Biomaterials (v.29, #11)

Functionalised amyloid fibrils for roles in cell adhesion by Sally L. Gras; Anna K. Tickler; Adam M. Squires; Glyn L. Devlin; Michael A. Horton; Christopher M. Dobson; Cait E. MacPhee (1553-1562).
We describe experiments designed to explore the possibility of using amyloid fibrils as new nanoscale biomaterials for promoting and exploiting cell adhesion, migration and differentiation in vitro. We created peptides that add the biological cell adhesion sequence (RGD) or a control sequence (RAD) to the C-terminus of an 11-residue peptide corresponding to residues 105–115 of the amyloidogenic protein transthyretin. These peptides readily self-assemble in aqueous solution to form amyloid fibrils, and X-ray fibre diffraction shows that they possess the same strand and sheet spacing in the characteristic cross-β structure as do fibrils formed by the parent peptide. We report that the fibrils containing the RGD sequence are bioactive and that these fibrils interact specifically with cells via the RGD group displayed on the fibril surface. As the design of such functionalized fibrils can be systematically altered, these findings suggest that it will be possible to generate nanomaterials based on amyloid fibrils that are tailored to promote interactions with a wide variety of cell types.
Keywords: Self-assembly; Nanotopography; XRD (X-ray diffraction); Fibrils; Bioactivity; RGD peptide;

The in vitro response of human osteoblasts to polyetheretherketone (PEEK) substrates compared to commercially pure titanium by Karen B. Sagomonyants; Marcus L. Jarman-Smith; John N. Devine; Michael S. Aronow; Gloria A. Gronowicz (1563-1572).
Polyetheretherketone (PEEK) is used as an alternative to titanium in medical devices. Previous in vitro studies examining PEEK have differed in their choice of polymer variant [PEEK or carbon-fiber reinforced PEEK (CFR-PEEK)], source of polymer (some of which are no longer available or for implantation) and cell type. While all studies demonstrated favorable cytocompatibility of the PEEK material, no studies are available which reflect the current state of the art of the material. Here, we use different forms of the only implantable grade PEEK available. These are compared with commercially pure titanium (cpTi) Grade 1 using a human primary osteoblast model. Sample materials were presented as industrially relevant surfaces. Machined or injection molded PEEK and CFR-PEEK were evaluated along with polished (Ra = 0.200 μm) and rough (Ra = 0.554 μm) cpTi. Osteoblast adhesion at 4 h on injection molded variants of PEEK (Ra = 0.095 μm) and CFR-PEEK (Ra = 0.350 μm) material was comparable to titanium. Machined variants of PEEK (Ra = 0.902 μm) and CFR-PEEK (Ra = 1.106 μm) materials were significantly less. Proliferation at 48 h determined by [3H]-thymidine incorporation was the greatest on the smoothest of all materials, the injection molded unfilled PEEK, which was significantly higher than the rough titanium control. The machined unfilled PEEK had the lowest DNA synthesis. RT-PCR for alkaline phosphatase, Type I collagen and osteocalcin normalized to glyceraldehyde-3-phosphate dehydrogenase revealed different patterns of mRNA levels. High mRNA levels for Type I collagen showed that CFR-PEEK stimulated osteoblast differentiation, whilst injection molded unfilled PEEK was less differentiated. Machined unfilled PEEK had comparable message levels of bone matrix proteins as rough titanium. All material variants permitted a degree of mineralization. Scanning electron microscopy at 3 days and 2 weeks in differentiation medium showed that human osteoblasts were well spread on all the different substrates. The varied response reported here at different time points during the study suggests that material formulation (unfilled PEEK or CFR-PEEK), subjection to industrial processing, surface roughness and topography may all influence the cellular response of osteoblasts to PEEK. Thus, differences in human osteoblast responses were found to the various samples of PEEK, but implantable grade PEEK, in general, was comparable in vitro to the bone forming capacity of rough titanium.
Keywords: Bone tissue engineering; Osteoblast; Polyetheretherketone (PEEK); Calcification; Cell proliferation; Titanium;

