10 for type I collagen from rat tail tendon, and χ zzz/χ zxx = 1.14 ± 0.09 and χ xzx/χ zxx = 0.29 ± 0.11 for type II collagen from rat trachea cartilage. We also applied this methodology on the label-free imaging of engineered cartilage tissue which produces type I and II collagen simultaneously. By displaying the χ(2) tensor ratios in the image format, the variation in the χ(2) tensor ratios can be used as a contrast mechanism for distinguishing type I and II collagens.
Keywords: Second harmonic generation (SHG); Susceptibility tensor analysis; Second order susceptibility microscopy; Collagen; Cartilage;

Phosphorylation-dependent mineral-type specificity for apatite-binding peptide sequences by William N. Addison; Sharon J. Miller; Janani Ramaswamy; Ahmad Mansouri; David H. Kohn; Marc D. McKee (9422-9430).
Apatite-binding peptides discovered by phage display provide an alternative design method for creating functional biomaterials for bone and tooth tissue repair. A limitation of this approach is the absence of display peptide phosphorylation – a post-translational modification important to mineral-binding proteins. To refine the material specificity of a recently identified apatite-binding peptide, and to determine critical design parameters (net charge, charge distribution, amino acid sequence and composition) controlling peptide affinity for mineral, we investigated the effects of phosphorylation and sequence scrambling on peptide adsorption to four different apatites (bone-like mineral, and three types of apatite containing initially 0, 5.6 and 10.5% carbonate). Phosphorylation of the VTKHLNQISQSY peptide (VTK peptide) led to a 10-fold increase in peptide adsorption (compared to nonphosphorylated peptide) to bone-like mineral, and a 2-fold increase in adsorption to the carbonated apatite, but there was no effect of phosphorylation on peptide affinity to pure hydroxyapatite (without carbonate). Sequence scrambling of the nonphosphorylated VTK peptide enhanced its specificity for the bone-like mineral, but scrambled phosphorylated VTK peptide (pVTK) did not significantly alter mineral-binding suggesting that despite the importance of sequence order and/or charge distribution to mineral-binding, the enhanced binding after phosphorylation exceeds any further enhancement by altered sequence order. Osteoblast culture mineralization was dose-dependently inhibited by pVTK and to a significantly lesser extent by scrambled pVTK, while the nonphosphorylated and scrambled forms had no effect, indicating that inhibition of osteoblast mineralization is dependent on both peptide sequence and charge. Computational modeling of peptide–mineral interactions indicated a favorable change in binding energy upon phosphorylation that was unaffected by scrambling. In conclusion, phosphorylation of serine residues increases peptide specificity for bone-like mineral, whose adsorption is determined primarily by sequence composition and net charge as opposed to sequence order. However, sequence order in addition to net charge modulates the mineralization of osteoblast cultures. The ability of such peptides to inhibit mineralization has potential utility in the management of pathologic calcification.
Keywords: Phage display; Bone mineral; Peptides; Apatite; Osteoblast; Phosphorylation;

The endothelialization of prosthetic scaffolds is considered to be an effective strategy to improve the effectiveness of small-diameter vascular grafts. We report the development of a nanofibrous scaffold that has a polymeric core and a shell mimicking mussel adhesive for enhanced attachment, proliferation, and phenotypic maintenance of human endothelial cells. Polycaprolactone (PCL) was chosen as a core material because of its good biodegradability and mechanical properties suitable for tissue engineering. PCL was electrospun into nanofibers with a diameter of approximately 700 nm and then coated with poly(dopamine) (PDA) to functionalize the surface of PCL nanofibers with numerous catechol moieties similar to mussel adhesives in nature. The formation of a PDA ad-layer was analyzed using multiple techniques, including scanning electron microscopy, Raman spectroscopy, and water contact angle measurements. When PDA-coated PCL nanofibers were compared to unmodified and gelatin-coated nanofibers, human umbilical vein endothelial cells (HUVECs) exhibited highly enhanced adhesion and viability, increased stress fiber formation, and positive expression of endothelial cell markers (e.g., PECAM-1 and vWF).
Keywords: Endothelial cells; Nanofibers; Electrospinning; Mussel adhesives; Poly(dopamine); Vascular tissue scaffold;