Gallium nitride (GaN) has been developed for a variety of microelectronic and optical applications due to its unique electric property and chemical stability. In the present study, n-type and p-type GaN were used as substrates to culture cerebellar granule neurons to examine the effect of GaN on cell response for a long-term culture period. It was found that GaN could rapidly induce cultured neurons to exhibit a high phosphorylated Akt level after 20 h of incubation. It was assumed that the anti-apoptotic effect of Akt phosphorylation could be correlated with cell survival, neurite growth and neuronal function for up to 35 days of incubation. Morphological studies showed GaN induced larger neuronal aggregates and neurite fasciculation to exhibit a dense fiber network after 8 days of incubation. Western blot analysis and immunocytochemical characterization showed that GaN still exhibited the expression of neurite growth and function, such as high levels of GAP-43, synapsin I and synaptophysin even after 35 days of incubation. In addition, survival of cerebellar granule neurons on GaN was improved by the analysis of lactate dehydrogenase (LDH) release from damaged cells. These results indicated that neuronal connections were formed on GaN by a gradual process from Akt activation and cell aggregation to develop neurite growth, fasciculation and function. Therefore, GaN offers a good model system to identify a well-characterized pattern of neuronal behavior for a long-term culture period, consistent with the development of a neurochip requiring the integration of biological system and semiconductor material.
Keywords: Gallium nitride (GaN); Cerebellar granule neurons; Long-term culture;

Fibroblast and macrophage are 2 dominant cell types respond cooperatively to degrade implanted biomaterials. Using an electrospun Dextran/Poly-lactide-co-glycolide (PLGA) scaffold as a model, an in vitro fibroblast/macrophage co-culture system was developed to investigate the degradability of implantable biodegradable materials. SEM showed that both fibroblasts and macrophages were able to degrade the scaffold, separately or cooperatively. Under the synergistic coordination of macrophages and fibroblasts, scaffolds showed faster degradation rate than their counterparts incubated with a single type of cells as well as in PBS or cell culture medium. Lysozyme, non-specific esterase (NSE), gelatinase, hyaluronidase-1 and α-glucosidase were up-regulated in the presence of the scaffold, suggesting their roles in the cell-mediated scaffold degradation. In addition, the expressions of cell surface receptors CD204 and Toll like receptor 4 (TLR4) were elevated 1 week after cell seeding, implying that these receptors might be involved in scaffold degradation. The results of in vivo subdermal implantation of the scaffold further confirmed the biodegradability of the Dextran/PLGA scaffold. The fibroblast/macrophage co-culture model adequately mimicked the in vivo environment and could be further developed into an in vitro tool for initial biomaterial evaluation.
Keywords: Fibroblasts; Macrophages; Co-culture; Biodegradation; Enzyme; Receptor;

Effects of FGF-2 release from a hydrogel polymer on bone mass and microarchitecture by Guillaume Mabilleau; Eric Aguado; Izabella C. Stancu; Corneliu Cincu; Michel F. Baslé; Daniel Chappard (1593-1600).
Bone substitutes are widely used for filling and restoring bone defects. Among them, methacrylic polymers are employed in load-bearing bones to seal hip prostheses. Incorporation of growth factors into a polymer device could be a way to enhance bone growth. In the present study, we evaluated the capacity of poly(2-hydroxyethyl methacrylate) – pHEMA – copolymerized with 2-vinyl pyrrolidone – VP – to release proteins. Fibroblast growth factor-2 (FGF-2) was incorporated into cylinders of p(HEMA-co-VP). FGF-2 release was studied by ELISA in vitro and cylinders were implanted in the femoral condyle of white New Zealand rabbits. After 2 months post-surgery, FGF-2 was able to enhance bone formation by increasing bone volume; this effect was evidenced by an increase in trabecular number and bone gain was mainly in the form of woven bone. At 3 months post-surgery, no difference could be evidenced between animals receiving vehicle or FGF-2. Animals receiving vehicle exhibited bone mass higher than at 2 months and woven bone was replaced by mature bone with a lamellar matrix. The hydrogel polymer allowed the release of FGF-2, which in return enhanced bone regeneration soon after surgery but the effect vanished rapidly.
Keywords: Controlled release; FGF-2; Histomorphometry; Growth factor; pHEMA;

Peripheral nerve regeneration within an asymmetrically porous PLGA/Pluronic F127 nerve guide conduit by Se Heang Oh; Jun Ho Kim; Kyu Sang Song; Byeong Hwa Jeon; Jin Hwan Yoon; Tae Beom Seo; Uk Namgung; Il Woo Lee; Jin Ho Lee (1601-1609).
Asymmetrically porous tubes with selective permeability and hydrophilicity as nerve guide conduits (NGCs) were fabricated using poly(lactic-co-glycolic acid) (PLGA) and Pluronic F127 by a modified immersion precipitation method. The inner surface of the tube had nano-size pores (∼50 nm) which can effectively prevent from fibrous tissue infiltration but permeate nutrients and retain neurotrophic factors, while the outer surface had micro-size pores (∼50 μm) which can allow vascular ingrowth for effective supply of nutrients into the tube. From the animal study using a rat model, the hydrophilized PLGA/F127 (3 wt%) tube showed better nerve regeneration behavior than the control silicone or hydrophobic PLGA tubes, as investigated by immunohistochemical observation (by fluorescent microscopy with anti-neurofilament staining), histological observations (by light microscopy with toluidine blue staining and transmission electron microscopy), and electrophysiological evaluation (by compound muscle action potential measurement). This is probably owing to the effective permeation of nutrients and prevention of fibrous scar tissue invasion as well as the good mechanical strength of the tube to maintain a stable support structure for the nerve regeneration.
Keywords: Peripheral nerve regeneration; Nerve guide conduit (NGC); Asymmetrically porous tube; Selective permeability; Hydrophilicity;

Response of mesenchymal stem cells to the biomechanical environment of the endothelium on a flexible tubular silicone substrate by Eoin D. O'Cearbhaill; Marie A. Punchard; Mary Murphy; Frank P. Barry; Peter E. McHugh; Valerie Barron (1610-1619).
Understanding the response of mesenchymal stem cells (MSCs) to forces in the vasculature is very important in the field of cardiovascular intervention for a number of reasons. These include the development of MSC seeded tissue engineered vascular grafts, targeted or systemic delivery of MSCs in the dynamic environment of the coronary artery and understanding the potential pathological calcifying role of mechanically conditioned multipotent cells already present in the vessel wall. In vivo, cells present in the coronary artery are exposed to the primary biomechanical forces of shear stress, radial stress and hoop stress. To date, many studies have examined the effect of these stresses in isolation, thereby not presenting the complete picture. Therefore, the main aim of this study is to examine the combined role of these stresses on MSC behaviour. To this end, a bioreactor was configured to expose MSCs seeded on flexible silicone substrates to physiological forces – namely, a pulsatile pressure between 40 and 120 mmHg (5.33–1.6 × 104  Pa), radial distention of 5% and a shear stress of 10 dyn/cm2 (1 Pa) at frequency of 1 Hz for up to 24 h. Thereafter, the ‘pseudovessel’ was assessed for changes in morphology, orientation and expression of endothelial and smooth muscle cell (SMC) specific markers. Hematoxylin and eosin (H&E) staining revealed that MSCs exhibit a similar mechanosensitive response to that of endothelial cells (ECs); they reorientate parallel with direction of flow and have adapted their morphology to be similar to that of ECs. However, gene expression results show the cells exhibit greater levels of SMC-associated markers α-smooth muscle actin and calponin (p  < 0.05).
Keywords: Mesenchymal stem cell; Silicone; Bioreactor; Endothelial cell; Smooth muscle cell; Soft tissue biomechanics;