Efficacy of hESC-MSCs in knitted silk-collagen scaffold for tendon tissue engineering and their roles by Jia Lin Chen; Zi Yin; Wei Liang Shen; Xiao Chen; Boon Chin Heng; Xiao Hui Zou; Hong Wei Ouyang (9438-9451).
Human embryonic stem cells (hESC) and their differentiated progenies are an attractive cell source for transplantation therapy and tissue engineering. Nevertheless, the utility of these cells for tendon tissue engineering has not yet been adequately explored. This study incorporated hESC-derived mesenchymal stem cells (hESC-MSCs) within a knitted silk-collagen sponge scaffold, and assessed the efficacy of this tissue-engineered construct in promoting tendon regeneration. When subjected to mechanical stimulation in vitro, hESC-MSCs exhibited tenocyte-like morphology and positively expressed tendon-related gene markers (e.g. Collagen type I & III, Epha4 and Scleraxis), as well as other mechano-sensory structures and molecules (cilia, integrins and myosin). In ectopic transplantation, the tissue-engineered tendon under in vivo mechanical stimulus displayed more regularly aligned cells and larger collagen fibers. This in turn resulted in enhanced tendon regeneration in situ, as evidenced by better histological scores and superior mechanical performance characteristics. Furthermore, cell labeling and extracellular matrix expression assays demonstrated that the transplanted hESC-MSCs not only contributed directly to tendon regeneration, but also exerted an environment-modifying effect on the implantation site in situ. Hence, tissue-engineered tendon can be successfully fabricated through seeding of hESC-MSCs within a knitted silk-collagen sponge scaffold followed by mechanical stimulation.
Keywords: hESC-MSCs; Silk scaffold; Mechanical stress; Engineered tendon; Tendon regeneration;

Osteogenesis and angiogenesis of tissue-engineered bone constructed by prevascularized β-tricalcium phosphate scaffold and mesenchymal stem cells by Le Wang; Hongbin Fan; Zhi-Yong Zhang; Ai-Ju Lou; Guo-Xian Pei; Shan Jiang; Tian-Wang Mu; Jun-Jun Qin; Si-Yuan Chen; Dan Jin (9452-9461).
Although vascularized tissue-engineered bone grafts (TEBG) have been generated ectopically in several studies, the use of prevascularized TEBG for segmental bone defect repair are rarely reported. In current study, we investigated the efficacy of prevascularized TEBG for segmental defect repair. The segmental defects of 15 mm in length were created in the femurs of rabbits bilaterally. In treatment group, the osteotomy site of femur was implanted with prevascularized TEBG, which is generated by seeding mesenchymal stem cells (MSCs) into β-TCP scaffold, and prevascularization with the insertion of femoral vascular bundle into the side groove of scaffold; whereas in the control group, only MSC mediated scaffolds (TEBG) were implanted. The new bone formation and vascularization were investigated and furthermore, the expression of endogenous vascular endothelial growth factor (VEGF) which might express during defect healing was evaluated, as well. At 4, 8, and 12 weeks postoperatively, the treatment of prevascularized TEBG led to significantly higher volume of regenerated bone and larger amount of capillary infiltration compared to non-vascularized TEBG. The expression of VEGF in mRNA and protein levels increased with implantation time and peaked at 4 weeks postoperatively, followed by a slow decrease, however, treatment group expressed a significant higher level of VEGF than control group throughout the whole study. In conclusion, this study demonstrated that prevascularized TEBG by insertion of vascular bundle could significantly promote the new bone regeneration and vascularization compared to non-vascularized TEBG, which could be partially explained by the up-regulated expression of VEGF.
Keywords: Tissue engineering; Bone defect; Vascularization; Tricalcium phosphate;