The clinical application of autologous bioengineered skin based on a hyaluronic acid scaffold by Nicolò Scuderi; Maria G. Onesti; Giovanni Bistoni; Simona Ceccarelli; Sabrina Rotolo; Antonio Angeloni; Cinzia Marchese (1620-1629).
The aim of this work was to generate an in vitro skin substitute harbouring autologous fibroblasts, keratinocytes and melanocytes, to establish a new one-step clinical method in problems associated with skin disorders. Here we present a case of a nine-year-old girl with a congenital giant nevus treated by surgical approach, with primary co-cultures of keratinocytes, melanocytes and fibroblasts obtained from autologous skin biopsy. Generally these lesions need to be removed to avoid the risk of transformation into malignant melanoma. With this purpose we analyzed the melanocytes contained in the new skin substitute for the presence of genetic alterations correlated to increased risk for melanoma. The organotypical cultures were designed including an engineered scaffold of a non-woven mesh of hyaluronic acid (HYAFF®11). This biomaterial has been previously demonstrated to be the most suitable to maintain polarity and to support the in vitro constructs. Six dermal–epidermal skin substitutes were transplanted and 14 days after surgery the re-epithelialized area was about 90%. Our results suggest that this new dermal–epidermal construct not only reduces hospitalization time and ameliorates scar retraction, but might also represent a solution for the high risk of developing a tumour derived from the original nevus.
Keywords: Autologous cell; Co-culture; Hyaluronic acid scaffold; Transplantation;

Preparation and rheological characterization of a gel form of the porcine urinary bladder matrix by Donald O. Freytes; Jeffrey Martin; Sachin S. Velankar; Annie S. Lee; Stephen F. Badylak (1630-1637).
Biologic scaffolds composed of extracellular matrix (ECM) have been used to facilitate the repair and reconstruction of a variety of tissues in clinical and pre-clinical studies. The clinical utility of such scaffolds can be limited by the geometric and mechanical properties of the tissue or organ from which the ECM is harvested. An injectable gel form of ECM could potentially conform to any three-dimensional shape and could be delivered to sites of interest by minimally invasive techniques. The objectives of the present study were to prepare a gel form of ECM harvested from the urinary bladder (urinary bladder matrix or UBM), to characterize the rheological properties of the gel, and finally to evaluate the ability of the gel to support in vitro growth of smooth muscle cells. Following enzymatic solubilization with pepsin, UBM was induced to self-assemble into a gel when brought to physiological conditions. The UBM gel supported the adhesion and growth of rat aortic smooth muscle cells when cultured under static in vitro conditions. The present study showed that an intact form of UBM can be successfully solubilized without purification steps and induced to repolymerize into a gel form of the UBM biologic scaffold material.
Keywords: Extracellular matrix; Viscoelasticity; Rheology; Scaffold; Gel;

Inflammatory cytokine removal by an activated carbon device in a flowing system by Susan R. Sandeman; Carol A. Howell; Sergey V. Mikhalovsky; Gary J. Phillips; Andrew W. Lloyd; J. Graham Davies; Stephen R. Tennison; Anthony P. Rawlinson; Oleksaudr P. Kozynchenko (1638-1644).
A prototype in-line filtration/adsorption device has been developed using novel synthetic pyrolysed carbon monoliths with controlled mesoporous domains of 2–50 nm. Porosity was characterized by SEM and porosimetry. Removal of inflammatory cytokines TNF, IL-6, IL-1β and IL-8 was assessed by filtering cytokine spiked human plasma through the walls of the carbon modules under pressure. The effect of carbon filtration on plasma clotting response and total plasma protein concentration was also assessed. Significant removal of the cytokines IL-6, IL-1β and IL-8 was observed. Initially marked TNF removal diminished over time. The coagulation studies indicated that the carbon device does not exacerbate the propensity of blood plasma to clot. The total plasma protein concentration remained constant. The device offers a broader approach to the treatment of systemic inflammatory response syndrome (SIRS) by the removal of inflammatory mediators central to its progression.
Keywords: Adsorption; Cytokine; Clotting; Inflammation;