The role of the interplay between polymer architecture and bacterial surface properties on the microbial adhesion to polyoxazoline-based ultrathin films by Bidhari Pidhatika; Jens Möller; Edmondo M. Benetti; Rupert Konradi; Ekaterina Rakhmatullina; Andreas Mühlebach; Ralf Zimmermann; Carsten Werner; Viola Vogel; Marcus Textor (9462-9472).
Surface platforms were engineered from poly(l-lysine)-graft-poly(2-methyl-2-oxazoline) (PLL-g-PMOXA) copolymers to study the mechanisms involved in the non-specific adhesion of Escherichia coli (E. coli) bacteria. Copolymers with three different grafting densities α (PMOXA chains/Lysine residue of 0.09, 0.33 and 0.56) were synthesized and assembled on niobia (Nb2O5) surfaces. PLL-modified and bare niobia surfaces served as controls. To evaluate the impact of fimbriae expression on the bacterial adhesion, the surfaces were exposed to genetically engineered E. coli strains either lacking, or constitutively expressing type 1 fimbriae. The bacterial adhesion was strongly influenced by the presence of bacterial fimbriae. Non-fimbriated bacteria behaved like hard, charged particles whose adhesion was dependent on surface charge and ionic strength of the media. In contrast, bacteria expressing type 1 fimbriae adhered to the substrates independent of surface charge and ionic strength, and adhesion was mediated by non-specific van der Waals and hydrophobic interactions of the proteins at the fimbrial tip. Adsorbed polymer mass, average surface density of the PMOXA chains, and thickness of the copolymer films were quantified by optical waveguide lightmode spectroscopy (OWLS) and variable-angle spectroscopic ellipsometry (VASE), whereas the lateral homogeneity was probed by time-of-flight secondary ion mass spectrometry (ToF-SIMS). Streaming current measurements provided information on the charge formation of the polymer-coated and the bare niobia surfaces. The adhesion of both bacterial strains could be efficiently inhibited by the copolymer film only with a grafting density of 0.33 characterized by the highest PMOXA chain surface density and a surface potential close to zero.
Keywords: Poly(2-methyl-2-oxazoline); Nonfouling surfaces; Bacteria adhesion; Escherichia coli (E. coli); Fimbriae; Polymer brush; Ionic strength; Surface charge; Streaming current; Non-specific adhesion;

The influence of dipalmitoyl phosphatidylserine on phase behaviour of and cellular response to lyotropic liquid crystalline dispersions by Hsin-Hui Shen; Jonathan G. Crowston; Florian Huber; Simon Saubern; Keith M. McLean; Patrick G. Hartley (9473-9481).
Lyotropic liquid crystalline nanoparticles (cubosomes) have the potential to act as amphiphilic scaffolds for the presentation of lipids and subsequent application in, for example, bioseparations and therapeutic delivery. In this work we have formulated lyotropic liquid crystalline systems based on the synthetic amphiphile 1,2,3-trihydroxy-3,7,11,15-tetramethylhexadecane (phytantriol) and containing the lipid dipalmitoyl phosphatidylserine (DPPS). We have prepared a range of DPPS-containing phytantriol cubosome formulations and characterized them using Small Angle X-ray Scattering and Cryo-transmission electron microscopy. These techniques show that increased DPPS content induces marked changes in lyotropic liquid crystalline phase behaviour, characterized by changes in crystallographic dimensions and increases in vesicle content. Furthermore, in vitro cell culture studies indicate that these changes correlate with lipid/surfactant cellular uptake and cytotoxicity. A model cell membrane based on a surface supported phospholipid bilayer was used to gain insights into cubosome–bilayer interactions using Quartz Crystal Microgravimetry. The data show that mass uptake at the supported bilayer increased with DPPS content. We propose that the cytotoxicity of the DPPS-containing dispersions results from changes in lipid/surfactant phase behaviour and the preferential attachment and fusion of vesicles at the cell membrane.
Keywords: Phytantriol; Small angle X-ray scattering; Phase behaviour; Cubosomes; Cytotoxicity; Confocal microscopy;

Gadonanotubes as magnetic nanolabels for stem cell detection by Lesa A. Tran; Ramkumar Krishnamurthy; Raja Muthupillai; Maria da Graça Cabreira-Hansen; James T. Willerson; Emerson C. Perin; Lon J. Wilson (9482-9491).
Stem cell-based therapies have emerged as a promising approach in regenerative medicine. In the development of such therapies, the demand for imaging technologies that permit the noninvasive monitoring of transplanted stem cells in vivo is growing. Here, we report the performance of gadolinium-containing carbon nanocapsules, or gadonanotubes (GNTs), as a new T1-weighted magnetic resonance imaging (MRI) intracellular labeling agent for pig bone marrow-derived mesenchymal stem cells (MSCs). Without the use of a transfection agent, micromolar concentrations of GNTs can deliver up to 109 Gd3 + ions per cell without compromising cell viability, differentiation potential, proliferation pattern, and phenotype. Imaging 10 × 106 GNT-labeled MSCs demonstrates a nearly two-fold reduction in T1 relaxation time when compared to unlabeled MSCs at 1.5 T in a clinical MRI scanner, which easily permits the discrimination of GNT-labeled MSCs in a T1-weighted MR image. It is anticipated that GNTs will allow in vivo tracking of GNT-labeled MSCs, as well as other mammalian cell types, by T1-weighted imaging with greater efficacy than other current technologies now allow.
Keywords: Stem cell; MRI (magnetic resonance imaging); Nanoparticle; Cell labeling; Carbon nanotubes; Gadonanotubes (GNTs);

Two-photon microscopy powered by a femtosecond laser is a promising tool for luminescence imaging and localized microsurgery of cancers. However, the high energy required to destruct cells limits its medical applications. In this work, gold nanorods were conjugated with transferrin for efficient targeting, two-photon luminescence imaging and enhanced microsurgery of cancer cells. Due to the large two-photon excitation cross section of gold nanorods, gold nanorods are a hundred times more efficient than Fluorescein isothiocyanate (FITC), a common molecular dye, in three-dimensional imaging of cancer cells. The enhanced light absorption and energy conversion by gold nanorods enable treatment of cells with energy fluences two orders of magnitude below that in the absence of gold nanorods. By manipulating the energy fluence, apoptosis of cancer cells has been achieved. At a same power density, the energy fluence for apoptosis induction is less than 20% of that for necrosis. Gold nanorods-enhanced luminescence imaging coupled with apoptosis induction of cancer cells provides a medically safe femtosecond laser-based imaging and microsurgery system for cancer diagnosis and treatment.
Keywords: Apoptosis; Gold; Nanoparticle; Surface modification; Laser ablation;

Implant-assisted targeting of magnetic particles under the influence of an external magnetic field has previously been verified through mathematical modeling, in vitro studies, and in vivo studies on rat carotid arteries as a feasible method for localized drug delivery. The present study focuses on the development of nanoparticles for the treatment of in-stent thrombosis. Magnetic nanoparticles in the size-range 10–30 nm were synthesized in a one-pot procedure by precipitation of ferrous hydroxide followed by oxidation to magnetite. The nanoparticles were silanized with tetraethyl orthosilicate in the presence of triethylene glycol and/or polyethylene glycol. The surface coated magnetite nanoparticles were activated with either N-hydroxysulfosuccinimide or tresyl chloride for covalent immobilization of tissue plasminogen activator (tPA). Hysteresis loops showed saturation magnetizations of 55.8, 44.1, and 43.0 emu/g for the naked nanoparticles, the surface coated nanoparticles, and the tPA-nanoparticle conjugates, respectively. The hemolytic activity of the nanoparticles in blood was negligible. An initial in vivo biocompatibility test in pig, carried out by intravascular injection of the nanoparticles in a stented brachial artery, showed no short-term adverse effects. In vitro evaluation in a flow-through model proved that the nanoparticles were captured efficiently to the surface of a ferromagnetic coiled wire at the fluid velocities typical for human arteries. A preliminary test of the tPA-nanoparticle conjugates in a pig model suggested that the conjugates may be used for treatment of in-stent thrombosis in coronary arteries.
Keywords: Enzyme immobilization; Implant-assisted magnetic drug targeting; In-stent thrombosis; In vivo pig model; Magnetic nanoparticles; Thrombolysis;