Bioprosthetic valves are used in thousands of heart valve replacement surgeries. Existing glutaraldehyde-crosslinked bioprosthetic valves fail due to either calcification or degeneration. Glutaraldehyde crosslinking does not stabilize valvular glycosaminoglycans (GAGs). GAGs, predominantly present in the medial spongiosa layer of native heart valve cusps, play an important role in regulating physico-mechanical behavior of the native cuspal tissue during dynamic motion. The primary objective of this study was to identify the role of cuspal GAGs in valve tissue buckling. Glutaraldehyde-crosslinked cusps showed extensive buckling compared to fresh, native cusps. Removal of GAGs by treatment with GAG-degrading enzymes led to a marked increase in buckling behavior in glutaraldehyde-crosslinked cusps. We demonstrate that the retention of valvular GAGs by carbodiimide crosslinking together with chemical attachment of neomycin trisulfate (a hyaluronidase inhibitor), prior to glutaraldehyde crosslinking, reduces the extent of buckling in bioprosthetic heart valves. Furthermore, following exposure to GAG-digestive enzymes, neomycin-trisulfate-bound cusps experienced no alterations in buckling behavior. Such moderate buckling patterns mimicked that of fresh, untreated cusps subjected to similar bending curvatures. Thus, GAG stabilization may subsequently improve the durability of these bioprostheses.
Keywords: Tissue buckling; GAGs; Neomycin; Carbodiimide;

This paper presents for the first time a mathematical model for a mechanism of controlled drug release involving both ion exchange and transient counter diffusion of a drug and counterions. Numerical analysis was conducted to study the effect of different factors on drug release kinetics including environmental condition, material properties, and design parameters. The concentration profiles of counterions and drug species, the moving front of ion exchange, and three distinct regions inside a microsphere, namely unextracted region, ion-exchange region and drug diffusion region, were revealed by model prediction. The numerical results indicated that the rate of drug release increased with an increase in the initial drug concentration in the microspheres, the salt concentration in the external solution, or the valence of the counterions, whereas it decreased with increasing Langmuir isotherm constant. The mathematical and experimental procedures for determination of the equilibrium constant and the usefulness of the model were demonstrated using verapamil hydrochloride and sulfopropyl dextran microsphere system as an example. This work has provided a very useful mathematical tool for predicting kinetics and equilibrium of drug release and for optimizing the design of ion-exchange drug delivery systems.
Keywords: Ion-exchange microspheres; Mathematical modeling; Numerical analysis of factors; Concentration profiles; Drug release kinetics; Experimental verification;

Preparation and cytotoxic activity of poly(ethylene glycol)-modified poly(amidoamine) dendrimers bearing adriamycin by Kenji Kono; Chie Kojima; Nobuyuki Hayashi; Eiko Nishisaka; Katsuyuki Kiura; Shinobu Watarai; Atsushi Harada (1664-1675).
We have developed poly(amidoamine) (PAMAM) dendrimers that have poly(ethylene glycol) (PEG) grafts at all dendrimer chain ends. To obtain PEG-modified dendrimers with sites for conjugation of anticancer drugs for this study, we prepared PAMAM G4 dendrimers that have a glutamic acid (Glu) residue at every chain end of dendrimer; PEG chains were attached to amino groups of Glu residues. We then combined the anticancer drug adriamycin to side chains of the Glu residues using an amide bond, [PEG–Glu(ADR)-G4], or hydrazone bond, [PEG–Glu(NHN–ADR)-G4]. For the dendrimers bearing adriamycin through amide linkage, adriamycin was released only slightly at pH 7.4 and 5.5. Although a negligible level of release occurred at pH 7.4 for dendrimers with adriamycin via hydrazone linkage, a remarkable extent of adriamycin release was induced at pH 5.5, which corresponds to the pH of late endosome. These adriamycin-bearing dendrimers showed much lower toxicity to HeLa cells than did free adriamycin. However, compared to PEG–Glu(ADR)-G4, PEG–Glu(NHN–ADR)-G4 exhibited 7 times higher cytotoxicity, suggesting the importance of pH-sensitive hydrazone linkage for high cytotoxicity. Furthermore, the PEG-modified dendrimers exhibited an equivalent level of toxicity to that of adriamycin-resistant SBC-3/ADR100 cells and their parent adriamycin-sensitive SBC-3 cells.
Keywords: Poly(amidoamine); Dendrimer; Drug delivery; Adriamycin; Poly(ethylene glycol); Chemotherapy;