Serum heat inactivation affects protein corona composition and nanoparticle uptake by Anna Lesniak; Abigail Campbell; Marco P. Monopoli; Iseult Lynch; Anna Salvati; Kenneth A. Dawson (9511-9518).
Nanoparticles are of an appropriate size to interact with cells, and are likely to use a range of cellular machinery for internalisation and trafficking to various sub-cellular compartments. It is now understood that once in contact with biological fluids, the nanoparticle surface gets covered by a highly specific layer of proteins, forming the nanoparticle protein corona. This protein layer is stable for times longer than the typical time scale of nanoparticle import, and thus can impact on particle uptake and trafficking inside the cells. In this work, the effect of the corona composition on nanoparticle uptake has been investigated, by studying the impact of serum heat inactivation and complement depletion on the load of nanoparticles accumulated inside the cell. For the same material and nanoparticle size, cellular uptake was found to be significantly different when the nanoparticles were dispersed in medium where the serum was heat inactivated or not heat inactivated, even for non-specialized cells, suggesting that different sera can lead to different nanoparticle doses. The fact that uptake was correlated with the amount of protein bound into the nanoparticle corona suggests the need for commonly agreed dispersion protocols for in vitro nanoparticle–cell studies.
Keywords: Nanoparticles; Complement; Protein adsorption; Flow cytometry; Confocal microscopy;

Antimicrobial peptides on calcium phosphate-coated titanium for the prevention of implant-associated infections by Mehdi Kazemzadeh-Narbat; Jason Kindrachuk; Ke Duan; Håvard Jenssen; Robert E.W. Hancock; Rizhi Wang (9519-9526).
Prevention of implant-associated infections has been one of the main challenges in orthopaedic surgery. This challenge is further complicated by the concern over the development of antibiotic resistance as a result of using traditional antibiotics for infection prophylaxis. The objective of this study was to develop a technique that enables the loading and local delivery of a unique group of cationic antimicrobial peptides (AMP) through implant surfaces. A thin layer of micro-porous calcium phosphate (CaP) coating was processed by electrolytic deposition onto the surface of titanium as the drug carrier. The broad spectrum AMP Tet213 (KRWWKWWRRC) was selected and loaded onto the CaP coating. SEM, XRD and FTIR analyses confirmed the CaP coating to be micro-porous octacalcium phosphate. By using a luminescence spectrometer technique, it was demonstrated that a 7 μm thick porous CaP coating could load up to 9 μg of AMP/cm2 using a simple soaking technique. The drug-loaded CaP coating (CaP-Tet213) was not cytotoxic for MG-63 osteoblast-like cells. The CaP-Tet213 implants had antimicrobial activity against both Gram-positive (Staphylococcus aureus) and Gram-negative (Pseudomonas aeruginosa) bacteria with 106-fold reductions of both bacterial strains within 30 min as assessed by measuring colony-forming units (CFU). Repeated CFU assays on the same CaP-Tet213 specimen demonstrated retention of antimicrobial activity by the CaP-Tet213 surfaces through four test cycles. The susceptibility of bacteria to the CaP-Tet213 surfaces was also evaluated by assessing the inhibition of luminescence of P. aeruginosa containing a luxCDABE cassette at 4 h and 24 h with ∼92% and ∼77% inhibition of luminescence, respectively. It was demonstrated that CaP-Tet213 was a more efficient antimicrobial coating than CaP-MX226, CaP-hLF1-11 or CaP-tobramycin following incubation of CaP implants with equimolar concentrations of Tet213, the commercially developed antimicrobial peptide MX-226, hLF1-11 or tobramycin. A device coated with CaP-Tet213 could be a potential solution for the prevention of the peri-implant infection in orthopaedics.
Keywords: Calcium phosphate coating; Electrolytic deposition; Antimicrobial peptide; Titanium; Infection; Orthopaedic implants;

The effect of protein structure on their controlled release from an injectable peptide hydrogel by Monica C. Branco; Darrin J. Pochan; Norman J. Wagner; Joel P. Schneider (9527-9534).
Hydrogel materials are promising vehicles for the delivery of protein therapeutics. Proteins can impart physical interactions, both steric and electrostatic in nature, that influence their release from a given gel network. Here, model proteins of varying hydrodynamic diameter and charge are directly encapsulated and their release studied from electropositive fibrillar hydrogels prepared from the self-assembling peptide, MAX8. Hydrogelation of MAX8 can be triggered in the presence of proteins for their direct encapsulation with neither effect on protein structure nor the hydrogel’s mechanical properties. Bulk release of the encapsulated proteins from the hydrogels was assessed for a month time period at 37 °C before and after syringe delivery of the loaded gels to determine the influence of the protein structure on release. Release of positively charged and neutral proteins was largely governed by the sterics imposed by the network. Conversely, negatively charged proteins interacted strongly with the positively charged fibrillar network, greatly restricting their release to <10% of the initial protein load. Partition and retention studies indicated that electrostatic interactions dictate the amount of protein available for release. Importantly, when protein encapsulated gels were delivered via syringe, the release profiles of the macromolecules show the similar trends as those observed for non-sheared gels. This study demonstrates that proteins can be directly encapsulated in self assembled MAX8 hydrogels, which can then be syringe delivered to a site where subsequent release is controlled by protein structure.
Keywords: Peptide; Hydrogel; Delivery; Protein; Syringe;

A synthetic cantharidin analog for the enhancement of doxorubicin suppression of stem cell-derived aggressive sarcoma by Chao Zhang; Yi Peng; Fengchao Wang; Xu Tan; Nan Liu; Song Fan; Dechun Wang; Lilong Zhang; Dengqun Liu; Tao Wang; Shaojun Wang; Yue Zhou; Yongping Su; Tianmin Cheng; Zhengping Zhuang; Chunmeng Shi (9535-9543).
Failure to cure many cancers once they are disseminated has been attributed to the presence of resistant cancer stem cells. Cantharidin, a natural compound isolated from the beetles and other insects has been traditionally used as anticancer agent, but limited by its significant toxicity. It has shown that cantharidin can force cancer cells prematurely into cell cycle and subsequently induce apoptotic cell death through the inhibition of protein phosphatase 2A (PP2A). In this study, we showed that a synthesized analog of cantharidin, LB1, with significant PP2A inhibition activity but without apparent toxicity, greatly enhanced the effectiveness of the standard anti-sarcoma chemotherapeutic agent, doxorubicin (DOX), in the xenograft growth inhibition and lung metastases prevention of an aggressive sarcoma derived from transformed mesenchymal stem cells in syngeneic rats. We report here on the possibility of, pharmacologic inhibition of PP2A with low toxicity cantharidin derivatives may be a useful strategy to enhance the effectiveness of DNA-damaged chemotherapeutic drugs against stem cell-derived cancer.
Keywords: Cantharidin derivative; PP2A inhibitor; Doxorubicin; Sarcoma; Metastasis; Enhanced chemotherapy;

Entrapment and release of drugs by a strict “on-off” mechanism in pullulan microspheres with pendant thermosensitive groups by Gheorghe Fundueanu; Marieta Constantin; Ionela Oanea; Valeria Harabagiu; Paolo Ascenzi; Bogdan C. Simionescu (9544-9553).
Here, we report a new method to predict the appropriate size of drugs which can be entrapped in and released from a hydrogel with pendant thermosensitive units by a strict “on-off” mechanism. Moreover, the valve-type action of the thermosensitive arms has been investigated. Inverse size exclusion chromatography (ISEC) and environmental scanning electron microscopy (ESEM) have been used to characterize the extension and collapse of the pendant thermosensitive units, below and above the lower critical solution temperature (LCST) under physiological conditions, confirming the hypothesis postulated by the “arid” theoretical models.The functionalized pullulan (Pul) microspheres, here prepared, were coupled with thermoresponsive oligomers by reaction between the –NH2 end-group of oligomers and chlorine present on Pul microspheres. The Pul microspheres with temperature sensitive moieties were packed in a glass column and the elution volume of standard molecule with well-known molecular weights (radius of gyration) was determined below and above the LCST. FITC-Dextran 4000 diffused through the pores of Pul microspheres with short thermosensitive arms (Mw = 1500 g/mol) both below and above the LCST of the thermosensitive units. In contrast, Pul microspheres with long thermosensitive arms (Mw = 3300 g/mol) allowed the diffusion of FITC-Dextran 4000 only above the LCST of the thermosensitive units. Indeed, the long thermosensitive arms are extended below the LCST and FITC-Dextran 4000 is completely excluded from the pores. The loading/release profile of this model molecule follows an “on-off” mechanism, confirming the results obtained by ISEC. ESEM was used as a new technique, taking images of the surface of the thermosensitive pullulan microspheres in their natural swollen state, with no prior specimen preparation, below and above the LCST. The low toxicity of pullulan microspheres observed below and above the LCST of thermosensitive units at high concentrations (10 mg/ml) recommends their potential use for controlled drug delivery applications.
Keywords: Pullulan; Thermoresponsive polymers; “on-off” release mechanism; Drug delivery;