Affinity manipulation of surface-conjugated RGD peptide to modulate binding of liposomes to activated platelets by Guofeng Huang; Zhongmin Zhou; Rekha Srinivasan; Marc S. Penn; Kandice Kottke-Marchant; Roger E. Marchant; Anirban S. Gupta (1676-1685).
Platelet adhesion, activation and fibrinogen-mediated aggregation are primary events in vascular thrombosis and occlusion. An injectable delivery system that can carry thrombolytics selectively to the sites of active platelet aggregation has immense potential in minimally invasive targeted therapy of vascular occlusion. To this end we are studying liposomes surface-modified by fibrinogen-mimetic RGD motifs that can selectively target and bind integrin GPIIb–IIIa on activated platelets. Here we report liposome surface-modification with a conformationally constrained high affinity cyclic RGD motif to modulate the GPIIb–IIIa-binding capability of the liposomes. Such affinity enhancement is important for practical in vivo applications to compete with native fibrinogen towards binding GPIIb–IIIa. The platelet-binding of RGD-modified liposomes was studied by fluorescence and scanning electron microscopy, and flow cytometry, in vitro. Binding of RGD-modified liposomes was also tested in vivo in a rat carotid injury model and analyzed ex vivo by fluorescence microscopy. The results from all experiments show that cyclic RGD-liposomes bind activated platelets significantly higher compared to linear RGD-liposomes. Hence, the results establish the feasibility of modulating the platelet-targeting and binding ability of vascularly targeted liposomes by manipulating the affinity of surface-modifying ligands.
Keywords: Cyclic RGD; Liposomes; Peptide modification; Platelet-targeting;

Osteotropic β-cyclodextrin for local bone regeneration by Xin-Ming Liu; Andrew T. Wiswall; John E. Rutledge; Mohammed P. Akhter; Diane M. Cullen; Richard A. Reinhardt; Dong Wang (1686-1692).
An osteotropic alendronate-β-cyclodextrin conjugate (ALN-β-CD) was developed as a bone-targeting delivery system for improved treatment of skeletal diseases. The conjugate shows very strong binding to hydroxyapatite (HA, main component of the skeleton). Its ability in forming molecular inclusion complex with prostaglandin E1 (PGE1, a potent bone anabolic agent) was confirmed by phase solubility experiments and differential scanning calorimetry (DSC). In a bilateral rat mandible model, ALN-β-CD/PGE1 molecular complex was shown to stimulate strong local bone anabolic reaction. In the control study, ALN-β-CD itself was also found to be bone anabolic. To investigate this finding, other control groups were studied. The histomorphometry data suggest that ALN-β-CD itself could generate more new bone at the injection site than its complex with PGE1. Alendronate (ALN) injection could also cause new bone formation, which locates peripheral to the site of injection. PGE1, saline or ethanol injections do not have anabolic effect. These findings were also confirmed by micro-CT evaluation of mandibular bones. It is clear that the bone anabolic effect of ALN-β-CD is independent of mechanical stimuli of the periosteum or ALN injection alone. Further studies are warranted to understand the working mechanism of ALN-β-CD as a bone anabolic agent.
Keywords: Bone regeneration; Osteoporosis; Hydroxyapatite; Drug delivery;

Hydrophobically derivatized hyperbranched polyglycerol as a human serum albumin substitute by Rajesh K. Kainthan; Johan Janzen; Jayachandran N. Kizhakkedathu; Dana V. Devine; Donald E. Brooks (1693-1704).
There is a huge clinical demand for Human Serum Albumin (HSA), with a world market of ∼$1.5B/year. Concern over prion and viral transmission in the blood supply has led to a need for safer substitutes and offers the opportunity for development of materials with enhanced properties over the presently available plasma expanders. We report here the synthesis and testing of a new synthetic plasma expander that can replace not only the osmotic and volume expansion properties of HSA but, uniquely, its binding and transport properties. We have synthesized several hyperbranched polyglycerols derivatized with hydrophobic groups and short poly(ethylene glycol) (PEG) chains. The hydrophobic groups provide regions for binding fatty acids and other hydrophobic materials while PEG imparts the necessary protection from host defense systems and enhances circulation longevity. These polymers, being hyperbranched, have only a small effect on plasma viscosity. We have shown in vitro that our materials bind 2–3 moles palmitic acid per mole, do not activate the platelet, coagulation or complement systems and do not cause red cell aggregation. In mice these materials are non-toxic with circulation half-lives as high as 34 h, controllable by manipulating the molecular weight and the degree of PEG derivatization.
Keywords: Plasma expander; Albumin substitute; Biocompatibility; Hyperbranched polyglycerols; Poly(ethylene glycol); Animal toxicity;

Reversible on-demand cell alignment using reconfigurable microtopography by Mai T. Lam; William C. Clem; Shuichi Takayama (1705-1712).
Traditional cell culture substrates consist of static, flat surfaces although in vivo, cells exist on various dynamic topographies. We report development of a reconfigurable microtopographical system compatible with cell culture that is comprised of reversible wavy microfeatures on poly(dimethylsiloxane). Robust reversibility of the wavy micropattern is induced on the cell culture customized substrate by first plasma oxidizing the substrate to create a thin, brittle film on the surface and then applying and releasing compressive strain, to introduce and remove the microfeatures, respectively. The reversible topography was able to align, unalign, and realign C2C12 myogenic cell line cells repeatedly on the same substrate within 24 h intervals, and did not inhibit cell differentiation. The flexibility and simplicity of the materials and methods presented here provide a broadly applicable capability by which to investigate and compare dynamic cellular processes not yet easily studied using conventional in vitro culture substrates.
Keywords: Surface topography; Micropatterning; Compression; Silicone elastomer; Poly(dimethylsiloxane); Muscle;

Incorporation of a matrix metalloproteinase-sensitive substrate into self-assembling peptides – A model for biofunctional scaffolds by Ying Chau; Ying Luo; Alex C.Y. Cheung; Yusuke Nagai; Shuguang Zhang; James B. Kobler; Steven M. Zeitels; Robert Langer (1713-1719).
Controlling and guiding cell behavior requires scaffolding materials capable of programming the three-dimensional (3-D) extracellular environment. In this study, we devised a new self-assembling peptide template for synthesizing nanofibrous hydrogels containing cell-responsive ligands. In particular, the insertion of a matrix metalloproteinase-2 (MMP-2) labile hexapeptide into the self-assembling building blocks of arginine-alanine-aspartate-alanine (RADA) was investigated. A series of peptides, varied by the position of the MMP-2 hexapeptide substrate and the length of RADA blocks, were prepared by parallel synthesis. Their self-assembling capabilities were characterized and compared by circular dichroism spectroscopy and dynamical mechanical analysis. Among all the different insertion patterns, the sequence comprising a centrically positioned MMP-2 substrate was flanked with three RADA units on each side self-assembled into a hydrogel matrix, with mechanical properties and nanofiber morphology comparable to the native material built with (RADA)4 alone. Exposure of the new gel to MMP-2 resulted in peptide cleavage, as confirmed by mass spectroscopy, and a decrease in surface hardness, as detected by nanoindentor, indicating that the enzyme mediated degradation was localized to the gel surface. The new design can be used for introducing biological functions into self-assembling peptides to create scaffolding materials with potential applications in areas such as tissue engineering and regenerative medicine.
Keywords: Self-assembly; Peptide; Biomimetic material; Matrix metalloproteinase; Nanofiber; Hydrogel;