A doxycycline loaded, controlled-release, biodegradable fiber for the treatment of aortic aneurysms by A. Yamawaki-Ogata; R. Hashizume; M. Satake; H. Kaneko; S. Mizutani; T. Moritan; Y. Ueda; Y. Narita (9554-9564).
The pathogenesis of aortic aneurysm (AA) is characterized by degradation of extracellular matrix with increased matrix metalloproteinases (MMPs) and inflammatory reaction. Doxycycline (DOXY) has been reported to control the extension of AA by regulation of MMP. However, systemic administration may cause adverse side effects. In this study, we demonstrated the possibility of local administration of DOXY controlled-release biodegradable fiber (DCRBF) for AA in mice. DCRBF was fabricated by biodegradable polymer (polylactic acid; PLA) mixed with DOXY using an electrospinning technique. DCRBF was cocultured with SMCs, macrophages and aortic tissue, and placed on an abdominal aortic aneurysm which induced apolipoprotein E-deficient mice. We evaluated gene and protein expression of proteases, elastin and inflammatory markers. In the presence of DCRBF, MMP-12 was significantly decreased, TGF-β1 and Lox were significantly increased in SMC gene expression, MMP-9 and -12 significantly decreased gene expression of macrophages. The DCRBF preserved elastin content and decreased MMP-2 and -9 in aortic tissue. In addition, IGF-1 and TIMP-1 were significantly increased and IL-6 and TNF-α were significantly decreased with DCRBF in vivo. In conclusion, our results suggested that local administration of DCRBF may become a promising alternative therapeutic strategy for AA.
Keywords: Aortic aneurysm; Doxycycline; Drug delivery system; ECM; Elastin; Matrix metalloproteinase;

Long-term stability of cell micropatterns on poly((3-(methacryloylamino)propyl)-dimethyl(3-sulfopropyl)ammonium hydroxide)-patterned silicon oxide surfaces by Woo Kyung Cho; Bokyung Kong; Hyung Ju Park; Jinkyu Kim; Won Chegal; Joon Sig Choi; Insung S. Choi (9565-9574).
In this work, we compared the long-term stability and integrity of cell patterns on newly reported, zwitterionic poly((3-(methacryloylamino)propyl)dimethyl(3-sulfopropyl)ammonium hydroxide) (poly(MPDSAH)) films with those on widely used, poly(poly(ethylene glycol) methyl ether methacrylate) (poly(PEGMEMA)) ones. The micropatterns of both polymers were formed on a silicon oxide surface by a combination of micropattern generation of a photoresist, vapor deposition of a silane-based polymerization initiator, and surface-initiated, atom transfer radical polymerization (SI-ATRP) of each monomer, MPDSAH or PEGMEMA. The successful formation of the silane initiator SAMs, and poly(MPDSAH) and poly(PEGMEMA) micropatterns was confirmed by X-ray photoelectron spectroscopy (XPS) and imaging ellipsometry. Onto each substrate patterned with poly(MPDSAH) or poly(PEGMEMA), NIH 3T3 fibroblast cells were seeded, and the cell micropatterns were generated by the selective adhesion of cells on the cell-adhesive region of the patterned surfaces. The cell pattern formed on the poly(MPDSAH)-patterned surface was observed to have a superior ability of finely maintaining its original, line-shaped structure up to for 20 days, when compared with the cell pattern formed on the poly(PEGMEMA)-patterned surface. In order to verify the relationship between the integrity of the cell micropatterns and the stability of the underlying non-biofouling polymer layers, we also investigated the long-term stability of the polymer films themselves, immersed in the cell culture media, for one month, in the aid of ellipsometry, contact goniometry, and XPS.
Keywords: Micropatterning; Surface modification; Cell adhesion; anti-Adhesion; Polymerization;

Recombinant spider silk as matrices for cell culture by Mona Widhe; Helena Bysell; Sara Nystedt; Ingrid Schenning; Martin Malmsten; Jan Johansson; Anna Rising; My Hedhammar (9575-9585).
The recombinant miniature spider silk protein, 4RepCT, was used to fabricate film, foam, fiber and mesh matrices of different dimensionality, microstructure and nanotopography. These matrices were evaluated regarding their suitability for cell culturing. Human primary fibroblasts attached to and grew well on all matrix types, also in the absence of serum proteins or other animal-derived additives. The highest cell counts were obtained on matrices combining film and fiber/mesh. The cells showed an elongated shape that followed the structure of the matrices and exhibited prominent actin filaments. Moreover, the fibroblasts produced, secreted and deposited collagen type I onto the matrices. These results, together with findings of the matrices being mechanically robust, hold promise not only for in vitro cell culturing, but also for tissue engineering applications.
Keywords: Biomaterial; Cytocompatibility; Collagen production; Fibroblasts; Serum free; Tissue engineering;

Hydroxyapatite (HA) is a widely-used biomaterial for bone repair due to its high degree of osteoconductivity. However, strategies for improving HA performance by functionalizing surfaces with bioactive factors are limited. In this study, we explored the use of a HA-binding domain (heptaglutamate, “E7”) to facilitate coupling of the collagen mimetic peptide, DGEA, to two types of HA-containing materials, solid HA disks and electrospun polycaprolactone matrices incorporating nanoparticulate HA. We found that the E7 domain directed significantly more peptide to the surface of HA and enhanced peptide retention on both materials in vitro. Moreover, E7-modified peptides were retained in vivo for at least two months, highlighting the potential of this mechanism as a sustained delivery system for bioactive peptides. Most importantly, E7-DGEA-coupled HA, as compared with DGEA-HA, enhanced the adhesion and osteoblastic differentiation of mesenchymal stem cells, and also increased new bone formation and direct bone-implant contact on HA disks implanted into rat tibiae. Collectively, these results support the use of E7-DGEA peptides to promote osteogenesis on HA substrates, and further suggest that the E7 domain can serve as a universal tool for anchoring a wide variety of bone regenerative molecules to any type of HA-containing material.
Keywords: Bone repair; Hydroxyapatite; Collagen; Peptide; Mesenchymal stem cell;

Extracellular matrix derived from human and animal tissues is being used to repair and reconstruct a variety of tissues clinically. The utility of such constructs is limited by the geometry, composition and constitutive properties of the tissue or organ from which the ECM is harvested. To address this limitation, we have developed an approach to isolate extracellular matrix in bulk from populations of living cells grown in culture on three-dimensional substrates. Human biopsy derived fibroblasts were seeded within open-cell foams and cultured in-vitro for periods up to three weeks, after which the synthetic component was removed by incubation in a water miscible solvent. After several wash steps and lyophilization, a white, lacy, multi-molecular construct was isolated. Tandem mass spectroscopy showed that it contained 22 extracellular matrix constituents, including such proteins and proteoglycans as collagen type I and type III, fibronectin, transforming growth factor beta, decorin and biglycan among others. On average 47 mg of construct was isolated for each gram of synthetic substrate initially seeded with cells. The biomaterial harvested from human tracheal fibroblasts had an elastic modulus (250 kPa) and a composition similar to that of human vocal fold tissue, and supported reseeding with human tracheal derived fibroblasts. An important finding was that the approach was useful in isolating ECM from a variety of cell lineages and developmental stages including skin fibroblasts, brain derived astrocytes and mesenchymal stem cells. The results, together with the archival literature, suggest that the approach can be used to produce a range of cell derived constructs with unique physical and chemical attributes for a variety of research and medical applications.
Keywords: ECM (extracellular matrix); Fibrous tissue; Fibroblast; Mesenchymal stem cell; Polyurethane; Scaffold;