The role of actively released fibrin-conjugated VEGF for VEGF receptor 2 gene activation and the enhancement of angiogenesis by Martin Ehrbar; Steffen M. Zeisberger; George P. Raeber; Jeffrey A. Hubbell; Christian Schnell; Andreas H. Zisch (1720-1729).
A major challenge for therapeutic delivery of angiogenic agents such as vascular endothelial growth factor (VEGF) is to achieve sustained, low dose signaling leading to durable neovessel formation. To this end, we recently created a variant of VEGF121, TG-VEGF121 that directly binds to fibrin and gets released locally in proteolysis-triggered manner. Here we combined noninvasive biophotonic monitoring of VEGF receptor 2 gene activation in transgenic VEGFR2-luc mice and histomorphometry to compare endothelial activation and long-term neovascularization by actively released TG-VEGF121 versus passively released, diffusible wild-type VEGF121 in subcutaneous fibrin implants. Monitoring in real-time over 3 weeks of luciferase signal driven by the VEGFR2 promoter revealed endothelial activation in skin exposed to wild-type VEGF121, but no detectable elevation over fibrin alone by TG-VEGF121. Histology at 3 weeks, however, demonstrated that TG-VEGF121 promoted vessel growth significantly more effectively and reliably than wild-type VEGF121. The majority of vessels surviving to 3 weeks contained stabilizing smooth muscle cells. Yet, by 6 weeks, no extra vessels induced by exogenous VEGF were left. In conclusion, release of fibrin-conjugated variant TG-VEGF121 elicited lower VEGFR2-luc activation than wild-type VEGF121 yet significantly more vascularization. In the absence of true physiological demand, even stabilized vessels are ultimately regressed.
Keywords: Therapeutic angiogenesis; Vascular endothelial growth factor; Fibrin; Biomimetic materials; VEGF receptor 2 gene activation;

The response of bone to nanocrystalline hydroxyapatite-coated Ti13Nb11Zr alloy in an animal model by Adriana Bigi; Milena Fini; Barbara Bracci; Elisa Boanini; Paola Torricelli; Gianluca Giavaresi; Nicolò N. Aldini; Alessandro Facchini; Fausto Sbaiz; Roberto Giardino (1730-1736).
An in vivo study was carried out on uncoated and hydroxyapatite (HA)-coated nanostructured Ti13Nb11Zr alloy in comparison with high-grade Ti6Al4V, to investigate the effect of the different surfaces on osteointegration rate. A highly effective method to obtain a fast biomimetic deposition of a thin layer of nanocrystalline HA was applied to coat both substrates. Cylindrical pins were implanted in rabbit cortical bone and evaluated at 4 and 12 weeks by histomorphometry and microhardness tests. The results confirmed the ability of the slightly supersaturated Ca/P solution to induce a fast deposition of nanocrystalline HA on Ti alloys' surfaces. HA-coated Ti13Nb11Zr had the highest osteointegration rate at 4 and 12 weeks. Both HA-coated surfaces showed an affinity index significantly higher than those of native surfaces at 4 weeks (Ti13Nb11Zr + HA: 37%; Ti6Al4V + HA: 26%). Microhardness test showed a significantly higher bone mineralization index of HA-coated Ti13Nb11Zr in comparison with that of HA-coated Ti6Al4V surface. The study suggests that the HA coating on both alloys enhances bone response around implants and that there is a synergic effect of Ti–Nb–Zr alloy with the HA coating on bone remodeling and maturation.
Keywords: Titanium alloys; Niobium; Biomimetic material; Hydroxyapatite; Osteointegration;